f Of y%%S\ 
 
 Division of Agricultural Sciences 
 UNIVERSITY OF CALIFORNIA 
 
 i 
 
 15 
 
 j. 
 
 The AUTORADIOGRAPHY 
 of PLANT MATERIALS 
 
 ALDEN S. CRAFTS • SHOGO YAMAGUCH 
 
 CALIFORNIA AGRICULTURAL 
 Experiment Station 
 Extension Service 
 
 UNIVERSITY OF CALIFORNIA 
 DAVIS 
 
 JUL 2 1964 
 U&RARY 
 
 MANUAL 35 
 
 Price $1.75 
 
The work described here was supported by The Atomic Energy 
 Commission under contract AT (11-1) -34 Project Nos. 9 and 38. 
 
The AUTORADIOGRAPHY 
 of PLANT MATERIALS 
 
 ALDEN S. CRAFTS • SHOGO YAMAGUCHI 
 UNIVERSITY OF CALIFORNIA • DIVISION OF AGRICULTURAL SCIENCES 
 
 THE AUTHORS: 
 
 A. S. Crafts is Professor of Botany and Botanist in the Experiment Station, 
 Davis; Shogo Yamaguchi is Assistant Research Botanist in the Department of 
 Botany, Davis. 
 
 >F CALIFORNIA 
 
 PAV55 
 
CONTENTS 
 
 Preface 3 
 
 Chapter I. Introduction 4 
 
 SECTION A: METHODS 
 
 Chapter II. Methods of Autoradiography 7 
 
 Chapter III. Autoradiography: Interpretation of Results .... 34 
 
 SECTION B: PROBLEMS AND SOLUTIONS 
 
 Chapter IV. Mechanism of Translocation 39 
 
 Chapter V. Evidences for Mass Flow in the Phloem 42 
 
 Chapter VI. Comparative Movement of Tracers 52 
 
 Chapter VII. Root Absorption and Xylem Transport 62 
 
 Chapter VIII. Comparative Movement of Tracers 
 
 Via Shoot and Root 71 
 
 Chapter IX. Effects of time and Dosage on Transport 82 
 
 Chapter X. Interaction of Herbicidal Molecules 87 
 
 Chapter XI. Symplastic and Apoplastic Movement 96 
 
 Chapter XII. Studies on Herbicide Movement in Woody Plants. . . 104 
 
 Chapter XIII. Translocation in Grasses and Coffee Plants .... 113 
 
 Chapter XIV. Translocation of Some Amino Acids 
 
 In Two Barley Varieties 120 
 
 Chapter XV. Role of Formulation Additives in 
 
 Absorption and Translocation of Herbicides .... 126 
 
 Chapter XVI. Conclusions 133 
 
 Glossary of Terms 136 
 
 Literature Cited 138 
 
 Unless otherwise noted, all illustrations of mounted plants and their 
 autographs in this publication show autographs on top and mounts below. 
 
 JUNE, 1964 
 
PREFACE 
 
 liant autoradiography is a new and 
 revolutionary scientific technique, and 
 the authors of this publication are proud 
 and grateful that they have been able to 
 contribute to it by developing the freeze- 
 drying method of plant preparation and by 
 discovering refinements in technique that 
 make critical studies on intact plants 
 possible. The ability to study distribu- 
 tion patterns of labeled tracers in such 
 plants has resulted in research of out- 
 standing importance, and it is these 
 techniques and studies which are the sub- 
 ject of the present volume. 
 
 The work described herein provides 
 strong evidence for a mass-flow type of 
 mechanism to explain food and tracer 
 movement in plants. This evidence, to- 
 gether with the new anatomical informa- 
 tion furnished by electron microscopy, 
 provides a picture of the mechanics of 
 solute and water movement in plants as 
 illuminating to botanists as were the data 
 supplied to animal workers by the blood 
 circulation studies instituted by Harvey 
 and extended by others. 
 
 Botanists have struggled for 100 
 years trying to rationalize the structure- 
 function relations of the phloem of plants. 
 This tissue, ramifying throughout the 
 organized plant body and extending within 
 microns of the apical meristems, provides 
 for the distribution of foods to all liv- 
 ing cells. Evidence for a correlated 
 movement of solutes and solvent throughout 
 this tissue system has been strengthened 
 by autoradiographic studies on whole 
 intact plants. Evidence from the electron 
 microscope indicates that the last ser- 
 ious barrier to acceptance of this mech- 
 anism - the nature of the protoplasmic 
 strands that traverse the sieve plates - 
 has been removed; Esau and her students 
 have demonstrated that these strands, in 
 the functioning condition, are tubular. 
 
 Interpretations presented throughout 
 the volume and stressed in chapters III 
 and XVI are derived not only from the 
 autoradiographic studies but also from 
 observations of plot work and physiologi- 
 cal information gleaned from the litera- 
 ture. This is the result of the broad 
 field of study undertaken at the Univer- 
 sity of California at Davis, and the con- 
 stant attempt being made to integrate lab- 
 oratory research and field studies. It is 
 sincerely hoped that plant physiologists 
 will find satisfaction in the use made of 
 their science in the field of herbicide 
 mechanism, that researchers in weed sci- 
 ence will find elucidation of some per- 
 plexing problems of herbicide physiology, 
 and that agriculturists will be aided 
 and encouraged by the valuable informa- 
 tion obtained from modern isotope techni- 
 ques. 
 
 The writers wish to acknowledge with 
 gratitude the help from colleagues and 
 students, including Charles McCarthy, 
 Barbara Kean, Douglas Steward, Otto Ander- 
 son, Mahmood Clor, Myra Mihajlovic, Ches- 
 ter Foy, Henry De Stigter, Fred Slife, 
 Joe Key, Bernard Forde, Jose Pereira, 
 George Mason, Ri chard Hull, and Fred Boyd. 
 
 We also appreciate the donation of 
 labeled compounds by the following organi- 
 zations: Am Chem Products, Inc., American 
 Cyanamid Co. , Chipman Chemical Co. , Dia- 
 mond Alkali Co., Dow Chemical Co., E. I. 
 du Pont de Nemours and Co. , Geigy Agricul- 
 tural Chemicals, Naugatuck Chemical Divi- 
 sion of the U. S. Rubber Co. , Pineapple 
 Research Institute , Rohm and Haas Co. , 
 Spencer Chemical Co. , Stauffer Chemical 
 Co. 
 
 Davis, California 
 December 19, 1963 
 
SECTION A -- Methods 
 
 Chapter I. Introduction 
 
 A, 
 
 icquainting young people with some 
 of the exciting discoveries that man has 
 made in the exploration of his world is 
 both a privilege and a challenge for the 
 teacher. On several occasions it has 
 been our pleasure to explain to young 
 people what botany is and what botanists 
 do - and always these young people are 
 eager to learn more of the world of 
 plants and of useful products of forest 
 and range and cultivated field. The 
 story of photosynthesis, the process by 
 which plants capture the sun's energy and 
 utilize it to produce chemical products 
 essential to the whole biological world, 
 is always thrilling to the scientifically 
 inclined. A description of the processes 
 of absorption and translocation by means 
 of which plants are able to acquire min- 
 erals from their environment and utilize 
 them to develop their own bodies is sti- 
 mulating to students with aptitudes for 
 chemistry or engineering. And, finally, 
 to describe the history of early research 
 on transpiration, the rise of sap in tall 
 trees, and the movement of food in plants, 
 is always to awaken interest in those who 
 like mechanics. 
 
 In these days of higher specializa- 
 tion, it is well to remember that the 
 food we eat and the clothes we wear are 
 largely the result of the coordinated 
 activities of whole plants. Therefore, 
 research which seeks a complete picture 
 of plant functions through study of the 
 integrated activities of particulates, 
 organelles, and grosser plant structures, 
 is of the first importance. This volume 
 describes discoveries that have filled 
 many gaps in our knowledge of whole-plant 
 function, and documents a new technique 
 for the study of plants. 
 
 In all probability plants originated 
 as single cells in the sea. The first 
 
 cell involved a staggering number of 
 structure- function relationships, and 
 modern higher plants have all of these 
 relationships plus countless more. It is 
 through the development of diverse struc- 
 tures and their impact on plant function 
 that many plants in the sea grew huge, 
 that certain sea plants invaded the land, 
 and that, on land, plants have been able 
 to live in the tropical and the polar re- 
 gions, in the osmotic concentration of 
 saline lakes, and in montane environments. 
 
 Botanists were early intrigued by 
 the great variety of form found in plants; 
 later they delved into the functions of 
 these myriad structures; now they pursue 
 structure to its ultimate molecular state 
 as they study the fundamental chemical and 
 physical bases of function. In these 
 studies one of the most useful of the 
 newer tools is the radioactive isotope. 
 By using isotopic labeling, innumerable 
 natural and synthetic compounds may be 
 converted to tracers that enable the 
 biochemist and plant physiologist to ex- 
 plore areas beyond the reach of older 
 methods. 
 
 The use of naturally-occuring radio- 
 active isotopes in medicine is well known. 
 The wider use of artificially-produced 
 isotopes awaited their large-scale pro- 
 duction in accelerators of various designs. 
 By using neutrons to bombard elements, E. 
 Fermi and his associates had contributed 
 many new isotopes to science by the mid- 
 19 30's; by the end of 1935 about 100 
 artificial radioactive elements had been 
 discovered. Since then, the development 
 of the cyclotron has resulted in produc- 
 tion of many radioactive isotopes, and by 
 19H7 over 1,000 induced radioactive iso- 
 topes were known. The attainment of the 
 self-sustaining nuclear chain reaction 
 with uranium marked another milestone in 
 
the field of artificial radioactivity. 
 
 Ruben and Kamen (1940, 1941) repor- 
 ted the discovery of carbon- 14 in 1940- 
 41, but this useful isotope was not gener- 
 ally available until an inexpensive method 
 of production by the use of the uranium 
 reactor was perfected. By 1947 the Uni- 
 ted States Atomic Energy Commission Price 
 List No. 2 listed 74 radioactive isotopes 
 and the price of a millicurie of carbon- 
 14 as barium carbonate was down to $50.00. 
 
 Today there are a great number of 
 radioactive isotopes available for re- 
 search, and progress in their application 
 in a multitude of uses has been rapid. 
 In addition to carbon- 14 with its extreme- 
 ly long half- life of about 5,000 years, 
 sulfur- 35 , chlorine- 36 , phosphorus- 32 , 
 calcium-45, and zinc-65 have proved very 
 useful as tracers in plant physiological 
 research. These materials have enabled 
 scientists to make outstanding advances 
 in such fields as photosynthesis, respi- 
 ration, ion uptake and transport, trans- 
 location of organic nutrients, and in the 
 synthesis and use of labeled compounds of 
 physiological activity, including enzymes, 
 co- factors, growth regulators, and inhi- 
 bitors . 
 
 When used on plants dried in the fro- 
 zen condition, autoradiography gives an 
 accurate picture of the distribution of 
 a given tracer in a plant that was intact 
 at the time of treatment and subject to 
 no more drastic treatment than the appli- 
 cation of a droplet of solution to a leaf 
 or stem, or inclusion of the tracer in the 
 culture solution around the roots (Crafts 
 and Yamaguchi, 1960b). So far as the 
 authors have been able to determine, 
 radioactive tracers in the quantities 
 used have no deleterious effect upon the 
 plant tissues because of their radioac- 
 tivity. On the other hand, stimulators 
 or inhibitors of growth, or physiologi- 
 cal agents that cause chlorosis, inhibi- 
 tion of oxygen evolution, cessation of 
 mitosis, or contact injury, seem to be 
 unchanged in their activity by radioacti- 
 vity of tracer elements included in their 
 composition. 
 
 Here it should be emphasized that 
 autoradiography is only one method for 
 studying plants; for a complete record of 
 plant behavior, counting, extraction, 
 
 chromatography and other methods should 
 be used. When long-lived tracer elements 
 are used it is possible to autograph 
 plants and then to carry out more accurate 
 quantitative determinations upon them. 
 When short-lived tracers are used, paral- 
 lel treatments may be made so that count- 
 ing or chromatography can be carried out 
 while autoradiographs are being exposed. 
 And, finally, where a detailed determina- 
 tion of the exact locus of uptake and 
 transport is desired, histoautoradiography 
 (autoradiography of sectioned materials) 
 can be used to pinpoint the channels of 
 transport and the final site of action of 
 a labeled compound. 
 
 While the autograph of a treated plant 
 gives a complete picture of the distribu- 
 tion of a mobile compound in the plant, 
 quantitative interpretation of the picture 
 obtained must be made with caution. As 
 explained by Yamaguchi and Crafts (1958), 
 the autographic image ranges from a light 
 trace to an opaque black area. Foy (1958) 
 found a close correlation of intensity 
 with counts within the range of visible 
 differences. Beyond the first opaque im- 
 age, no further estimate of quantity of 
 tracer can be made from a given autograph. 
 By using different exposure times on the 
 film, varying intensities of image may be 
 had. Counting on the plant materials may 
 also be used to determine radiation den- 
 sity. With most plant materials, incin- 
 eration or extraction is not necessary; 
 by construction of a standard self-absorp- 
 tion curve and standardization of tech- 
 nique, quite accurate counting can be 
 done on ground plant materials (Foy, 
 1960). 
 
 The techniques used in plant autora- 
 diography are relatively simple; they are 
 easily taught, and equipment is relatively 
 inexpensive. The screen trays, the vacuum 
 chamber, the plant press and the materials 
 used in the final exposure of the X-ray 
 films are easily obtained and rather 
 simply fabricated. The original steel 
 vacuum tank (Yamaguchi and Crafts, 1958, 
 fig. 14) has recently been supplemented 
 by tanks of light metal, and the original 
 simple wooden press has been replaced by 
 a steel screw press. Additionally, the 
 original sponge rubber separators used in 
 the autographing bundle may be replaced 
 with polyethylene sponge sheets, and the 
 plywood separators with aluminum sheets. 
 
Other such improvements will be explained 
 later in this publication. 
 
 Results obtained by autoradiography 
 are striking, and often exciting: evi- 
 dence has been produced for hydrolysis of 
 esters (Crafts, 1959a), for apoplastic 
 and symplastic movement of assimilates 
 and tracers (Crafts, 1961b), for general 
 vs. localized distribution of compounds 
 in plants (Yamaguchi and Crafts, 1958, 
 fig. 10 vs. fig. 17), for a correlation 
 of tracer movement with food movement, 
 and for widely differing patterns of dis- 
 tribution of different tracer molecules. 
 As new labeled compounds are synthesized 
 their behavior in the plant can readily 
 be studied, and in the field of pesticide 
 research the role of translocation in 
 systemic action can be rapidly determined. 
 Thus new and valuable information is con- 
 stantly being supplied by scientists 
 working with isotopic labeled compounds. 
 Indeed, use of labeled tracers in plants 
 is proving to be one of the major advan- 
 ces in scientific technology. 
 
 The technique of interpreting plant 
 autoradiographs has made rapid strides. 
 Just as the physician studying X-rays 
 may find evidence of injuries, abnormali- 
 ties, malignancy, and disease, so the 
 investigator studying plant autoradio- 
 graphs has learned to recognize many sig- 
 nificant signs of important physiological 
 
 processes. The bypassing of mature leaves 
 by tracers undergoing symplastic movement 
 in the phloem, the intense labeling of 
 buds, shoot tips, and root tips, the inter- 
 interaction of one compound upon movement 
 of a second labeled compound, the rela- 
 tion of acre-petal to basipetal movement 
 in stems, the accumulation of some com- 
 pounds and the fixing of others in non- 
 living tissues, and finally, the faint 
 labeling of untreated leaves by compounds 
 that characteristically migrate from 
 phloem to xylem - all are examples of the 
 types of response that lend themselves to 
 such interpretations. 
 
 Other techniques are also relevant 
 in plant studies: the evolution of 
 C1402 from tracer- treated plants, the de- 
 termination of metabolic products of tra- 
 cer denaturation by chromatography, and 
 the use of 0-^02 to study disturbed paths 
 of metabolism in pesticide-treated plants, 
 are some examples. Histoautoradiography 
 is useful in studying the increase of 
 various constituents in developing tissues 
 and cells. We have found its application 
 to problems of uptake, distribution, and 
 metabolism very difficult. The point 
 which we wish to emphasize here is the 
 great usefulness of gross autoradiography 
 in studying the initial penetration, the 
 rapid translocation, and the ultimate dis- 
 tribution of various compounds in plants. 
 
Chapter II. Methods of Autoradiography 
 
 PLANT MATERIALS 
 
 A: 
 
 .lmost any plant species that lends 
 itself to greenhouse culture can be used 
 for autoradiography. Where it was desir- 
 able to use small intact plants, the 
 authors grew them from seed in nutrient 
 culture. The seeds may be germinated in 
 moist, washed sand or on cheese cloth 
 suspended above a culture solution. As 
 soon as the roots are long enough (usual- 
 ly 2-3 inches) the seedlings, held in 
 holes in wide corks by cotton, are arran- 
 ged in culture jars (fig. 11,1) or, if 
 details of the root systems are desired, 
 are attached with masking tape to filter 
 paper supported by glass plates (fig. II, 
 2). Barley, oats, corn, bean, cotton, 
 soybean, nutgrass, sugar beet, tomato, 
 Bermudagrass , rye grass, quackgrass, 
 alfalfa, Zebrina pendula and Tradescantia 
 fluminensis have proved convenient, Zeb- 
 rina has hypos tomatous leaves, so that 
 cuticular vs. stomatal absorption can be 
 readily studied; tradescantia comes in a 
 number of variegated varieties and has 
 been used to study translocation from 
 green vs. chlorotic leaves (Crafts, 1961 
 b). Both are very easily propagated in 
 the greenhouse, their growth can be readi- 
 ly regulated by the nutrient level provi- 
 ded, and they endure long periods of 
 relative inactivity without loss of 
 leaves when nutrients are depleted; they 
 grow readily in soil, sand, or water cul- 
 ture. 
 
 Woody plants have been used both in 
 the greenhouse and in the field (Clor, 
 1959; Leonard and Crafts, 1956; Yamaguchi 
 and Crafts, 1959). Seedlings of oaks and 
 coffee have been grown and treated in the 
 greenhouse. Large shrubs and trees have 
 been used in the field, and here careful 
 
 handling of the labeled compounds is es- 
 sential. (In working in state and na- 
 tional forests, permission should be ob- 
 tained from the proper authorities before 
 using isotopes). 
 
 In treating plants for autoradiogra- 
 phy, thought should be given to convenient 
 film sizes. The standard 10 x 12-inch 
 X-ray film is very convenient and seed- 
 lings of many plants lend themselves to 
 these proportions. When larger plants are 
 needed, two 10 x 12-inch films can be 
 placed end to end to accommodate a plant 
 that is 24 inches long, or can be placed 
 side to side to give an area 12 x 20 in- 
 
 Fig. II, 1. Solution cultures of bar- 
 ley, cotton, and soybean. Culture solu- 
 tions have been reduced to a volume of 
 100 ml each in preparation for root treat 
 ment with a labeled tracer. 
 
Fig. II, 2. Culture of Zebrina pendula roots on filter paper. Plants are grown to 
 proper size (left). Transfer to paper is made by floating roots in water, inserting 
 paper-covered glass sheet, tipping tray to leave roots on paper, fastening plant to 
 glass-backed paper with masking tape (center), and inserting in darkened glass cylinder 
 (right) which contains culture solution. 
 
 ches. A 14 x 17-inch film commonly used 
 for chest X-rays is also available. 
 
 PL/WT CULTURE 
 
 Convenience of culture is often a 
 factor in the selection of plant species 
 for experimental use. One-pint jars are 
 very useful, and careful control of solu- 
 tion level, with the upper portion of the 
 root system being exposed, may eliminate 
 the need for forced aeration; such aera- 
 
 tion is to be avoided in cultures having 
 tracer applied via the culture solution, 
 as the resultant splattering causes conta- 
 mination. Plants which have proved useful 
 here are red kidney bean, soybean, barley, 
 cotton, corn, wild buckwheat, field bind- 
 weed, tradescantia, and zebrina; undoubt- 
 edly many other species will do as well. 
 
 In starting an experiment, two or 
 more times the required number of seeds 
 is used, and at the time of transfer to 
 
culture jars rigorous selection is made 
 in order to have uniform material. Care- 
 ful examination for uniformity of vigor 
 and development and equality of root 
 growth reduces replication to a minimum. 
 Where series experiments are performed 
 using concentration, dosage, time, locus 
 of treatment, or similar factors as vari- 
 ables, single plants are often used with 
 perfectly satisfactory results. 
 
 In transferring plants from sand to 
 culture jars, roots 2 or 3 inches long 
 are preferable - if shorter, roots may be 
 too small to be handled conveniently; if 
 longer, they become intertwined and break 
 when separated. Figure II, 3, shows bean 
 cultures in the most convenient stage for 
 establishment. 
 
 Plants may be treated at any stage of 
 development. Bean plants (Phaseolus vul- 
 garis var. Red Kidney) usually have the 
 primary leaves fully expanded 11 to 14 
 days after planting; barley commonly re- 
 quires 14 to 20 days to reach a proper 
 stage for treatment. Under greenhouse 
 conditions the culture solution may not 
 require replenishing. If the treatment 
 period is to be over 10 days the culture 
 
 solution should be renewed just prior to 
 treatment . 
 
 Nutrient-solution culture assures 
 uniformity of plant growth, ease of har- 
 vest, freedom from root breakage and con- 
 venient control of inorganic nutrition. 
 Hoagland's formula has been used in our 
 work; barley grows well in full strength 
 solution; beans and cotton do best in 
 half-strength solution. Iron is added 
 as Fe2S04« 
 
 FILTER PAPER CULTURE 
 
 Filter paper culture is used to 
 study details of root structure or accur- 
 ate location of a tracer in roots follow- 
 ing foliar application. Barley, zebrina, 
 and rape plants have been employed, and 
 others would probably be suitable. Plants 
 for these cultures are started as usual, 
 then transferred to culture solution and 
 grown until the roots are 3 to 4 inches 
 long. At the time of transfer to filter 
 paper culture, filter paper of double or 
 treble thickness is taped to long glass 
 sheets that fit the culture jars. These 
 culture plates are then partially im- 
 mersed in a flat container of water, the 
 
 Fig. II, 3. Bean plants at proper stage for transfer from sand to water culture, 
 
plant to be mounted is placed in the 
 water, and the roots are spread in the 
 proper position. The culture plate is 
 then slowly raised from the water, leav- 
 ing the roots adhering to the filter 
 paper, and the base of the plant is af- 
 fixed to the culture plate with masking 
 tape. When the roots have been properly 
 arranged, the culture plate carrying the 
 plant is carefully lowered into its cul- 
 ture jar and enough culture solution 
 added to come to within an inch or two of 
 the root tips. The solution continues to 
 moisten the filter paper by capillary 
 action, and must be replenished to make 
 up for loss from plant transpiration. 
 
 The culture jar should be covered 
 with aluminum foil, and a sheet of alu- 
 minum foil (split so as to surround the 
 plant stem) is used to close the jar on 
 top. If the culture is to be maintained 
 for an extended period, the filter paper 
 should be sterilized in a 1 per cent 
 sodium hypochlorite solution for 10 min- 
 utes and thoroughly rinsed; this will 
 restrain bacterial and fungal growth. 
 
 TREATMENT 
 
 LABELED COMPOUNDS 
 
 Organic compounds labeled with C^ 4 , 
 S 35 , or Cl 3 ^ are satisfactory for auto- 
 radiography. The inorganic isotopes P32 9 
 Ca.^ 9 Znb5, Fe59 s As?? , and others, are 
 also adaptable for autoradiographic use. 1 
 Although most manufacturers of labeled 
 compounds provide materials of high puri- 
 ty, purity cannot be taken for granted; 
 purity and specific activity may be as- 
 sayed by paper chromatography, autoradio- 
 graphy of the chromatograms , and GM 
 counting, direct or after extraction. 
 
 For ascending chromatography, 1 ul 
 volumes of stock or treatment solutions 
 are spotted 1 inch from the edge of 
 Whatman #1 filter paper measuring 10 x 16 
 inches, and the paper is joined into a 
 cylinder and placed in a glass vessel 
 measuring 6 x 18 inches. A solvent system 
 useful for several herbicidal compounds 
 is isopropanol-NHL|Ac 20 per cent in water 
 glacial acetic acid, 79:20:1. Fifty to 
 100 ml of solvent are required and devel- 
 
 opment time at 72 degrees Fahrenheit is 
 18 to 24 hours. 
 
 Counting is done on 1 jul samples of 
 stock or treatment solutions slowly 
 pipetted onto discs of lens paper in plan- 
 chets. One-minute counts are usually 
 adequate . 
 
 For most purposes, a specific activi- 
 ty of 0.5 to 1.0 mc/mmole is adequate for 
 autoradiography. With highly toxic com- 
 pounds, such as the chlorophenoxy acids, 
 a higher activity ranging to 10.0 mc/- 
 mmole may be required if physiological 
 injury is to be kept to a minimum and a 
 satisfactory autograph obtained. Where 
 a number of compounds are being used com- 
 paratively it is best to standardize the 
 specific activity; here, the compound 
 having the lowest activity determines 
 the dosage. An alternative method for 
 using compounds of differing specific 
 activity is to vary the exposure time of 
 the treated plants on the film, using an 
 inverse relation between activity and 
 time. 
 
 Many labeled compounds are available 
 for autoradiographic use. Isotopes or 
 labeled compounds, or both, are sold by 
 several chemical companies, by a number 
 of organizations specializing in work 
 with isotopes, and by the United States 
 Atomic Energy Commission at Oak Ridge, 
 Tennessee. These materials are distrib- 
 uted in various quantities; common pack- 
 aged quantities are millicurie lots and 
 50 microcurie quantities, although P 3 ^, 
 Cl4-barium carbonate, and other materials 
 may be obtained in much greater quantities 
 In plant autoradiography individual doses 
 often range from 0.5 uc to 0.05 juc; these 
 doses correspond to 0.5 jumole to 0.05 
 Aimole of a material having 1 mc per mmole 
 (a common specific activity). Figure 
 II, 4, gives the range of activity for 
 Cl4« containing compounds. 
 
 VOLUME 
 
 Application for many of the treat- 
 ments reported herein is in the form of 
 a 10 Ail droplet. Volumes may vary, but 
 if too great in a given application the 
 solution may run and cover an undefined 
 
 1 See GLOSSARY, page 136, for description of technical terms and trade names 
 used in this publication. 
 
 10 
 
Autoradiography with Carbon-l4 
 
 Fig. II, k. Range of activity (shaded 
 region) most useful for autoradiography 
 with carbon-14. Range runs from 14-days 
 exposure for a solution producing 3 counts 
 per minute, to 1 day for a solution pro- 
 ducing 4200 counts per minute. Activities 
 outside this range may be used, but are 
 i nconvenient . 
 
 area. Treatments are usually placed with- 
 in a lanolin ring about 0.5 cm in dia- 
 meter; under warm weather conditions, the 
 lanolin is thickened with dry starch or 
 water so that it will not flow. Where 
 a larger dose is desired, high specific- 
 activity tracer may be used; another 
 method is to apply several drops at 
 intervals, allowing time for drying or 
 absorption of droplets between applica- 
 tions . 
 
 STOCK SOLUTIONS 
 
 Because only a few micrograms of 
 chemicals may be used in one experiment, 
 it is convenient to store the original 
 compounds in stock solutions involving, 
 often, several milligrams of the compound, 
 and to make up treatment solutions from 
 these by dilution. Because some labeled 
 compounds break down as a result of self- 
 irradiation, our practice is to prepare 
 them in stock solutions in acetone and to 
 store the solutions in sealed ampules in 
 a refrigerator (fig. II, 5). The com- 
 pounds are weighed on a microbalance 
 using micro-weighing dishes and a micro- 
 spatula, or on an analytical balance hav- 
 ing a special support to hold the con- 
 tainer (fig. II, 6). To weigh, set the 
 
 balance at approximately the desired 
 amount, remove container top and care- 
 fully transfer tracer to the aluminum 
 boat until the balance tips. Weigh the 
 amount in the boat, dissolve in the proper 
 volume of solvent and store in an ampule. 
 From the stock solution, small aliquots 
 may be taken with a micropipette to make 
 up treatment solutions. 
 
 Some stock solutions may be made up 
 in alcohol, some in acetone, some in 
 water, etc. , depending upon the solubil- 
 ity of the compounds. If not objection- 
 able, 50 per cent alcohol is preferred 
 over aqueous solutions because droplets 
 of the alcoholic solvent do not adhere 
 to the walls of the container, nor does 
 this solvent support bacterial growth. 
 If a stock solution is to be kept in a 
 stoppered tube the stopper should be 
 thoroughly coated with high-vacuum sili- 
 cone grease to prevent loss of solvent. 
 Stock solutions are usually made up to 
 50 uc per ml in quantities of 1 to 2 ml 
 and labeled with the name of the chemi- 
 cal, its specific activity, its concen- 
 tration in ppm, its weight and number of 
 microcuries per unit volume, the name of 
 the solvent, and the date; stock solutions 
 are also often made up to solute concen- 
 tration of 10~lM, or 100 yumole/ml. Stock 
 solutions are kept in a test-tube holder 
 stored in a refrigerator. 
 
 TREATNENT SOLUTIONS 
 
 Two aspects of the treatment solution 
 are important: the quantity of chemical 
 in the treatment dose, and the intensity 
 of the radiation in the treatment dose. 
 Where penetration and translocation with 
 a minimum of physiological response 
 (growth regulation, toxicity) are being 
 studied the tracer should be at the maxi- 
 mum specific actiyity obtainable. For 
 example, if 2,4-D" (radioactive 2,4-D, 
 MW 221) of a specific activity of 10 mc 
 per mmole is used and the treatment dosage 
 is to be 0.05 /uc per treatment in 10 wl 
 of solution, the treating solution should 
 contain 1.105 yug per 10 jul or 110.5 /ig per 
 ml. Applied at a rate of 1.105 jug per 
 treatment 2,4-D will cause a slight but 
 perceptible formative effect on young cot- 
 ton plants, leaf malformations may be de- 
 tectable on bean seedlings, but barley, 
 oat, and zebrina plants will show no 
 effect. Such dosages are not injurious - 
 
 11 
 
ig. II, 5. Ampules of C' H -1abe1ed phenylacetic acid (standing in block) and 
 barium carbonate (right side of block) as received from distributor. Stock solutions 
 in acetone are stored in ampules (in pint jar, right). 
 
 they do not inhibit uptake and distri- 
 bution within 24 hours and plants will 
 recover from them in time. If 2,4-D* 
 having a specific activity of 1.0 mc per 
 mmole is used, the same microcurie dose 
 involving 11.05 ug per treatment will 
 cause drastic formative changes in cot- 
 ton, severe changes in bean, but no de- 
 tectable changes in barley. Where only 
 minor injury may be involved, if pene- 
 trability is low it may be necessary to 
 increase dosage of the chemical. For ex- 
 ample, a compound having the same molecu- 
 lar weight as 2,H-D and having a specific 
 activity of 0.5 mc per mmole, 0.10 >uc in 
 10 Jul, will involve H4.2 jug - sufficient 
 to bring about greater penetration and to 
 cause drastic injury if the compound has 
 the same toxicity as 2,4-D. If toxicity 
 is low, there may be little or no injury. 
 
 Experience indicates that a dosage 
 of 0.1 umole per treatment in 10 pi of 
 
 treatment solution usually assures ade- 
 quate penetration. If the specific acti- 
 vity of the compound is lower than 1.0 mc 
 per mmole, exposure time on the film may 
 be lengthened to compensate for the lower 
 activity. 
 
 When making comparative tests using 
 labeled compounds the radioactivity of 
 the various treatments and the chemical 
 dosage should be uniform. Under these 
 conditions, the researcher is limited by 
 the lowest specific activity among the 
 compounds being used. In studies invol- 
 ving a large number of labeled compounds 
 we have used treatment solutions stand- 
 ardized at 0.5 mc per mmole; a specific 
 activity of 1.0 mc per mmole gives more 
 satisfactory results. In preparing 
 treatment solutions from stock concen- 
 trates, unlabeled compounds of comparable 
 purity are used, and these are used in 
 making all tracer solutions to the common 
 
 12 
 
Fig. II, 6. Support for tracer con- 
 tainer used in weighing. 
 
 specific activity. It is most convenient 
 to make two stock solutions, one of the 
 labeled compound and one of the unlabeled; 
 these are mixed to give the required spe- 
 cific activity and then diluted to treat- 
 ment strength. Self-filling micropipettes 
 are handy for preparing treatment sol- 
 utions, and solutions of the desired vol- 
 ume can be made by careful calculation. 
 
 Using 2,4-D" as an example, and 
 starting with a stock solution containing 
 1.105 mg per ml of the labeled material: 
 if the treatment solution is to contain 
 0.05 fjc per 10 pi with a specific activity 
 of 0.5 (instead of 10.0 as in the previous 
 case ) , to prepare 1 ml of treatment sol- 
 ution would require 100 pi of the stock 
 solution plus 100 pi of a stock solution 
 of unlabeled 2,4-D containing 19 times 
 1.105, or 20.995 mg per ml. After evapo- 
 ration of the volatile solvent these ali- 
 quots, totaling 2,210 jug or 10 yumole of 
 2,4-D, are made up to 1 ml with the sol- 
 vent used for the treatment solution. 
 The authors usually use 50 per cent alco- 
 hol containing 0.1 per cent Tween 20, but 
 the choice of formulating materials de- 
 pends upon the labeled compounds being 
 used and the specific requirements of the 
 problem. Preparation of 1 ml of treatment 
 solution in the above example is a matter 
 of the requirements of the experiment; 
 10 pi of each stock solution could be 
 made to 100 pi of treatment solution if 
 desired. Recent work on surfactants 
 (Jansenj et at. , 1961) indicates the need 
 for more research UDon these important 
 formulating materials. 
 
 Occasionally, small treatment vol- 
 umes are desired - as in a case where 
 treatments were made to single leaves of 
 
 bennudagrass. Here, the leaves were 
 fastened in a horizontal position and 
 2 Ml droplets of stock solution of sever- 
 al labeled herbicides were used. If sur- 
 factant is called for, droplets in 50 per 
 cent alcohol may be applied and allowed 
 to dry prior to application of the tracer 
 solutions . 
 
 PREPARATION OF TREATMENT SOLUTIONS 
 
 In preparing treatment solutions, 
 a clean working space covered with heavy 
 wax- lined disposable paper is needed. 
 Also needed are proper stock solutions, 
 a box of facial tissue, aluminum foil for 
 wrapping waste facial tissue, culture 
 tubes 10 x 75 mm, a test-tube support 
 block, silicone stopcock grease, 50-ml 
 beakers, medicine droppers, a slender 
 tipped pipette, a set of self- filling 
 micropipettes, a 1/4-oz. capacity syringe 
 bulb to fit the micropipettes, and a grad- 
 uated 1/10-ml pipette fitted with a con- 
 trol made from a 2-ml syringe (having a 
 barrel lubricated by lanolin) attached by 
 a thick-walled rubber tube (fig. II, 7); 
 the 1/4-oz. syringe bulb should be per- 
 forated on top with a red-hot needle. 
 (By careful manipulation of the finger 
 tip on the perforation one soon learns 
 to fill and empty the pipette at will). 
 The 1/10 ml pipette is used to transfer 
 the stock solutions to the 10 x 75 mm 
 
 Fig. II, 1. Equipment for treating 
 plants with radioactive tracers. Wooden 
 block holds 10 x 75 mm test tubes, a 20 x 
 150 mm test tube of washing solvent, and 
 a l/4-oz. syringe bulb and pipette stand- 
 ing in a 20 x 1 50 mm tube of washing sol- 
 vent. Self-filling micropipettes are in 
 front; a l/10-ml pipette with a 2-ml 
 attached syringe is immediately behind 
 them. 
 
 13 
 
test tubes for dilution to treatment 
 solution strength. Pipettes should be 
 rapidly rinsed with 95 per cent alcohol 
 and blown dry after each use; traces of 
 phenoxyacetic acids are removed by flush- 
 ing with hydrochloric acid. Facial 
 tissue may be used to take up the rinsing 
 alcohol; when a particular operation or 
 treatment is finished, all such tissue 
 should be wrapped in aluminum foil and 
 placed in a proper container for disposal. 
 
 In all transfer and washing oper- 
 ations, care must be taken to avoid 
 spillage, to avoid formation of bubbles 
 at the pipette tip, and to apply all sol- 
 utions and washing alcohol to the sides 
 of the containers. The bursting of bub- 
 bles and dropping of solutions from pip- 
 ettes causes splattering and is bound to 
 result in contamination. 
 
 Sealed ampules should be wrapped 
 with masking tape, scored with a file 
 and opened with care, using pliers and 
 rubber gloves (fig. II, 5). If acetone 
 is used as a diluent for storing solutions 
 of labeled compounds the ampule is opened 
 to allow the acetone to vaporize, and the 
 chemical is then dissolved in 95 per cent 
 ethyl alcohol and transferred to the 
 treatment solution container, using a 
 slender-tipped pipette. With care, one 
 may transfer the original solution plus 
 washings and still use only one-half the 
 volume of the final treatment solution; 
 water is then added if the final solvent 
 is to be 50 per cent alcohol. Formulation 
 is done in this way because the chlorc- 
 phenoxy compounds are slowly soluble in 
 50 per cent alcohol, but are rapidly sol- 
 uble in 95 per cent alcohol. Surfactant, 
 if used, should be made up as a stock 
 solution and added before bringing the 
 treatment solution to final volume. 
 
 Some compounds require special sol- 
 vents. EPTC has been dissolved in methyl 
 cellosolve as a carrier; this solvent is 
 apparently of low phytotoxicity. For 
 root treatment, EPTC has been dissolved 
 in a mixture of equal volumes of Tween 20 
 and acetone and then dispersed in the 
 culture solution. 
 
 The butoxyethanol ester of 2,4-D is 
 not sufficiently soluble in 50 per cent 
 alcohol; it has been handled in pure 
 acetone as a solvent with the container's 
 
 stopper (which is coated with silicone 
 stopcock grease) taped in place. The 
 treatment solution is kept in the refri- 
 gerator, and solvent lost during storage 
 is replaced by bringing the level back to 
 a predetermined mark on the container. 
 Water and water-alcohol, acetone-water, 
 and other mixtures and solvents have been 
 used in preparing treatment solutions of 
 some 25 labeled compounds. Volatile sol- 
 vents such as pure acetone and viscous 
 oils as solvents are to be avoided be- 
 cause they make it difficult to handle 
 and clean the equipment. 
 
 TREATMENT METHODS 
 
 Selection of the treatment area is 
 important. Leaves or stems may be 
 treated by applying solution to a small 
 area, to several areas, or by spray or 
 multi-droplet application. Figure II, 8, 
 left, shows single-droplet treatments on 
 barley and bean, and, right, multiple- 
 drop treatments on soybean and cotton; 
 figure II, 9, shows treatment on the 
 phloem tissue on the side of a tree trunk, 
 For study of phloem translocation from 
 leaves, a 10 ul droplet over the midrib 
 close to the base of the leaf gives the 
 best results; application to a leaf tip 
 or on spots around the edges of a leaf is 
 much less effective (Crafts, 1956a). 
 
 Treatments are best made on a sunny 
 day in the greenhouse or field, or under 
 illumination in control cabinets. If 
 dark treatment is used to show lack of 
 translocation when photosynthesis is 
 lacking the starch and sugar content of 
 the leaves should be determined, as some 
 species evidently continue to export 
 foods for several days after being placed 
 in the dark; others cease movement with- 
 in 24 hours and studies on coffee plants 
 indicate that about 96 hours are required 
 to deplete carbohydrate reserves in their 
 leaves . 
 
 Temperatures should also be watched. 
 Below 70 degrees Fahrenheit, absorption 
 and translocation by beans may be slow, 
 while that by barley is faster. Temper- 
 atures around 80 degrees Fahrenheit are 
 more satisfactory for transport studies; 
 above 90 degrees Fahrenheit evaporation 
 of the treatment droplet is rapid, but 
 repeated application of solvent, or 
 enclosure of the plant in a high-humidity 
 
 14 
 
W0 
 
 ■wi^ ^Bj 
 
 ^^HBBBj '■V^^L fc ^__^BBB KB^BlttB^. 
 
 pi * • . I w 
 
 -jH j 
 
 -' bYbotbI iP^f 
 
 ^BB 
 
 ft! 
 
 BJ ; ^B BFV 
 
 
 Fig. II, 8. Single-droplet treatments on barley (left), bean (center); multiple- 
 drop treatments on soybean and cotton plants (right) with lanolin rings and dams 
 used to localize droplets. 
 
 chamber, will prolong treatment time. 
 
 For treatment with labeled compounds 
 via the roots, plants are most easily 
 handled by growing them in nutrient cul- 
 ture. Beans, barley, soybeans, and simi- 
 lar plants do well in one-quart Mason 
 jars. The quantity of labeled tracer 
 used to treat a leaf will usually suffice 
 to treat roots if only 100 ml of culture 
 solution is used at treatment time. In- 
 jury to roots should be avoided. 
 
 Small barley and oat seedlings have 
 been successfully treated when their 
 roots were contained in 4 ml of solution 
 in 12 x 75 mm culture tubes. When held 
 for treatment times of up to 8 days, 
 2 ml sufficed to keep the plants thrifty. 
 Three-week-old barley plants starting to 
 tiller were treated by holding the roots 
 in 250 ml of culture solutions contain- 
 ing the labeled tracer. Root treatments 
 of a variety of types have been carried 
 out in volumes between these extremes. 
 
 Extensive replication is not usual- 
 ly required in plant autoradiography. 
 Where the degree of difference is not 
 very great in the independent variable of 
 
 an experiment, four replicates are often 
 used; where wider differences are involved 
 three or two plants may be used, and where 
 time series are used and trends can be 
 followed only one plant per variable may 
 be used. In all experiments control 
 plants should be included, as an untreated 
 plant frequently produces a faint image 
 due to volatile substances given off 
 during film exposure. (Such effects must 
 be taken into consideration in interpret- 
 ing the images produced by treated plants. ) 
 
 Treatment time may vary from a min- 
 ute to a month or more, depending upon the 
 objective of the experiment. At approxi- 
 mately 80 degrees Fahrenheit in the 
 greenhouse, 2,U-D' : treatment of a bean 
 plant on a sunny day may result in move- 
 ment into the stem within one hour, and 
 into the roots within 3 hours. Movement 
 of MH >: in barley may be into roots in 
 3 hours, and may continue for 2*4 hours or 
 more; movement into zebrina may be slower 
 but thorough if sufficient time is allowed. 
 Twenty- four hours proved sufficient time 
 for appreciable movement of several 
 tracers in woody stems and young woody 
 plants (Yamaguchi and Crafts, 1959). 
 
 15 
 
Fig. II, 9« Treatments on phloem tis- 
 sue of a tree trunk. 
 
 The effects of local accumulation 
 are important in the use of labeled 
 tracers. The pattern of distribution 
 usually develops quite fully in 4 to 16 
 days. Some compounds remain mobile, how- 
 ever, and redistribute from mature to 
 young growing-tissues for even longer 
 periods. With 2,4-D, accumulation in 
 parenchymatous tissues along the trans- 
 port route may limit the extent of dis- 
 tribution; with amitrole, maleic hydra- 
 zide (MH), and dalapon, accumulation is 
 transitory and redistribution in the sym- 
 plast persists for many days. 
 
 Migration of a tracer from phloem to 
 xylem and vice versa may be an important 
 factor in determining the fina^ distribu- 
 tion pattern. MH and dalapon" are known 
 to move laterally in stems; hence, when 
 the primary leaf of a bean is treated 
 and sufficient time is allowed, the op- 
 posite leaf will show an image. Potas- 
 sium and phosphorus distribute this way 
 in many plants, while 2,4-D*, amitrole* 
 and calcium- H 5 do not show the same trans- 
 fer pattern. 
 
 After plants have been treated by 
 droplet application (figs. II, 8, 11), 
 the treated area should be covered with 
 masking tape (fig. II, 1H). This may be 
 done at any time after the droplet has 
 dried but is usually done just prior to 
 the start of the freeze-drying process. 
 Covering of the treated spot prevents 
 contamination during mounting of the 
 plants and gives a permanent record of 
 the locus of treatment. Finally, all 
 plants should be clearly labeled with 
 labels that can survive the freeze-drying 
 process; small cardboard labels attached 
 with string, or masking tape tags with 
 code numbers in pencil are satisfactory. 
 
 PREPARATION FOR AUTOGRAPHY 
 
 Freeze-drying has proved essential 
 in studies where labeled tracers are 
 employed to study absorption and trans- 
 location (Crafts, 1956a; Pallas and 
 Crafts, 1957; van der Zweep, 1961). 
 Treated spots are cleansed with facial 
 tissue to remove lanolin rings and, in 
 the case of foliar applications, are 
 covered with masking tape to prevent con- 
 tamination when mounting the dried plants, 
 
 Following leaf and root treatments, 
 roots are removed from the culture jars 
 and rinsed under running water. (Conta- 
 mination of the unexposed portions of the 
 root systems with the culture solution 
 must be avoided. ) Roots are thoroughly 
 rinsed with tap water, placed on sheets 
 
 Fig. II, 10. Apparatus for treating 
 with a volatile labeled compound. 
 
 16 
 
of facial tissue to drain, and plants 
 are then placed in the drying tray. If 
 plants have had root treatment via the 
 culture solution, the roots must be 
 wrapped individually in waxed paper or 
 facial tissue to avoid contamination. 
 
 The screen drying- tray should be 
 placed on a sheet of stiff aluminum foil 
 bent up to a height of about 5 inches on 
 all sides to hold in the CO2 from the dry 
 ice. Plants are arranged in the tray by 
 alternating the position of tops and 
 roots; as plants are added, pulverized 
 dry ice is poured over them through a 
 1/2-inch mesh screen covering the tray 
 (fig. II, 15). When full, the tray is 
 placed in the precooled metal freeze-dry 
 tank (figs. II, 12, 13). The tank is 
 then closed, placed in a deep freeze set 
 at -15 degrees Centigrade and then hooked 
 up to a vacuum pump. Only enough dry ice 
 to freeze the plants should be used, as 
 
 excessive ice prolongs the freeze-drying 
 process and may fracture leaves. After 
 removing plants from the culture medium, 
 processing should be done quickly to 
 avoid wilting, which prolongs the drying 
 process. 
 
 When this process was first used, 
 calcium hydride lumps were placed in the 
 bottom of the vacuum tank to hasten dry- 
 ing; the spent material had to be re- 
 moved each day. We now find that a high 
 capacity 1/2 horsepower vacuum pump, pro- 
 tected by two large-capacity vapor traps 
 connected in parallel and a smaller one 
 in series, extends drying time slightly 
 but eliminates the need for the dessicant. 
 
 Figure II, 16, illustrates the inside 
 arrangement of the deep- freeze box now in 
 use by the authors. The temperature con- 
 trol is set to maintain -15 degrees Centi- 
 grade. Defrosting is accomplished during 
 
 Fig. II, 11. Droplet application to leaf of Bermudagrass. The stem is fastened 
 with masking tape, the leaf taped into a horizontal position, a band of lanolin ap- 
 plied to prevent creeping of the solution, and a droplet of treating solution applied, 
 
 17 
 

 c 
 a> 
 
 (L) 
 i_ 
 U 
 (/) 
 
 • 
 >* E 
 +j D 
 
 Q- 3 
 E U 
 
 <u to 
 
 > 
 
 O 
 
 4-< — 
 
 ■eg 
 
 +J 
 
 •i- o 
 
 U +J 
 
 <D O 
 
 -O _* 
 
 E </> 
 
 03 (D 
 -C O) 
 O 
 
 1_ 
 E 0) 
 3 -Q 
 =J .Q 
 O 3 
 0J 1- 
 > I 
 0) 
 >v C 
 l- O 
 XI o 
 
 tt) JI 
 
 N ••- 
 <U (/> 
 <D 
 
 -C 
 E -t- 1 
 
 3 »- 
 
 c 2 
 
 E X) 
 
 => <L> 
 
 •— +-> 
 
 < •»-> 
 
 1_ 
 0) 
 
 > 
 o 
 o 
 
 18 
 
Round the corner 
 by 3/16" 
 
 ALUMINUM VACUUM TANK 
 
 Fig. II, 13* Structural details of the aluminum freeze-dry vacuum chamber. 
 
 A. Gasket groove, 3/16-inch deep and 3/8-inch wide. 
 
 B. Corner of groove is rounded so that rubber gasket can be flared around the corner 
 in the groove. Corner is rounded by 3/16-inch on the line of the hypotenuse; in- 
 side corners of the vacuum-seal plate are similarly rounded. 
 
 C. Skids on the bottom may be of aluminum (or other metal) 1/4 x 1/U inch, and at- 
 tached entire length of tank, about 1 inch from edge. 
 
 D. Runners inside tank are 1/8- inch thick and 3/8-inch wide; they are placed exact- 
 ly in the middle of the side walls. Runners must support a tray weighing 2 to 5 
 pounds. End wall has no runners. 
 
 E. Vacuum-seal face is 1-11/32 inches wide; this allows an extra sealing surface of 
 1/8-inch on the inside of rubber gasket when lid is secured. Center of bolt shaft 
 should be about 29/64-inch in from outer edge of the face. 
 
 F. Vacuum- releasing device. This consists of a 1/2-inch section of 1/ 8-inch water 
 pipe welded over an 1/8-inch hole on a 3/4- inch section of 3/4-inch water pipe. 
 The hole through the tank should be same diameter as inside diameter of 3/U-inch 
 section so that hole can be used as housing for retaining something to soften the 
 stream of air coming in from the vacuum release (vacuum-release valve is attached 
 to the opening). 
 
 G. This arc should have an upward inclination of about 10 degrees. 
 H. This arc should be about 30 degrees. 
 
 I. Short arrow represents a distance of about 3/U-inch from top surface of tank to 
 base of the "arm." 
 
 Outside overall dimensions of the tank are not given because they vary with the 
 thickness of material selected. Total length from the back end to the vacuum-seal 
 plate should not exceed 15 inches. 
 
 19 
 
Fig. II, 14. Treated areas on barley 
 and bean leaves covered with masking tape 
 after droplets have been absorbed. 
 
 slack periods, as automatic defrosting 
 would allow fluctuation in temperature 
 and possible thawing of plants. If the 
 door of the deep freeze has to be left 
 open for extended periods while freeze- 
 drying is in progress, blocks of dry ice 
 applied to the vacuum tanks will hold 
 them at the required freezing tempera- 
 ture. 
 
 Each vacuum tank is equipped with a 
 vacuum-release mechanism consisting of a 
 short pipe welded into the chamber, to 
 which a 1/8-inch stopcock is attached 
 with a piece of thick-walled pure gum 
 rubber tubing (the stopcock should be 
 lubricated with low- temperature silicone 
 lubricant). Vacuum must be released 
 slowly to avoid shattering the plant 
 materials and to avoid too-rapid a tem- 
 perature rise of the chamber and its 
 contents . 
 
 One of the most difficult problems 
 in the use of the vacuum tanks has been 
 that of securing a tight seal between 
 the lid and the tank. The lid has a 
 routed groove (fig. II, 12, 13) into 
 which is fitted a gasket of silicone 
 rubber which retains its elasticity at 
 -15 degrees Centigrade; if the groove 
 is properly designed (fig. II, 13) the 
 
 Fig. II, 15« Plants (left) folded to fit the screen tray in which they are to be 
 killed and f reeze-dried . Center tray has received the pulverized dry ice; tray on 
 the right is ready to place in the deep-freeze chamber. Aluminum foil retains the 
 cold CO2 atmosphere and assures even freezing. 
 
 20 
 
gasket should stay in place indefinite- 
 ly. Silicone grease is sometimes used 
 on the smooth face of the tank to which 
 the gasket is applied to lessen conden- 
 sation and ice formation. If the stud- 
 bolts that hold the lid in place are 
 tapped into the face of the tank, a 
 speed wrench may be used to secure the 
 lid in place and to remove it when open- 
 ing the tank. 
 
 The rubber tubing used in the 
 vacuum line (fig. II, 16) has an inside 
 diameter of 13/16-inch and walls 7/16- 
 inch thick. It is soft and flexible at 
 -15 degrees Centigrade and is slipped 
 over the pipes, which are smooth but need 
 not be tapered; the tubing is pure gum, 
 has high elasticity, high tensile 
 
 strength, and is long-lived. (A manifold 
 to connect two or more tanks to the pump 
 and pressure gauge was made of standard 
 3A-inch water pipe fittings, smoothed 
 to take the rubber tubing . ) 
 
 The large vapor (FLO) traps are de- 
 signed to fit into one-gallon Dewar 
 flasks, each having the capacity to trap 
 about a pound of water. When in use the 
 flasks are charged daily with small lumps 
 of dry ice and covered with soft insula- 
 ting material. About a pint of methanol 
 or commercial acetone is poured over the 
 lower one-third to one-half of the dry 
 ice, causing a uniform condensation of 
 moisture in the trap. Traps may be 
 cleaned out each time the vacuum tanks are 
 opened; the trap flask is held under run- 
 
 Fig. II, 16. Arrangement of freeze-dry chambers in the deep-freeze box, showing 
 vacuum hoses, moisture traps, and vacuum pump. 
 
 21 
 
ning water until the ice melts free 
 enough to be taken out - if a trap flask 
 is allowed to warm up gradually when 
 loaded with ice the expanding ice will 
 cause the flask to burst. Traps may be 
 cleaned at any time without releasing 
 the vacuum by clamping the rubber tube 
 connections between the tanks and traps, 
 thus releasing the vacuum in the traps, 
 and melting the ice under running water 
 (individual tanks may be isolated for 
 servicing by placing clamps on the rubber 
 vacuum tube). After replacing traps, 
 clamps should be released slowly until 
 the vacuum has been reestablished. 
 
 The third small trap is used as in- 
 surance against the possible loss of the 
 dry ice in the larger traps. Because it 
 traps little or no moisture during normal 
 operation, the dry ice in this trap lasts 
 longer and provides a safety trap. 
 
 If moisture passes all three traps 
 and reaches the vacuum pump the oil will 
 appear cloudy in the window; when this 
 occurs the oil must be changed and the 
 pump cleaned of all moisture before fur- 
 ther use. These operations can be per- 
 formed conveniently if traps are connec- 
 ted to freezer tanks and pump with suf- 
 ficient lengths of flexible tubing. 
 
 Experience has proved that freeze- 
 drying of plants is not successful unless 
 the vacuum is below 2 mm of mercury. A 
 range of 500 to 200 microns of vacuum has 
 proved effective; the equipment pictured 
 in figure II, 16 has reached 20 microns 
 under freeze-drying conditions, and the 
 pump now in use will bring the vacuum 
 within this range in about 10 minutes. 
 Use of silicone stopcock lubricant on 
 the vacuum chamber lids and on all tube 
 connections has helped in attaining such 
 results. 
 
 Freeze-drying in a vacuum tank pro- 
 ceeds quite differently than does drying 
 under unfrozen conditions. Thin leaves 
 dry rather rapidly, but succulent stems 
 and inner leaves of grasses dry more 
 slowly and at different points: drying 
 starts at the leaves, nodes and roots, 
 and progresses along the stems. A par- 
 tially freeze-dried plant may be com- 
 pletely dry in some regions and scarce- 
 ly starting to dry in others. 
 
 Different plants require different 
 
 drying times. Small cereal seedlings, 
 young bean, and cotton seedlings and 
 similar plants may dry completely in 
 7 to 10 days; large cereal plants, zebri- 
 na stems, and similar succulent tissues 
 may require from 2 to 3 weeks. Storage 
 of frozen plants in freezer compartments 
 of refrigerators (approximately -12 
 degrees Centigrade) for 2 days, followed 
 by normal freeze-drying, resulted in a 
 longer drying time in the case of bean 
 plants; similar treatment of cucumber 
 plants had no effect. The nature of 
 the drying of leafy plants suggests that 
 water molecules escape via the stomata, 
 and hence high vacuum is essential to 
 rapid drying. 
 
 Bark samples from treatments of 24 
 hours or more have been dried in the 
 open air. Samples of thin bark may be 
 pressed flat between screens and dried 
 in the open air, or in a forced- draft 
 drying oven; a sheet of paper should 
 separate the cambium side of the bark 
 from the screen. With thick bark, it is 
 best to pare away the outer part, leaving 
 live inner tissue from 1/8-inch to 
 1/16-inch thick; this tissue may be dried 
 between screens. Since the removal of the 
 bark from the tree damages the transloca- 
 tion system and there is a slight move- 
 ment of fluids, a slight artifact results 
 (Leonard and Crafts, 1956; Yamaguchi and 
 Crafts, 1959). 
 
 MOUNTING DRIED PLANTS 
 
 When the drying process is complete 
 plant materials should be removed from 
 the vacuum chamber, remoistened slightly 
 (fig. II, 17) and mounted on white, sized 
 paper, using casein or polyvinyl glue. 
 Remoistening facilitates separation of 
 plants that have dried in a single screen 
 tray and helps avoid shattering the leaves, 
 which are quite fragile when first removed 
 from the vacuum chamber. Remoistening is 
 most easily done in a closed chamber or 
 covered pan with the plants resting in a 
 screen tray which is open below to a 
 standing-water surface. At 72 degrees 
 Fahrenheit, barley plants are adequately 
 humidified in 1 to 2 hours; large bean 
 plants require 3 to 4 hours; and cotton 
 plants may need 6 or 8 hours. Humidifying 
 time can be shortened by using warm water, 
 but condensation inside the humid chamber 
 should be avoided. 
 
 22 
 
Fig. II, 17. Screen trays holding 
 f reeze-dn'ed plants are placed in enam- 
 eled pans (left) with water below, and 
 are then covered with sheet plastic. Re- 
 moistening helps keep plants from fractur- 
 ing when handled. 
 
 When plants are sufficiently moist 
 they should be removed from the moisten- 
 ing chamber, arranged on a sheet of white, 
 sized paper, and glued down. For routine 
 work the plant may be tacked down by glu- 
 ing the main leaves and the roots and 
 other organs (fig. II, 18); where mounted 
 plants are to be used in demonstrations, 
 all plant parts should be glued down 
 
 (this is time consuming and not ordinari- 
 ly done). The treated leaf should always 
 be mounted with the treated side down, 
 and information from the label on the 
 plant should be copied on the lower edge 
 of the mount. The mounted plants should 
 not be dry enough to be brittle at the 
 time of pressing. Pressing consists of 
 covering the exposed side of the mounted 
 plant with a piece of wax paper or alumi- 
 num foil, placing this covered face 
 against a hard flat surface such as ply- 
 wood or Masonite, backing the mount with 
 several thicknesses of dry felt, and 
 sandwiching a whole series so that all 
 faces are against hard surfaces and all 
 backs are against felt. A heavy (3/H- 
 inch) piece of plywood is then placed on 
 each side of the bundle and the whole 
 placed in the press. Fig. II, 19, shows 
 a simple wooden press; fig. II, 20, shows 
 a press built from a screw-jack. 
 
 Pressure should be great enough to 
 compress the stems and completely flatten 
 the face of the plants - a force of 50 or 
 more pounds per square inch may be re- 
 quired; plants should be left in the 
 press overnight or, preferably, for 24 
 hours (during which time the plant must 
 dry thoroughly). After plants are re- 
 moved from the press the thin cover 
 sheets should be removed from the faces; 
 these should peel off easily, but if 
 there is trouble in removing them, Saran- 
 wrap (or other plastic wrapping material) 
 should be tried next time. 
 
 Fig. II, 18. A freeze-dried Tropaeol urn 
 maj us plant placed for mounting. 
 
 Fig. II, 19 
 wooden bars. 
 
 Press made of 2" x 2 1 
 
 23 
 
On removal from the press the plant 
 should be flat, smooth, and dry, and 
 the thickness of stems, veins, etc. 
 should be impressed into the mounting 
 paper (fig. II, 21) due to the deforma- 
 tion of the felt backing. If sap (from 
 incomplete drying) is squeezed from suc- 
 culent stems, buds, or young grass 
 leaves onto the paper, the contaminated 
 areas should be carefully cut out and 
 the mounting repaired. It is advisable 
 to dry the plants so that this does not 
 occur, but occasionally one may misjudge 
 the drying; in such cases it is best to 
 excise blotched areas, as they usually 
 show in the final autograph. 
 
 AUTOGRAPHING 
 
 After removal from the press, the 
 mounted plant materials may be allowed 
 to stand in the laboratory to equilibrate 
 with the atmosphere; they are then ready 
 for exposure on the film. The authors 
 use Kodak Royal Blue Medical X-ray film, 
 a double-coated film either side of which 
 may be placed against the mounted plant. 
 In the dark room, the mounted plant is 
 inserted with its face against the film, 
 inside the folder in which the film is 
 packed (fig. II, 22). It is advisable to 
 pencil-mark each film to help in matching 
 
 Fig. II, 20. Screw-jack press. 
 Aluminum sheets or plywood boards may be 
 used as press plates; plant mounts should 
 be backed with blotters to allow for the 
 thickness of the stems and leaves. 
 
 Fig. II, 21. Back view of a plant 
 mount showing how backing with blotters 
 allows the thickness of the plant mater- 
 ial to be pressed into the mounting 
 paper. 
 
 films and plants after the films are de- 
 veloped; in large experiments, radioactive 
 ink can be used on the mounts. To prevent 
 the control plant (without radioactive 
 material) from producing a pseudoauto- 
 graph from natural labile emanations, we 
 have used a Saranwrap covering - this has 
 kept out such emanations while permitting 
 passage of 50 to 60 per cent of the beta 
 particles. 
 
 Fig. II, 22. Inserting the mounted 
 plant beneath the X-ray film in its fol- 
 der. Exposure bundle of plants and films 
 is shown on the right. 
 
 24 
 
Fi 
 of C 
 k hours. 
 
 ig. II, 23. Autoradiography of a mounted plant having an unusually high content 
 '^. Film exposure times were (top left to bottom right) 1/8, 1/4, 1/2, 1, 2 and 
 
 When all plants are so placed, the 
 folders should be arranged with the film 
 side against a separator of plywood with 
 aluminum foil, or aluminum sheet of 1/32- 
 inch thickness; this is to give the film 
 a firm flat backing. With the folder on 
 top of the separator film side down, a 
 piece of 1/4-inch sponge rubber or poly- 
 thene foam should be placed on the upper 
 side to back the plant mount; the next 
 folder should then be placed mount side 
 down against the foam, and another sepa- 
 rator put on top to back the film. The 
 
 films and plants are sandwiched between 
 separators and foam sheets until all 
 plants are included after which stiff 
 boards (1/4-inch plywood boards will do) 
 are placed on both sides of the bundle 
 and elastic belts are cinched around to 
 hold plants and films firmly together; 
 the bundle (fig. II, 22) is then placed 
 in a light-tight box and left for the 
 proper exposure time. 
 
 If plywood sheets are used for sepa- 
 rators in the autoradiograph bundle they 
 
 25 
 
should be covered with aluminum foil, as 
 bare plywood may cause a pseudoautograph. 
 A single thickness of aluminum-covered 
 1/4-inch plywood and 1/4-inch sponge rub- 
 ber sheets provides adequate shielding 
 between successive mounts when Ca 1 ^, S35, 
 Cl36, or d^ are being used; P32 and Zn^o 
 require more shielding - such as several 
 aluminum-covered separators and several 
 1/4-inch sponge rubber sheets. Equiva- 
 lent stocks of newspapers will do for 
 extra shielding. 
 
 Normal exposure of the plants on the 
 film is 2 weeks, but exposure may be 
 varied to bring out different features of 
 the autograph. Figure II, 23, shows a 
 bean plant that was exposed to a large 
 amount of d 1 ^, allowed a time period 
 of several hours before being freeze- 
 dried, mounted and autographed. Expo- 
 sures on the six films were as follows: 
 top left to right bottom: 1/8, 1/4, 1/2, 
 1 , 2 , and 4 hours . 
 
 DEVELOPMENT 
 
 When exposure is complete, the bundle 
 is opened, the films are removed, clipped 
 in hangers and developed (following in- 
 
 Fig. II, 24. Developing and fixing 
 tanks, sink, and film rack for drying 
 X-ray f i 1ms . 
 
 structions in the Eastman Kodak Company's 
 publication, The Fundamentals of Radio- 
 graphy), Optimum developing temperature 
 is 68 degrees Fahrenheit, and develop- 
 ment time is based on this; if this tem- 
 perature cannot be maintained, develop- 
 ment time must be changed accordingly, as 
 explained in the above publication. We 
 have used Kodak liquid X-ray developer 
 and replenisher, and Kodak liquid fixer 
 and replenisher, with complete satisfac- 
 tion. Plastic and stainless steel devel- 
 oping, fixing, and washing tanks are 
 available in sizes to fit the needs of 
 the operation. An acetic acid stopbath 
 is used between developer and fixer. 
 
 The standard recommended development 
 time for the Royal Blue X-ray film pro- 
 duces a dense gray background; we use 
 only one-half this time as we want only 
 a faint background for easy viewing - 
 also, a dense background makes photo- 
 graphic reproduction difficult. 
 
 In removing films from the bundle 
 in which they are exposed it is best to 
 keep the plants and separators in the ori- 
 ginal order until the films have been pro- 
 cessed - thus, if any stray marks or radio 
 images appear they can be traced to their 
 source. Sponge rubber sheets and separa- 
 tors occasionally become contaminated but 
 such contamination can be readily detect- 
 ed if the development bundle is kept in 
 order. 
 
 By careful planning one may save 
 considerable time in the developing and 
 fixing of films. We develop four at a 
 time for 2.5 minutes. The films are 
 placed carefully into the developing tank 
 in order; after development, they are 
 removed and dipped for 1 minute each in 
 a stop-bath, then placed in the fixative 
 and while there (for a period of approxi- 
 mately 10 minutes) four more are placed 
 in the developer. Washing requires an 
 hour, and drying usually requires 4 to 5 
 hours (fig. II, 24); after washing, each 
 side of the film is wiped carefully with 
 a wet sponge to remove dust or debris. 
 When dry, each film is reinserted in its 
 folder with the plant upon which it was 
 exposed; for future use, it is advisable 
 to put in a piece of the sized mounting 
 paper at the same time. In photographing, 
 this backing with shiny white paper will 
 give the image maximum contrast. Material 
 
 26 
 
from each experiment can be stored in a 
 manila folder, and several experiments 
 can be combined in each empty file box. 
 
 REPRODUCTION 
 
 In photographing autographs and 
 mounts we use an easel as illustrated in 
 figures II, 25, 26. The great contrast 
 between the background of the autograph 
 and the white backing of the mount pre- 
 sents a problem in reproduction. If the 
 autograph and plant are to be photographed 
 together, it is advisable to have a set 
 of evenly exposed X-ray films, that have 
 been developed, fixed and dried, to place 
 over the plant mounts. The film used with 
 each plant-autograph pair should be lighter 
 
 Fig. II, 25. Easel used in photo- 
 graphing plants and autoradi ographs . 
 Glass cover is raised for placing mounts 
 and films in position. The chain and 
 treadle enable operator to open or close 
 the cover and to snap easel into position 
 with the foot while holding cover with 
 both hands. 
 
 than the background of the autograph - 
 without such matching, different exposures 
 must be used for plant mount and autograph 
 or the latter will be much darker than 
 the mount. 
 
 The plants and autographs are placed 
 on the padded surface of the easel (figs. 
 II, 25, 26) and the glass front is closed 
 to hold them in place. Reflected incident 
 light from two or more photoflood lamps, 
 placed on each side of the easel, gives 
 satisfactory results. The easel used by 
 the authors can be placed in a horizontal 
 position while the plants and autographs 
 are arranged; after clamping the cover- 
 ing glass in position the easel is ro- 
 tated into a vertical position for photo- 
 graphing. A view camera is used for 
 black and white photos, and a 35 mm camera 
 is used for Kodachrome. By using a black 
 curtain, with an aperture for the camera 
 lens, glare on the glass of the easel 
 can be avoided. Panatomic-X 4x5 inch 
 film is used for making negatives, and 
 Kodabromide paper is used for printing. 
 Exposure is judged by a light-meter read- 
 ing of the blank grey areas of the auto- 
 radiograph. 
 
 Fig. II, 26. Easel in position for 
 photographing enclosed materials. 
 
 27 
 
STORING OF PLANTS AND AUTORADIOGRAPHS 
 
 For future reference, the usual 
 10 x 12-inch mounts and films can be 
 stored in an ordinary steel filing case. 
 Each plant mount and its autographic film 
 is kept in the film folder; the folders 
 are grouped into manila folders, one for 
 each experiment, and the folders labeled 
 and stored in the filing case. The 
 double-length films and mounts illustrated 
 in figures X, 1 to 4 , are laid horizon- 
 tally in a filing case drawer; the 
 14 x 17 inch films and mounts are stored 
 in special filing cabinets made for stor- 
 ing X-rays in hospitals. 
 
 Freeze- dried plants require no spe- 
 cial care in storage. We have plants 
 stored for 10 years that look as well as 
 they did when first stored; those in which 
 Cl4_ labeled compounds were used still 
 produce autoradiographs indistinguishable 
 from the originals. During storage over 
 several years many compounds sublime into 
 the covering materials; when this occurs, 
 the mounted plants may be re-autographed, 
 and placed in new folders; new backing 
 sheets for the films should be used. The 
 discarded folders and paper should be 
 handled as contaminated materials. These 
 plants may be ground and counted to quan- 
 tize the autoradiographs if desired. The 
 plants may be extracted and chromato- 
 graphed to study breakdown of the labeled 
 compounds . 
 
 HARVEST I NG, MOUNTING AND DRYING 
 OF DIFFICULT PLANT MATERIAL 
 
 Not all problems involving autoradio- 
 graphy can be handled by the routine green- 
 house and laboratory methods described. 
 In some cases, plants growing wild or in 
 the field are required; these may include 
 shrubs or full-grown trees. Any plants, 
 herbaceous or woody, large or small, in 
 the greenhouse, the field, or in a wild 
 state, may be submitted to treatment with 
 labeled compounds followed by autoradio- 
 graphic analysis of absorption and trans- 
 location processes. 
 
 Large melon and cucumber plants have 
 been grown in the greenhouse with their 
 roots cultured on dark cloth over which 
 the culture solution flowed slowly and 
 constantly (de Stigter, 1961). These 
 plants were freeze-dried, mounted (fig. 
 
 II, 27), and autographed on 14 x 17- inch 
 film, the foliar portion on one film 
 (fig. II, 28) and the roots on another 
 (fig. II, 29). The problem being studied 
 involved compatibility of grafts, and 
 radiographic analysis was critical in its 
 solution. 
 
 With woody plants, cross-sections of 
 stems and rings of bark removed from tree 
 trunks have been used (Leonard and Crafts, 
 1956; Yamaguchi and Crafts, 1959). Such 
 samples may be air-dried or oven-dried if 
 the sampling techniques are designed to 
 minimize artifacts - for example, in 
 taking cross sections, the original cut 
 should be at the base of the stem and 
 subsequent cuts should be made toward 
 the tip. In taking bark samples, simul- 
 taneous ringing in two positions with 
 removal of the bark sample between cuts 
 reduces the error of sap movement during 
 the sampling process. 
 
 Stem tips and small tree seedlings 
 (Clor, 1959) have been used effectively. 
 The tree seedlings were grown and treated 
 in the greenhouse; they were oven-dried, 
 mounted and autographed whole. Care 
 should be taken to avoid excessive pres- 
 sure during the exposure of the mounted 
 plant on film as deformation of the film 
 will cause a pseudoautograph (fig. 11,30). 
 In work on grape vines in the field, 
 samples have included leaves, stem sec- 
 tions, and stem tips; occasionally bark 
 samples are taken at a time of year when 
 bark will peel. In the case of large 
 leaves such as grape, a section of the 
 leaf involving the midrib and a centi- 
 meter or two of tissue on each side may 
 be used; this section can be examined to 
 distinguish between areas of accumula- 
 tion - for example the veins, interveinal 
 areas, or leaf margin. 
 
 In the case of the grape studies 
 whole shoots were taken to the laboratory 
 and sampled consistently from base to tip, 
 taking cross sections of each internode 
 and portions of each leaf. Samples were 
 fastened to heavy filter paper with 
 narrow strips of tape, freeze-dried, 
 mounted (using casein glue), pressed, and 
 autographed. 
 
 Stem sections from large woody 
 plants, cut with a fine-toothed band saw, 
 air-dried, sanded smooth, mounted and 
 
 28 
 
Fig. II, 27. Mounted cucurbit plant ready to autoradiograph. 
 
 autographed have also given satisfactory 
 results. (A vacuum dust-catcher should 
 be used to avoid contamination from the 
 dust, and a dust mask should be worn.) 
 This work has been limited to plants 
 treated with small doses of C^- labeled 
 compounds . 
 
 Large herbaceous plants are difficult 
 to handle, but excellent results have been 
 obtained with tobacco, cabbage and sugar 
 beets. With the latter, sections approxi- 
 mately 2 mm thick were removed, placed on 
 filter paper, frozen with dry ice, dried 
 in the vacuum tank, mounted, pressed and 
 
 29 
 
Fig. II, 28. Autor adiograph of plant in fig. 
 
 autographed. Both cross- and longitudinal 
 sections have been prepared successfully. 
 
 Plants with complex, fragile leaves 
 have been placed between sheets of light 
 filter paper, rolled into open rolls, 
 treated with pulverized dry ice and 
 
 freeze-dried in the usual fashion (fig. 
 II, 31). After remoistening, the freeze- 
 dried plants are carefully unrolled, 
 arranged on mounting paper and pasted 
 down; thus, complicated leaf structures 
 may be preserved through the freeze- 
 drying process. 
 
 30 
 
Fig. II, 29. Autoradiograph of the root system of plant in fig. II, 27. 
 
 31 
 
m Fig. II, 30. Pseudoautographs of the 
 stems of a woody plant mount caused by 
 excessive pressure in the exposure bundle, 
 The only radioactivity in these samples 
 was in the four blurred tips. 
 
 '^'Fig. II, 31. A tray of freeze-dried 
 tomato plants, and one plant spread out 
 on the filter paper in which it was 
 rol led. 
 
 32 
 
HEALTH HAZARDS IN HANDLING 
 LABELED COMPOUNDS 
 
 With care in handling, health hazards 
 from using C^ 4 , S35 } and Cl36 in the quan- 
 tities used in autoradiography are neg- 
 ligible. Protective materials such as 
 lead bricks, special aprons and film 
 badges are not required, but rubber gloves 
 and good hood facilities are needed. 
 Radiation can be detected at the outer 
 surface of the glass tubes in which the 
 labeled materials are shipped, but can 
 be eliminated by dilution when stock solu- 
 tions are made up. Treatment solutions 
 in bacterial culture tubes show no detec- 
 table radiation on the outer surface. 
 
 Because spillage may occur in trans- 
 ferring the labeled compounds during 
 weighing, the area over which this is done 
 should be protected with wax-surfaced 
 paper or aluminum foil (distance of trans- 
 fer may be minimized by the arrangement 
 shown in figure II, 6). Transfer and 
 weighing should be done in a closed weigh- 
 ing room, and all motions should be slow 
 and careful. 
 
 Some labeled compounds are sticky, 
 
 some are fine powders that adhere to all 
 surfaces, and some are liquids. To faci- 
 litate handling, the total amount of the 
 compound is dissolved in a given volume 
 of solvent and made into a stock solu- 
 tion. Rubber gloves must be worn and 
 work must be done under a hood. Portions 
 of the stock solution may be transferred 
 to small ampules and sealed, using a pip- 
 ette with syringe control for such trans- 
 fers. Stock solutions are kept in a re- 
 frigerator; those having volatile com- 
 pounds or solvents are kept in sealed 
 ampules, while others may be kept in test 
 tubes with corks coated with silicone 
 grease and fastened with masking tape or 
 cellophane tape. 
 
 In handling the more hazardous mat- 
 
 >32 
 
 65 
 
 erials, such as P^ or Zn D0 , shielding 
 should be used; rubber gloves and hood 
 or glove boxes are necessary. Storing 
 of such materials requires shielding; 
 containers holding C^ 1 *- labeled compounds 
 in quantities of several millicuries 
 should be shielded. Film should be 
 stored well away from radioactive chemi- 
 cals; storage in a separate room is 
 advisable. 
 
 33 
 
Chapter III. Autoradiography: 
 Interpretation of Results 
 
 A 
 
 autoradiography is unique as a 
 method for studying plants, as it allows 
 the researcher to investigate processes 
 occurring inside the intact plant. Plow- 
 ever, because the Lnages obtained indi- 
 cate distribution processes and chemical 
 reactions inside living cells or within 
 organized cell systems, interpretation of 
 autoradiographic data is an important 
 phase of the over-all method. 
 
 In the uptake and distribution of 
 pesticides or tracers by plant foliage, 
 a number of processes are involved. 
 These processes include the penetration 
 of the cuticular layers, either on leaf 
 surfaces or within the stomatal chambers; 
 they also involve the migration of ions 
 or molecules from the epidermal wall to 
 the chlorenchyma and conducting cells of 
 the vascular systems. They require the 
 passage of the molecules across the cell 
 wall-cytoplasm interface that represents 
 the external surface of the living plant 
 body, and they include the movement of 
 the molecules through living cells from 
 epidermis to chlorenchyma to border par- 
 enchyma and into the sieve tubes of the 
 phloem. Finally, they involve rapid 
 transport from treated leaves and stems 
 to remote regions of shoots and roots. 
 
 It has been proposed (Crafts, 1961a; 
 Crafts and Foy, 1962) that in traversing 
 the cuticle chemicals follow two routes: 
 one lipoid, through which non-polar com- 
 pounds move by diffusion, and one aqueous 
 (made up of micropores) through which 
 polar compounds may come into contact 
 with the water continuum of the plant and 
 move by diffusion or convection into the 
 
 cell wall-cytoplasm interface. Inside 
 the cuticle the moving molecules encoun- 
 ter the primary pectin-cellulose phase of 
 the epidermal or chlorenchyma cell-wall 
 system, and here they must move through 
 a highly aqueous medium. Having tra- 
 versed this medium, the molecules come 
 in contact with the outer surface of the 
 cell protoplast, a living membrane of 
 lipo-protein composition having an energy 
 supply enabling it to move and accumulate 
 some molecules against a gradient. 
 
 Two terms introduced by Munch (1930) 
 are extremely useful in considerations of 
 the entry and translocation of molecules 
 in plants. These are "symplast", the sum 
 of the interconnected living protoplasm 
 of the plant, and the "apoplast", the 
 non-living cell-wall continuum that sur- 
 rounds and contains the symplast. The 
 concept of the symplast implies that all 
 of the living cells of a higher plant are 
 united into a continuum by means of proto- 
 plasmic connections, a fact now recognized 
 by most plant scientists. Symplastic mov- 
 ment of substances is defined as movement 
 confined to the living body of the plant. 
 In contrast, apoplastic movement is move- 
 ment via the interconnected non-living 
 cell-wall and xylem phases. Symplastic 
 movement is capable of carrying labeled 
 sugar, derived from Cl^-labeled urea, 
 from the leaf where the urea is applied, 
 to the utmost extremities of the shoot 
 tip and of the root system and even into 
 root hairs (figs. Ill, 1, 2). 
 
 It has been proposed (Crafts, 19612?) 
 that the sieve tubes of the phloem are a 
 
 34 
 
ramifving, integrated system of conduits, 
 permeable to longitudinal movement and 
 constituting a functional part of the 
 symplast system. The tracheary elements 
 of the xylem compose a specialized per- 
 meable system of conduits constituting a 
 functional part of the apoplast complex. 
 Evidence from autoradiography indicates 
 that some tracer molecules enter and move 
 in the symplast system with ease, while 
 others move much less freely. The substi- 
 tuted urea and symmetrical triazine herbi- 
 cides seem unable to enter and move in 
 the phloem along with the assimilate 
 stream. In contrast, most water-soluble 
 compounds enter roots with ease but some, 
 notably 2,4-D and benzoic acid, are moved 
 to the foliar portion of the plant slowly 
 and in small amount. These contrasting 
 behaviors pose some extremely challenging 
 problems both to plant physiologists 
 (with respect to the mechanics of solute 
 uptake and movement) and to agricultural 
 technologists who wish to employ these 
 
 mechanisms in using agricultural chemicals. 
 The contrasting and unpredictable beha- 
 viors of many new chemicals pose a con- 
 stant problem to those engaged in the 
 use of pesticides and mineral nutrients, 
 and this field is one in which plant 
 autoradiography seems capable of making, 
 some outstanding contributions. 
 
 Early work with Cl 1 *- labeled 2,4-D, 
 in which treated plants (killed by quick- 
 freezing and dried between hot, dry blot- 
 ters) gave indications of phenomenal and 
 unprecedented rates of transport, was 
 soon found to involve an artifact 
 (Crafts, 1956a; Pallas and Crafts, 1957). 
 The fact that the opposite leaf of treated 
 bean plants receiving this treatment had 
 labeling of the veins (fig. Ill, 3) led 
 to the answer to this problem of trans- 
 port. Theoretically, if 2,4-D* was being 
 exported from one primary leaf with the 
 assimilates it should not enter the oppo- 
 site leaf via phloem. Critical studies 
 
 Fig. Ill, 1. Root of zebrina grown on filter paper, as shown in Fig. II, 2. 
 
 Fig. Ill, 2. Autoradiograph of root shown in fig. Ill, 1. This plant was leaf- 
 treated with C^-labeled urea; C lZ +02 released by hydrolysis has been converted to 
 sucrose and moved in the transport system to the roots. 
 
 35 
 
±w 
 
 111,3 
 
 2 4.-D, 4V7../UH 
 14 Rll Ki»a(V SCAN. 
 
 iD«oo US 3Hw. 
 
 Fncut-Kiuie «u blotti* - 
 {tea*. 
 
 111,4 
 
 Rep Ki»mcv Bcan. I Dhop OS 3<fcHftS. Fueeze-Dkicd. 
 
 Fig. Ill, 3. Autoradiograph of bean plant treated on one leaf with a droplet of 
 2,4-D* then killed between blocks of dry ice and blotter-dried. Presence of the tra- 
 cer in the opposite leaf proved that rapid movement of tracer was taking place during 
 drying; this occurred in xylem because of evaporation from the leaf. 
 
 Fig. Ill, k. Autoradiography of bean plants treated on one leaf with 2,4-D* (left) 
 and MH* (right), f reeze-ki 1 1 ed and f reeze-dried. In this critical 3.5-hour treatment 
 all movement was in the phloem; opposite leaves do not show. 
 
 proved this conclusion to be true (fig. 
 Ill, 4); the label was moving in the 
 transpiration stream and hence the mech- 
 anism was not truly symplastic. Thus, 
 correct interpretation of data revealed 
 by autoradiography led to the solution 
 of this vexing problem (Pallas and 
 Crafts, 1957). 
 
 In comparative studies with three 
 herbicides, 2,4-D* moved a relatively 
 short distance, amitrole* moved through 
 the root system and accumulated in root 
 and shoot tips where assimilate utiliza- 
 tion was high, and mature untreated 
 leaves were bypassed with no leakage 
 from roots. In this test MH' ,C moved freely 
 throughout the symplast; a faint image 
 could be seen on the filter paper on 
 which the roo£s were growing, and all 
 mature leaves showed light labeling. 
 This was interpreted to mean that MH" was 
 migrating from phloem of roots to the 
 
 culture medium and to the xylem. Thus 
 again, interpretation of autographs led 
 to a realization of the importance of the 
 concepts of symplastic and apoplastic 
 movement, and to a recognition of the 
 widely differing behaviors of different 
 chemicals . 
 
 Plant autoradiography makes it pos- 
 sible to study a number of plant processes 
 in intact plants not previously subject to 
 critical investigation. A few of these 
 processes are: (1) absorption of solutes; 
 (2) translocation of solutes; (3) symplas- 
 tic vs. apoplastic movement; (4) selective 
 accumulation by tissue systems; (5) the 
 relative mobility of contrasting molecular 
 types; (6) the hydrolysis of esters in 
 plant leaves; (7) the activity of urease 
 and other enzymes in leaves; (8) the role 
 of photosynthesis in food transport; 
 (9) the effects of metabolic inhibitors 
 on uptake and distribution of compounds; 
 
 36 
 
(10) the localization of auxins, inhibi- 
 tors and plant nutrients in the plant 
 body. 
 
 Because in work on translocation by 
 either the phloem system or the xylem 
 conduits any injury of the plant may lead 
 to erratic results, it seems apparent that 
 such work must be done on intact plants. 
 Before the introduction of isotopes, stu- 
 dies on phloem transport of viruses came 
 nearest to meeting this situation. Now 
 it is possible to study the penetration 
 and translocation of labeled compounds 
 without the injury caused by insect feed- 
 ing or the risk of upset of metabolism and 
 transport mechanisms by virus injury. 
 Thus the use of autoradiography seems 
 ideal for studies on absorption and trans- 
 location. 
 
 In working with physiologically ac- 
 tive compounds it is important to know 
 where a compound accumulates with respect 
 to its true site of action. Autoradio- 
 graphy may be used to find the distribu- 
 tion of a labeled compound in the plant, 
 to discover regions of accumulation, and 
 to gain insight into its possible mode 
 of action. Collection of C-^C^ may give 
 a clue to the breakdown of the compound, 
 and extraction and chromatography may be 
 used to discover evidence for chemical 
 change in its composition. 
 
 By using compounds labeled in two or 
 more positions, evidence of decomposition 
 of a molecule inside plants may be readi- 
 ly obtained. By using labeled and un- 
 labled forms of two compounds, interac- 
 tion of one on the other can be investi- 
 gated. Thus, synergism or antagonism 
 between two components of a formulation 
 may be studied not only in the test tube 
 but inside the living plant. 
 
 Ashton, Zweig and Mason (1960) used 
 C^Oo to study the effects of triazine 
 compounds on CO2 fixation by bean plants. 
 Fixation was almost completely blocked 
 at 1 ppm and above, and time studies at 
 1 ppm showed that three triazines all 
 blocked Cl 1 ^ fixation in 6 hours whereas 
 at 3 hours simazine inhibited, methoxy- 
 simazine had no net effect, and trieta- 
 zine stimulated Cl 1+ 02 fixation. In fur- 
 ther studies Zweig and Ashton (1962) found 
 that atrazine greatly modified the course 
 of organic synthesis in bean leaves, and 
 
 that sucrose synthesis was completely in- 
 hibited, whereas aspartic acid increased 
 and glycine disappeared. Since citric 
 acid, serine, and asparagine were affec- 
 ted, these workers concluded that atra- 
 zine was interfering with the tricarboxy- 
 lic acid cycle and that respiration and 
 dark CO2 fixation were not being inter- 
 fered with. Thus, by isotope treatment, 
 chromatography and counting, basic studies 
 can be made on plants intact during the 
 treatment and reaction period. 
 
 Mason (1960) used tritiated 2,3,6- 
 trichlorobenzoic acid (TBA) to study the 
 uptake and movement of this herbicide. 
 By autoradiography, he found a high con- 
 centration of label in shoot tips, and 
 he proved by Warburg analysis that TBA 
 stimulates respiration; he concluded that 
 the high accumulation in meristems resul- 
 ted from a sort of autocatalytic reaction: 
 the herbicide moved freely to the meristem 
 where it increased the rate of respira- 
 tion, using up available substrate; this 
 steepened the gradient of foods into the 
 meristem and this in turn stimulated 
 rapid transport which in turn increased 
 the concentration of the herbicide, etc. 
 
 These examples indicate the type of 
 problems that lend themselves to study by 
 means of labeled tracers ; in all such 
 studies autoradiography is a major tool. 
 
 In the use of autoradiography certain 
 experimental designs have proved useful: 
 
 1. Time series have proved essential 
 in studies on absorption, translocation 
 and redistribution. Initial penetration 
 can be studied using short times, minutes 
 and hours. Translocation via phloem or 
 xylem starts only when the tracers have 
 reached these vascular tissues; usually 
 
 1 to 3 hours are required; time series 
 from this time to about 4 days will give 
 a picture of the continuing process. 
 
 2. Redistribution following initial 
 transport has been effectively illustra- 
 ted by time series in barley and bean 
 plants running 4 and 16 days. Even longer 
 times can be used with tracers having long 
 half- lives. 
 
 3. Concentration series have proved 
 helpful in studying penetration rates. 
 These can involve wide differences only 
 
 37 
 
when compounds of high solubility are 
 being studied. 
 
 4. Dosage series are excellent for 
 studying the capacity of a transport sys- 
 tem to distribute a tracer. Dosage series 
 with 2,4-D* have been used to show the 
 interrelation between concentration as 
 related to uptake and total dosage as it 
 brings about contact injury. In one 
 2,4-ET study with leaf application, a 
 dosage of 88. 4 ug or 0.4 mmole per treat- 
 ment proved optimum in the series 0.02, 
 0.1, 0.2, 0.4 and 0.8 jumole. 
 
 5. Parallel comparisons are useful for 
 studying the relative permeation and 
 mobility of different compounds. In 
 these trials many compounds are used on 
 one or a few species. 
 
 6. Selectivity trials are useful in 
 testing herbicides; in these, one com- 
 pound is used on many species. In the 
 latter two types of studies (4 and 5, 
 above) many plants are grown under the 
 same conditions, and treatments are made 
 rapidly during a short time period so 
 that time, dosage, and environmental 
 variables are minimized. 
 
 7. Comparative trials, in which appli- 
 cations are made to the leaves of some 
 plants and to the roots of others, drama- 
 tically show the great differences exist- 
 ing in the permeability of these organs, 
 the selectivity of the phloem and xylem 
 systems, and the role of migration and 
 interchange between them in ultimate 
 distribution. 
 
 8. Loci of application series on 
 plants having many leaves have given 
 strong confirming evidence that a mass- 
 flow type of mechanism in phloem trans- 
 port exists. 
 
 9. Treatment with tracers at different 
 stages in the growth of plants has shown 
 the division of labor among organs, the 
 differing processes of synthesis, trans- 
 port and storage of foods, the changing 
 permeability of cuticle and root tissues, 
 and the roles of maturity and senescence 
 in plant function. 
 
 10. Uptake and distribution trials of 
 extended duration have given valuable in- 
 formation on the locations, concentrations 
 and decomposition of pesticide residues 
 
 in plants. 
 
 38 
 
SECTION B 
 
 Some Typical 
 
 Problems and Their Solutions 
 
 o* 
 
 Chapter IV. Mechanisms of 
 Translocation 
 
 .For almost 2000 years research has 
 been done on transport mechanisms of 
 plants. In the 17th and 18th centuries 
 Marcellus Malpighi, the Italian anatomist, 
 and Stephen Hales, the British plant phys- 
 iologist, ringed plants in attempts to 
 solve the riddle of sap movements, but 
 it was not until 18 94 that Dixon and 
 Joly in Ireland elaborated a satisfactory 
 theory to explain the rise of sap to the 
 tops of tall trees. Their "cohesion 
 theory", though attacked on all sides, 
 has stood the test of time and is now 
 generally accepted by most plant physio- 
 logists (Crafts, 1961b). Tracer research 
 to be described in ensuing chapters has 
 provided confirming evidence on the per- 
 formance of this mechanism. 
 
 While the cohesion theory of water 
 movement in plants accounts for a major 
 portion of the transport accommodated by 
 the apoplast system, the phenomena of 
 active root absorption, root pressure, 
 guttation, and xylem exudation require 
 an additional mechanism. The root- 
 pressure mechanism of Priestley (1920), 
 as altered and elaborated by Crafts and 
 Broyer (1938), seems adequate to cover 
 the grosser aspects of active root uptake. 
 The root-pressure mechanism, plus the 
 cohesion mechanism of xylem transport, 
 account for the major uptake and movement 
 of water and inorganic salts in plants. 
 
 These mechanisms are described by 
 Dixon (1914) and Crafts (1961b). Briefly, 
 the root-pressure theory proposes that 
 with the germination of the seed and 
 growth of the radicle into the soil, salts 
 are actively absorbed by the cortical 
 
 cells of the expanding root, including the 
 root hairs. As the ions of these salts 
 enter the symplast system they are picked 
 up by the streaming protoplasm and carried 
 throughout the lumina of the cortex cells; 
 they move by diffusion from cell to cell 
 along the protoplasmic connections. Thus 
 they traverse the cortex, cross the endo- 
 dermis, pass through the pericycle and 
 permeate the ground parenchyma of the 
 stele. 
 
 The cells of the epidermis and cortex 
 are well-aerated by the soil atmosphere 
 and the elaborate intercellular space sys- 
 tem of the cortex, hence they have ample 
 opportunity to take up oxygen. But the 
 cells of the stele are small, closely 
 packed, and separated from the oxygen 
 supply by the endodermis with its suber- 
 ized Casparian strip in its radial walls. 
 This strip constitutes an effective bar- 
 rier JDetween the cortex and the stele of 
 the primary root, both for the influx of 
 oxygen and the efflux of sap. 
 
 Accompanying this decreasing supply 
 of oxygen between cortex and stele is an 
 increasing amount of CO2 liberated in the 
 processes of respiration that provide 
 energy for accumulation, protoplasmic 
 streaming, cell division and cell differ- 
 entiation. Thus, the cells of the cortex 
 and the cells of the stele in the primary 
 root find themselves in two different 
 environments. The cortical tissue has an 
 ample oxygen supply and a low CO2 concen- 
 tration; the stelar cells have just the 
 opposite . 
 
 Concomitant with a decreasing oxygen 
 
 39 
 
and an increasing CO2 gradient from cortex 
 to stele is an uptake and migration of 
 salts that by diffusion and protoplasmic 
 streaming are seeking an equilibrium of 
 concentration. But, as the concentration 
 in the stele approaches that in the cor- 
 tex where active accumulation is taking 
 place, the cells in this low C^-high CO2 
 environment are unable to hold the solutes, 
 which escape from the symplast to the 
 apoplast. With the gradient of solute 
 concentration between the soil solution 
 and the apoplast of the stele established 
 by active accumulation in the cortex and 
 release within the stele, water moves 
 osmotically, volume increases, sap accumu- 
 lates and - being unable to pass back 
 across the Casparian strip - migrates into 
 the xylem conduits and procedes upward 
 in the plant. This process makes root 
 pressure; it is responsible for xylem ex- 
 udation from excised roots, for guttation 
 from leaves, and for a form of apoplastic 
 solute movement. 
 
 The above processes take place in 
 young seedlings, in many herbaceous 
 plants during the night, and in woody 
 plants at the onset of root growth in the 
 spring. As leaves expand and transpira- 
 tion increases, however, the water bal- 
 ance in the apoplast system passes gra- 
 dually to the negative side and water is 
 moved (due to its cohesive force) in a 
 state of tension. Thus, in most plants 
 during summer days, in all plants grow- 
 ing in soils having a soil-moisture con- 
 tent approaching the permanent wilting 
 percentage, and in all tall trees above 
 the height of atmospheric rise (around 
 30 feet), water exists and moves in a 
 subatmospheric or tensile state. While 
 seeming to defy the common laws of the 
 liquid state, this water is maintained 
 in a metastable condition of super-heating 
 by its internal pressure, its cohesion to 
 itself and its adhesion to the thoroughly 
 wet lignocellulose walls of the xylem 
 capillaries, and also by the fact that 
 within the apoplast system there are no 
 unwet surfaces upon which vapor-phase may 
 become initiated. 
 
 While the mechanisms described above 
 seem completely adequate to account for 
 water and salt movements within plants, 
 the transport of foods is a different 
 matter. Ever since the pioneering stu- 
 dies of Mason, Maskell and Phillis it 
 
 seems well established that food movements 
 in plants follow a source-sink pattern 
 (Mason and Phillis, 1937). There has 
 been long and heated controversy, how- 
 ever, on the details of the mechanism in- 
 volved, one group (including Mason and 
 his associates) maintaining that movement 
 partakes of the nature of an activated 
 diffusion, and a second group (exemplified 
 by Munch) holding out for a mass- flow 
 process. 
 
 It seems obvious that in most plants 
 food substances are synthesized in the 
 leaves and utilized in meristematic re- 
 gions of the shoots and roots. Thus, the 
 source-sink pattern of food distribution 
 seems inherent in the physiological ana- 
 tomy of the plant. With developing proof 
 that the carbohydrates of plants move 
 principally in the form of sucrose, at 
 least during their transport via the 
 phloem, it also seems obvious that within 
 the plant there is a gradient of osmoti- 
 cally active solute paralleling the 
 source-sink gradient. It is now gener- 
 ally accepted that the sieve tubes of the 
 phloem constitute the common conduits 
 through which rapid food distribution 
 takes place, and it is known that the 
 phloem and xylem are parallel systems 
 within the vascular strands. Hence, with 
 a ready and constant supply of water 
 available in the xylem it seems obvious 
 that the osmotic gradient inherent in the 
 phloem system should result in a hydro- 
 static gradient between source and sink, 
 and that, if the sieve tubes constitute 
 a system of physiologically open conduits, 
 a mass-flow of solution should occur in 
 the phloem from source to sink. 
 
 What sort of evidence may be marshal- 
 led to substantiate this mass-flow mecha- 
 nism in plants? The following points will 
 serve to answer: (1) If one carefully 
 cuts into the phloem there invariably 
 occurs an exudation from this tissue which 
 contains sugars and small amounts of 
 nitrogenous nutrients. (2) Virus parti- 
 cles inoculated into the phloem by insects 
 follow the same source-sink pattern of 
 distribution as do foods. (3) If insects 
 feeding on sieve tubes of the phloem 
 (Mittler, 1957a, b\ Zimmerman, 1961) are 
 anaesthetized and cut free from their 
 mouth parts, prolonged phloem-exudation 
 occurs. This exudate contains the same 
 compounds that are found in phloem-exudate 
 
 40 
 
from cut stems. (4) When tracer mole- 
 cules are applied to intact plants they 
 may penetrate into the phloem and move 
 with the assimilate stream. These tra- 
 cers follow the same source-sink distri- 
 bution pattern as food substances. They 
 bypass mature leaves that are exporting 
 foods, enter younger leaves that have 
 not reached compensation, and concentrate 
 in meristems of stems, roots, and shoots. 
 
 A good many experiments to be de- 
 scribed in the following chapters throw 
 new light on the relations of various 
 cell functions to the translocation mech- 
 anisms, and several of them emphasize the 
 
 complex interrelations between the sym- 
 plast, the apoplast, the intercellular 
 space system, and the conducting tissues 
 that constitute specialized phases of 
 these systems. Often overlooked in re- 
 searches on vascular components of plants 
 is the fact that these components consti- 
 tute distribution systems adapted to the 
 functions of total dissemination of sol- 
 utes and not simply to movement from one 
 locus to another. Viewed in this latter 
 sense, it appears that the tracer studies 
 included here provide a wealth of infor- 
 mation on the distribution function in 
 plants . 
 
 41 
 
Chapter V. Evidences for Mass Flow 
 in the Phloem 
 
 A, 
 
 LUtoradiography has been used for 
 some 20 years to study uptake and distri- 
 bution of labeled compounds in plants. 
 Colwell (1942) supplied P 32 to leaves of 
 squash and used counting and autoradio- 
 graphy to study its location in various 
 plant parts. Heavy labeling of the phloem 
 groups of the bi collateral bundles indica- 
 ted that P32 } when properly applied, moved 
 out of the leaves via the phloem. Cross- 
 sections of fruits indicated accumulation 
 of the tracer in seeds, and P32 was also 
 present in phloem exudate. Colwell con- 
 cluded that movement of P 32 in phloem 
 paralleled movement of foods. Autoradio- 
 graphic work on tomato by Arnon, Stout 
 and Sipos (1940) showed accumulation of 
 p32 in the seeds of this plant. 
 
 These early studies indicated the 
 possible use of autoradiography for de- 
 tailed investigation of the absorption 
 and translocation of pesticides. Day 
 (1952) showed that 2,4-D applied to the 
 leaf of bean brought about bending of the 
 epicotyl, and by careful studies he 
 arrived at linear rates of presumed trans- 
 port of this regulator. With the produc- 
 tion of Cl4- labeled 2,4-D it became ob- 
 vious that a tool was available for use 
 in critical studies on the absorption and 
 translocation of this important compound 
 by intact plants. 
 
 To attack this problem we posed two 
 questions: Does 2,4-D as such get into 
 a plant? If 2,4-D is absorbed, is it 
 translocated into the regions of growth 
 response unchanged? 
 
 Our first tracer experiment involved 
 the use of carboxy- labeled 2,4-0-014, and 
 provided for a 4-hour treatment time and 
 
 two methods of killing the treated plants: 
 quick-freezing between blocks of dry ice 
 followed by drying between hot dry blot- 
 ters, and killing between hot dry blotters 
 by the method commonly used with herbarium 
 specimens. Several plants were handled by 
 each method and they were used to test ex- 
 posure times of the dried plants on Kodak 
 No-screen X-ray safety films. The experi- 
 ment indicated the 4-week exposure period 
 to be best, and proved t that in the 4-hour 
 treatment period 2,4-D* penetrated the 
 leaf and was translocated throughout the 
 stem and well into the roots. It proved 
 also that the quick killing caused an 
 artifact - the uptake and rapid transport 
 of the labeled compound via the xylem to 
 regions where drying was taking place 
 (figs. Ill, 3, 4). 
 
 The artifact introduced by the 
 quick-killing treatment is reminiscent of 
 the early work with the acid- arsenical 
 method for killing weeds (Crafts, 1933). 
 In this method, an acid solution of arsen- 
 ic is applied as a spray to perennial 
 weeds; with the rapid killing of the meso- 
 phyll tissues of the leaves, the leaf -sap, 
 plus arsenic which had diffused into it, 
 is rapidly sucked back through the stem 
 into the roots. This is typical apop las- 
 tic movement - it is the phenomenon that 
 takes place during drying when tissues of 
 the treated plant are killed by quick 
 freezing and then allowed to thaw out; 
 the solution moves along hydrostatic gra- 
 dients to regions where drying is taking 
 place (Pallas and Crafts, 1957). 
 
 Our next experiment involved a time 
 series in which treatment times were as 
 follows: 1/4, 1/2, 1, 2, 3, 4, 5, 6, 7, 
 8, 12, 24, 36, 48, 72, 96, 120, and 144 
 
 42 
 
hours. The quick-killing method was used. 
 The plant receiving the lA-hour treatment 
 had tracer in its opposite leaf, the 1/2- 
 hour treatment plant had tracer in its 
 opposite leaf and roots, and the 3-hour 
 treatment plant was bent and had tracer 
 in its opposite leaf and roots. Day 
 (1952) had found that absorption through 
 the cuticle and movement into the epico- 
 tyl takes somewhat over an hour; pres- 
 ence of 2,4-D* in the roots and opposite 
 leaf of the plant treated for 1/2-hour in- 
 dicated again the artifact of xylem move- 
 ment. However, since the tracer was pre- 
 sent in the stems and roots of five repli- 
 cates in the 3-hour test and four of the 
 plants showed the bean bend in the epico- 
 tyls, it seems evident that our first 
 questions had received a positive answer: 
 2,4-D* was present in the bent epicotyl, 
 the hypocotyl and the roots of all plants 
 treated 5 hours or more. Movement into 
 terminal buds, which could be possible 
 only via phloem, required 6 hours or more. 
 
 Experiments of the type described 
 were continued, using bean, cotton, and 
 cucumber seedlings (Crafts, 1956a). Ef- 
 fects of water balance, droplet placement, 
 leaf location and various methods of kill- 
 ing showed definitely that quick killing 
 followed by rapid drying between blotters 
 results in the artifact of xylem transport. 
 Drying alone between hot blotters was 
 slower, but the plants seemed to show a 
 true picture of phloem transport. The 
 greatest problem in this method is the 
 lack of a definite treatment time, for 
 translocation undoubtedly continues for 
 some time after the plants are put in the 
 press. On the other hand, freeze-drying 
 in a vacuum chamber proved effective 
 (fig. Ill, 4) and after further trials 
 this method has been adopted for all work 
 with small plants. From these early 
 trials a few definite conclusions stand 
 out: 
 
 (1) Following application in solution 
 as a droplet, 2,4-D" penetrates the cuti- 
 cle, migrates through the mesophyll, en- 
 ters the phloem tissue and moves from 
 treated leaf to growing roots and young 
 shoots . 
 
 (2) Under some conditions 2,4-D" will 
 move from leaf to roots ; under other con- 
 ditions from leaf to shoot tip; under 
 certain conditions to both roots and 
 shoot tips. 
 
 (3) Whenever the bean epicotyl bends 
 as a result of 2,4-D"" treatment the com- 
 pound has penetrated the leaf, petiole 
 and stem, and is present in epicotyl and 
 hypocotyl. 
 
 Although our original conclusions 
 were drawn on the basis of the presence 
 of the tracer in the affected regions 
 only, more recent work by bio-assay and 
 chromatography has substantiated our view 
 that 2,4-D attains this distribution. 
 
 TRACER DISTRIBUTION 
 
 Meanwhile, tests using 2,4-D" on 
 seedlings of a number of plants were pos- 
 ing new problems. For example, when seed- 
 lings of field bindweed {Convolvulus ar- 
 vensis) were treated on one cotyledon, 
 2,4-D* moved down into the roots without 
 labeling the opposite cotyledon in some 
 plants; in others it moved both downward 
 into roots and upward into the. shoot tip, 
 while the opposite cotyledon was free of 
 label (Crafts, 19562?). When tracer was 
 applied farther up the stem on seedlings 
 having several leaves, translocation still 
 carried the tracer to the roots and mature 
 leaves along the stem were bypassed. On 
 the other hand, when a young growing 
 leaf at the tip of the plant was treated 
 the tracer did not move downward from 
 the area of treatment but did move to the 
 tip of the leaf (Crafts, 19562?, figs. 32, 
 33, 34, 35). 
 
 For over a hundred years there has 
 been controversy as to the mechanism of 
 movement of foods in plants. Two diver- 
 gent views are: (1) translocation in the 
 phloem system is a diffusion- like process 
 in which each molecular species moves 
 along its own independent gradient un- 
 affected by movement of other substances , 
 including solvent water; and (2) trans- 
 location in the sieve-tube system is a 
 pressure or mass-flow in which food mole- 
 cules , tracers , or any other molecules 
 present in the conduits move together with 
 the solvent water from the source of syn- 
 thesis of osmotically active solutes to 
 the sink, or sinks, where assimilates are 
 utilized in growth, respiration and stor- 
 age. 
 
 By a dif fusion- like movement, tracer 
 applied to a leaf should presumably move 
 out through the petiole, and then tra- 
 verse the stem to enter the entire plant. 
 
 43 
 
The fact that 2,4-D* moving out from a 
 newly treated leaf might move solely to 
 the root, or to shoot and root, and that 
 it consistently bypasses mature leaves 
 that are themselves exporting foods, but 
 enters young growing leaves that are con- 
 suming foods, indicates that this tracer 
 moves in a stream with assimilates. Its 
 failure to move out of young leaves that 
 are importing foods implies the same sort 
 of mechanism. Because this indicates the 
 existence of a flow-type of mechanism, 
 further confirmation of such a type of 
 mechanism seemed desirable. 
 
 Brown, 1950). Addition of sugar to the 
 2,4-D applied under these conditions in- 
 duced movement as did illumination. 
 
 Treatment of chlorotic leaves of 
 variegated Tradescantia fluminensis 
 plants in the greenhouse with 2,4-D*, 
 amitrole", and MH" resulted in no move- 
 ment, while treatment on green leaves re- 
 sulted in extensive movement. The lat- 
 ter two compounds are more freely mobile 
 than 2,4-D* (figs. V, 1, 2, 3). " 
 
 THE SINK 
 
 THE SOURCE 
 
 If mass-flow requires an active 
 source of synthates, there should be a 
 correlation between translocation and 
 food synthesis. Earlier work with cold 
 2,4-D on bean had shown that this compound 
 would not move out of a leaf starved by 
 darkening until all food reserves were 
 exhausted (Mitchell and Brown, 1946; 
 Rohrbaugh and Rice, 1949; Weintraub and 
 
 * 
 
 M 
 
 't ■ T 
 
 V, 1 
 
 Having established the requirement 
 for photosynthesis or other supply of 
 food at the source, sink activity was 
 next examined. 2,4-D" movement from the 
 treated leaf of a plant that had been 
 growing for 30 days in tap water was neg- 
 ligible; the root was long but it had 
 ceased to grow and the plant had low ac- 
 tivity. Treatment of a similar plant 
 recently removed from a nutrient solution 
 showed movement of tracer into roots; a 
 
 V^ 
 
 •i 
 
 
 kJCJ 
 
 Fig. V, 1. Movement of 2,4-D* from green leaves (left) and a chlorotic leaf (right) 
 
 of variegated Tradescantia fluminensis . Photosynthesis activates the assimilate stream 
 
 which carries the tracer out of the green leaves; there is no movement from the chloro- 
 tic leaf. 
 
 Fig. V, 2. Movement of amitrole* from green leaves (left), and a chlorotic leaf 
 (right) of variegated tradescantia. 
 
 44 
 
plant still actively growing in a 1/2- 
 
 Hoagland nutrient solution showed strong 
 labeling of roots (Crafts, 19612? , fig. 
 8-5). Obviously, the more active the 
 growth process in the sink, the more 
 tracer moves into it. 
 
 To further study direction of move- 
 ment from treated leaves of zebrina, 
 several plants growing in a 1/2-Hoagland 
 solution were pruned to leave three healthy 
 leaves at the base and two to four young 
 growing leaves at the tip. The two upper- 
 most of the lower leaves were treated with 
 a high specific activity 2,4-D* (6 mc per 
 mmole totaling 1.1 uc per plant). Plants 
 so treated for 9 days translocated tracer 
 throughout their roots as well as into 
 their tip leaves (Crafts and Yamaguchi, 
 1958, fig. 8). 
 
 The location of source and sink were 
 also reversed in order to demonstrate that 
 tracer movement is polarized not with re- 
 spect to the organization of the plant but 
 with respect to source and sink. Tall 
 zebrina plants that had been growing vig- 
 orously in a 1/2-Hoagland solution for 1 
 month were pruned, removing the growing 
 stem-tip and leaving two fully expanded 
 leaves at the upper extremity of the 
 stem. The two tip leaves were treated, 
 using 1.1 juc of 2,4-D* with a treatment 
 time of 9 days; here, movement took place 
 through the total length of stem in a 
 basipetal direction with ample labeling 
 in the roots and in basal shoots. Again 
 there was a two-way movement at the base 
 of the plants, the assimilate stream 
 dividing and carrying the tracer into two 
 active sinks (Crafts and Yamaguchi, 1958, 
 fig. 9). It seems obvious from these 
 experiments that translocation proceeds 
 from a source of assimilates to a sink, 
 or sinks, where assimilates are being 
 utilized in growth; meanwhile, other 
 sources such as the lower leaves (fig. X, 
 1) are bypassed. (For further observa- 
 tions of similar response see Jackson, 
 1962.) Such distribution cannot be ex- 
 plained on the basis of a diffusion type 
 of movement, and in 11 years we have 
 failed to find an exception to this type 
 of tracer distribution in plants. In 
 cases where leakage occurs into the tran- 
 spiration stream, leakage also takes place 
 into the external medium of the roots. 
 
 \ 
 
 •v: 
 
 
 
 Fig. V, }. Movement of maleic hydra- 
 zide* from green leaf (left) and a chloro- 
 tic leaf (right) of variegated tradescan- 
 tia . 
 
 ROLE OF LEAF LOCATION IN TRACER MOVEMENT 
 
 The mass-flow concept of phloem 
 transport in plants requires that foods 
 manufactured in leaves low on the axis 
 should move into roots, that foods far- 
 ther up might move both downward to roots 
 and upward to sinks in young leaves, buds 
 and the shoot tip, that those even far- 
 ther up should move exclusively upward, 
 and that young rapidly expanding leaves 
 should be importing rather than exporting 
 foods. If tracers are moved in this same 
 stream they should undergo the same gener- 
 al distribution, save that they should be 
 subject to accumulation in varying degrees 
 according to their molecular properties. 
 A common expression of this mechanism is 
 the bypassing of mature leaves by tracers 
 in movement from active sources to active 
 sinks. Here we know that the phenoxy- 
 acetic acids and the benzoic acids are 
 strongly retained in living cells, that^ 
 amitrole is intermediate, and that maleic 
 
 45 
 
1U . 
 
 *L 
 
 V,4 
 
 1 
 
 Fig. V , k. Results of treatment with 2,4-D* on one cotyledon of 
 Pol ygonum convol vul us (left), leaf 2 (center), leaf h (right). 
 
 Fig. V, 5» Results of treatment with 2,4-D* on leaf 6 of P. con - 
 volvulus (left), leaf 8 (center), leaf 10 (right). 
 
 46 
 
hydrazide and dalapon are freely mobile 
 and may migrate from phloem to xylem. 
 The author ■ s early work (Crafts, 1956k) 
 with 2,4-D" in field bindweed (Convolvu- 
 lus arvensis) indicated that the above 
 pattern of distribution was to be expec- 
 ted, and subsequent studies with zebrina 
 plants gave many indications of the same 
 pattern, with bypassing of mature leaves 
 being a common phenomenon. In order to 
 document this concept more completely, 
 two experiments on the relation of leaf 
 location to tracer movement have been 
 conducted. 
 
 The first experiment involved a 
 series in which Polygonum convolvulus 
 plants growing in Hoagland's solution 
 were treated with 1/10 yumole of 2,4-D""' 
 of specific activity 1.24 mc per mmole on 
 various leaves and given a 1-day treat- 
 ment. These plants had been growing in 
 the greenhouse, had passed through the 
 rosette stage, and were making rapid 
 length-growth, but roots were apparently 
 not making rapid growth since 2,4-D* did 
 not move into them in more than trace 
 amounts . 
 
 From the cotyledon (fig. V, 4 ? left) 
 which was green and healthy, 2,4-D" moved 
 principally downward but was present in 
 the roots at a distance of 1 inch or more 
 only in trace amounts. From leaf 1, 
 2,4-D" moved downward in the stem and 
 upper root system; upward, it entered two 
 shoots in the axils of leaves 2 and 3, 
 and in small quantity it traversed the 
 total length of the shoot to the tip. 
 From leaf 2, (fig. V, 4, center), 2,4-D* 
 moved into the shoot in the axil of leaf 
 2; it moved downward in the main stem 
 into the upper roots, bypassing leaf 1, 
 and in lower concentration ascended the 
 stem to the tip, labeling two expanding 
 leaves and bypassing three full-grown 
 leaves. From leaf 3 downward, movement 
 was still strong into the upper roots; at 
 intermediate concentration, 2,4-D* moved 
 along the upper stem to its tip, labeling 
 two expanding leaves and bypassing four 
 fully expanded ones. 
 
 From leaf 4 (fig. V, 4, right), the 
 tracer went in both upward and downward 
 directions in approximately equal concen- 
 trations; it reached the roots and tra- 
 versed the shoot to its tip, labeling 
 three expanding leaves, slightly marking 
 
 a fourth, and bypassing three mature ones. 
 Uptake by the fifth leaf was strong and 
 2,4-0" moved downward past four mature 
 leaves into the roots; it moved in equal 
 concentration up the stem to its tip, 
 labeling one expanding leaf and bypassing 
 two. From leaf 6 (fig. V, 5, left), 
 2,4-D" movement was predominantly upward; 
 downward labeling faded within 2 inches, 
 and upward there was strong labeling to 
 the stem tip; one maturing leaf was almost 
 completely bypassed, and a young one was 
 strongly labeled. From leaf 7, movement 
 downward was about 1 inch, and upward it 
 was dense to the shoot tip; one mature 
 leaf was bypassed and one young one had 
 medium labeling. From leaf 8 (fig. V, 5, 
 center), movement was upward; leaf 9, 
 which was not fully grown, was lightly 
 labeled. From the tenth leaf on one 
 plant (fig. V, 5, right) and the eleventh 
 on another, labeling from the point of 
 application to the tip of the leaf was 
 very dense; there was some movement basi- 
 petally along the midvein but no tracer 
 left the treated leaf - in both cases the 
 treated leaves were not completely expan- 
 ded and were very evidently still import- 
 ing assimilates. In all but the two 
 uppermost leaf treatments, tests were run 
 in triplicate with remarkable uniformity, 
 and shorter series in which applications 
 were made to stems at various levels gave 
 the same general pattern of 2,4-D* dis- 
 tribution. Had amitrole, a more freely 
 mobile tracer, been used there would 
 probably have been more labeling of the 
 roots. 
 
 The second experiment in this series 
 was on nasturtium plants; amitrole*, 
 1/10 A-imole at 0.94 specific activity was 
 used in 10.0 ul droplets with 1/10 per 
 cent Tween 20 in the formulations, and 
 treatment time was 4 days. Nasturtium 
 discards its cotyledons upon germination; 
 in our plants the epicotyl averaged about 
 4 inches and there were a few roots at its 
 base. The first two leaves come out as a 
 pair from a single node, but above these 
 the leaves alternate. From one of the 
 first pair of leaves (fig. V, 6, left) 
 amitrole* moved predominantly downward; 
 roots were strongly labeled whereas label- 
 ing in the tops was faint to medium among 
 the three replicates. From leaf 4 
 (fig. V, 6, right), movement was about 
 equal judging from the density of stem 
 labeling. In this plant, amitrole* 
 
 47 
 
w. 
 
 2Z g|| * 
 
 Fig. V, 6. Distribution of ami t role" applied to one of the lower pair of leaves of 
 Tropaeol urn maj us (left) and to leaf k (right). 
 
 leaked from phloem to xylem in small 
 amounts; a faint image of all leaves could 
 be detected. Density of labeling of 
 leaves along the axis of nasturtium can 
 be used as a measure of compensation; 
 leaves that were still enlarging were 
 
 labeled, density being in proportion to 
 their growth rate. In the plant treated 
 on leaf 2, three young leaves at the 
 shoot axis were densely labeled, and four 
 leaves between the treated leaf and the 
 first strongly labeled leaf were faintly 
 
 48 
 
Fig. V, 7 
 of T. ma jus . 
 
 Distribution of amitrole* applied to leaf 6 (left) and to leaf 9 (right) 
 
 labeled, apparently due to leakage. 
 
 From leaf 3, movement was about 
 equally divided, the root system and three 
 young leaves having dense labeling with 
 the first pair and other mature leaves 
 being virtually bypassed. Leaves 4, 5 and 
 6 (fig. V, 7, left) showed similar distri- 
 bution of the tracer, with the balance of 
 distribution shifting upward but the roots 
 still showing strong labeling of the tips 
 and late- formed adventitious members. Two 
 
 plants were treated, each on leaf 9; the 
 one on which this leaf was approaching 
 full size showed two-way movement, 
 although the roots, lower stem, and lower 
 leaves were lightly labeled. The other 
 plant (fig. V, 7, right) was treated on a, 
 small leaf; although it enlarged consider- 
 ably during the 4-day treatment period all 
 movement was acropetal, with two younger 
 leaves being labeled. Most of the tracer 
 was localized within the treated leaf. 
 
 49 
 
vv 
 
 4 
 
 EFFECT OF SHADING ON 2,4-D" 
 TRANSLOCATION FROM LEAVES AND ROOTS 
 
 Work with cold 2,4-D soon after the 
 discovery of its herbicidal effects proved 
 that shading leaves to deplete them of 
 stored foods will inhibit movement from 
 them. In order to check this, an experi- 
 ment using labeled 2,4-D on bean plants 
 was carried out. Dosage was 1/10 /umole 
 of tracer at 1.24 mc per mmole specific 
 activity in 10 jul droplets; the same 
 tests and dosage were also run with root 
 treatments in 100 ml of Hoagland's solu- 
 tion. All plants were in the dark through 
 the 24-hour treatment period, and the 
 radiographs show that translocation from 
 leaves was appreciably inhibited by pre- 
 treatment in the dark. Figure V, 8, shows 
 plants pre treated for 6 hours (left) and 
 18 hours (right) in the dark. Figure V, 
 9, shows 30- and 42-hour dark pre treat- 
 ments. Similar pre treatments had a 
 slight but detectable effect on root up- 
 take, but no effect on xylem movement 
 from roots to tops ; this last observation 
 applied also to cotton, soybean and 
 barley plants. 
 
 If one leaf of bean is kept in the 
 dark and the opposite leaf treated, more 
 tracer will enter the shaded leaf. When 
 two bean plants ware disbudded to hold 
 down apical growth the treatment induced 
 
 continued growth of the primary leaves. 
 These plants were leaf-treated with 
 1/10 (umole of amitrole* of specific acti- 
 vity 0.94, a 10 fjl droplet each. One 
 plant was in the open greenhouse with 
 both leaves exposed, and the untreated 
 leaf of one plant was in the dark for 3 
 days before treatment and during the 
 4-day treatment period; evidently it be- 
 came depleted, as more tracer entered it 
 than entered the unshaded leaf (fig.V,10). 
 Anderson (1958) has shown this effect in 
 using amitrole on Cyperus rotundas. 
 
 The present data are offered as evi- 
 dence that a mass- flow mechanism is re- 
 sponsible for rapid longitudinal movement 
 in the phloem. How molecules moving by 
 diffusion, or by any other mechanism in- 
 volving random distribution, could con- 
 sistently pass along stems while bypass- 
 ing mature leaves and entering young ex- 
 panding leaves and growing shoot tips or 
 root systems, is difficult to comprehend. 
 The distribution patterns noted above so 
 obviously follow those of assimilated 
 foods in the plant that the only plausible 
 explanation seems to be a concomitant 
 movement of tracer and food and water 
 molecules in the assimilate stream, via 
 the phloem, from the source of foods to 
 the various sinks. 
 
 50 
 
V.10 
 
 Fig. V, 8, opposite, right: effect of dark pretreatment on translocation 
 of 2,4-D* in bean. From left: plant 1, root treatment; plant 2, leaf treat- 
 ment 6 hours in dark; plant 3, 18 hours in dark; plant k, 2k hours in dark. 
 A1 1 plants treated in dim light and given 24-hours treatment time in dark. 
 
 Fig. V, 9, opposite, left: effect of dark pretreatment on translocation 
 of 2,4-D in bean. Left pair of plants 30 hours in dark; right pair k2 hours 
 in dark. All plants, 2k hours treatment in the dark. 
 
 Fig. V, 10, above: effect of dark treatment on translocation of amitrole- 
 in bean. Plants debudded; left plant, opposite leaf in light; right plant, 
 opposite leaf in dark. 
 
 51 
 
Chapter VI. Comparative Movement of 
 Tracers 
 
 With the synthesis of a number of 
 Cl 4 - labeled herbicides, tracer studies 
 comparing the mobility of these compounds 
 became possible. The first such experi- 
 ment using 2,4-D*, amitrole*, and MH* has 
 been described in a number of publications 
 (Crafts and Yamaguchi, 1958; Crafts, 
 1961a, b). Figure VI, 1, shows the 
 mounted plants and the autoradiographs of 
 this experiment. The plants were growing 
 in depleted soil and were about at a 
 standstill, although healthy- looking. 
 Two treatment times were used - 7 hours, 
 and 4 days. The 7-hour treatments resul- 
 ted in very little movement from treated 
 leaves; the 4-day plants showed the in- 
 teresting differences seen in figure VI, 
 1. Treatments in this experiment involved 
 droplet applications of 10 jul each on 
 three leaves of each plant; total dosage 
 per plant was 0.5 juc 
 
 The autographs show the inherent 
 differences between these three compounds 
 with respect to mobility in plants. The 
 2,4-D" remained accumulated mainly in the 
 three treated leaves; little tracer was 
 present in the stem beyond the points of 
 attachment of these leaves. In these 
 rather inactive plants 2,4-D* was rela- 
 tively immobile, but in 4 days the ami- 
 trole" had moved throughout the plant and 
 was present at low concentration in all 
 stems and in the roots. It accumulated 
 in three separate shoot tips and in one 
 fruit at concentrations above that of the 
 intervening stems ; it bypassed many 
 mature leaves. 
 
 In the 4-day treatment period, the 
 MH* had moved throughout the plant; it 
 was present at medium concentrations in 
 
 the stems from shoot tips to roots, de- 
 creasing from the treated leaves toward 
 the base of the plant. Three shoot tips 
 were intensely labeled, indicating normal 
 phloem transport to active sinks. In 
 contrast to amitrole*, MH* was present in 
 all mature leaves in varying concentra- 
 tions depending upon proximity to treated 
 leaves. Intensity of labeling was lower 
 in treated leaves than was the case in the 
 2,4-D*-treated plant 
 
 From these results it seems obvious 
 that MH* moves most freely in a plant, 
 amitrole* is intermediate, and 2,4-D* is 
 least mobile. Since MH* moved so freely 
 it seems that the limited movement of 
 2,4-D* reflects not a failure of the 
 translocation mechanism but rather, a 
 strong affinity for retention of these 
 molecules by cells of the living leaves 
 and stems. Accumulation apparently im- 
 mobilized the chemical before it had op- 
 portunity to translocate any great dis- 
 tance in the slow-moving assimilate 
 stream of these inactive plants. 
 
 Amitrole" moved throughout the plant 
 body in this experiment but was limited 
 to the phloem in the stem and roots. MH* 
 apparently migrated from phloem to xylem 
 and, carried by the transpiration stream, 
 appeared in all leaves. The uniform 
 labeling of mature untreated leaves indi- 
 cates little tendency for MH* to be accu- 
 mulated by living cells. 
 
 When a comparative experiment using 
 2,4-D*, amitrole*, MH*, urea* and monuron* 
 was conducted on rape plants, the 2,4-D* 
 moved more extensively, reaching the main 
 stem in high concentration and the roots 
 in amounts diminishing with distance. 
 
 52 
 
» 
 
 I 
 
 
 '<% 
 
 ■ 2j 
 
 ■> 
 
 • ■ - 
 
 r 
 
 *m • 
 
 f J 
 
 Ol 
 O 
 
 > 
 
 "o 
 > 
 
 c 
 
 ■!-> 
 
 c 
 a; 
 
 E 
 
 -4-> 
 
 <0 
 
 c • 
 
 ..- </> 
 
 a» -o 
 
 N 
 
 J- 
 
 c 
 
 * e 
 
 31 •-- 
 
 C C 
 
 E 
 -!< ■»-» 
 
 — <u 
 
 O i- 
 
 
 e * 
 
 CO X 
 
 2: 
 
 \ \ 
 
 O C 
 1 fO 
 
 *, 
 
 -3- 
 
 M- *o 
 
 t 1 
 
 i- 
 
 ■M »r- 
 C E 
 
 J 
 
 a) 01 
 
 6" * 
 
 ' ; 
 
 E O 
 
 •\ 
 
 4) — 
 > 
 
 •1— * 
 
 %j 
 
 Compara 
 f 2,4-D 
 
 ^ 1^ 
 
 > 
 
 c • 
 
 Fig. 
 plant in 
 
 53 
 
VI, 2 
 
 \ 
 
 \ 
 
 < / 
 
 I 
 
 I * 
 
 ■ 
 
 V 
 
 I I 
 
 VI, 3 
 
 
 
 I// 
 
 I 
 
 M 
 
 54 
 
Amitrole" moved throughout roots and 
 showed high accumulation in root tips; it 
 bypassed one mature leaf, as did 2,4-D*. 
 MH" moved effectively throughout the plant 
 and was present in medium intensity in 
 two mature leaves; again this is evidence 
 for migration to the xylem. Urea" is 
 rapidly hydrolyzed by urease in rape 
 leaves; the Cl^C>2 is synthesized to 
 sugars, and chromatography shows the 
 labeled compounds in roots to be sucrose. 
 Mature leaves on this plant were bypassed; 
 apparently sucrose is retained within the 
 symplast. Monuron" apparently failed to 
 enter the symplast; its distribution in- 
 dicates apoplastic movement only, in 
 treated leaves of tomato (Haun and Peter- 
 son, 1954), rape, barley (Crafts, 19592?) 
 and several other plants. 
 
 The more extensive movement of 
 2,4-D in the rape plant may be accounted 
 for by two facts: the plants were young 
 and actively growing, and the treatment 
 time was 2.5 days - a shorter time than 
 in the case of the zebrina. 
 
 An additional study was made using 
 barley plants grown in nutrient cultures 
 in a greenhouse. When they were at a 
 stage in which there were four expanded 
 leaves, 2,4-D ft , IAA*, amitrole*, MH", 
 urea*, and monuron* were applied to leaf 
 1 on two plants and to leaf 4 on two 
 plants. Dosage was 0.5 juc per plant of 
 solutions standardized at 0.5 mc per 
 mmole; treatment time was 27 hours. 
 
 Figure VI, 2, 3, show the mounted 
 plants and autographs of this experiment. 
 
 Fig. VI, 2. Barley plants given 27- 
 hour treatments with 2,4-D* (left 4 plants 
 plants); IAA* (center 4 plants); amitrole* 
 (right 4 plants). Of the four plants re- 
 ceiving each chemical, the left-hand pair 
 were treated on leaf 1, the right hand 
 pair on leaf 4. Dosage, 0.05 AJC, volume 
 10 ajI, specific activity, 0.5 mc per 
 mmo 1 e . 
 
 Fig. VI, 3. Barley plants given 27- 
 hour treatments with maleic hydrazide- 
 (left 4 plants); urea''' (center 4 plants); 
 monuron''" (right 4 plants). Of each group 
 of four plants the left-hand pair were 
 treated on leaf 1; the right-hand pair on 
 leaf 4. Dosage 0.05 >uc, volume 10 jul, 
 specific activity 0.5 mc per mmole. 
 
 It is apparent that the first four com- 
 pounds constitute a mobility series, in- 
 creasing in mobility in the order given 
 above. It is evident from the autographs 
 that the older leaves provide nourishment 
 for the roots, whereas the youngest 
 expanded leaf provides for its own nour- 
 ishment and exports to the younger leaves 
 and inflorescence. Even in the case of 
 MH* there is little evidence for leakage 
 into the xylem, as the mature leaves show 
 only in the case of the treatments on 
 leaf 1. Amitrole* moved from leaf 1 to 
 younger leaves, but bypassed all mature 
 leaves . 
 
 Autographs of the urea* treatment 
 indicate that urea is hydrolyzed during 
 penetration, and that the C02" is rapidly 
 incorporated into assimilates of which the 
 principal constituent is sucrose which 
 moves in the assimilate stream to regions 
 of growth where food utilization is active. 
 In the autographs of Leaf 4 it is apparent 
 that accumulation is high in the interca- 
 lary meristem and in the root tips, and 
 low in the intermediary mature tissue. 
 
 Monuron* is not split by urease; it 
 failed to move out of the treated leaves, 
 and showed apoplastic movement only. 
 
 Fig. VI, 4. Barley plants treated with 
 dalapon* on leaf 3 (left) and leaf 6 
 (right). Dosage was 0.1 yuc per treatment. 
 
 55 
 
Figure VI, 4, shows results of a 
 similar experiment with dalapon* on some- 
 what older barley plants. Evidently this 
 compound moves freely in the phloem; it 
 also migrates to the xylem and shows apo- 
 plastic distribution, particularly from 
 treatment on the older leaf. Noticeable 
 also is the fact that dalapon* does not 
 accumulate in the mature roots of the 
 barley plants. 
 
 Since most of our work with labeled 
 tracers had utilized whole intact plants 
 where both absorption and translocation 
 are involved, it seemed important to 
 carry out tests in which these processes 
 could be distinguished. To study absorp- 
 tion uncomplicated by rapid transport, a 
 test using cubes of potato tuber tissue 
 was employed. 
 
 Cubes were cut 1-1/2 inches square 
 and placed on filter paper saturated with 
 water in a metal container; a loose lid 
 of cardboard was used as a cover. Lano- 
 lin rinp;s about 7.0 mm in diameter were 
 centered on the tops of the blocks and 
 treatments were made using 10 jul per drop- 
 let in each ring, with 0.05 uc per appli- 
 cation with solutions standardized at 0.5 
 mc per mmole. The chemicals used were 
 2,4-D*, amitrole*, MH*, urea*, monuron*, 
 and IAA* ; treatment times were 2 , 4 , 8 , 
 and 16 days. Each chemical was applied 
 to two tuber blocks; at the end of the 
 treatment time one block was split ver- 
 tically and a 2 mm slice through the 
 middle of the treatment was taken. The 
 second block had three 2 mm horizontal 
 slices removed, starting at the top. All 
 slices were freeze-dried, mounted, pressed 
 and autographed. In mounting, the three 
 horizontal slices were inverted so that 
 the autograph faces represented location 
 of the chemicals at levels of 2, 4, and 
 6 mm in the tuber block. Figure VI, 5, 
 shows the autographs and the tuber slices 
 for the 2,4, and 8-day tests (the verti- 
 cal slices are on the left and the hori- 
 zontal slices starting from the left are 
 arranged in the order of 2 mm, 4 mm, and 
 6 mm) . 
 
 In this storage parenchyma tissue in 
 which phloem transport is not involved, 
 the same mobility relation holds for 
 2,4-D*, IAA*, amitrole*, and MH*. 2,4-D* 
 moved for the first two periods and then 
 seemed to be immobilized. IAA* moved 
 
 through the first three periods though 
 not rapidly - in 16 days it had moved 
 along the isolated phloem strands, pre- 
 sumably in phloem parenchyma because the 
 sieve tubes were blocked (Crafts, 1933). 
 Amitrole" continued to move slowly and by 
 the 8th day the diameter of the occupied 
 circle in the 2mm slice was about 2.5 cm. 
 The MH" moved even farther, reaching the 
 borders of the slice at the 2mm and 4mm 
 level. Urea in this non-green tissue in 
 the dark was apparently hydrolyzed and 
 the C02* lost to the atmosphere. Monuron* 
 showed typical apoplastic movement, tra- 
 veling rapidly to the outer borders of the 
 
 slices, where it accumulated as a^ result 
 of evaporation of the aqueous medium. 
 This is especially noticeable in the ver- 
 tical slices, which show increasing con- 
 centration from the lower to the upper 
 region as a result of the fact that the 
 blocks stood on a water film from which 
 movement caused by evaporation was upward 
 and outward toward the exposed surface. 
 
 The experiments with potato tuber 
 tissue show that differences in mobility 
 between chemical compounds are not the 
 result of differences in phloem transport 
 per se but are differences in the ability 
 of the molecules to migrate through rela- 
 tively undifferentiated tissues such as 
 the mesophyll of the leaf, the parenchyma 
 of the root cortex, and the parenchyma of 
 the vascular tissues. Apparently, paren- 
 chyma cells have different affinities for 
 these tracer molecules; 2,4-D" seems to be 
 absorbed with avidity and retained. This 
 probably explains its failure to move from 
 roots. IAA" moves a bit more freely; ami- 
 trole* moves relatively freely but is re- 
 tained within the symplast; MH* is freely 
 mobile. In whole plants it even leaks 
 from phloem to xylem and ascends to the 
 foliage in the transpiration stream; there 
 is evidence that P32 may do this and thus 
 circulate in plants (Biddulph, et at., 
 1958). 
 
 If 2,4-D* can be retained by the 
 chlorenchyma of the leaf it may be 
 absorbed from phloem along the transloca- 
 tion route ; this probably explains its 
 limited distribution. When the assimilate 
 stream is moving rapidly, as in the case 
 of the plants used in experiments on leak-' 
 age from roots, transport may be effective 
 in moving 2,4-D into roots. When it is 
 slow, the tracer is all absorbed and re- 
 
 56 
 
tained within the treated leaves and 
 stems and none reaches the root. 
 
 These conclusions have important 
 implications in the use of 2,4-D for 
 controlling perennial weeds; the recom- 
 mendation of treatment at the early 
 flower bud stage probably relates to the 
 physiological condition when root carbo- 
 hydrates are low (Weise and Rea, 1962), 
 photosynthesis is high, and translocation 
 rapid. Dosage of 2,4-D must pass through 
 a maximum; Weise and Rea found this to be 
 1 pound per acre for field bindweed. The 
 reason is that increasing dosage brings 
 about increasing penetration, and trans- 
 location in the assimilate stream in- 
 creases up to a point where contact in- 
 jury impairs the source and transport 
 stops". Optimum dosages for treatment of 
 
 perennials with 2,4-D may vary widely 
 because of differences in cuticle thick- 
 ness, toughness of leaves, partition ef- 
 fects, saturation levels, and specific 
 sensitivity to phytotoxicity , but opti- 
 mum treatment levels have been found for 
 many weed species. 
 
 MECHANISM OF ESTER TRANSPORT 
 
 In order to throw some light on the 
 problem of how esters of the phenoxy 
 herbicides move in plants, isopropyl 
 esters of 2,4-D labeled on the carboxyl- 
 carbon and on the alcohol chain with Cl4 
 were synthesized. When applied to cotton 
 and barley plants (Crafts and Foy, 1959, 
 Crafts, 1959a) it was shown that the es- 
 ter is hydrolyzed during penetration of 
 the leaf because the alcohol label was 
 
 • • 
 
 Fig. VI, 5. Tuber slices (lower) and autoradiography (upper) of potato tuber blocks 
 treated for 2, 4, and 8 days with, top to bottom, 2,4-D*, ami trole*, MH*, urea*, monu- 
 ron*, and IAA*; no replication. Slices on the left of each treatment were taken verti- 
 cally, those in the number 2, 3, and 4 positions are horizontal samples from another 
 treated block taken 2, 4, and 6 mm from top surface. 
 
 57 
 
subject only to apoplastic movement with- 
 in the leaves, whereas the carboxy- labeled 
 2,4-D ion moved to the crown of the plant 
 via the phloem just as did the ion from 
 2,4-D* acid treatment. Szabo (1963) has 
 shown that hydrolysis of 2,4-D esters may 
 take place in a solution in contact with 
 the exterior leaf surface of some plants. 
 
 Labeling of molecules in different 
 positions has proved extremely valuable 
 in biochemical studies on metabolic path- 
 ways in plants; it was useful in studies 
 on 2,4-D metabolism as employed by Wein- 
 traub, et at., (1952a, b) ; it proved useful 
 in this study on 2,4-D ester transport in 
 plants, and has many more uses in studies 
 on the uptake, distribution, and meta- 
 bolic fate of herbicides in plants. 
 
 COMPARATIVE MOVEMENT OF LABELED TRACERS 
 IN POTATO TUBER CYLINDERS 
 
 As new labeled compounds became 
 available, it seemed desirable to study 
 their movement into and through tissues 
 of different types. Since movement of 
 labeled compounds through parenchyma tis- 
 sue may play an important role in their 
 subsequent distribution in the plant, 
 studies were made using cylinders of 
 potato tuber tissue. Twelve Cl^-labeled 
 compounds plus P32 were applied to the 
 upper ends of potato tuber cylinders 
 within lanolin rings. Movement was deter- 
 mined by removing slices from the centers 
 of the cylinders, freeze-drying, mounting 
 and autographing. Table VI, 1, presents 
 data on the penetration of the compounds 
 into the cylinders. 
 
 Tissue from the tuber cylinder not 
 used for autograph slices was freeze- 
 dried, ground and extracted. From 96 to 
 97 per cent of the activity of 2,4-D* 
 was extracted in alcohol and from one- 
 fifth to one-sixth of the activity in the 
 extract was not partitionable into chloro- 
 form - this was not 2,4-D*, and chromato- 
 graphic studies indicated it to be a com- 
 plex of 2,4-D adsorbed to an alcohol- 
 extractable substance or in weak conjuga- 
 tion; the amount of this substance was 
 constant from 2 through 16 hours. 
 
 Studies on the amitrole* content of 
 the dried potato tissue showed decreased 
 extract ability in alcohol with time; 
 chromatography showed decreasing activity 
 
 in the spot coincident with amitrole* with 
 time. Only about 10 per cent of the 
 alcohol-extracted activity was free ami- 
 trole*. A major fraction of the extrac- 
 ted activity remained near the origin and 
 was associated with a dark-colored sub- 
 stance ; this substance remained constant 
 with time, being present at zero time. 
 The other major fraction of the extract- 
 able activity was found in the extract 
 from the residue of the treated samples 
 and of the control ( time ) , after par- 
 titioning with chloroform. 
 
 Mi*" was largely extracted with alco- 
 hol and the extract chromatographed as a 
 spot coincident with stock MH-Cl^. This 
 compound seems to be almost completely 
 immune from breakdown or conjugation. 
 
 Monuron" showed a drop in extracta- 
 bility, with time from 100 per cent at 
 zero time to 91 per cent after 4 days. 
 Monuron"" was readily freed from movement 
 interference by dark-colored materials ; 
 when the front was e luted and the eluate 
 co-chromatographed with stock monuron*"* 
 on reversed phase paper, the only spot of 
 significance was monuron*"". 
 
 Small amounts of simazine" moved into 
 the potato tissue; simazine-Cl^ decreased 
 with treatment time from nearly 100 per 
 cent at zero time to about 25 per cent 
 at 16 days. The remaining activity oc- 
 curred in three other spots with lower 
 Rf values; one of these was associated 
 with dark-colored materials, and a third 
 spot was coincident with a radioactive 
 impurity present in the original sima- 
 zine". 
 
 Alcoholic extracts of IAA*-treated 
 tissue showed no IAA-C14 after 4 days. 
 
 COMPARATIVE UPTAKE AND DISTRIBUTION 
 OF TRACERS APPLIED TO ROOTS 
 
 Because tracers applied to leaves are 
 subject to such wide differences in dis- 
 tribution in plants it seemed logical to 
 study comparatively the uptake by roots. 
 Following a preliminary trial with barley 
 seedlings growing in small culture tubes, 
 an extensive test using 2,4-D*. IAA*, 
 amitrole*, MH*, urea*, dalapon , monuron*, 
 simazine*, and P32 was set up. Each 
 plant used was growing in 4 ml of culture 
 solution and dosage was standardized at 
 
 58 
 
0.037 mc per plant. The following were 
 the treatment times: 1/2-hour, 1 hour, 
 2 hours, 4 hours, 8 hours, 16 hours, 1 
 day, 2 days, 4 days, and 8 days. The 
 roots were rinsed and the plants freeze- 
 dried, mounted and autographed (Crafts 
 and Yamaguchi, 1960a). Figures VI, 6, 7, 
 8, show the results of this experiment. 
 
 The 2,4-D*, monuron*, simazine*, and 
 IAA* were absorbed sufficiently in 30 min- 
 utes to give strong images of the roots. 
 In 2 hours amitrole* and P32 had also 
 
 entered the roots sufficiently to give 
 good autographs. Monuron* and simazine* 
 started moving upward in 2 hours and by 
 8 hours the tops were all labeled. In 
 16 hours, amitrole*, dalapon*, and p32 
 had concentrated sufficiently in tops to 
 autograph well; monuron* and simazine* 
 continued to accumulate. p32 built up to 
 high concentrations in tops and roots so 
 that in 2, 4, and 8 days the images of 
 the roots became broad and blurred (this 
 results from the high- intensity radiation 
 of P 32 ). After 4 days very little 2,4-D* 
 
 TABLE VI, 1 
 Penetration of Labeled Compounds into Median Slices 
 From Potato-tissue Cylinders 
 
 Compound 
 
 Penetration (millimeters) 
 
 
 Days 
 
 Average 
 penetration 
 
 
 2 
 
 4 
 
 8 
 
 16 
 
 
 2 
 2 
 
 1 
 3 
 
 1 
 5 
 
 1 
 5 
 
 1.25 
 
 
 3.75 
 
 
 5 
 
 5 
 
 5 
 
 6 
 
 5.25 
 
 
 5 
 
 6 
 
 5 
 
 5 
 
 5.25 
 
 2,4-D 
 
 7 
 7 
 
 7 
 
 6 
 
 6 
 8 
 
 7 
 8 
 
 6.75 
 
 
 7.25 
 
 Sodium benzoate .... 
 
 8 
 
 6 
 
 8 
 
 7 
 
 7.25 
 
 IAA f 
 
 5 
 8 
 
 6 
 10 
 
 10 
 8 
 
 11 
 10 
 
 8.0 
 
 Sodium pheny lacetate . . 
 
 9.0 
 
 
 9 
 
 11 
 
 15 
 
 20 
 
 13.5 
 
 IAA 1 
 
 10 
 12 
 
 11 
 10 
 
 15 
 20 
 
 20 
 18 
 
 14.0 
 
 P320i+ 
 
 15.0 
 
 
 12 
 
 15 
 
 20 
 
 23 
 
 17.5 
 
 MH 
 
 10 
 23 
 
 15 
 34 
 
 20 
 45 
 
 35 
 55 
 
 20.0 
 
 
 39.0 
 
 t Penetration in symplast 
 
 t Penetration in phloem strands 
 
 § Penetration in apoplast 
 
 59 
 
VI. 6 
 
 \ 
 
 
 VI. 7 
 
 \ 
 
 j \ 
 
 S 
 
 
 
 II 
 
 # c 
 
 1 
 
 
 60 
 
and urea" had entered the tops, and after 
 8 days these tracers and IAA" were still 
 in lew concentration in the foliage. 
 Urea" was still low in intensity in the 
 roots; IAA"" and 2,4-D* were concentrated 
 in roots. 
 
 From these studies it seems obvious 
 that even though different tracer mole- 
 cules enter leaves and roots at different 
 rates and attain different distributions, 
 the patterns of movement reflect the two 
 basic mechanisms - that is, symplastic 
 movement via parenchyma cells and phloem, 
 and apoplastic movement via cell walls 
 
 Fig. VI, 6. Comparative uptake and trans 
 The tracers in each set as follows, left to 
 monuron*, dalapon--'-, simazi ne*, P 32^ anc | IAA* 
 each plant with roots in k ml of Hoagland's 
 series, left to right: l/2 hour, 1 hour, 2 
 
 and xylem. Although different molecules 
 such as 2,4-D, IAA, monuron and simazine 
 enter roots rapidly, it does not follow 
 that they migrate into and move in the 
 xylem with ease. 2,4-D and IAA, which 
 are avidly accumulated by living paren- 
 chyma, were the slowest to enter the xylem 
 of roots and move to tops; monuron and si- 
 mazine, which follow an apoplastic pattern, 
 moved most rapidly to the tops. Just how 
 these latter molecules are able to tra- 
 verse the root cortex, endodermis and 
 pericycle so rapidly is unknown; they must 
 enter and cross living cells in order to 
 do this. 
 
 location of nine tracers by barley plants, 
 right: 2,4-D*, amitrole*, MH*, urea-, 
 . All tracers except P32, Cl^-labeledj 
 solution with 0.037 wc of tracer. Time 
 hours. 
 
 Fig. VI, 7. Comparative uptake and translocation of nine tracers by barley plants. 
 Chemicals and conditions same as in fig. VI, 6. Time series, left to right; k hours, 
 8 hours, and 16 hours. 
 
 - V 
 
 / 
 
 M 
 
 r ) 
 
 s 
 
 K [ 
 
 r 
 
 Fig. VI, 8. Comparative uptake and translocation of nine tracers by barley 
 plants. Chemicals and conditions same as in fig. VI, 6. Time series, left to 
 rightj 2 days, k days, and 8 days. 
 
 61 
 
Chapter VII. Root Absorption and 
 Xylem Transport 
 
 MOVEMENT OF 2,4-D" ABSORBED BY ROOTS 
 
 leaving determined to our own satis- 
 faction the nature of the mechanism re- 
 sponsible for the translocation of 2,4-D" 
 applied to leaves, it seemed important to 
 study uptake and distribution by root 
 absorption. Accordingly, plants of bar- 
 ley, bean, cotton and zebrina were grown 
 in nutrient cultures; when they had de- 
 veloped a number of leaves per plant they 
 
 \3SfUi m 3SO„l 3.1, j) 
 
 were treated by adding 2,4-D* to the cul- 
 ture solution at a rate of 1.25 pc per 
 250 ml, each plant having its roots limi- 
 ted to 250 ml. The 2,U-D* had a specific 
 activity of 5.0 mc per mmole. Treatment 
 time was 10 days; at the end of this time 
 the roots were rinsed in tap water and 
 the plants freeze-dried and autographed. 
 Figure VII, 1, shows barley plants and 
 
 Fig. VII, 1. Autoradiography (left) and plants (right) of barley grown 10 days in 
 250 ml of solution containing 1.25 juc of Cl^-labeled 2,4-D. 
 
 62 
 
TABLE VII, 1 
 Radioactivity Recovered from Culture Solutions 
 In which 2,4-D*-treated Cotton Plants Were Grown 
 
 
 Radioactive counts per minute 
 at various time intervals t 
 
 Replication 
 
 0-2 days 
 
 2-4 days 
 
 4-8 days 
 
 8-16 days 
 
 1. . . 
 
 2,235 
 
 13,165 
 
 2,378 
 
 1,254 
 
 2 . . . 
 
 1,948 
 
 12,944 
 
 3,990 
 
 1,358 
 
 3 . . . 
 
 1,875 
 
 10,760 
 
 7,700 
 
 1,120 
 
 4 . . . 
 
 2,602 
 
 10,892 
 
 2,103 
 
 921 
 
 5 . . . 
 
 1,928 
 
 8,660 
 
 2,728 
 
 845 
 
 6 . . . 
 
 2,332 
 
 14,524 
 
 3,428 
 
 1,304 
 
 7. . . 
 
 2,744 
 
 16,635 
 
 4,280 
 
 440 
 
 Average 
 
 2,238 
 
 12,511 
 
 2,801 
 
 1,035 
 
 ' Activity is shown as counts per minute per plant within 
 given time intervals. Plants were transferred to new cul- 
 ture solution at the end of each interval. Dosage 0.5 jjc 
 of 2,4-D s ' { was applied to upper surface of one cotyledon. 
 
 autographs from the experiment. 
 
 In the 10-day period, 2,4-D* had 
 accumulated on or in the roots but not 
 much had translocated to the tops. Bean 
 and zebrina plants apparently translocated 
 none at all, barley translocated enough 
 to show traces in the leaves, and cotton 
 plants showed clear but faint images. 
 This indicates that 2,4-D is not readily 
 moved from roots to tops of plants, and 
 may provide a reason why 2,4-D, when used 
 in the soil, must be applied pre-emergence 
 so that the compound comes in contact 
 with the roots as soon as they emerge 
 from the seeds; the results may also help 
 explain why a high dosage of 2,4-D is 
 required when it is used to kill grown 
 plants. 
 
 LOSS OF 2,4-D" TO THE CULTURE MEDIUM 
 
 In 1951, Clor observed that when one 
 of two cotton seedlings growing in a com- 
 mon culture jar was treated on a lower 
 leaf with 2 ,4-D the other plant showed 
 2 , 4-D symptoms within about 2 weeks 
 (Crafts, 1956a, fig. 1). To further 
 
 check this phenomenon, barley, bean, 
 cotton and zebrina plants were grown 
 in water culture, with cotton seedlings 
 included in the culture jars of barley, 
 bean, and zebrina. Barley, bean, cotton, 
 and zebrina plants in the cultures were 
 treated with 2,4-D* at 0.5, 1.0 and 2.0 
 juc per plant, leaving the cotton seedlings 
 untreated to serve as indicators of 2,4-D" 
 leakage. All plants were freeze-dried 
 and autographed after 24 hours. A similar 
 set was left for 15 days in the case of 
 barley, 20 days for bean, cotton, and zeb- 
 rina. In all cases faint autographs were 
 obtained for the 24-hour treatment times, 
 and somewhat darker autographs for longer 
 treatment times. The untreated cotton 
 test plants in the latter experiment 
 showed 2,4-D symptoms, and significant 
 counts of the 2 , 4-D* concentration in 
 some culture solutions were recorded. 
 Clor (1959) has confirmed these results, 
 as shown in tables VII, 1, 2. 
 
 It seems obvious from these experi- 
 ments that 2,4-D* is absorbed by leaves 
 of plants, that it moves to the roots and, 
 under some conditions, may leak from the 
 
 63 
 
roots into the culture medium. Later 
 experiments have shown that maleic hydra- 
 zide will do this but that amitrole and 
 sucrose will not; this indicates that 
 roots respond differently to different 
 compounds. Experiments cited in chapter 
 VI prove that roots also respond differ- 
 ently to different compounds applied to 
 them via the culture medium. 
 
 TRANSLOCATION OF LABELED HERBICIDES 
 IN XYLEM AS INDICATED BY ACTIVITY 
 OF GUTTATION FLUID 
 
 In order to further study the com- 
 parative responses of plants to a number 
 of herbicides, an experiment was set up 
 to find out if these compounds are 
 altered during their absorption by roots 
 and translocation to tops via the xylem. 
 Barley seedlings were grown in culture 
 solution in small tubes and, after the 
 solutions containing the chemicals were 
 introduced, guttation fluid was collected 
 and droplets from ten plants were pooled 
 to provide enough for chromatography. 
 The plants were covered with bell jars 
 in order to obtain sufficient fluid; at 
 the end of the collection period the 
 plants were freeze-dried, mounted and 
 autoradiographed. Each plant was in a 
 tube containing 4 ml of culture solution, 
 and dosage was 0.05 /uc per plant. The 
 collection period was from 4 to 6 days. 
 
 The effects of the herbicides on 
 plants were recorded at time of harvest. 
 Plants treated with amitrole* and MH* 
 were still guttating, those treated with 
 2,1-D*, monuron* and simazine* had stop- 
 ped. In general, 2,4-D* stunted the 
 plants, amitrole* caused chlorosis on the 
 lower halves of the first leaf, and on the 
 whole second leaf; MH* had no visible ef- 
 fect at the time and concentration used; 
 monuron* reduced the size and growth- 
 rate of all plants (the leaves were light 
 green and wilted at the tips); simazine* 
 had effects similar to those of monuron*". 
 
 2 , 1-D* was absorbed by roots but 
 moved slowly to the tops. MH* accumu- 
 lated slowly in roots but moved to tops 
 in somewhat greater amount than did 2,4-D*. 
 Amitrole* moved to tops in even greater 
 amounts, the images attaining a dense 
 black tone. All plants treated with mon- 
 uron*, and simazine* were heavily labeled 
 throughout, with a noticeable tendency 
 
 for accumulation at the leaf tips in old 
 leaves in contrast to young. This dis- 
 tribution pattern, a reverse of that of 
 2, 1-D*, MH*, and amitrole*, is an indica- 
 tion of apoplastic movement. 
 
 Guttate was chromatographed from all 
 collections. In the case of 2, 1-D* treat- 
 ment there was no spot at the normal Rf of 
 2, 1-D* as shown on the control; there was 
 a small, faint image at the solvent front 
 and this corresponded with a similar spot 
 on the control, indicating a small impur- 
 ity in the 2, U-D* - this impurity came 
 through in the exudate, whereas 2, 1-D* 
 did not. Amitrole* produced spots in all 
 replicates and these corresponded to the 
 amitrole* spot on the control; evidently 
 it came through the plant unchanged. In 
 the case of MH* some replicates produced 
 no spots; three separate replicates gave 
 faint images of the Rf value of the ori- 
 ginal compound. All replicates from the 
 monuron* experiment gave images of moder- 
 ate density; one replicate gave the 
 strongest spot of all the samples. In 
 the case of simazine*, chromatography of 
 the original compound gave three distinct 
 spots on the control; three replicates 
 from the treated plant samples produced 
 the same three spots. 
 
 Here, there did not seem to be any 
 relation between the volume of guttate 
 and the image density on the chromatograph, 
 
 TABLE VII, 2 
 Radioactivity Recovered from Culture Solutions 
 In which 2,1-D*-treated Bean Plants Were Grown 
 
 
 Radioactive counts per minute 
 at various time intervals t 
 
 Replication 
 
 0-2 days 
 
 2-6 days 
 
 6-11 days 
 
 1 . . . 
 
 220 
 
 1,268 
 
 286 
 
 2 . . . 
 
 18U 
 
 928 
 
 116 
 
 3 . . . 
 
 195 
 
 1,151 
 
 366 
 
 4 . . . 
 
 312 
 
 1,016 
 
 325 
 
 5 . . . 
 
 378 
 
 1,38H 
 
 223 
 
 + Activity as counts per minute per plant 
 within given time intervals. Plants were 
 transferred to new culture solutions at the 
 end of each interval. Dosage 0.5 juc of 
 2,4-D* applied to the upper surface of one 
 primary leaf. 
 
 64 
 
2,4-D* failed to come through the plants, 
 but the other four compounds traversed the 
 roots and tops and appeared in the gutta- 
 tion fluid. There was no evidence for 
 metabolic change of any compound despite 
 the fact that they must have moved through 
 the living cells of the endodermis. These 
 observations confirm evidence from many 
 sources that herbicides in general are 
 stable compounds and that they probably 
 cause their characteristic effects with- 
 out being altered in the process. 
 
 THE MECHANISM OF ACCUMULATION 
 OF 2,4-D BY ROOTS 
 
 The evidence presented above, and 
 the work of Blackman (1955, 1957), indi- 
 cates that 2,4-D accumulation by roots 
 may be reversible. To study this, ex- 
 cised root systems of nutrient-grown 
 barley plants were exposed to varying 
 concentrations of labeled 2,4-D in aer- 
 ated culture solution. After a 6-hour 
 absorption period, concentration of 2,4-D* 
 in the roots was from two to four times 
 that of the external solution. A time 
 series showed that under the temperature 
 conditions of the experiment (approxi- 
 mately 70 degrees Fahrenheit) absorption 
 was rapid and reached its maximum in 30 
 minutes. From washing and exchange 
 experiments conducted at this time it 
 seems that 2,4-D is rather loosely bound 
 and can be readily removed by leaching. 
 However, not all of the absorbed 2,4-D* 
 came off so easily - possibly, some is 
 adsorbed to the outer root surface and 
 some is held metabolically by parenchyma 
 cells of the root cortex. 
 
 Recent studies in which DNP (dini- 
 trophenol) treatment was used in connec- 
 tion with the uptake of 2,4-D* by roots 
 throw more light on the subject. Where 
 barley roots had absorbed 2,4-D* from a 
 solution of 0.1 jumole per 100 ml for 4 
 days, DNP applied simultaneously at 
 1 x 10~ 4 M caused a slight release to the 
 tops; at 1 x 10~3 a large release to the 
 tops occurred. With bean plants in a 
 similar 2,4-D* solution, 1 x 10" 5 M DNP 
 released some 2,4-D* to the stem, and 
 1 x 10- 4 released considerably more; a 
 similar response was found in soybean. 
 Figure VII, 2, shows plants and auto- 
 graphs from these experiments. 
 
 Since DNP is known to uncouple oxi- 
 
 dative phosphorylation, thus reducing 
 energy supply to the cells, it seems that 
 2,4-D accumulation in roots is an active 
 process; lowering the energy supply and 
 membrane integrity apparently allows 
 2,4-D to move across the cortex and endo- 
 dermis and enter the xylem (apoplast) of 
 the stele along with the transpiration 
 stream. Current studies with high dosages 
 indicate that 2,4-D applied to roots in 
 injurious amounts will bring about the 
 same free movement into tops. Figure 
 VII, 3, shows an experiment with a 2-hour 
 absorption time, the roots of the plant 
 on left having been in a solution having 
 0.1 juM concentration, and those of the 
 plant on the right in a solution having 
 a 10.0 /jM concentration per 20 ml. Fig- 
 ure VII, 4 shows a similar experiment 
 with a 24-hour exposure time. Monuron* 
 and simazine*, being less subject to 
 active accumulation by living cells, can 
 apparently pass through the root with 
 little restraint. Hence, these compounds 
 readily move through roots and stems and 
 accumulate in leaves where, if in suffi- 
 cient concentration, they enter green 
 cells and block oxygen evolution in photo- 
 synthesis (Crafts, 1961a). The plants in 
 this experiment were alive at the end of 
 the 24-hour absorption period; evidently 
 2,4-D* was being metabolized and lost 
 from the leaves. 
 
 DINITROPHENOL EFFECTS 
 ON HERBICIDE TRANSPORT 
 
 Many tests on the uptake of herbi- 
 cides by roots and translocation to tops 
 have proved that there is a wide variation 
 in distribution. Although 2,4-D* is 
 highly accumulated by roots with little 
 or no movement into tops, monuron*, sima- 
 zine*, calcium* and phosphorus* are rapid- 
 ly translocated following a short accumu- 
 lation period. Experiments with DNP 
 proved that this respiration inhibitor, 
 when used at a concentration of around 
 10-4 m $ would affect roots in such a way 
 that 2 ,4-D* was released for transport to 
 the foliar region of the plant. 
 
 Repetition of the above tests with 
 sufficient replication to assure signifi- 
 cant results proved that DNP at 10"^ M 
 will allow for transport of 2,4-D* to 
 the tops of soybean plants, and that it 
 has a slight but detectable effect in 
 barley and little effect in bean. At 
 
 65 
 
10-3 m concentration it has a strong re- 
 leasing effect on 2,4-D* in all three 
 species, but it also produces injury 
 symptoms; at 10-5 m DNP, soybean is the 
 only species tested that gives a detec- 
 table difference - and it is at the lower 
 limit of significance. 
 
 DNP had no effect upon the movement 
 of amitrole* from the roots of soybean at 
 
 10- 4 M; MH* movement was slightly inhib- 
 ited in soybean and barley, as was P32 
 and Ca 1 ^ movement in barley; monuron* 
 transport was unaffected in soybean and 
 barley. Figure VII, 2, shows a 6-hour 
 treatment of soybean plants cultured in 
 Hoagland's solution with 2,U-D*, at 
 1/2 yumole per 100 ml, with DNP at lO" 4 M 
 compared with no DNP. In a series of 
 barley cultures, sodium benzoate* at 
 
 Fig. VII, 2. Effect of di ni trophenol on the movement of 2,4-D* from roots of soy- 
 beans. Left, soybean cultured 6 hours in 2,4-D* solution at 0.1 pmole per 100 ml. 
 Right, soybean cultured 6 hours in 2,4-D* at 0.1 ;umo1e per 100 ml plus DNP at 10"^ M, 
 
 66 
 
L 
 
 s * * 
 
 I 'I 
 
 'ym**s 
 
 VII. 3 
 
 Fig. VII, 3. Effect of high concentration of 2,4-D* on movement through the 
 root system. Left, bean plant cultured in a solution of 2,4-D* at 0.1 jumole; 
 right, the same plant in a solution of 2,4-D* at 10 juM concentration per 20 ml. 
 Treatment time, 2 hours. 
 
 Fig. VII, 4. Bean plant in a solution of 2,4-D*. Effect of high concentration 
 of 2,^-D* on movement through the root system. Plants and treatments as shown in 
 fig. VII, 3 } but with a 1-day treatment. The lower concentration of tracer in leaves 
 may result from decarboxylation of the 2,4-D*, f rom translocation back to the roots, 
 or from both. 
 
 1/10 jumole in 100 ml for 4 days showed 
 no upward movement; 2,4-D* at the same 
 dosage shows detectable movement; monu- 
 ron* shows strong upward transport but 
 no response to DNP; p32and Ca 1 ^ show 
 strong upward movement inhibited by DNP; 
 MH* shows weak upward movement slightly 
 enhanced by DNP, but accumulation in the 
 roots is noticeably reduced. Figures 
 VII, 5, 6, 7, show these results. Sodium 
 azide affects 2,4-D movement, as does 
 DNP. 
 
 Repeated trials with bean, barley, 
 soybean and buckwheat failed to give any 
 
 positive results when DNP was applied 
 with 2,4-D-, 2,4,5-T* or monuron* to 
 leaves. Apparently, movement from cor- 
 tex to stele in roots is governed by dif- 
 ferent factors than is migration from 
 cuticle to symplast in leaves; the com- 
 parative behavior of the substituted urea 
 and triazine herbicides when applied to 
 leaves and roots substantiates this be- 
 lief. Trials with sodium fluoride proved 
 that this inhibitor has no effect on 
 2,4-D* uptake by roots or movement from 
 roots within the concentration range of 
 10-2 m to 10-4 m in soybean. Other 
 metabolic inhibitors are being tested. 
 
 FIGURES VII, 5, 6, AND 7 FOLLOW. | } | 
 
 67 
 

 Fig. VII, 5- Effect of DNP on the uptake and transport of sodium benzoate* (left), 
 
 and 2,*+-D* (right). Plant on left in each set of three plants is the control, 
 other two were treated for 1 day with DNP at 10"^ M. DNP had no effect on sodi 
 benzoate-, but released 2,4-D* for movement to tops. 
 
 the 
 urn 
 
 68 
 
Fig. VII, 6. Effect of di ni trophenol on uptake and transport of maleic hydrazide* 
 (left), and monuron''' (right). Plants and treatments as in fig. VII, 5» MH», like 
 2,4-D", was released to the tops; there was no observable effect on monuron-'' movement. 
 
 69 
 
Fig. VII, 7. Effect of di ni trophenol on uptake and transport of P^ 2 (left), and 
 Ca^5( right) . Plants and treatments as in fig. VII, 5- DNP apparently inhibits the 
 uptake and movement of these inorganic elements. 
 
 70 
 
Chapter VIII. Comparative Movement 
 of Tracers via Shoot and Root 
 
 MOBILITY OF FOUR TRACERS IN BARLEY 
 APPLIED TO LEAVES AND ROOTS 
 
 X 
 
 ^o compare leaf and root application 
 and to detect root leakage in barley, an 
 experiment using 2,4-D*, IAA*, amitrole*, 
 and simazine* was carried out. To study 
 initial absorption, translocation and re- 
 distribution, the plants were given 1-day, 
 4-day, and 16-day treatment times. To 
 test for root leakage, three plants were 
 grown in a single culture jar; two re- 
 ceived applications on their leaves, and 
 one was left to indicate leakage. 
 
 In the foliage treatments, 2,4-D* 
 moved very little below the root crowns 
 even in the 16-day period, while IAA" 
 was appreciably more mobile, giving light 
 images of roots from the leaf treatments 
 at 4 days and similar images in 16 days. 
 MH* was quite mobile, giving heavy images 
 after 1 day and continuing to move even 
 up to 16 days; there was ample evidence 
 that MH* retrans located within the plants 
 during the 16-day treatment period. 
 Simazine" failed to move basipetally from 
 the treated spots on the leaves at any 
 treatment time; movement in foliage was 
 wholly apoplastic. 
 
 The untreated test plants were free 
 of tracer except in the MH* leaf treat- 
 ments; the roots of the test plant pro- 
 duced a light image in 4 days; after 16 
 days the whole plant was labeled. 
 
 In root treatments, 2,4-D* failed 
 to leave the roots, while IAA" moved 
 upward in 4 days producing an image of 
 medium intensity; in 16 days the whole 
 
 plants were heavily labeled. MH* failed 
 to leave the roots in 1 day; light images 
 of tops were produced in 4 days; in 16 
 days, larger tops were thoroughly labeled. 
 but only at low intensity. Simazine" 
 moved to the tops, and produced intense 
 images in all treatment times. Figure 
 VIII, 1, shows the 1- and 16-day plants 
 and autographs of the 2,4-D*, IAA*, and 
 simazine" experiments. 
 
 Bean and barley plants were next 
 treated by leaf application with 2,4-D*, 
 amitrole*, MH*, dalapon*, P32, Zn65 s and 
 Ca 1 ^ using treatment times of 1, 4, and 16 
 days. In this experiment the relative 
 mobility of 2,4-D*, amitrole*, and MH* 
 was consistent with previous tests. Dala- 
 pon* proved very mobile; like MH*, it 
 migrated from phloem to xylem and thus the 
 untreated opposite leaves of bean were 
 lightly labeled. From leaf treatment as 
 shown by figure VIII, 4, right, dalapon 
 entered the roots but failed to accumu- 
 late. Resembling MH*, it probably leaked 
 into the ambient culture medium and migra- 
 ted to the xylem - as evidenced by the 
 labeling of untreated mature leaves. Be- 
 cause grass roots are unable to produce 
 shoot buds, and hence are not vegetatively 
 reproductive from root tissue, this re- 
 sponse to dalapon may explain its selec- 
 tivity against grasses; in grasses, kill- 
 ing of stems and buds is all that is 
 necessary to destroy the plant. 
 
 p32 and Zn65 proved to be phloem- 
 mobile in barley and bean; they trans lo- 
 
 Fig. VIII, 1. Autoradiography (top) and mounted plants (bottom) showing the distri- 
 bution of 2,4-D- (a, d), IAA* (b, e), and simazine* (c, f) applied to leaves (left) and 
 roots (right) of barley plants. Top row (a, b, c) 1 -day treatment times; bottom row 
 (d, e, f) 16-day treatment times. Dosage 0.1 ajc applied to the 4th leaf in each case; 
 1 .0 ajc in 200 ml of solution to roots of 3 plants in the root treatments. ^ 
 
 71 
 
72 
 
cated freely and redistributed to young 
 leaves in the 16-day period. Ca^S was 
 mobile only in the apoplast; like monuron* 
 and simazine* this element produced a 
 strong pattern of apoplastic movement in 
 
 leaves but failed to move out via phloem 
 even in 16 days. Figures VIII, 2, 3, 
 show the plants and autographs from the 1- 
 day part of this experiment; figure VIII, 
 4, illustrates the evidence for leakage 
 
 Fig. VIII, 2. Plants (below) and autoradiography (above) of bean treated for 1 day 
 with (left to right) 2,4-D*, amitrole*, MH*, and dalapon* Th^ 2,4-D*-treated plant 
 shows symplastic movement only; the other three display both symplastic and apoplas- 
 tic movement. 
 
 73 
 
of MH* and dalapon* in the U-day trial 
 with bean. 
 
 COMPARATIVE MOVEMENT OF 
 30 LABELED TRACERS IN BARLEY 
 AND BEAN PLANTS 
 
 As it became apparent that each new 
 labeled compound has a characteristic 
 translocation pattern in plants, it 
 seemed reasonable that a standardized 
 test using autoradiography as the princi- 
 pal tool could be devised to give infor- 
 mation concerning the pattern. Thus, a 
 technique was designed using leaf and 
 root application to barley and bean seed- 
 lings, with treatment times of 1, 4, and 
 16 days. Some twenty-seven organic com- 
 pounds and three mineral elements have 
 now been run through this test. The fol- 
 lowing paragraphs summarize the results ; 
 table VIII, 1. gives the distribution 
 patterns in quantitative terms and figures 
 VIII 5, 6, 7, illustrate the results of 
 16-day trials with 2,4-D*, MH*, amitrole*, 
 dalapon* and Zn&5, 
 
 ALANAP" In the leaf of bean, Alanap* 
 moves in both the apoplast and symplast. 
 Phloem movement to roots is weak and there 
 is no circulation via the xylem. 
 
 In barley, uptake by the leaf is 
 strong with medium phloem movement to 
 roots and no recirculation. 
 
 Uptake by bean roots is strong and 
 transport via xylem is medium with no re- 
 distribution. 
 
 Uptake by barley roots is low with 
 some transport to the tops, but there is 
 no accumulation. 
 
 AMI BEN : : Uptake and movement by bean leaf 
 is syrnplastic, with medium-strong trans- 
 port to roots and no retransport via the 
 xylem. 
 
 In barley leaf, uptake is medium 
 with appreciable movement to the roots. 
 
 Uptake by bean roots is strong with 
 no movement to tops via the xylem. 
 
 Uptake by barley roots is medium, 
 with only traces reaching the tops. 
 
 AMITROLE" This compound enters the bean 
 leaf readily and moves in both symplast 
 and apoplast (fig. VIII, 2). Transport 
 to roots and buds is strong, and there 
 is no recirculation via xylem. 
 
 Barley leaves absorb this compound 
 freely, and it moves freely to roots with 
 
 a bit of recirculation. 
 
 Bean roots absorb amitrole* in mod- 
 erate quantity and transport it to the 
 tops in increasing amounts with time. It 
 is re trans located from mature leaves to 
 the buds via phloem. 
 
 Barley roots absorb and transport 
 amitrole* in moderate quantity; the 
 amount moved increases with time. 
 
 AMMONIUM THIOCYANATE" This compound pene- 
 trates the leaf of bean and moves in both 
 apoplast and symplast. Phloem movement 
 is limited to traces in epicotyl and hypo- 
 cotyl in 4 and 16 days. 
 
 Barley leaves absorb and move this 
 compound in apoplast and symplast, and it 
 is transported to roots in low concentra- 
 tion. 
 
 Bean roots absorb and transport am- 
 monium thiocyanate* through the stem to 
 the leaves. 
 
 Barley roots also absorb and trans- 
 locate this compound, but in small quan- 
 tities . 
 
 ARSENATE As?7 ± s a short-lived isotope, 
 but we were able to obtain satisfactory 
 autographs in coffee seedlings. As 7 7 
 entered the mature leaf and moved sym- 
 plastically in fair concentration through 
 the stem and into the roots; absorbed by 
 roots, it concentrated to a high level. 
 It also ascended the stem in medium 
 amount, and there was light but thorough 
 labeling of the leaves. There was no evi- 
 dence of redistribution via the phloem. 
 
 ATRAZINE " Bean leaves absorb this com- 
 pound and it moves freely in the apoplast 
 but not at all in the symplast. 
 
 Barley leaves show only apoplastic 
 movement of atrazine* from the region of 
 application to the tips, with no syrnplas- 
 tic movement. 
 
 Roots of bean absorb atrazine* and 
 transport it to the top of the plant via 
 xylem. 
 
 Roots of barley absorb and transport 
 this compound in the same way. There is 
 no evidence of retransport via phloem. 
 
 BARBAN" This compound shows strong apo- 
 plastic movement in the bean leaf, no 
 syrnplastic movement, and no redistribu- 
 tion. 
 
 Barley leaves absorb and translocate 
 barban* in the apoplast, but only slightly 
 in symplast. 
 
 74 
 
Fig. VIII, 3- Plants and autoradiography of bean treated for 1 day with 
 (left to right) P32, Zn°5, and Ca^5. p32 anc j Zn°5 show symplastic move- 
 ment only; Ca^5 shows apoplastic movement only. 
 
 Bean roots absorb barban* in large 
 amounts, but move it to the tops in small 
 quantities only. 
 
 Barley roots absorb fair quantities 
 of barban*, but transport only a small 
 amount. 
 
 DACTHAL" Bean leaves show little tendency 
 to move this chemical via either symplast 
 or apoplast; it is present in stem and 
 roots in traces only. 
 
 Dacthal* is also only slightly mobile 
 
 in barley leaves and is present in roots 
 in traces only. 
 
 Roots of bean accumulate Dacthal" to 
 high concentration, but move very little 
 beyond the hypocotyl. 
 
 Roots of barley neither absorb nor 
 translocate Dacthal* in large quantity. 
 
 DALAPON" Bean leaves absorb and trans- 
 locate dalapon* principally via symplast 
 into the stem, but only traces stay in 
 the roots (fig. VIII, 2, H), Traces in 
 
 75 
 
opposite leaves indicate migration from 
 phloem to xylem and retransport via xylem. 
 
 Barley leaves absorb and move dala- 
 pon* rapidly, but this compound does not 
 accumulate in roots. 
 
 Bean roots absorb dalapon* only in 
 small quantities; this moves into the 
 stem but reaches the leaves in traces 
 only. 
 
 Barley roots likewise absorb and 
 move dalapon* in small quantities. 
 
 2,4-D" This compound is readily absorbed 
 by bean leaves and distributed throughout 
 the stem via the symplast. There is no 
 movement in the apoplast and no retrans- 
 port. Concentration in the roots is med- 
 ium (fig. VIII, 1, 2, 5). 
 
 Barley leaves absorb and transport 
 2,4-D* readily via symplast, not via 
 apoplast. 
 
 Bean roots absorb 2,4-D* to high con- 
 centration, but move little into the stem 
 and none into the leaves. 
 
 Barley roots absorb much 2,4-D*, but 
 transport little. 
 
 2, *J-DB" This compound is very immobile in 
 bean. It labels the treated spot on the 
 leaf, but scarcely moves beyond this. 
 
 In barley leaf, 2,H-DB* moves little 
 beyond the treated leaf. 
 
 2,4-DB* is strongly absorbed by bean 
 roots, but moves through the stem at very 
 low concentration and labels the leaves 
 very little. 
 
 Barley roots absorb 2,4-DB* strongly 
 and move a little to the tops. 
 
 DURASET" This compound penetrates the 
 bean leaf and produces a pattern of apo- 
 plastic movement; only traces arrive in 
 the stem and roots. 
 
 Barley leaves are heavily labeled 
 near the treated spot, but only a small 
 quantity of Duraset* reaches the roots. 
 
 Bean roots absorb Duraset*, but fail 
 to move much to foliage. 
 
 Barley roots absorb Duraset", and 
 appreciable quantities reach the tops in 
 4 to 16 days. 
 
 EPTAM :c This compound readily penetrates 
 the bean leaf; it moves acrope tally along 
 the veins (not via the apoplast). Basi- 
 petally, it moves only in traces, possi- 
 bly because of loss in the vapor form. 
 
 Barley leaves shew high concentra- 
 tion of Eptam* in the treated spots, and 
 
 little moves to roots. 
 
 Absorbed by bean roots, Eptam* moves 
 quite readily along the stem and into the 
 leaves. There is no redistribution. 
 
 Barley roots absorb only limited 
 quantities of Eptam*; it moves to leaves 
 in small amounts only. 
 
 ETHYL, ETHYL- N- BUTYL THIOLCARBAMATE" An 
 analogue of Eptam, this compound is, if 
 anything, less mobile in bean; it penet- 
 rates the leaf and moves acropetally in 
 the veins, but moves to stem and roots 
 in traces only. 
 
 In barley, this compound enters the 
 leaf but scarcely moves beyond the treated 
 spot. 
 
 This compound is absorbed in quan- 
 tity by roots of bean and moves in fair 
 amounts through the stem and into the 
 leaves. 
 
 Barley roots accumulate only a small 
 concentration of this compound, and it 
 moves to the leaves in like quantities. 
 
 MALE I C HYDRAZIDE" This is the most free- 
 ly mobile compound that we have studied. 
 It is absorbed by bean leaves and moves 
 acropetally via the apoplast, basipetally 
 via the symplast and phloem. It is pre- 
 sent throughout stems and leaves and 
 tends to recirculate, as shown by its 
 presence in opposite leaves (figs. VIII, 
 2, 4). 
 
 MH* is also very mobile in barley. 
 From the leaves it moves throughout tops 
 and roots of treated plants, showing high 
 concentration in young leaves. 
 
 Absorbed in medium quantities by 
 bean roots, MH* moves through the stems 
 and appears in leaves in lew quantities. 
 
 Barley roots absorb medium quanti- 
 ties of MH*, but pass it to the foliage 
 in traces only. 
 
 MONURON" This compound enters the bean 
 leaf readily and moves acropetally in the 
 apoplast; it seems unable to enter the 
 symplast and hence is missing from the 
 rest of the plant. 
 
 Monuron* moves in the barley leaf 
 only from the region of application to 
 the tip; no basipetal movement takes 
 place (fig. VI, 3). 
 
 Monuron* is readily absorbed and 
 translocated by bean roots; with time, 
 it builds up to high concentration 
 throughout the plant. 
 
 Barley roots also absorb monuron* 
 
 76 
 
Fig. VIII, k. Bean plants and autoradiographs after treatments with MH* 
 (left) and dalapon* (right). Treatment time k days. Leaves opposite treated 
 ones show as a result of leakage of tracers from phloem to xylem. Dalapon 
 fails to accumulate in old roots. 
 
 and transport it to leaves where it ac- 
 cumulates to high levels (fig. VII, 6). 
 
 PENTACHLOROFH ENOL" Tested in bean onlv, 
 this compound moves along the midrib and 
 side veins from the treated spot toward 
 the leaf tip; it shows no movement toward 
 the main stem. In root application it 
 accumulates to a medium concentration in 
 the contacted roots, moves in only a 
 light trace through the stem and petioles, 
 and does not reach the leaves. 
 
 N-PROPYL. DI-N-PROPYLTHIOL CARBAMATE" An- 
 other Eptam analogue, this material is 
 similar in distribution to the latter. 
 In bean leaf it is absorbed and moved 
 acropetally in the veins, basipetally its 
 movement takes place only in traces. 
 
 Barley shows apoplastic movement of 
 this compound in the treated leaf, but it 
 moves to roots in traces only. 
 
 Bean roots absorb this compound to 
 fairly high concentration but move it to 
 foliage in moderate amounts only. 
 
 77 
 
Barley roots absorb and move this 
 compound in small quantities. 
 
 3-CH LOROPHENY L- ALPHA PROPIONIC ACID" In 
 bean leaf, this compound is readily ab- 
 sorbed. It moves acropetally in veins, 
 basipetally in the symplast, concentrates 
 in the bud, and reaches the root in med- 
 ium to large quantities. 
 
 In barley leaf, this compound moves 
 both via the apoplast and the symplast; 
 it goes into the other leaves and reaches 
 the roots in low amounts. 
 
 Absorbed in medium quantities by bean 
 roots, this compound moves through the 
 stem and into the leaves in traces only. 
 
 Barley roots absorb only small 
 amounts of this compound and move it in 
 traces. 
 
 PROPYL, ETHYL- N- BUTYLTHIOL CARBAMATE" 
 This third analogue of Eptam is promising 
 in field trials. Absorbed and moved acro- 
 petally in veins of bean leaf, it is trans- 
 located in small to trace amounts. 
 
 In barley, this compound moves in the 
 apoplast, but scarcely enters and moves in 
 the symplast. The roots were almost de- 
 void of label. 
 
 In bean roots this compound is ab- 
 sorbed to high concentration, and it moves 
 in medium amount through stem and leaves. 
 
 In barley likewise, it is taken up 
 and moved to foliage in medium amounts. 
 
 SIMAZINE" The bean leaf absorbs simazine* 
 and moves it acropetally in the apoplast; 
 no simazine" enters or moves in the sym- 
 plast. 
 
 The barley leaf likewise absorbs and 
 moves simazine" via apoplast, but not via 
 symplast. 
 
 Applied to bean roots, simazine* 
 enters and moves readily throughout the 
 stem and leaves. 
 
 In barley also, simazine 5 '' enters 
 roots and moves freely to foliage. There 
 is no sign of recirculation, as shown in 
 figure VIII, 1. 
 
 SODIUM ACETATE- This metabolite is read- 
 ily absorbed by the bean leaf and it 
 moves acropetally in veins, basipetally 
 in the symplast; here, it traverses the 
 stem and reaches the roots in fair quan- 
 tity. It does not transfer to xylem nor 
 appear in the opposite leaf. 
 
 In barley, sodium acetate" enters 
 and moves freely throughout the foliage 
 
 and roots, where it may occur in high 
 concentration . 
 
 This compound is absorbed strongly 
 by bean roots, moves lightly into stems, 
 and fails to reach the leaves. 
 
 In barley it is strongly absorbed by 
 roots, and reaches the leaves only in 
 traces. 
 
 SODIUM BENZOATE" This compound enters 
 the bean leaf and traverses the lamina in 
 the veins; it moves to stems in low 
 amounts and reaches roots in low concen- 
 tration. 
 
 In barley leaves, sodium benzoate* 
 is strongly absorbed. It reaches the 
 other leaves in low concentration, and 
 appears in the roots in medium amounts. 
 
 In bean roots it is strongly ab- 
 sorbed; it moves in stems in traces, but 
 fails to reach the leaves. 
 
 In barley it builds to high concen- 
 tration in roots, but reaches the leaves 
 in small quantities only (fig. VII, 5). 
 
 2,^,5-T" This compound shows only sym- 
 plastic movement in the bean leaf. It 
 moves in moderate amount in the stems, 
 but reaches the roots in low concentra- 
 tion. 
 
 In barley, this compound is high in 
 concentration in the treated leaf, but 
 reaches the roots in traces only. 
 
 In bean roots, 2,4,5-T* enters in 
 medium amount, moves in small amount, 
 and fails to reach the leaves. 
 
 In barley, 2,4,5-T* is absorbed 
 slowly with time and builds to medium 
 concentration in 16 days; however, it 
 reaches the foliage only in traces from 
 root absorption. 
 
 2,4,5-T BUTOXYETHANOL ESTER- This heavy- 
 ester compound of 2,4,5-T" is absorbed 
 slowly. It builds to high concentration 
 in the treated leaf where it moves acro- 
 petally in the leaf veins and basipetally 
 in the symplast. It reaches the stem in 
 medium to high quantities and the roots 
 in medium quantities. 
 
 In barley it moves both acropetally 
 and basipetally in the treated leaf. It 
 enters the other leaves, particularly the 
 young ones , and reaches the root in small 
 to medium quantities. 
 
 In bean, the compound enters roots 
 in moderate amount, but moves in the stem 
 in traces only and fails to reach the 
 leaves. 
 
 78 
 
VIM, 5 
 
 VIII, 6 
 
 Fig. VIII, $. Autoradiography of bean plants after 16-day treatments with 2,4-D* 
 (right) and ami t role*'' (left). The amitrole treatment shows redistribution of the 
 tracer from old to young leaves as the plant grows. 
 
 Fig. VIII, 6. Autoradiography and plant mounts after 16-day treatments with MH* 
 and dalapon*. Redistribution is prominent; leakage is shown by the labeling of the 
 opposite primary leaf. 
 
 In barley root, absorption is medium; 
 the compound moves to leaves in low to 
 trace amounts. 
 
 TRICHLOROPROP IONIC ACID" This compound 
 enters the bean leaf readily, translocates 
 acropetally in the veins and basipetally 
 in the symplast. It moves through the 
 stem and into the roots in medium to small 
 quantities . 
 
 In barley it moves in both direc- 
 tions in the treated leaf, infiltrates the 
 untreated leaves, and moves into roots in 
 medium quantities. 
 
 In bean roots it builds to high con- 
 centration, but moves in stems in small 
 to trace amounts; it fails to reach leaves 
 in 1 and 4 days, and reaches them in tra- 
 ces only in 16 days. 
 
 In barley roots it accumulates to 
 medium quantities and moves to tops in 
 small quantities. 
 
 IND0LE- 3- ACETIC ACID" In barley, IM* 
 enters the leaves more freely than 2,4-D* 
 and moves into roots at low concentration 
 from leaf treatment. Applied to roots it 
 
 is strongly accumulated and moves into 
 tops in medium concentration. It has no 
 tendency to migrate from phloem to xylem - 
 at least not in a mobile form. Figure 
 VIII, 1, shows the 1- and 16-day results 
 in barley. 
 
 PHOSPHORUS P32 enters the leaf rapidly, 
 moves symplastically through petiole, epi- 
 cotyl and hypocotyl, and concentrates in 
 the terminal bud and in root tips (figs. 
 
 VII, 7; VIII, 3). 
 
 CALCIUM Ca45 enters the leaf in large 
 amounts and moves strongly in the apo- 
 plast; it moves scarcely at all in the 
 symplast. It does not reach the petiole 
 or stem, and it stays in the same location 
 for 16 days or more (figs. VII, 7; VIII, 3). 
 
 ZINC Zn65 enters the leaf rapidly, does 
 not move in the apoplast, moves about 
 like p32 i n the symplast, and accumulates 
 in the terminal bud and root tips (figs. 
 
 VIII, 3, 7). It retrans locates freely, 
 moving into young leaves as they grow; 
 
 in the stem it attains highest concentra- 
 
 79 
 

 c -~* 
 
 
 CO • 
 
 
 — « 
 
 
 Q- E 
 
 
 •^ 
 
 
 C 4-> 
 
 
 ID 
 
 
 V L. 
 
 
 CD O 
 
 
 (4- 
 
 
 C 
 
 
 •- u 
 
 
 
 
 10 <*_ 
 
 
 l- M- 
 
 
 o> u 
 
 
 U 0> 
 
 
 fl> Q- 
 
 
 l_ l/l 
 
 ^ 
 
 1- 
 
 
 *■» 
 
 M 
 
 TJ O 
 
 *— i 
 
 V C 
 
 r-i 
 
 ^- 
 
 >— i 
 
 0) V 
 
 => 
 
 XI U 
 
 
 <o c 
 
 a; 
 
 -1 0) 
 
 ^— 
 
 X 
 
 XI 
 
 U- 
 
 ID 
 
 o •> 
 
 h- 
 
 ■D 
 
 
 (/I 0) 
 
 
 C Ol 
 
 
 I- ro 
 
 
 0> L. 
 
 
 4J 0) 
 
 
 •M > 
 
 
 (S (D 
 
 
 a. 
 
 
 V 
 
 
 C L. 
 
 
 O <Q 
 
 
 •p- 
 
 
 4-> (/> 
 
 
 (D 4-> 
 
 
 O — 
 
 
 O 3 
 
 
 — i/> 
 
 
 Iff 0) 
 
 
 C £g 
 
 
 (0 — ' 
 
 
 l_ 
 
 'A 
 
 l 
 
 p 
 3 
 
 8" 
 
 P 
 
 O 
 
 P 
 
 613 
 p p 
 
 CO .o 
 
 w 
 p 
 
 a) 
 p 
 
 (A 
 
 "3 
 
 HJ 
 •5! 
 
 ! 
 
 3 
 
 •AS 
 
 + +. + fc fc ^ + H + ~ ~ + + ~ ~ ,> 
 
 + 0+ + + + + PPO+ + + + + P + o + o + + ooo 
 
 O O I I I 
 
 + O -f + 
 
 + U ++ + f_ + £. + u u u u u 
 
 + + + + + P+ + + + \ + + ±>+£>+ + ±>pppp\ I I 
 
 o I + o + + 
 
 + + + +• + fci -f k + u u u u u 
 
 + + + + + + + + + I++P+P++PPPPPI I I 
 
 + + + ■¥ •¥ ++ + 
 
 + + + •♦■ + + + + + + + + + + + + + + + + + + + + + + 
 + + + + + + + + + + + + + -♦■ + + + + + + + ■•■ + + + + + I I I 
 
 P++ 0+ OOPP+ OPPP++ OPP+PO+ + + + + o+ + 
 
 U + + u ~ ~ t* •*■ + ~^^F< + ..*-. f< + k _ 
 
 P+ + P+ OOP++ O P P P I + OPP+PO + 
 
 + + + „ + 
 + + + + o + 
 
 + + 
 
 + + P+ OOP++ O+PP I ♦ OOP+-PO+ + + + + O + + 
 
 p 
 
 S3 
 
 rH 
 CO 0. 
 
 i 
 
 U + {-> + ^ k + + k £■•++■ k + k ++ ^ + 
 
 P+ + P+ OOP++ OPOP++ OOP+PO+ + + + + o + 
 
 I 
 
 + + + + 
 
 ♦ o + oo++ o + oo++ o+ + + o++ o+ OOOO++ oo 
 
 
 + + F* + + H + + + ~ + ~ + ~ h> 
 + + 0+ 0+ + P+ + + O+ + +OOP+ + + o++ o+ + + OP 
 
 oSSe 
 
 cm cm a W CM 
 
 J CM H O CV.N 
 
 o 
 
 c 
 
 II 
 I 
 
 M 
 o 
 
 03 
 
 rH 
 
 XI 
 
 II 
 
 (0 
 
 >» 
 
 
 
 
 nj 
 
 
 
 
 (4 
 
 
 
 
 bo 
 
 
 
 
 +j 
 
 
 
 
 X! 
 
 
 
 
 bo 
 
 
 
 T3 
 
 •h 
 
 
 
 •H 
 
 rH 
 
 
 
 O 
 
 TO 
 
 II 
 
 
 
 U 
 
 + 
 
 
 
 •H 
 
 c 
 
 • * 
 
 
 
 o 
 
 a> 
 
 
 
 ■H 
 
 o 
 
 
 
 o. 
 
 aj 
 
 
 
 o c 
 
 U 
 
 
 
 b 10 
 
 *-> 
 
 
 
 1 -H X) 
 
 m 
 
 
 
 (0 u 
 x: n> • 
 
 ii 
 
 
 
 a. > 
 
 rH O H 
 
 u 
 
 
 
 (0 «H C 
 
 p 
 
 
 
 1 C o 
 
 •• 
 
 
 
 o a. c 
 
 (0 
 
 rH 
 
 
 
 COO) 
 
 4» U 6 
 
 o 
 
 I 
 
 
 
 roph 
 orop 
 re at 
 
 m 
 
 0) 
 
 > 
 
 OH P 
 
 
 0) 
 
 0) 
 
 rH x: 
 
 o 
 
 Mh 
 
 rH 
 
 x: o uh 
 
 +-• 
 
 4-i 
 
 u 
 
 o-h m 
 
 >> 
 
 0) 
 
 O 
 
 o 
 
 (13 
 
 m 
 
 i u v 
 
 CO H J 
 
 ■t— • 
 
 "r 
 
 <sr. u^ = 
 
 80 
 
Fig. VIII, 7. Redistribution 
 pattern in bean and barley treat- 
 ed for 16 days with Zn°5 # 
 
 tion in the epicotylar node. It contin- 
 ues to move into the plant and after 16 
 days the roots of bean were heavily 
 labeled with dense concentrations in root 
 tips. 
 
 In order to bring the important re- 
 sults of this study together in visual 
 form, table VIII, 1, has been compiled to 
 allow a quick comparison of the major 
 differences. 
 
 There are many mechanisms of herbi- 
 cidal action, and different and distinct 
 patterns of distribution are desirable in 
 different compounds. To control perennial 
 weeds one may use a highly mobile herbi- 
 cide (amitrole, for example) as a trans- 
 located spray in areas where valuable 
 crops are growing. On the other hand, on 
 roadsides, ditchbanks and other noncropped 
 agricultural and industrial areas it is 
 useful to have compounds of low water- 
 solubility (urea and triazine herbicides, 
 for example) which are readily absorbed 
 from the soil. 
 
 In cropped areas, residues are al- 
 
 ways a potential problem. For pre- and 
 post-emergence soil application, com- 
 pounds such as amiben, 3-CP, 2,4,5-T and 
 TCP, IPC and CIPC, which do not trans- 
 locate from roots to tops, can be used 
 on a no-residue basis. Others, such as 
 the triazines that are readily detoxified 
 by certain crop plants, may be used if 
 their decomposition products are harmless. 
 The advantage of the chlorophenoxy com- 
 pounds as pre-emergence materials is 
 their strong contact action against 
 emerging seedlings and the fact that they 
 concentrate in roots of larger plants and 
 move to tops in minimal amounts only. 
 Even the amounts shown as images in auto- 
 graphs may be largely converted to innoc- 
 uous compounds that do not appear in resi- 
 due analyses. Thus, it is obvious that 
 information gained from autoradiographic 
 studies must be interpreted in view of 
 the many mechanisms of herbicidal action 
 and in terms of residue problems. For 
 example, autographs of the herbicide 
 Dacthal used by root application proved 
 useful in registering it on a no-residue 
 basis. 
 
 81 
 
Chapter IX. Effects of Time and 
 Dosage on Transport 
 
 TIME RELATIONS 
 
 w. 
 
 hen using tracers in autoradiogra- 
 phy the time-dosage relations must be de- 
 termined for each isotope. An early ex- 
 periment with Cl^-labeled 2,4-D involved a 
 dosage series including 1, 5, 10, 20 and 
 50 >tig of radioactive 2,4-D having a speci- 
 fic activity of 1.2!+ mc per mmole with a 
 treatment time of 2 hours. This test 
 proved that 5 ;ug or more of this formula- 
 tion was required to produce a satisfac- 
 tory autograph. This amount is 0.028 juc, 
 a quantity just sufficiently radioactive 
 to provide labeling of a bean or barley 
 plant of a size to fit a 10 x 12-inch 
 X-ray film. We now use 0.10 jac as a 
 standard dosage for survey work with new 
 chemicals . 
 
 A comprehensive time series was next 
 conducted, using an arithmetical series 
 from 1/4 to 144 hours (Crafts, 1956a). 
 Because of the killing and drying methods 
 used, autographs showed the artifact of 
 xylem movement (Crafts, 1956a, figs. 10 
 through 14). By correlating stem bending 
 with tracer distribution they did prove 
 however that approximately 3 hours were 
 required for absorption and movement into 
 roots from the primary leaf of bean; 
 about 6 hours were required for transport 
 into apical buds. 
 
 Since these early tests, many experi- 
 ments have included a series of exposure 
 times so that a clear picture of the time 
 relations of tracer uptake could be ob- 
 tained. The tests described in this chap- 
 ter include 1-, 4-, and 16-day treatment 
 times designed to provide evidence on 
 initial uptake and rapid distribution 
 (1 day), continued uptake and distribution 
 (4 days), and pattern of redistribution in 
 time (16 days). 
 
 Figure IX, 1, shows a time series 
 involving MH* treatments on barley leaves 
 for periods of 3, 9, and 27 hours. Placed 
 on leaf 2 of these plants, MH* moved into 
 roots quite rapidly but into tops more 
 slowly. The droplets of formulated MH*, 
 containing 0.05 uc per treatment in 50 
 per cent ethyl alcohol and 0.1 per cent 
 Tween 20, dried in about 30 minutes; move- 
 ment into the leaf and around the plant 
 continued for hours. Drying of the drop- 
 lets evidently does not stop the uptake of 
 applied molecules, and their ultimate 
 destination depends upon their mobility 
 through the living symplast. 
 
 These tests prove that tracers formu- 
 lated with surfactant may continue to move 
 through cuticle and into living leaves 
 for days after application. This is par- 
 ticularly well illustrated in figure IX, 
 2, which shows zebrina treated with 1.0 
 mc per leaf for 1 and 20 days. Applica- 
 tions were made to the upper surfaces of 
 hypostomatous leaves, and the slow con- 
 tinued uptake of 2,4-D* proves that these 
 molecules may traverse the cuticle and 
 move through leaf, stem, and roots for 
 prolonged periods, providing they are 
 formulated so as to contact the leaf sur- 
 face from a liquid film. 
 
 THE EFFECTS OF DOSAGE UPON TRANSLOCATION 
 
 Early plot work with 2,4-D indicated 
 that there was a dosage optimum with this 
 herbicide when used as a systemic toxicant 
 on perennial weeds. When our autoradio- 
 graphic studies proved that 2,4-D*, and 
 other systemic herbicides, move through 
 the plant in the assimilate stream with 
 food materials, it was postulated that 
 high dosage, resulting in rapid-contact 
 injury, might knock out the phloem- 
 
 82 
 
\ 
 
 / j 
 
 I 
 
 / 
 
 . 
 
 • 
 
 Fig IX, 1. Transport of MH* in barley 
 2. Dosage was 0.05 Aic; specific activity 
 utions were dry after 30 minutes. 
 
 transport system and hence prevent sys- 
 temic distribution. In order to check 
 this hypothesis, a dosage-series experi- 
 ment was designed to explore a range of 
 dosages covering all normal application 
 rates for seven herbicides. 
 
 The dosage series used was 0.02 
 Jumole, 0.05 jumole, 0.1/jmole, 0.2 AJmole, 
 0.U jumole and 0.8 jumole per application 
 applied to one primary leaf of bean. The 
 same amount of chemical dissolved in 100 
 ml of culture solution was used to treat 
 root systems of individual culture solu- 
 tion plants. The results of this experi- 
 ment were interpreted as follows. 
 
 2,^-D ; : All dosages caused stunting and 
 deformation of the plants. The labeling 
 in the hypocotyls increased from 0.02 
 jumole through . 1 /jmole and then de- 
 
 after 3, 9> and 27 hours; treatment on leaf 
 0.5 mc per mmole. Droplets of treating sol- 
 creased. It was assumed then that dosage 
 of 2,^-D on bean should not exceed this 
 amount for optimum results. The root 
 treatment showed a regular increase in 
 concentration with dosage, and a consis- 
 tent reduction in size of the trifoliate 
 leaves that developed during the treat- 
 ment. Swollen tips of treated roots had 
 high labeling; there was little accumu- 
 lation in the hypocotyls. Subsequent 
 treatments at 10 jumole per 20 ml culture 
 solution per plant resulted in increasing 
 transport from roots to tops (figs. VII, 
 3, U). This last dosage is roughly equi- 
 valent to the actual absorption of around 
 2 pounds of 2,4-D acid per acre of bean 
 plants - a dosage which would be used 
 only to control a perennial weed by soil 
 application. It is considerably above 
 the dosage range used in pre-emergence 
 applications for controlling seedlings 
 of annual weeds. 
 
 83 
 
3 W«« 
 
 X 
 
 I 
 
 JO itSR.™ IB 
 
 Ik O* if^ 
 
 3.1.1 s^f-M 
 
 n Jo» l-Cyu ^( 
 
 
 ►*"*•. 
 
 Fig. IX, 2. Translocation of 2,4-D* in ze 
 (right). Tracer from this formulation in 50 
 apparently continued to move into the plants 
 leaves. 
 
 DALAPON" This compound showed a consis- 
 tent movement from treated leaf to bud 
 and expanding trifoliate leaf, increasing 
 with increasing dosage. No optimum was 
 observed. Dalapon* moved through the 
 roots into hypocotyls and terminal buds 
 to high levels. Concentration was high 
 in root tips, but only medium in older 
 roots. 
 
 MONURON" This compound showed strong apo- 
 plastic movement in bean leaves, the in- 
 tensity of labeling increasing with dosage, 
 
 brina after 1 day (left) and after 20 days 
 per cent alcohol and 0.1 per cent Tween 20 
 long after drying of treatment solution on 
 
 There was no symplastic movement into the 
 stem and roots; a slight trace appeared in 
 trifoliate leaves and buds at dosages of 
 0.4 and 0.8 umole. Monuron* enters roots 
 and moves in the transpiration stream very 
 readily. At 0.02, 0.05 and 0.1 AJmole 
 dosages, the primary leaves showed more 
 labeling than the trifoliates; at the 
 higher dosage rates all leaves were black. 
 The shoots were much inhibited through the 
 dosage range of 0.2 to 0.8 pmole, follow- 
 ing the series in intensity. Concentra- 
 tions in roots were medium to high through 
 
 84 
 
the series; in hypocotyls, lew to high. 
 Figure IX, 3, shows the results of this 
 treatment for 1 day; figure IX, 4, shows 
 the 4-day results. 
 
 AMITROLE" Both symplastic and apoplastic 
 movement occurred in bean with amitrole*. 
 Concentrations were high in buds at all 
 dosage levels; there was no growth inhi- 
 bition. Concentrations were high in root 
 tips at all levels; opposite primary 
 leaves had light labeling at dosages of 
 0.2, 0.4 and 0.8 wmole. 
 
 Amitrole* applied to roots showed 
 a graded series of concentrations with 
 dosage ranging from medium to high; in 
 hypocotyls, the range was from low to high 
 There was no inhibition of shoot growth, 
 and buds showed accumulation to higher 
 levels than subtending internodes. 
 
 2,4,5-T" This compound had little tenden- 
 
 cy to move in the apoplast, and its sym- 
 plastic movement was less than that of 
 2,4-D*. Inhibition of buds and trifoliate 
 leaf growth followed the dosage level, as 
 did tracer concentration in the buds. As 
 with 2,4-D*, the level of tracer in roots 
 from leaf treatment was low, and was inde- 
 pendent of dosage; hypocotylar concentra- 
 tions followed the series, but were not 
 high at any level. 
 
 In root uptake, 2,4,5-T* concentra- 
 tions in roots and amounts translocated 
 to tops follow the dosage series. Move- 
 ment up the hypocotyl increased with 
 dosage and both primary leaves carried the 
 label at 0.8 /umole dosage. 
 
 MALEIC HYDRAZIPE* This compound is very 
 mobile in plants, and distribution 
 patterns in leaves are both apoplastic and 
 symplastic. MH* concentrates in the young 
 leaves and buds and varies from light to 
 
 Fig. IX, 3. Autoradiography showing results of treating bean plants with a dosage 
 series of monuron for 1 day. Dosages, left top to right bottom, 0.02, 0.05, 0.1, 0.2, 
 0.4, and 0.8 umole; there is increased uptake with increased dosage. 
 
 85 
 
Fig. IX, 4. Autoradiography showing results of treatments as in fig. IX, 3, but 
 left for k days. The higher dosages inhibited growth from the second day until the 
 fourth. 
 
 medium in the hypocotyls. MH* was present 
 in the roots from light to medium through 
 the dosage series and some reached the 
 opposite leaves, indicating transfer from 
 phloem to xylem. 
 
 MH* enters roots and moves upward in 
 the transpiration stream. 
 
 Concentration in the primary leaves 
 increased through the dosage series, being 
 higher in the young leaves and buds than 
 in stem and primary leaves. This indi- 
 cates symplastic movement from the pri- 
 mary leaves after arrival in the transpi- 
 ration stream. 
 
 In conclusion, only 2,4-D showed an 
 optimum dosage; 2,4,5-T penetration limits 
 the amount of this chemical that enters 
 the symplast. Monuron does not enter the 
 symplast. Amitrole penetrates slowly and 
 translocates fast and apparently does not 
 build up to a toxic concentration. The 
 same relation may explain the lack of an 
 optimum for dalapon and MH. Solubility, 
 penetration rate, speed of transport, in- 
 herent toxicity, and accumulation in the 
 symplast all enter into this problem. 
 Within the range of dosages and the series 
 of compounds used, 2,4-D seems to be the 
 only herbicide that causes acute injury 
 to the transoprt system. . 
 
 86 
 
Chapter X. Interaction of Herbicidal 
 Molecules 
 
 SYNERGISM, ANTAGONISM, INTERACTION 
 
 X 
 
 .he terms "synergism" and "antagon- 
 ism" are often used in reports and dis- 
 cussions of herbicidal action. These 
 terms, while convenient, have little 
 basic meaning in terms of chemical mech- 
 anism; we choose to use the term "inter- 
 action" to express both concepts, and we 
 realize the need to study such responses 
 by chemical and physiological means. 
 
 In studies on large zebrina plants 
 growing with their roots spread on filter 
 paper saturated with culture solution 
 (fig. II, 2), it was shown that amitrole* 
 moved throughout the stem and root system 
 and accumulated in root tips in a 4-day 
 treatment period (fig. X, 1). Labeled 
 sucrose formed from the Cl402 from urea* 
 showed a similar distribution (fig. X, 2). 
 On the other hand, labeled 2,4-D moved 
 into the upper portion of the root system 
 but did not reach the lower growing re- 
 gions (fig. X, 3). The question natural- 
 ly arises as to whether this failure to 
 move is the result of accumulation of 
 2,4-D* along the route of transport, or 
 whether it is caused by some physiologi- 
 cal effect on the phloem. To answer this 
 question 2,4-D* was applied to similar 
 zebrina plants, and 4 days later amitrole* 
 was applied to some plants, while urea* 
 was applied to others. In all cases the 
 autographs produced were similar to the 
 original 2,4-D* autographs: they showed 
 little or no tracer in the root tips 
 (fig. X, 4). This indicated that 2,4-D* 
 was having an effect on the phloem and 
 was inhibiting distribution of the ami- 
 trole* and the sucrose* from urea*. 
 
 EFFECT OF 2,4-D ON PHLOEM 
 
 Because application of 2,4-D before 
 
 application of amitrole* inhibited distri- 
 bution of the latter in zebrina, it was 
 decided to explore the dosages and pre- 
 treatment times that effectively block 
 the phloem. Three experiments were con- 
 ducted: one in which 2,4-D and amitrole* 
 were applied to one primary leaf of bean, 
 one in which 2,4-D was applied to one pri- 
 mary leaf and amitrole* applied to the 
 opposite leaf, and one in which 2,4-D was 
 applied to roots and amitrole* to one pri- 
 mary leaf. Dosage was varied through the 
 series - 1/100 jumole, 1/10 Aimole and 
 1 yumole. Each experiment in which leaf 
 application was made involved application 
 of both chemicals at the same time, appli- 
 cation of 2,4-D 24 hours before the ami- 
 trole* and application of 2,4-D 48 hours 
 before the amitrole*. 
 
 The most obvious response followed 
 application of amitrole* 48 hours after 
 the 1.0 umole dosage of 2,4-D. In this 
 case the amitrole* was limited to the 
 bud, primary leaves, and epicotyl; none 
 reached the hypocotyl and roots (fig. X, 
 5). The 1-day interval at 1.0 umole dos- 
 age showed a slightly lower transport of 
 amitrole* to the roots; all other dosages 
 and time intervals showed no effect. In- 
 hibition was greater where both applica- 
 tions were made to the same leaf, and the 
 there were no differences where roots 
 were treated. 
 
 This experiment suggests that only 
 when dosage is high, and only when an 
 appreciable time interval is left between 
 application of 2,4-D and application of a 
 second tracer, is there inhibition of 
 phloem transport of the second tracer. 
 These tests also prove that 2,4-D will 
 inhibit translocation of a separate tracer 
 
 87 
 
;vt ATA. 3*-*n */•• ' °*°* LS 4D * , ' , • P»i»"-t>«'W- 
 
 •»....*» TAP wtTta (Ut-TlMl. ONI MONTH. 
 
 
 Fig. X, 1. Translocation of ami trol e* in zebrina over a 4-day treatment time. The 
 tracer has undergone a thorough distribution by symplastic movement (for method of cul- 
 ture see fig. II, 2). 
 
 88 
 
( f 
 
 LXVI U««*. 25.1 
 
 T*m wAtu em-Timt, ow nttmt. 
 
 Fig. X, 2. Translocation of C as sucrose- derived from urea-' in zebrina over a 
 4-day period. All active sinks are labeled including root hairs (see fig III, 2). 
 
 89 
 
applied after the 2,4-D has had time to 
 act. Presumably not only the movement of 
 a second tracer, but movement of the 
 2,4-D and of foods and all other consti- 
 tuents of the assimilate stream is 
 blocked. This may well be a key to the 
 toxic action of 2,4-D on whole plants. 
 
 Eames (1949, 1950) found that 2,4-D 
 caused phloem collapse in nutsedge and 
 bean, and Muni (1959) showed that 2,4-D 
 applied in lethal concentration to both 
 susceptible and resistant species of 
 monocotyledonous and dicotyledonous plants 
 caused tissue proliferation, formation 
 of galls and adventitious roots, and 
 crushing of the phloem. Continued action 
 also resulted in plugging of the xylem 
 and complete disorganization of the vas- 
 cular tissues. 
 
 In the zebrina plants, stoppage of 
 transport occurred in the more mature 
 vascular tissues of the stem and older 
 roots. This is a somewhat different case 
 than those studied by Muni, as the action 
 of 2,4-D here is more subtle. The re- 
 searcher may well ask if there is a key 
 to this puzzle in the known physiology 
 of phloem. 
 
 Anatomical (Esau, 1953) and physio- 
 logical (Crafts, 19612?) work on phloem 
 indicates that the sieve tubes (the spe- 
 cialized conduits through which rapid 
 transport takes place) are highly spe- 
 cialized elements (Esau and Cheadle, 
 1961). Starting much like normal young 
 parenchyma cells, they soon go through a 
 series of characteristic and unique devel- 
 opmental changes and become longitudinally 
 permeable conduits through which assimi- 
 lates apparently move in a stream. This 
 highly permeable functional period may 
 last for only a short time in the case of 
 protophloem sieve tubes, longer in the 
 case of metaphloem elements, and even 
 longer for secondary sieve tubes - possi- 
 bly through a single, or, in a few cases, 
 two annual functioning cycles. Eventual- 
 ly, however, every sieve tube enters a 
 period of reduced functional activity, 
 dies, and becomes obliterated, that is, 
 crushed, or at least nonfunctional. This 
 period of senescence may start after a few 
 days, a few weeks, or many months of 
 functioning, and it represents the final 
 stage of maturation. Usually, senescence 
 of sieve tubes is characterized by 
 
 callosing of the sieve plates, increased 
 thickening, and constriction of the sieve- 
 plate protoplasmic connections. 
 
 Recent studies with the electron 
 microscope indicate the possibility, long 
 questioned by plant physiologists (Crafts, 
 196 Lb), that the protoplasmic connections 
 of sieve plates actually constitute 
 intervacuolar tubules (Esau and Cheadle, 
 1961). If this proves true for many 
 species, the accretion of callose in the 
 form of cylinders around these sieve-plate 
 connections may very well constitute the 
 mechanism by which sieve tubes are grad- 
 ually closed off as they become oblitera- 
 ted. And when subject to injury by cut- 
 ting of the phloem, by virus invasion, or 
 by use of heat or inhibitors, this con- 
 striction of the sieve-plate connections 
 may be the means by which phloem exuda- 
 tion is rapidly checked and by which 
 translocation is hindered and stopped. 
 
 J. L. Key (1962) found that high 
 inhibitory concentrations of 2,4-D de- 
 crease the ascorbate activity of cells 
 and cause a shift in the oxidation state, 
 and that low stimulatory concentrations 
 cause increase in ascorbate activity and 
 an opposite shift in the oxidation state 
 (chart X, 1; Crafts, 1963). It is pos- 
 sible that this shift in the oxidation- 
 reduction state of the phloem tissues may 
 trigger the reaction that results in a 
 hastening of sieve-tube maturation and 
 obliteration. This would explain both 
 the subtle decrease in transport capa- 
 city brought about by intermediate con- 
 centrations of 2,4-D in phloem, and the 
 more obvious tissue proliferation that 
 causes crushing and death in the younger 
 roots brought about by high amounts of 
 2,4-D. Again, it becomes evident why the 
 exact physiological state of a plant is 
 so important in determining the extent of 
 damage that results from 2,4-D treatment - 
 and the capacity of shoot buds from roots 
 and rhizomes for vegetative reproduction 
 takes on added significance with respect 
 to the individual susceptibility of weed 
 species. 
 
 2,4-D PRETREATMENT AND INHIBITION 
 OF AMITROLE-Cl^ TRANSLOCATION 
 
 In our autoradiographic translocation 
 studies, 2,4-D always lagged behind ami- 
 trole in extent or distance of distribu- 
 
 90 
 

 ►■ oj *_j^r-« y 
 
 LXVI 2,4-6. li.i^ I,* idiwp LS *0*Y3. Fmm- 
 
 ZESRINA. TAP WATE" CULTUMK, ON* mowtm. 
 
 Fig. X, 3. Restricted distribution of 2,4-D* in zebrina in k days. Many roots 
 are unlabeled, and some are labeled in their more mature regions; root tips and root 
 hairs are free of C^. 
 
 91 
 
i 
 
 i 
 
 
 LXVl 
 
 *.*-». IU M **« iPm* LS *8*v«. 
 
 Fig. X, k. Restricted distribution of amitrole* applied k days after a 2,4-D* 
 application. Some roots are unlabeled and labeling is weak in some; root hairs lack 
 Oh. 
 
 92 
 
- JL 
 
 * 
 
 ' 
 
 
 Fig. X, 5. Results of 2,4-D application on translocation of amitrole*. Left, 
 2,4-D applied 2 days before the amitrole-; center, 1 day before; and right, 2,4-D 
 and ami trole'-' applied simultaneously. 
 
 tion, regardless of plant species. Later- 
 al movement and absorption by the tissues 
 surrounding the vascular tissue were 
 thought to be the cause. 
 
 To investigate this, it was decided 
 to pretreat with 2,4-D and to compare 24- 
 hour distribution patterns of amitrole- 
 Cl^ as affected by these pretreatments . 
 Dosages of 1/100, 1/10 and 1.0 micromole 
 of 2,4-D acid at 2210 ppm (1/100 micro- 
 mole per microliter) in 50 per cent ethan- 
 olic solution were used for pretreatments 
 of 0, 1, and 2 days on a primary bean leaf 
 or through the culture solution of 100 ml 
 in pint jars. One-tenth per cent of 
 Tween 20 was incorporated in 2,H-D and 
 ami trole* solutions, and 27 bean plants 
 in primary leaf stage were used. 
 
 The lowest dosage applied to the leaf 
 
 in no way affected the distribution of 
 ami trole* of 1-day treatment time. With 
 higher dosage the simultaneous application 
 of 2,4-D with ami trole*, whether to the 
 same or the opposite leaves, resulted in 
 a distribution essentially unaltered 
 because of 2,4-D. But with 1- and 2-day 
 pretreatments with 2,4-D on one leaf, and 
 with amitrole-Cl 1 * treatment on the same 
 or the other leaf, this distribution pat- 
 tern showed much less movement to the 
 major sinks, the root tips and the stem, 
 tip. Also, the stem traced much darker 
 than in the case of amitrole translocation 
 alone because the amitrole* penetrated 
 the cortex and epidermis; seemingly, the 
 total amount of amitrole-Cl 4 translocated 
 was not much altered by 2,4-D pre treatment 
 of either of the paired leaves, although 
 the distribution pattern was altered 
 (fig. X, 5). 
 
 93 
 
CU 
 
 > 
 
 o 
 o 
 
 <D 
 OS 
 
 rd 
 CU 
 
 CO 
 •H 
 CO 
 
 cu 
 
 x: 
 
 ■M 
 
 • CO +-> 
 
 < 
 
 2C bO 
 
 2 -h c 
 
 CU «H 
 
 • 4-> TJ 
 < O (0 
 ^ U V 
 
 O > 
 
 P. -H 
 
 4-» TJ 
 
 Sh 
 
 i 8 
 
 bO 
 C 
 
 P." 
 
 CO 
 
 U 
 0) 
 
 CO TJ 
 
 > -i 
 
 •H O 
 4-» 
 
 rd 4h 
 
 Pi O 
 CU 
 
 4n x: 
 
 •H -P 
 
 O O 
 
 P. P. 
 
 O 
 O 
 h 
 
 TJ 
 
 a co 
 rd cu 
 
 3 
 
 e co 
 at co 
 
 0. 60 CO +-> 
 
 c*h 
 
 CO 
 
 O 
 
 CO 
 
 
 •H 
 
 c 
 
 4-> 
 
 o 
 
 
 •H 
 
 P. 
 
 +J 
 
 rd 
 
 CX-H 
 
 3 
 
 3 
 
 u 
 
 O 
 
 CO 
 
 CO 
 
 •H 
 
 (fl 
 
 Q 
 
 > 
 
 +i 
 <3 
 
 " CO Cl, 
 
 £ *-» 
 
 PL, O 
 
 bO 
 C 
 
 o 
 
 • Pl- 
 +-» 
 a) 
 
 a> 
 > 
 
 •H 
 
 4-> 
 rd 
 
 S 
 P. 
 
 o 
 
 co 
 
 o 
 
 a) 
 
 ClH 
 
 cu 
 
 ■»*- 
 
 a> 
 
 
 4-> 
 
 > 
 
 H-> 
 
 C 
 
 o 
 
 O 
 
 rd 
 
 o 
 
 
 H 
 
 CU 
 
 
 Dh 
 
 « 
 
 
 
 a 
 i 
 
 J- 
 
 CNJ 
 
 o 
 
 +-> •(- 
 
 4- +J 
 03 O 
 -C < 
 
 o 
 4- 
 o 
 
 cu 
 
 TJ 
 
 o 
 
 TJ 
 
 •• c 
 
 co < rd 
 
 C < 
 •H 2 
 
 CO 
 0) 
 
 x: x: cu a 
 
 V MP •• 
 
 G "H O < 
 
 >,x: Pi z 
 
 CO w CX OS 
 
 
 t»H 
 
 
 
 O 
 
 
 o 
 
 
 
 •H TJ 
 
 CU 
 
 
 >> <3 
 
 •M 
 
 
 (TJ E 
 
 
 N 
 
 +J CO 
 
 
 G co 
 
 CO rd 
 
 TJ 
 
 cu cu 
 
 r-l 
 
 cu 
 
 •H 
 
 •h ex 
 
 o 
 
 TJ +J 
 
 rd o . 
 
 -»- c 
 
 CU -H 
 
 O 4-> 
 
 ^rd 
 
 Pi > 
 
 •H >> 
 
 x: 
 
 CU «H 
 
 co o 
 
 c 
 
 4-» +J 
 
 >> 
 
 w 
 
 H O 
 
 x: <+n 
 
 
 < f0 
 
 a. o 
 
 
 c 
 o 
 
 •H 
 
 rd O 
 Pi 
 
 +J a) 
 
 G -H 
 
 CU Ol 
 
 O -M 
 
 C CO 
 
 o 
 
 O G 
 
 O 
 
 C -H 
 
 •H -M 
 
 a 
 
 CU 3 
 
 CO TJ 
 
 rd cu 
 
 CU U 
 U 
 
 CJ TJ 
 
 C G 
 
 ►H (0 
 
 CU 
 
 G 
 O 
 •H 
 
 x: 
 
 ■M 
 <t 
 
 +J >-* 
 3 Pi -H 
 H 3H 
 bO Uh CU 
 
 -H O 
 
 • 3 
 
 TJ CO TJ 
 
 •H CU 
 
 O G O 
 
 fO »H 3 
 
 CU TJ 
 
 O •*-» CU 
 
 O Pi 
 
 Pi 
 
 a 2 
 O TJ O 
 
 wee 
 
 X) 
 P. 
 o 
 
 
 CU C 
 
 
 •M «H 
 
 
 nj 
 
 
 x: cu 
 
 
 O. CO *-s 
 
 
 CO ffl G 
 
 
 O CU o 
 
 
 x: f< «h 
 
 
 a o +j 
 
 
 o c o 
 
 
 G -H 3 
 
 
 O •— ' TJ 
 
 TJ 
 
 E O 
 
 CU 
 
 >» u 
 
 O 
 
 cu a) ex 
 
 c 
 
 CO 3 
 
 ro 
 
 o x: x 
 
 x: 
 
 X +-» 2 
 
 G 
 
 CU rd CU 
 
 W 
 
 x: o.e- 
 
 CU +J +J M-» 
 (X ^ H 4J >H -H rd XJ 
 3OrdC0>XlCU-t-' 
 £h G O -H »H »H H ;$ 
 co -h £4 -h x: O 
 
 QOrdgrd-HObO 
 
 G 
 
 
 CO 
 
 H 
 
 
 CU 
 
 
 M-< 
 
 TJ 
 
 cu 
 
 O 
 
 •H 
 
 w 
 
 
 CU -M 
 
 rrj 
 
 >» 
 
 G O 
 
 <u 
 
 H 
 
 •H CU 
 
 U 
 
 a 
 
 G <H 
 
 
 
 a 
 
 0) O 
 
 c 
 
 3 
 
 TJ 3 
 
 H 
 
 CO 
 
 rd G 
 
 
 
 
 rH 
 
 
 
 
 rd 
 
 
 TJ 
 
 
 CO 
 
 
 CU 
 
 
 CU «H 
 
 E 
 
 CO 
 
 
 M 10 ^ 
 
 CO 
 
 rd „ 
 
 CU * 
 
 TJ 
 
 O +J >> O 
 
 rd 
 
 CU 
 
 •h »h x: 
 
 H 
 
 S 
 
 £ 
 
 E > ex cu 
 
 (X 
 
 
 
 cu 
 
 >>-h <-• 
 
 O 
 
 cu 
 
 4-» 
 
 N -M TJ rd 
 
 4-» 
 
 
 
 H 
 
 G O C -M 
 
 >^ 
 
 
 < 
 
 CU 
 
 fd rd co 
 
 O 
 
 
 
 I 
 
 cu 
 
 
 
 
 Ch 
 
 M 
 
 
 C 
 
 G 
 
 O 
 
 •rH 
 
 
 O 
 
 •H 
 
 CJ 
 
 TJ 
 
 
 •H 
 
 
 CO 
 
 •H 
 
 
 +-» 
 
 Q) 
 
 rd 
 
 X 
 
 
 rd 
 
 (O 
 
 
 
 
 
 TJ 
 
 rd 
 
 Mh 
 
 
 
 •H 
 
 cu 
 
 O 
 
 CU 
 
 
 X 
 
 £ 
 
 
 Pi 
 
 
 O 
 
 
 
 C 
 
 O 
 
 
 
 cu 
 
 O 
 
 E 
 
 
 c 
 
 TJ 
 
 •H 
 
 N*/ 
 
 
 •H 
 
 TJ 
 
 •M 
 
 rd 
 
 
 
 a> 
 
 C 
 
 U 
 
 TJ 
 
 
 to 
 
 <t) 
 
 +J 
 
 •H 
 
 
 rrj 
 
 
 C 
 
 O 
 
 
 cu 
 
 CU 
 
 0) 
 
 rd 
 
 /•** 
 
 U 
 
 •M 
 
 O 
 
 
 rH 
 
 O 
 
 rd 
 
 c 
 
 O 
 
 H 
 
 a 
 
 •H 
 
 
 
 •H 
 
 cu 
 
 i-i 
 
 CO 
 
 
 
 X» 
 
 
 
 SATioaioad 
 
 AioaxqTiiai 
 
 94 
 
In addition to phloem plugging a 
 point of interest in this experiment is 
 that a 24-hour pre treatment with 1.0 mmole 
 of 2,4-D per plant rendered the phloem 
 unable to retain amitrole. It is evident 
 from the distribution pattern that one 
 effect was on the permeability of the 
 sieve tubes rather than on increased ab- 
 sorption by the surrounding tissues. This 
 leakage along the route of translocation 
 is probably very important, particularly 
 when the movement is downward, because the 
 leaking tracer would be translocated up- 
 ward in the xylem. 
 
 Application of 2,4-D via the culture 
 solution, even at 2.2 ppm, did not stop 
 amitrole-Cl 4 translocation from the leaf 
 to the root tips, though the growth of the 
 bud was nearly completely halted and the 
 plant was slightly wilted. From other 
 experiments it is known that the inhibi- 
 tion of the bud growth in this case is not 
 because of the presence of 2,4-D; there is 
 no 2,4-D in the bud. 
 
 In a similar experiment with zebrina 
 plants cultured in pint jars, it was shown 
 that a 2,4-D dosage of 1.0 jumole, spot- 
 applied to a leaf 1 or 2 days prior to 
 amitrole-Cl^ application to the leaf 
 above, produced similar results; total 
 movement of amitrole-Cl4 (4-day treatment 
 time) out of the treated leaf was not 
 much altered. The distance of transloca- 
 tion was reduced to an internode or two. 
 The leakage of the amitrole-Cl 4 from the 
 phloem into the other tissues of the stem 
 greatly intensified the images of these 
 internodes adjacent to the treated leaf. 
 Additionally, leakage finding its way 
 into xylem was carried into a mature leaf. 
 
 From these 2,4-D pre treatment experi- 
 ments it is evident that tissue response 
 to 2,4-D of sublethal dosage is a part of 
 the limiting factor in 2,4-D translocation 
 and distribution. In fact, phloem failure 
 from 2,4-D seems to be intimately related 
 to the mode of action of this chemical 
 when used as a herbicide. 
 
 95 
 
Chapter XI. Symplastic and Apoplastic 
 Movement 
 
 reCHANICS OF ABSORPTION 
 
 L 
 
 .n the early chemical and physic- 
 logical studies on herbicides it was 
 suggested (Crafts and Reiber, 1945) that 
 absorption of the dinitro weed-killers 
 takes place via the lipoid phase of the 
 cuticle, and that buffering the spray 
 formulation of such a chemical would en- 
 hance uptake by presenting the toxicant 
 molecules in the undissociated state for 
 movement across this barrier. Because 
 the undissociated parent acid molecules 
 are more lipoid-soluble than the anions, 
 they should diffuse across cuticle more 
 rapidly. This idea was later applied to 
 2,4-D and other anionic toxicants (Crafts, 
 1948, 1956a). 
 
 Table XI, 1 presents data on absorp- 
 tion and translocation of 2,4-D in black- 
 eyed pea plants following application to 
 one unifoliolate leaf; variables are the 
 pH of the applied solutions and the time 
 in hours; values given are stem-bending 
 in degrees. 
 
 When MH became available as a plant 
 growth inhibitor it was used in a number 
 of experiments and proved to be an active 
 grass killer (Currier and Crafts, 1950). 
 However, later studies (Smith, et al. 9 
 1959; Crafts, Currier and Drever, 1958) 
 proved that the mode of chemical activa- 
 tion used for dinitro compounds and 2,4-D 
 did not apply in the case of MH; here, 
 the only effective means of enhancing up- 
 take was application in a saturated atmos- 
 phere. 
 
 Table XI, 2, presents data relating 
 growth of Bermudagrass cultures to pH of 
 applied MH solutions. It is obvious here 
 that the optimum toxicity lies in the 
 region of pH 6-8, with the greatest inhi- 
 bition at pH 7. 
 
 The fact that high relative humidity 
 might bring about a condition in the plant 
 leaf in which the water continuum of the 
 plant extends all the way to the leaf sur- 
 face led to the postulation of an alterna- 
 tive aqueous route for solute uptake 
 by plant leaves (Crafts, 1961a, b, <?); 
 such a mechanism obviously must involve 
 the apoplast of the treated leaf. 
 
 When amitrole* was first applied to 
 bean leaves a characteristic wedge-shaped 
 pattern of labeling became evident (Yama- 
 guchi and Crafts, 1958), and after consi- 
 derable study our interpretation was that 
 compounds causing this pattern were enter- 
 ing the leaf via the aqueous route and 
 were moving in the apoplast along with 
 the transpiration stream. Strugger (1938) 
 had observed this type of movement of 
 fluorescent dyes. 
 
 These patterns of apoplastic movement 
 are brought about by amitrole* (fig. VIII, 
 2), MH* (fig. Ill, 4, and VIII, 2), atra- 
 zine*, monuron*, barban*, alanap* and 
 Ca45(fi g . VIII, 3); even 2,4-D may cause 
 this pattern of distribution in the 
 cotyledon of cotton. Some of the above 
 compounds are phloem mobile (amitrole*, 
 MH*, alanap*, 2,4-d*) while others are not 
 (monuron*, simazine*, barban*", Ca 4b ). 
 Whether or not a compound will assume 
 this wedge-shaped pattern of distribution 
 seems to depend upon the avidity with 
 which it is absorbed into the symplast, 
 as contrasted with the rate of flow of 
 the transpiration water in the apoplast. 
 Such apoplastic distribution is of little 
 consequence in the practical use of herbi- 
 cides, but it has been extremely import- 
 ant in the interpretation of autoradio- 
 graphs and in our understanding of the 
 details of the process of absorption of 
 
 96 
 
TABLE XI, 1 
 Absorption and Translocation of 2,4-D in Black-eyed Peas 
 Treated by Application to One Unifoliolate Leaf 
 
 
 Stem-bending (in degrees) at end of: 
 
 pH of 
 solution 
 
 1 hr 
 
 2 hrs 
 
 3 hrs 
 
 4 hrs 
 
 5 hrs 
 
 6 hrs 
 
 7 hrs 
 
 8 hrs 
 
 24 hrs 
 
 0.5 
 
 
 
 
 
 1.0 
 
 3.0 
 
 1.5 
 
 2.5 
 
 7.0 
 
 10.0 
 
 • 
 
 11.0 
 
 1.0 
 
 
 
 
 
 2.0 
 
 2.5 
 
 1.5 
 
 1.5 
 
 4.5 
 
 5.5 
 
 7.0 
 
 2.0 
 
 
 
 
 
 5.5 
 
 21.5 
 
 65.1 
 
 100.5 
 
 102.5 
 
 94.0 
 
 91.5 
 
 3.0 
 
 
 
 
 
 4.5 
 
 27.0 
 
 58.0 
 
 88.5 
 
 94.0 
 
 87.5 
 
 92.5 
 
 4.0 
 
 
 
 
 
 0.0 
 
 15.0 
 
 54.5 
 
 94.5 
 
 96.0 
 
 81.0 
 
 84.5 
 
 5.0 
 
 
 
 
 
 0.0 
 
 11.0 
 
 46.0 
 
 87.5 
 
 100.0 
 
 88.0 
 
 86.0 
 
 6.0 
 
 
 
 
 
 1.5 
 
 12.0 
 
 44.5 
 
 90.5 
 
 100.0 
 
 91.0 
 
 79.5 
 
 7.0 
 
 
 
 
 
 0.0 
 
 1.0 
 
 9.0 
 
 32.0 
 
 70.5 
 
 56.5 
 
 82.5 
 
 8.0 
 
 
 
 
 
 0.0 
 
 1.0 
 
 1.0 
 
 10.0 
 
 24.5 
 
 72.5 
 
 76.5 
 
 9.0 
 
 
 
 
 
 0.0 
 
 0.0 
 
 1.5 
 
 10.5 
 
 39.0 
 
 70.0 
 
 91.5 
 
 10.0 
 
 
 
 
 
 0.0 
 
 0.0 
 
 1.0 
 
 12.5 
 
 40.0 
 
 68.0 
 
 86.5 
 
 applied compounds by the plant leaf. 
 Interpretations of the pattern of movement 
 of monuron* and simazine* in potato 
 slices (fig. VI, 5) and of the uptake 
 and movement of 2,4-D* (fig. VII, 1), 
 monuron* (fig. IX, 3, 4), and simazine* 
 (fig. VIII, 1) by plant roots have also 
 been illuminating. A continuing problem 
 is the ready entrance of monuron* and 
 simazine* into and through roots, pre- 
 sumably across the symplast of the endo- 
 dermis - in contrast to their consistent 
 failure to enter the symplast of leaves; 
 applied to stems of bean and soybean 
 seedlings they apparently bypass the 
 phloem, enter the xylem, .and move upward 
 in the transpiration stream (fig. XI, 1). 
 Histoautoradiographs using tritiated monu- 
 ron indicate that this tracer is present 
 in cell walls of the leaf but absent from 
 living cells. However, the substituted 
 urea and symmetrical triazine herbicides 
 are powerful inhibitors of oxygen evolu- 
 tion in photosynthesis (Hill reaction) , 
 as shown by Cooke (1956), Wessels and 
 van der Veen (1956), Moreland, et at. 
 
 (1959), Todd (1960), Jagendorf (1958), 
 Exer (1958), Gysin and Knusli (1958, 1960), 
 Ashton, Zweig and Mason (1960), Ashton, 
 Uribe and Zweig (1961), Roth (1958), 
 Sweetzer and Todd (1961), and Sweetzer 
 et at, (1961). Thus they must enter the 
 chlorenchyma cells of leaves and migrate 
 to the chloroplasts where photosynthesis 
 takes place. That this occurs at amaz- 
 ingly low concentrations is indicated by 
 the fact that these compounds are effec- 
 tive at dosages of from 1 to 4 pounds per 
 acre, corresponding to concentrations of 
 around 1 ppm in the soil solution. How- 
 ever, the dense labeling of foliage of 
 plants treated with monuron* and simazine* 
 by application to roots indicates a con- 
 centrating effect in the leaves resulting 
 from the continued supply through the 
 transpiration stream, the failure of re- 
 transport via phloem, and the loss of 
 water from the leaves by transpiration 
 (fig. VIII, 1; IX, 3, 4). 
 
 With regard to a possible aqueous 
 route for entry of solutes from the cuti- 
 
 97 
 
TABLE XI, 2 
 Weight in Grams of Growth of Bermudagrass Shoots 
 Sprayed January 11, 1951, with 0.5 per cent MH 
 Adjusted to a Series of pH Values 
 
 
 
 
 Weight in 
 
 grams * 
 
 
 
 
 
 
 
 Harvest 
 
 dates 
 
 
 
 
 pH 
 
 7/3/51 
 
 9/25/51 
 
 12/3/51 
 
 3/7/52 
 
 7/23/52 
 
 9/2/52 
 
 Average weight 
 
 (Un treated 
 control) 
 
 51.5 
 
 81.9 
 
 12.5 
 
 14.0 
 
 24.2 
 
 10.0 
 
 32.3 
 
 2.0 
 
 16.7 
 
 59.0 
 
 15.2 
 
 18.2 
 
 22.2 
 
 9.5 
 
 23.9 
 
 3.0 
 
 17.1 
 
 58.0 
 
 15.4 
 
 18.5 
 
 19.2 
 
 8.0 
 
 22.7 
 
 4.0 
 
 18.5 
 
 40.2 
 
 16.6 
 
 25.3 
 
 22.0 
 
 8.6 
 
 21.9 
 
 5.0 
 
 19.5 
 
 18.7 
 
 11.7 
 
 21.3 
 
 28.3 
 
 8.6 
 
 18.0 
 
 6.0 
 
 17.1 
 
 7.7 
 
 7.9 
 
 9.3 
 
 10.9 
 
 3.9 
 
 9.5 
 
 7.0 
 
 13.9 
 
 7.0 
 
 5.9 
 
 0.0 
 
 0.0 
 
 0.0 
 
 4.5 
 
 8.0 
 
 15.8 
 
 0.6 
 
 7.9 
 
 14.0 
 
 15.5 
 
 4.1 
 
 9.7 
 
 9.0 
 
 20.2 
 
 25.2 
 
 19.3 
 
 24.7 
 
 27.9 
 
 11.6 
 
 21.5 
 
 10.0 
 
 18.5 
 
 37.1 
 
 11.6 
 
 13.7 
 
 31.8 
 
 10.6 
 
 20.6 
 
 t Values are averages of duplicate cultures. 
 
 cle into the interior, it is plausible to 
 assume that the water in the leaf consti- 
 tutes a continuum. This water-phase may 
 reach the surface under saturated condi- 
 tions, and hence application of aqueous 
 sprays may present solutes for continuous 
 diffusion into the leaf. That such a con- 
 dition obtains in plants in saturated at- 
 mosphere is suggested by figure XI, 2, 
 which shows four cotton seedlings treated 
 with urea-C ll+ on one cotyledon. The two 
 plants on the left were treated and put 
 in a plastic bag to obtain a saturated 
 atmosphere; the right-hand pair stood in 
 the open greenhouse. The right-hand 
 plant of each pair was steam- ringed below 
 the cotyledons. In the greenhouse, only 
 synplastic movement took place; in the 
 plastic bags in saturated atmosphere 
 water condensed on the treated spots and 
 moved apoplastically to opposite cotyle- 
 dons and into mature primary leaves. 
 Symplastic movement was enhanced by the 
 high humidity conditions (Clor, Crafts 
 
 and Yamaguchi, 1962). 
 
 Figure XI, 3, shows coleus plants 
 treated with 2,4-D* and amitrole* and 
 placed in polyethylene bags to produce a 
 saturated condition. Although differences 
 in mobility of the two tracers are shown, 
 the humid atmosphere has promoted apoplas- 
 tic movement of amitrole* from the treated 
 portions of leaves to untreated portions, 
 to roots, and to growing axillary shoots. 
 These two herbicides are much more effec- 
 tive in humid regions than in semi-arid 
 regions . 
 
 Figure XI, 4, shows Tropaeolum majus 
 plants treated with 2,4-D* (left) and 
 amitrole* (right) in the greenhouse. 
 Here the 2,4-D* shows typical symplastic 
 movement whereas amitrole* has leaked 
 from phloem to xylem so that leaves below 
 the treated leaf show light labeling by 
 apoplastic movement. 
 
 98 
 
Fig. XI, 1. Apoplastic movement of monurori''" in bean. A droplet of monuron* solu- 
 tion (20 /u1 containing 0.1 jumole was applied to the midrib just above the insertion 
 of the petiole (left). The same amount was applied to the petiole, near the node 
 (right). 
 
 99 
 
When the leaf is under tension due to 
 low atmospheric humidity, menisci must 
 be drawn back for varying distances in the 
 cuticular pores and hence air blocks will 
 prevent union of an applied spray liquid 
 with the water continuum and will inhibit 
 entry of contained solutes. Crafts and 
 Foy (1962) have proposed that a surfactant 
 compatible with the cuticular lipoids may 
 bring about a wetting of pore walls, a 
 surrounding of the air bubbles, and hence 
 a solubilization or displacement of the 
 blocks. Effectiveness of a spray formu- 
 lation in surmounting such a block must 
 depend upon the amount of tension, and 
 the ability of the surfactant to wet the 
 specific cuticle. 
 
 Once union has been made between an 
 applied spray droplet and the water con- 
 tinuum of the leaf, drying of the droplet 
 must slow down or come to a halt and dif- 
 fusion of the applied solute must continue 
 for a considerable period of time. For a 
 lipoid-soluble solute, diffusion across 
 the cuticle and partition into the sym- 
 
 ■^?S#^! 
 
 w 
 
 plast should continue as long as the 
 molecules remain unbound in the leaf sur- 
 face. 
 
 The prolonged migration of solutes into 
 leaves is shown by many autoradiographic 
 studies. Figure IX, 1, shows barley 
 treated with MH* and given 3-, 9-, and 
 27-hour treatment times. Time-series 
 studies show the increased uptake and 
 distribution of IAA*, amitrole*, and MH* 
 that takes place during 1-, 4-, and 16- 
 day treatment times in barley and bean. 
 Jackson (1962) found this same effect in 
 treating oxalis plants with phenoxy herbi- 
 cides. 
 
 In view of these considerations it 
 seems obvious that the plant body consists 
 of two solid systems, the apoplast con- 
 tinuum and the symplast continuum, and 
 that each of these systems plays an impor- 
 tant role in the physiology of the plant. 
 The symplast makes up the living stuff of 
 the plant and hence is of critical impor- 
 tance in all plant functioning. The con- 
 cept of the symplast as an interconnected 
 continuum is all- important with respect 
 to translocation. Since foods are syn- 
 thesized in certain highly specialized 
 cells and transported from these to all 
 other living tissues, the distribution 
 function of the symplast is of utmost 
 significance. Many tracer molecules are 
 mobile in the symplast, while others are 
 subject to forces of retention within 
 living cells. The mobility factors of a 
 molecule determine whether it will have 
 local or systemic distribution within the 
 plant body. Thus, these factors must be 
 understood to properly evaluate pesticide 
 properties of new chemicals - and the 
 autoradiographic method is invaluable in 
 such studies. 
 
 The apoplast in turn is of critical 
 importance, both in the physiology of 
 plants and in the uptake and distribution 
 of exogenous chemicals. All such chemi- 
 
 ^ Fig. X, 2. Effects of a saturated at- 
 mosphere and steam-ri ngi ng on distribution 
 of sucrose-' from urea- in cotton plants. 
 Plants in photograph on left were enclosed 
 in a polyethylene bag to give a saturated 
 atmosphere; plants in photograph on right 
 were in the open greenhouse. The right- 
 hand plant of each pair was steam-ringed 
 below the cotyledons before treatment. 
 
 100 
 
n 
 
 Fig. XI, 3. Coleus plants treated with 2,4-D* (left) and amitrole* (right). The 
 left-hand leaf of each plant was treated on the lower surface (stomata present); the 
 right-hand leaf on the upper surface (stomata absent). After treatment, plants were 
 enclosed in polyethylene bags to develop high humidity. Treatment time was *t days 
 and dosage was 0.5 jumole per leaf; Tween 20, 0.1 per cent. 
 
 101 
 
Fig. XI, k. Nasturtium plants treated with 2,*+-D» (left) and amitrole* (right). 
 Dosage was 0.5 jumole per plant, time was k days; Tween 20, 0.1 per cent. The 2,^-D- 
 treated plant shows symplastic movement only; the ami trole^'-treated plant shows sym- 
 plastic movement plus some apoplastic, as a result of leakage from phloem to xylem. 
 
 102 
 
cals enter the plant via the apoplast; of the physiology of distribution, and 
 
 water, salts and many pesticides undergo from the standpoint of pesticidal action 
 
 their major distribution via the apoplast; and pesticide residues, the symplast- 
 
 the apoplast makes up a large portion of apoplast concept is illuminating and use- 
 
 the body of the living plant; a killed ful - in fact, it may be a more practical 
 
 plant approaches the apoplast in proper- concept than the cell concept in these 
 
 ties; most pesticide residues reside on particular fields, 
 or in the apoplast. From the standpoint 
 
 103 
 
Chapter XII. Studies on Herbicide 
 Movement in Woody Plants 
 
 Wh, 
 
 'hen 2,4-D was first used for con- 
 trol of deep-rooted perennial weeds it 
 became evident that a new tool was avail- 
 able for controlling woody plants on 
 range and forest areas. Trials along the 
 eastern seaboard proved that 2,4-D would 
 penetrate the foliage of woody plants and 
 would, in some cases, translocate to the 
 crowns, causing death of the meristematic 
 regions and hence resulting in control. 
 However, when the same type of applica- 
 tions were made in semi-arid regions, 
 treatments were not as effective. Contin- 
 ued trials soon proved the efficacy of the 
 basal-spray method, in which an ester for- 
 mulation of 2,4-D in diesel oil was 
 applied around the base of the tree in 
 sufficient volume to run down and cover 
 the crown where resprouting occurs. 
 
 Trials also proved the usefulness of 
 the injection or cut-surface method in 
 which the concentrated amine formulation 
 is poured into cuts through the bark near 
 the ground level. Many thousands of acres 
 of weed trees have been killed in forest 
 and range lands in the United States by 
 this method. Airplane spraying of weed 
 trees in cut- over forest to control broad- 
 leaved species, and hence to release the 
 young conifers in the stand, has also 
 proved successful and is being used an- 
 nually on thousands of acres in the 
 U.S.A. - particularly against alders and 
 willows, two species very susceptible to 
 2,4-D. A more recent method is to spray 
 trees in the bud-swelling stage with es- 
 ter formulations of 2,4,5-T; this has 
 proved effective against maples and oaks, 
 which are resistant to the toxic effects 
 of 2,4-D. 
 
 Only one of these methods - the 
 spraying of willows, alders and similar 
 susceptible species in early summer - 
 
 produces evidence for any extensive trans- 
 location of the herbicide; in the other 
 three methods the toxic formulations are 
 applied onto or very near the meristematic 
 tissue responsible for resprouting. In 
 the injection method, the chemical is 
 placed in the sapwood and is carried 
 principally into the tops via the trans- 
 piration stream; there is little evidence 
 of phloem transport in these treatments. 
 
 In attempting to control woody species 
 by means of translocated sprays a series of 
 problems arise: 
 
 1, In treating evergreen species, 
 application must be made at a time when 
 assimilates are moving into roots. 
 
 2. In the case of deciduous species, 
 the leaves must have passed the compensa- 
 tion point (exporting food materials), 
 and they must be mature enough to resist 
 contact action of the herbicide. 
 
 3 . To control deciduous species , 
 treatment must be made before soil mois- 
 ture is exhausted and root-growth stopped. 
 
 Thus, these requirements set limits 
 on time of application , concentration of 
 the formulation, polarity of the molecules 
 applied, and on the plant itself (with 
 respect to soil moisture, temperature and 
 growing conditions). The rules laid down 
 from tests on deep-rooted herbaceous per- 
 ennials must often be broken, and in the 
 case of mixed-brush stands susceptible 
 species may be controlled while resistant 
 ones survive. Experimental work on woody 
 plant species was undertaken with the 
 above conditions in mind. 
 
 AUTORADIOGRAPHY OF WOODY PLANTS 
 
 Some of the methods used in studies 
 on woody plants with labeled tracers are 
 
 104 
 
illustrated in figures XII, 1, 2. These 
 studies, using five toxicants and urea*, 
 have produced much information about ab- 
 sorption and translocation of herbicides 
 in such plants (Leonard and Crafts, 1956). 
 Figures XII, 1, 2, show plant materials 
 and autographs from the studies, and fig- 
 ure XII, 3, illustrates the method used 
 in presenting information in graphic form. 
 These studies on seven common woody plants 
 of western brush- lands emphasized a number 
 of points that were critical with respect 
 to the successful use of 2,M-D on woody 
 species in California. The following 
 were the principal findings. 
 
 COYOTE BRUSH (Baooharis pilularis) , an 
 evergreen shrub flowering in autumn. In 
 February, 2,4-D* was absorbed but moved 
 very little; translocation increased in 
 April, diminished in May and June, and 
 ceased in July. February and March treat- 
 ments resulted in movement downward from 
 treated leaves toward the roots. Tracer 
 applied in April and later moved in both 
 directions, with much more tracer located 
 in the upper foliage. 
 
 ARROYO WILLOW (Salix lasiplepis) , a deci- 
 duous tree. Little translocation of 
 2,4-D* took place in this species before 
 April 15, but from late April until late 
 summer translocation was continuous. 
 Translocation both upward and downward 
 from treated leaves continued from late 
 April until late September. Upward trans- 
 port was predominant in April; by October 
 all movement was downward. 
 
 WEDGE-LEAF CEANOTHUS (Ceanothus auneatus) , 
 an evergreen shrub flowering in spring. 
 There was ready absorption of 2,H-D* by 
 leaves but little translocation. Leaves, 
 which were treated on their surfaces, 
 were rapidly killed and this may explain 
 the failure of tracer to move. 
 
 MANZANITA (Arctostaphylos manzanita) , an 
 evergreen shrub flowering in midwinter 
 (fig. XII, 2). Translocation, starting 
 in March, was mostly downward. Bark of 
 this species could not be peeled after 
 June. 
 
 TOYON (Photinia arbuti folia) , an evergreen 
 
 Fig. XII, 1. Cross-sections of buckeye stem (left) mounted and ready to autoradio- 
 graph; (right) autoradi'ograph of the mount. Film was reversed in photographing. The 
 treatments appear as black spots; counterclockwise from left to right they are: 
 2,4,5-T*, monuron*, urea-, MH*, amitrole*, and 2,4-D*. At a level of 6 inches above 
 the treatment, 2,4,5-T*, amitrole* and 2,4-D* can be seen; at 1-1/2 inches below 
 2,4,5-T* and 2,4-D* are present. 
 
 105 
 
shrub or tree blossoming in early summer. 
 Absorption and translocation of 2,4-D* 
 was active from February through October. 
 Transport was predominantly downward dur- 
 ing February and March, upward in mid- 
 spring and summer, and downward again in 
 the fall. During late summer and early 
 fall the autographs were light, indica- 
 ting poor penetration of tracer. 
 
 BLUE OAK (Querous douglasii) , a deciduous 
 tree blossoming in early spring. Tracer 
 studies were mostly negative. From March 
 to June only five out of forty-two samples 
 showed any movement, and the average dis- 
 tance moved was only 5.2 inches. Blue oak 
 leaves are very sensitive to contact ac- 
 tion by 2,4-D; this may explain the poor 
 results in the experiments and the gener- 
 
 lil 
 
 i 
 
 / 
 
 UH CHU u*u 
 
 AT 1,<*-B 
 
 
 Fig. XII, 2. Autoradiographs (above) and mounted bark and wood (below) of manzanita 
 treated with six tracers. From left to right, the chemicals as shown in the bark and 
 wood autographs are: 2,^+, 5-T- v , monuron*, urea-, maleic hydrazide-'-, amitrole* and 2,k-D' 
 
 106 
 
ally poor results of field application of 
 this herbicide to blue oak. 
 
 LIVE OAK (Querous wislizenii) , an ever- 
 green tree blossoming in spring. Trans- 
 location was active from February through 
 September. Movement was entirely down- 
 ward in February, partially upward after 
 new growth started in March, and largely 
 downward during summer and fall. 
 
 These studies, carried on in the 
 field with native plants, emphasize some 
 important points. Because early trials 
 indicated that little 2,4-D* was penetra- 
 ting the cuticle on the upper surfaces of 
 leaves, later treatments were placed on 
 the lower surfaces. Careful studies of 
 the original autographs and of many later 
 ones showed, however, that tracer will 
 penetrate the cuticle in the absence of 
 stomata. Penetration studies showed that 
 the intact cuticle may protect the leaf 
 tissues from a too-rapid absorption which 
 might cause rapid injury of leaves and 
 inhibition of transport. We suspect that 
 this might explain the excellent results 
 obtained with airplane application of an 
 oil formulation of 2,4-D ester on wedge- 
 leaf ceanothus, whereas our tracer trials 
 placing the 2 , 4-D* on the lower surfaces 
 of leaves resulted in little movement, 
 and field application of 2,4-D ester in 
 oil to blue oak usually fails because of 
 excessive contact toxicity. This conclu- 
 sion reemphasizes the significance of 
 penetration and the relation of contact 
 injury to uptake and transport. Many 
 failures in the use of herbicides on 
 woody plants may result not from lack of 
 absorption but from too rapid an uptake, 
 with resultant injury and breakdown of 
 tissues providing the source of energy 
 for translocation. 
 
 The relation of 2,4-D transport to 
 food movement in plants is evident 
 throughout the woody plant work, and the 
 fact that only growing cells and tissues 
 respond to 2,4-D is also important. It 
 is evident that a thorough knowledge of 
 the physiology of plants is necessary if 
 one is to treat woody species with success, 
 
 Translocation in deciduous species 
 is going on during much of the year, and 
 probably year-round in evergreen plants; 
 as long as plants are synthesizing foods 
 and using them in growth, flowering, 
 
 fruiting, and storage, translocation pro- 
 cesses are going on. Failure to kill 
 active plants having green foliage seldom 
 results from failure of translocation; 
 rather, it may result from lack of absorp- 
 tion, from translocation to the wrong 
 tissues (flowers, fruits, or vegetative 
 shoots), or from the inability of root 
 cells to respond to 2,4-D. Because root 
 response to 2,M~D involves active root 
 growth, available soil moisture is essen- 
 tial to successful treatment, and this is 
 true for all hormone herbicides. 
 
 The period for successful treatment 
 of deciduous woody species may be rela- 
 tively brief, occurring in spring or early 
 
 Fig. XII, 3. Translocation of six cJ^- 
 labeled tracers in the trunks of toyon 
 through an annual cycle. Readings were 
 made on rings of bark removed 2k hours 
 after treatment and then dried and auto- 
 graphed. (Methods used are described in 
 Yamaguchi and Crafts, 1959.) 
 
 mt(r>-z_i l9 tt- , ->oz 
 
 UJ <a <333UlOOuJ4 
 2 < 2 -j -> < Vt O Z Q -> 
 
 UJ<0u<333UJOOuj< 
 
 u..s,< ; a.->.-».<,cp,o.z,Q,-». 
 
 u 
 
 2 
 
 UJ 
 
 > 
 o 
 
 2 
 
 -I 
 < 
 Z 
 
 a 
 
 3 
 
 © 
 
 Z 
 
 o 
 
 -I 
 
 u. 
 o 
 
 to 
 
 UJ 
 
 ■ VERY INTENSE RADIOACTIVITY 
 
 £53 MEDIUM INTENSITY 
 
 HD LIGHT INTENSITY * IMAGE AT BARK SURFACE 
 
 E3 PERIOD OF GROWTH FLUSH O TREATED SPOT 
 
 107 
 
simmer when leaves have fully expanded and 
 are mature enough to absorb actively yet 
 are not too heavily cutinized. Weather 
 conditions should be favorable, with mod- 
 erate temperatures and adequate humidity. 
 In seasons of low rainfall such periods 
 may last a few weeks or only a few days; 
 in extremely dry periods there may be no 
 such time and chemicals will be ineffec- 
 tive, as was shown by the failure of chem- 
 ical treatment of mesquite in Texas and 
 parts of Arizona during a succession of 
 dry years. 
 
 Continued spray programs on many spe- 
 cies have proved that, in contrast to blue 
 oak, arroyo willow - which thrives only 
 in moist habitats - may be treated suc- 
 cessfully from the time leaves expand in 
 spring until late autumn. Several ever- 
 green species may be treated in late win- 
 ter or early spring while the previous 
 season's leaves are still green and ac- 
 tive, and at a time when soil moisture 
 is amply available. Toyon and wedge- 
 leaf ceanothus are two such species 
 
 With the introduction of amitrole, 
 translocation studies soon proved this 
 herbicide to be highly mobile in plants. 
 Because its mode of action does not nec- 
 essarily involve growing tissues it may 
 be used as a translocated chemical during 
 summer, when soil moisture may be rela- 
 tively unavailable. Under conditions 
 common in the American West, this consid- 
 erably extends the period when woody 
 plants may be controlled. 
 
 COMPARATIVE STUDIES ON TRACER UPTAKE 
 AND TRANSPORT IN WOODY PLANTS 
 
 With the demonstration that differ- 
 ent labeled tracers show different and 
 characteristic behaviors in plants (pages 
 71 to 81) , it became apparent that an ex- 
 tension of these studies of woody plants 
 should open new paths to control of these 
 weeds (Yamaguchi and Crafts, 1959). Ac- 
 cordingly, studies were set up to examine 
 comparatively the uptake and distribution 
 of 2,4-D*, 2,4,5-T*, amitrole*, maleic 
 hydrazide*, monuron*, and urea*. Table 
 XII, 1, shows the molecular weights and 
 concentration of treatment solutions of 
 chemicals used in these comparative 
 studies. 
 
 Three tree species were used - manza- 
 
 Table XII, 1 
 
 Molecular Weights and Concentrations 
 
 Of Chemicals Used in Treating Woody Plants 
 
 Chemical 
 
 MW 
 
 Concentration, ppm 
 
 2,4-D 
 
 221.0 
 
 2,500 
 
 2,4,5-T 
 
 255.5 
 
 2,890 
 
 Amitrole 
 
 84.5 
 
 956 
 
 MH 
 
 112.0 
 
 1,267 
 
 Urea 
 
 60.0 
 
 679 
 
 Monuron 
 
 198.0 
 
 2,240 
 
 nita, toyon, and buckeye (Aesculus eali- 
 fornica)\ the first two are evergreens, 
 while buckeye is a deciduous species 
 blossoming in early summer. Applications 
 were to the active phloem of the trunk 
 after removal of the outer bark (fig. II, 
 9). The autographed materials consisted 
 of rings of bark and strips of wood taken 
 in the region of treatment (fig. XII, 2), 
 sections of stem (fig. XII, 1), and 
 shoots. Experiments were carried on for 
 1 year. Figure XII, 3, shows a graph of 
 the combined results of the whole test on 
 toyon. 
 
 In this study, 2,4-D* and 2,4,5-T* 
 moved downward in March, April, and May 
 in buckeye; in March, April, May and June 
 in manzanita, and June through August in 
 toyon. In March and April the movement 
 was mostly or entirely downward in manza- 
 nita and buckeye. From May until midsum- 
 mer the downward movement gradually low- 
 ered and upward movement became prominent, 
 especially in manzanita and buckeye. In 
 late summer, fall and winter, movement in 
 these two species came to a standstill, 
 probably because of water stress. Season- 
 al trends were similar in toyon, except 
 that some upward movement occurred 
 throughout the year. Toyon, unlike man- 
 zanita and buckeye, grew at the bottom of 
 a canyon where water was available and 
 root growth could have continued through- 
 out the summer months and into the fall. 
 
 Amitrole* movement downward was gener- 
 ally less than that of the phenoxyacetic 
 acids, and was somewhat inconsistent. 
 
 108 
 
Movement was most extensive in manzanita 
 and continued from January to July; down- 
 ward movement during these months was 
 equal to, or less than, upward movement - 
 from August through December movement was 
 predominantly upward. 
 
 In toy on, downward movement of ami- 
 trole* was less than that of the phenoxy 
 compounds and occurred mostly from Feb- 
 ruary to April. In buckeye, movement was 
 also restricted and took place mainly in 
 June. Amitrole* had a strong tendency 
 to fix in the xylem tissues and movement 
 was only in the upward direction; this was 
 true of all three species. 
 
 Maleic hydrazide* and urea* had simi- 
 lar distribution patterns. They moved 
 downward 12 inches or more in manzanita 
 during January and February and again in 
 May; upward movement was even more erratic. 
 MH* and urea* are both highly water sol- 
 uble; the treated spots in many cases were 
 low in radiation intensity; probably these 
 compounds diffused readily along the apo- 
 plast and were flushed up the xylem in low 
 concentration in the transpiration stream. 
 This indicates, in contrast to 2,4-D*, 
 2,4,5-T* and amitrole*, a very low tissue- 
 retention and relatively free mobility. 
 In toyon and buckeye, MH* and urea* were 
 almost completely carried away. 
 
 In the January treatment of toyon, 
 light images of radiation from MH* and 
 urea* were found at the outer surface of 
 the bark up to 6 inches or more above the 
 treated spots. Transpiration was low at 
 this time, and apparently tracers were 
 carried laterally from the vascular chan- 
 nels across phloem and cortex and depos- 
 ited at the surface where water loss by 
 evaporation was occurring. Later, when 
 transpiration increased, tracers were 
 probably dissipated over so much area that 
 they could not be detected by the autora- 
 diographic method. Evidently, free mobi- 
 lity of a tracer in such tissues is an 
 indication of lack of retention by living 
 cells. 
 
 Monuron* showed essentially no down- 
 ward movement in these tree trunks. Up- 
 ward movement was moderate in toyon, less 
 in buckeye, and almost lacking in manza- 
 nita; even the treated spots were faint. 
 This compound apparently moved with great 
 freedom in the apoplast and was largely 
 
 carried away to the tops of the trees in 
 the transpiration stream; it did not en- 
 ter the phloem and move in a downward 
 direction. 
 
 Following the studies on tracer 
 movement in tree trunks, an experiment was 
 set up using toyon and manzanita seedlings 
 (Yamaguchi and Crafts, 1959). Here, chem- 
 icals were confined to single small plants 
 so that the distribution of each could be 
 determined. Applications were made to 
 stems in two ways: by making a small cup 
 of aluminum foil around the stem and fill- 
 ing it with treatment solution, and by 
 cutting away the outer bark (as had been 
 done in the previous study) and treating 
 by means of an aluminum foil cup which 
 surrounded the bared area (fig. II, 9, 
 right). The cups were sealed in place 
 with lanolin and 60 microliters of treat- 
 ment solution were used in each treatment. 
 
 Autographs of these plants show the 
 distribution obtained by this method 
 (fig. XII, 4, 5). The autographs also 
 showed one consistent difference between 
 the treatment on old tree trunks and on 
 young seedlings: a predominance of up- 
 ward movement in the young plants. The 
 young trees were growing rapidly and they 
 might be compared with the older trees 
 during their spring flush of growth. In 
 both cases 2,4-D* showed movement both 
 upward and downward; in the seedlings, 
 downward movement was much less prominent. 
 Removal of outer bark considerably enhan- 
 ced absorption of the tracers in toyon. 
 
 LATERAL MOVEMENT OF TRACERS IN WILLOW 
 
 Knowledge of the relative mobility of 
 different compounds both longitudinally 
 and laterally in woody stems is needed 
 in order to interpret the distribution of 
 pesticides in plants treated via the foli- 
 age and via the roots. To obtain infor- 
 mation on these processes, experiments 
 were performed with willow stems of 3/4- 
 inch diameters. The autographs of the 
 phloem and xylem tissues immediately 
 above and below treated spots show that 
 monuron*, MH*, and amitrole* move princi- 
 pally upward. Although such movement 
 could have occurred only in the xylem, 
 evidence for presence of the tracers is 
 present in both tissues. In the case of 
 2,4-D* there is evidence for both upward 
 movement in the xylem and downward move- 
 
 109 
 

 Fig. XII, k. Toyon shoots treated on the stem with 2,4-D* Shoot on the left was 
 treated by applying tracer solution through a cut in the bark; shoot on right was 
 treated by application of tracer to the intact bark. 
 
 110 
 
Fig. XII, 5- Toyon shoots treated with monuron". The left-hand shoot was intact, 
 the right-hand shoot was treated through the cut bark. In contrast to leaves in fig. 
 XII, k, the very young leaves here are lighter than the old - an indication of apoplas- 
 tic movement of monuron*. 
 
 Ill 
 
merit in phloem, and both tissues contained 
 the tracer. This confirms the evidence 
 presented by Stout and Hoagland (1939) for 
 rapid lateral movement of tracers across 
 the cambium. When cuts were made across 
 the bark at positions 1 inch apart and 
 treatment was applied between the cuts, 
 all four compounds moved upward; none 
 moved downward. This proves that the 
 tracers penetrated into the xylem and 
 moved in the transpiration stream; because 
 the phloem was blocked by the cut below 
 the treatment no downward movement of 
 2,4-D* occurred. 
 
 Autographs were made of stems from 
 which the bark was pried loose over a 
 distance of 4 inches and then pressed back 
 and bound in place with masking tape. The 
 autographs, when compared with those of 
 treatments on stems with intact bark, show- 
 ed that there is almost no difference be- 
 tween movement of monuron* and MH* through 
 stems in which the bark was separated and 
 through intact stems. In the case of ami- 
 trole*, there was labeling in the phloem 
 and xylem and upward movement in xylem 
 only. The results with 2,4-D* are most 
 illuminating. Here, movement took place 
 in the phloem in both directions but was 
 strongest in the downward direction. In 
 the xylem there was little upward move- 
 ment; below the treated spot there was 
 little evidence of tracer in the region 
 where the bark was lifted, and there was 
 a strong labeling below this region. 
 Evidently the downward movement took place 
 in the phloem, but in the regions of in- 
 tact bark lateral movement to the xylem 
 was very rapid. Separation of the bark 
 (phloem) from the xylem evidently injur- 
 
 ed the symplast so that even though the 
 tissues were bound together again later- 
 al movement was inhibited. This is a 
 complementary picture to the one obtained 
 by Stout and Hoagland (1939) for movement 
 via the xylem. 
 
 These studies show that movement may 
 take place in three distinct ways: (1) 
 upward in the transpiration stream (aque- 
 ous continuity in the apoplast), (2) down- 
 ward by way of the phloem (specialized 
 channels of the symplast), and (3) later- 
 ally via the cell walls (apoplast) from 
 phloem to xylem and viae versa , and into 
 living parenchyma (accumulation). The 
 more polar compounds bypass the symplast 
 and move via the apoplast and the special- 
 ized xylem conduits; the more lipophilic 
 ones are absorbed and moved in the sym- 
 plast. These studies show clearly the 
 possible relations between the three 
 above modes of transport. It is well to 
 keep in mind that all applications, prac- 
 tical and experimental, are to the apo- 
 plast. 
 
 Much knowledge of transport phenomena 
 in woody plants has been acquired, and 
 already some of it has been applied to 
 field practice. Meanwhile there is much 
 room for more studies and for amplifica- 
 tion of our knowledge of tree physiology 
 through autoradiography. Already, hun- 
 dreds and thousands of acres are sprayed 
 in various tree- farming and range-manage- 
 ment operations, and in the future millions 
 of acres will be sprayed annually. Thanks 
 to such practices, utilization of our for- 
 est and range lands will be greatly im- 
 proved. 
 
 112 
 
Chapter XIII. Translocation in Grasses 
 and Coffee Plants 
 
 TRANSLOCATION STUDIES ON GRASS SPECIES 
 
 B< 
 
 because some grass species are high- 
 ly important crops, and because some are 
 obnoxious weeds, studies on certain of 
 them were initiated early in our program. 
 Our earlier studies were designed to 
 elucidate the mode of action of certain 
 herbicides. For example it has been shown 
 that 2,4-D* moved about as readily in 
 barley as in bean and hence that the se- 
 lectivity of 2,4-D was not directly rela- 
 ted to the uptake and transport of this 
 compound (figs. VI, 2, 3; VIII, 1, 2). 
 These studies proved, furthermore, that 
 amitrole* moves more readily than 2,4-D* 
 in barley, and that MH* moves very freely 
 and actually migrates from phloem to 
 xylem and hence may circulate. Monuron*, 
 on the other hand, does not enter and 
 move in the phloem (fig. VI, 3). Barley 
 was used in these experiments as an ex- 
 ample of a monocotyledonous species - one 
 that has scattered vascular bundles and a 
 unique nodal anatomy as contrasted with 
 dicotyledonous species. 
 
 Peterson (1958) applied droplets of 
 2,4-D* solution to all of the main leaves 
 of barley plants in various stages of 
 growth; a consistent change in the pattern 
 of distribution occurred at about the 
 four- to five- leaf stage, and far less 
 2,4-D* reached the root system than in 
 treatment at earlier stages. Van der 
 Zweep (1961), studying 2,4-D* movement in 
 barley, also found restricted movement to 
 roots after the three- to four- leaf stage. 
 This restriction was not related to the 
 change from vegetative to reproductive 
 growth of the growing point; it was more 
 probably the result of a shift in the 
 activity of various sinks, those in the 
 roots being superseded by the growing 
 leaves and inflorescences. 
 
 A more recent study on movement of 
 Cl 1 *- labeled assimilates in wheat is that 
 of Quinlan and Sagar (1962). In the early 
 stages of development of the wheat plant 
 assimilate from leaves on the main shoot 
 was transported throughout the plant but 
 accumulation occurred in meristematic 
 regions. Assimilates produced by leaves 
 of newly-formed tillers were distributed 
 mainly to the meristematic regions of 
 those tillers, although sometimes there 
 was movement into the rest of the plant. 
 In the later stages of development of 
 each tiller the labeled assimilate was 
 restricted to the tiller itself and to 
 any very young tillers attached to it. 
 After emergence of the head, the distri- 
 bution pattern from the youngest and 
 second-youngest leaves changed: movement 
 was predominantly toward the head from 
 the flag leaf and toward the roots from 
 the leaf below the flag leaf. 
 
 Quinlan and Sagar found little evi- 
 dence for a block in the movement of as- 
 similate from top to roots in the wheat 
 plant. Crafts (1959&) found free move- 
 ment of amitrole* from leaves to roots in 
 barley, some restriction on the part of 
 IAA*, and almost no movement of 2,4-D*; 
 he concluded that the block was physio- 
 logical, and that it was related to the 
 avidity with which meristematic cells 
 accumulated the latter two tracers. 
 
 Quinlan and Sagar also found evi- 
 dence for movement of labeled compounds 
 from young into older leaves. While con- 
 centrations were low, evidence for their 
 presence is certainly definite (Quinlan 
 and Sagar, 1962, plates 20a, b, d) . 
 They attempted to increase and decrease 
 transpiration rate and to prove that this 
 
 113 
 
is related to xylem movement. While their 
 methods should have changed the rate of 
 water loss, even surrounding the leaf with 
 water will not prevent loss if the leaf is 
 in the light. Similar labeling of old 
 leaves in our experiments has been attri- 
 buted to the movement of degradation pro- 
 ducts of assimilate metabolism in the 
 transpiration stream. Instead of attempt- 
 ing to stop transpiration in order to 
 solve this problem, xylem sap should be 
 analyzed for Cl 1 *- labeled compounds - 
 organic acids, for example. 
 
 The work described below was on the 
 basic organic nutrition of different grass 
 species as it is related to the growth of 
 forage plants. Compounds labeled with 
 Cl 14 were used and an attempt was made to 
 discover how various plant parts fit into 
 the economy of the plant as a whole. 
 Earlier work with perennial ryegrass (Lo- 
 lium perenne) had shown that the translo- 
 cation of Cl^, introduced into the leaf 
 as Cl 4 02 under photosynthetic conditions, 
 was very rapid and that the isotope moved 
 to all growth-centers of a plant in about 
 an hour (Forde, 1959). A most striking 
 feature of ryegrass is the absence of 
 internodal elongation during the vegeta- 
 tive stage. In the present study it was 
 desired to contrast translocation in rye- 
 grass with that in Bermudagrass (Cynodon 
 daatylon) , which has internode elongation 
 above ground by a nonstorage organ, quack- 
 grass (Agropyron repens) which has inter- 
 node elongation in a storage organ, the 
 rhizome, and corn (Zea mays) which has 
 vertical internodal elongation. (After 
 initial investigation the last species 
 was omitted because when at all mature it 
 was inconvenient for this type of study.) 
 
 Under controlled conditions (75 de- 
 grees Fahrenheit and 70 per cent relative 
 humidity) a 2 Jul drop of radioactive sol- 
 ution was applied to the lamina of the 
 leaf at a point between 1/2 and 1 centi- 
 meter above the ligule (fig. II, 11). The 
 compounds used were urea*, amitrole* and 
 MH*. Carbon- labeled ammonium thiocyanate 
 was used in one series but unless its 
 name is mentioned, it can be assumed that 
 only the first three compounds were used. 
 Clor (1959) showed that urea" is rapidly 
 
 split by the urease present in cotton to 
 give ammonia and, under photosynthetic 
 conditions, the products of normal Cl 1 ^ 
 fixation by photosynthesis, the labeled 
 compound translocated being sucrose. 
 Urea* has a number of advantages over 
 C1402 in application, the principal one 
 being that urea* can readily be applied 
 as an aqueous solution. The translocation 
 pattern of the products of urea* breakdown 
 is typical of phloem movement, with little 
 xylem movement. In earlier studies ami- 
 trole* had been found to move with the 
 assimilate stream in the phloem, movement 
 being typically from the point of synthe- 
 sis of photosynthate (the source) to the 
 point of utilization (the sink). Maleic 
 hydrazide* follows this pattern to a con- 
 siderable extent, but also has been shown 
 to move in xylem to a marked degree after 
 leaf application, presumably due to leak- 
 age from the phloem to the xylem. 
 
 Unless otherwise stated, plants in 
 the translocation studies were grown in 
 sand culture with periodic waterings with 
 normal Hoagland's solution. Treatments 
 lasted from 1 to 96 hours, after which 
 time the plants were carefully harvested 
 and killed with powdered dry ice. The 
 frozen plant material was freeze-dried, 
 mounted on paper, and an autoradiography 
 was then prepared. 
 
 After observing the translocation 
 pattern in the whole intact plant, experi- 
 ments were conducted in which parts of the 
 plant were either blacked-out completely 
 (using black paper screen) or defoliated. 
 The radioactive solution was always ap- 
 plied to a nonscreened or nondefoliated 
 leaf. The purpose of these experiments 
 was to see whether other sinks could be 
 created by interfering with the production 
 of photosynthate, thus modifying the trans- 
 location pattern. 
 
 EXTRACTION OF THE PRODUCTS OF 
 UREA" APPLICATION 
 
 Corn and ryegrass plants were treated 
 with urea* using the methods described by 
 Clor; after some hours (3 and 6 hours for 
 corn; 3, 9, and 27 hours for ryegrass) 
 plants were harvested, the treated spot 
 
 2. Work carried out by Bernard Forde and incorporated in a thesis for the partial 
 satisfaction of requirements for the Ph.D. degree, University of California, Davis, 
 California (Forde, 1963). 
 
 114 
 
discarded, and the remainder of the plant 
 divided into treated leaf, the rest of the 
 shoot, and the roots. These fractions were 
 homogenized in boiling 80 per cent ethanol 
 and the supernatant, following concentra- 
 tion by evaporating under reduced pressure, 
 was spotted on chromatography paper. Urea* 
 and sucrose* were spotted singly on the 
 same paper and also on top of a spot of 
 extract. The chromatograms were run in 
 butanol-acetic acid/water (4/1/1.5) dried, 
 and autoradiographed. With corn, sucrose 
 was the predominant labeled compound ex- 
 tracted (as evidenced from a comparison 
 of the Rf of the known sucrose and the 
 extract spot) , and urea* was not detected 
 in any of the fractions isolated. With 
 ryegrass it was found that some urea* 
 could be detected in the extract from the 
 treated leaf though it could not be detec- 
 ted in the remainder of the plant, and 
 sucrose was the main compound identified 
 by cochromatography. 
 
 Extracts from the treated ryegrass 
 leaf were also run in two dimensions using 
 first phenol/water (80/20) and, after 
 drying, butanol/propionic acid/water 
 (62 3/310/437). Following autoradiography 
 the compounds detected in the 3- and 9- 
 hour treatments were compared with the 
 compounds detected from a red kidney bean 
 plant treated with Cl 1 ^. Apart from the 
 presence of urea* in the ryegrass extract, 
 no marked qualitative or quantitative dif- 
 ference could be detected between the urea* 
 fixation pattern and a C1402 fixation 
 pattern. Apparently it is safe to assume 
 that the labeled products of urea* appli- 
 cation translocated from the treated leaf, 
 under photosynthetic conditions, are simi- 
 lar or identical to those from normal 
 photosynthesis . 
 
 CORN EXPERIMENTS 
 
 Young plants with about four mature 
 leaves were used and the solution of 
 labeled compound (including Tween 20) was 
 applied as a drop to the youngest mature 
 leaf, or to the next leaf below it. No 
 consistent difference was discernible in 
 the amount of translocation from either 
 leaf. The Cl^ derived from urea* moved 
 into the roots and into the meristematic 
 regions in 3 hours, though in fairly small 
 amounts. This pattern of phloem movement 
 to the active sinks was accentuated after 
 6 hours and even more so after 24 and 48 
 
 hours. Amitrole* showed a similar pattern 
 of movement, but moved more slowly than 
 the compounds derived from urea*. 
 
 The movement of MH* was somewhat ir- 
 regular, but appeared to be slower than 
 the sucrose* from urea*. There was also 
 some evidence of xylem movement, as shown 
 by the presence of the isotope in mature 
 leaves. 
 
 PERENNIAL RYEGRASS EXPERIMENTS 
 
 Plants with about seven mature leaves 
 on the main tiller were treated on the 
 designated leaf with either labeled urea, 
 amitrole, or MH for 3, 6, 12, 24 or 48 
 hours. The following leaves on different 
 plants were treated at each of the times 
 noted: the youngest mature leaf on the 
 main tiller, the leaf two below the young- 
 est mature leaf on the main tiller, the 
 youngest mature leaf on a large side tiller 
 and the youngest mature leaf on a small 
 side tiller. In addition, one tiller was 
 either shaded or defoliated 24 hours be- 
 fore an unshaded or undefoliated young 
 mature leaf was treated. Movement was 
 rapid, with the first two compounds being 
 largely restricted to the phloem and the 
 third compound moving in both the phloem 
 and the xylem. 
 
 BERMUDAGRASS EXPERIMENTS 
 
 Plants raised from cuttings and with 
 the main stolon about 2-1/2 feet long were 
 treated for 3, 6, 12, 24 and 48 hours on 
 one of a number of leaves. The plants 
 were growing vigorously and had many side 
 tillers. The following leaves were 
 treated: the third mature leaf counting 
 from the tip of the main stolon, the fifth 
 mature leaf behind the main stolon tip, 
 and a leaf inserted directly on the main 
 stolon somewhere between at least two 
 rooted nodes. Leaves were also treated on 
 a large side tiller, on a small side 
 tiller which was rooted below its point of 
 attachment to the main stolon, and on a 
 small side tiller not rooted below its 
 point of attachment to the main stolon. 
 Shading and defoliation experiments were 
 made with all three chemicals for 12 and 
 48 hours following the pretreatment for 
 24 hours - that is, plant parts were 
 shaded or defoliated 24 hours before 
 treatment. 
 
 The most striking effect was the 
 
 115 
 
strongly polarized movement of compounds 
 towards the growing point of the main 
 stolon. Except where a portion of the 
 stolon itself was meristematic proximal 
 to the treated leaf, there was virtually 
 no movement of C^ back towards the main 
 
 root system. This effect is illustrated 
 in figure XIII, 1. Where a small tiller 
 on the main stolon was treated, it trans- 
 located material strongly into the main 
 stolon and some activity passed into all 
 tillers progressing towards the stolon 
 
 «is ti« 
 
 Fig. XIII, 1. Autoradiography (above) and mounted plants (below) of bermudagrass; 
 treatment with urea*. Plant on left was treated for 12 hours on a mature leaf inserted 
 on the main stolon between two rooted nodes. Center plant had similar treatment on the 
 sixth mature leaf from the tip of a minor stolon. Plant on right was treated for kQ 
 hours on a mature leaf on the main stolon between two rooted nodes. 
 
 116 
 
tip. Shading or defoliation for 24 hours 
 before treatment did not appear to affect 
 this pattern but it was found possible to 
 reverse the pattern by shading or defolia- 
 ting for 4 days before treatment with the 
 isotope, with shading continued during 
 
 period of treatment. Defoliation consis- 
 ted of cutting off the laminae of all 
 mature leaves 4 days before treatment and 
 removing any regrowth 2 days before treat- 
 ment and on the day of treatment. Figure 
 XIII, 2, shows translocation of sucrose 
 
 Fig. XIII, 2. Autoradiographs and mounted plants of bermudagrass; treatment, urea" 
 for 48 hours. Plant on left had all but the treated stolon darkened for 4 days before 
 and during treatment. Plant on right had all but the treated stolon defoliated 4 and 2 
 days before treatment, and again at time of treatment. 
 
 117 
 
in a shaded and in a defoliated plant. 
 
 QUACKGRASS EXPERIMENT 
 
 Plants with well-developed rhizome 
 systems were treated for either 12 or 48 
 hours with one of the three compounds. 
 Leaves treated were as follows: the 
 youngest mature leaf on the main tiller, 
 the leaf two below the youngest mature 
 leaf on the main tiller, the youngest mat- 
 ure leaf on a medium-sized side shoot. 
 Shading and defoliation treatments were 
 also carried out. Translocation occurred 
 fairly freely within a treated tiller and 
 from a main tiller through a rhizome to a 
 small tiller. However, little or no move- 
 ment occurred in the reverse direction. 
 There was strong movement of the deriva- 
 tives of urea*, and of amitrole* into the 
 young leaves, the roots, and into the 
 actively growing rhizomes. In addition 
 to phloem movement, MH* shewed consider- 
 able movement in the xylem. Shading or 
 defoliation of a single shoot 24 hours 
 before treatment did not appear to greatly 
 alter the pattern of movement. Peterson 
 (1962) has studied the movement of ami- 
 trole* and dalapon* in quackgrass. 
 
 TRANSLOCATION STUDIES. ON THE 
 INTERCALARY MERISTEM 
 
 In earlier studies it had been found 
 that when Cl 1 ^ was applied to an immature 
 leaf of perennial ryegrass, the isotope 
 moved strongly to the region of the inter- 
 calary meristem but did not pass across 
 it to the remainder of the plant unless 
 the leaf was almost mature. In view of 
 statements in the literature of this sub- 
 ject about intercalary meristems being a 
 block to translocation, an attempt was 
 made to study movement of photosynthate 
 in this region. A blockage or constric- 
 tion might be caused by a partial or en- 
 tire discontinuity in the phloem, or by 
 metabolic activity in the area using photo- 
 synthate before it has time to pass across 
 the meristem, or by a combination of the 
 two. To investigate anatomical constric- 
 tion or blockage t serial transverse sec- 
 tions were made of paraffin-embedded rye- 
 grass material. Sections were made of the 
 youngest mature leaf, and of all younger 
 leaves, from their insertion into the main 
 stem to a point about 7 mm above the level 
 of insertion. The sections were stained 
 with Shaman's tannic acid/iron alum 
 stain which distinctly shows the phloem. 
 
 The numbers of active phloem elements in 
 the midrib and the lateral bundles on one 
 side of the leaf were counted; an active 
 phloem element was arbitrarily defined as 
 one in which the blue stain was visible 
 around the entire periphery of the cell, 
 and one in which the cell was not crushed. 
 The results indicate a considerable con- 
 striction of the phloem in the region of 
 the intercalary meristem, and marked de- 
 crease in the numDer of phloem elements 
 as compared with the more distal sections. 
 In one midrib the number of phloem ele- 
 ments decreased from about fourteen cells 
 to three. Some bundles are represented 
 in the meristem by procambium only, while 
 higher up they contain protophloem, and 
 yet smaller bundles are present higher up 
 and absent in the meristematic region. 
 
 These studies indicate that the re- 
 striction of the phloem conduits in the 
 region of the intercalary meristem may 
 constitute a physical block. However, the 
 dense labeling of this region with Cl4- 
 labeled sucrose (derived from urea*) - in 
 contrast to less intense labeling in more 
 mature regions (fig. VI, 3) - seems to 
 indicate an intense fixation of sugar by 
 the growth processes, and this would slow 
 down the linear rate of translocation. 
 It seems likely that restricted phloem and 
 high meristematic activity may both serve 
 to block translocation in this region. 
 
 TRANSLOCATION IN COFFEE PLANTS 
 
 Translocation of a number of tracers 
 in coffee plants has been shown to follow 
 certain specific patterns (Pereira, Crafts 
 and Yamaguchi, 1963). Amitrole* is high- 
 ly phloem-mobile, quite readily moved via 
 the xylem, subject to redistribution in 
 the phloem, and absorbed slowly by roots. 
 Dalapon* is readily phloem mobile, is 
 slowly absorbed by roots, is retained by 
 root tips but not by mature roots, and 
 shows only restricted apoplastic movement 
 in leaves. 
 
 Maleic hydrazide* is readily trans- 
 located with foods in the phloem, is 
 somewhat restricted in the xylem, and is 
 absorbed and transported to the xylem 
 slowly by roots. MH* tends to leak from 
 phloem to xylem and is readily redistri- 
 buted from old to young leaves via phloem. 
 2,4-D* is avidly accumulated and retained 
 by living cells, hence its distribution 
 
 118 
 
in normal plants may be limited; only un- 
 der optimum conditions of leaf penetration 
 and food movement may it be moved from 
 foliage to roots. 2,4-D* is not readily 
 moved from roots to foliage via the xylem. 
 Monuron* is non-phloem-mobile, but readily 
 xylem mobile. It displays^ strong apop las- 
 tic movement in leaves and moves readily 
 through roots and into tops when applied 
 to roots in the culture medium. 
 
 The Cl 4 label of urea was readily 
 moved via the phloem in coffee; it was 
 transported from illuminated leaves, pro- 
 bably in the form of sucrose. In shaded 
 leaves, labeled urea moved mainly in the 
 apop last. Calcium 1 ^ was not translocated 
 via the phloem; it was readily absorbed 
 by roots and moved via the xylem. It is 
 accumulated in leaves and immobilized, 
 showing no redistribution. Phosphorus 32 
 did not penetrate coffee leaves readily, 
 hence its distribution via phloem could 
 not be determined with certainty. Phos- 
 phorus 32 was accumulated by roots and 
 moved only slowly to tops. Zinc^S was 
 less mobile in coffee than in barley and 
 bean; it was less actively absorbed and 
 moved by roots than was monuron. 
 
 The translocation of pentavalent ar- 
 senic as arsenic acid-As ' was studied 
 in coffee. Leaves at various positions 
 were given 4-day treatments by droplet 
 application. Autographs showed the move- 
 ment of small quantities both acropetally 
 and basipetally from mature leaves. Ac- 
 tive sinks in buds and small growing 
 
 leaves and in root tips accumulated the 
 tracer; all mature leaves were bypassed. 
 Treatment via the culture solution resul- 
 ted in movement to all mature leaves; most 
 of the As 77 applied to roots was retained 
 in these organs however. General distri- 
 bution of As 77 resembled that of P32 and 
 amitrole*. 
 
 These studies with coffee reveal in- 
 dividual idiosyncrasies of the different 
 tracers, but also show patterns remark- 
 ably similar to those established in bar- 
 ley, bean, grass species, and zebrina. 
 The repeated observation of the relatively 
 free phloem mobility of amitrole, the re- 
 stricted movement of 2,4-D as related to 
 metabolic accumulation, the free phloem 
 mobility and slow leakage to xylem of MH 
 and dalapon, the lack of symplastic move- 
 ment of monuron and calcium - these and 
 many other characteristic behavior pat- 
 terns have been found in coffee. Grad- 
 ually there is evolving a consistent pic- 
 ture of solute transport in plants indi- 
 cating the existence of a source-to-sink 
 pattern of distribution of foods and tra- 
 cers via phloem in plants, a variable pat- 
 tern of uptake rates by roots, a consis- 
 tent view of root- transport mechanism re- 
 lated to solute species, and a redistri- 
 bution system keyed to the supply of 
 needed nutrients to all living cells. 
 Pesticide molecules foreign to plants fall 
 into the various patterns, and their dis- 
 tributions often provide keys to their 
 local or systemic action. 
 
 119 
 
Chapter XIV. Translocation of Some 
 Amino Acids in Two Barley Varieties 
 
 I, 
 
 .n studies on the connion barley var- 
 iety Atlas and a mutant Atsel selected 
 from a dalapon- treated population of Atlas, 
 Wijewantha and Stebbins (1964) found that 
 adding arginine to the culture of the mu- 
 tant barley in mineral solution culture 
 shifted the expression of the phenotype of 
 the mutant to almost normal. Since this 
 implies uptake and distribution of the 
 amino acid from the culture medium into 
 the growing organs, it seemed useful to 
 initiate a study using Cl 4 - labeled amino 
 acids by the technique of autoradiography. 
 
 Studies have been made using trypto- 
 phan in a dosage series involving appli- 
 cations of 0.05 umole, 0.2 umole and 0.8 
 umole of tryptophan-Cl 4 having a specific 
 activity of 6.64 mc per /umole to both 
 Atlas and Atsel barleys. One set of 
 plants in this 1-day series indicated in- 
 sufficient uptake for satisfactory com- 
 parison; a second set which ran for 4- 
 days proved satisfactory (fig. XIV, la,b) . 
 This preliminary trial showed that trypto- 
 phan was absorbed and translocated via 
 both leaves and roots with about equal 
 
 \H\ 
 
 
 la 
 
 
 Fig. XIV, la, lb. Autographs and mounted plants showing distribution of trypto- 
 phan''- in Atlas (left) and Atsel barley (right) treated via the leaves (la) and via 
 the roots (lb). Dosages in each treatment were, left to right, 0.05 pmole, 0.2 
 ^imole and 0.8 pmole per plant; treatment time was h days. 
 
 120 
 
TABLE XIV, 1 
 Label Locations, Specific Activities and Exposure Times 
 Of Amino Acids Used in Translocation Studies 
 On Atlas and AtseT Barley 
 
 Acid 
 
 Label location^ 
 
 Specific activity 
 mc/mmole 
 
 Exposure , 
 days 
 
 DL-arginine 
 
 quanido 
 
 2.2 
 
 6.4 
 
 Glycine 
 
 C-2 
 
 3.0 
 
 4.7 
 
 L-histidine 
 
 uniform 
 
 9.3 
 
 1.5 
 
 DL-lysine 
 
 C-l 
 
 8.95 
 
 1.5 
 
 DL-phenylalanine 
 
 C-3 
 
 1.3 
 
 11.0 
 
 DL- tryptophan 
 
 C-3 
 
 6.64 
 
 2.1 
 
 L-valine 
 
 C-l 
 
 5.73 
 
 2.5 
 
 DL-valine 
 
 C-l 
 
 6.05 
 
 2.3 
 
 ' Experimental time periods were 1, 4, and 14 days. 
 Applications were made to leaves and roots. (Cf. chapter 8.) 
 
 facility; of the various chemicals tested 
 so far, tryptophan most resembles amitrole 
 and is quite different from the phenoxy- 
 acetic acids and the substituted ureas 
 and symmetrical triazines. 
 
 In application by both methods the 
 resulting images show a good correlation 
 between dosage and amount of tracer absor- 
 bed and translocated. Comparing the 
 images produced by the three dosages it 
 seems that any dosage from 0.05 to 0.2 
 jumole would prove satisfactory. For the 
 following comparative study with eight 
 amino acids, a dosage of 0.1 umole was 
 chosen. 
 
 Table XIV, 1, lists the amino acids 
 used and specifies the location of the 
 labels and the specific activities. 
 Treatments involved 0.1 jumole per plant, 
 with exposure times on the films adjusted 
 to give standard images. 
 
 COMPARATIVE MOVEMENT OF EIGHT AMINO ACIDS 
 IN TWO BARLEY VARIETIES 
 
 ARGININE Uptake of this amino acid by 
 barley leaves was strong, with rapid 
 phloem movement and light apoplastic 
 
 movement. 
 
 GLYCINE Uptake of glycine by barley 
 leaves was strong, with rapid and continued 
 phloem movement into growing leaves and 
 roots. Evidence for continuing distribu- 
 tion in the 4-day plants was positive; in 
 the 14-day plants labeling weakened in the 
 young leaves, probably because of ex- 
 haustion of supply of mobile tracer. In 
 the 14-day plant, concentration was medium 
 in mature roots and high in young roots. 
 Apoplastic movement in the treated leaf 
 was medium in intensity. 
 
 Root application of glycine resulted 
 in medium movement to tops in Atlas bar- 
 ley, and light movement in Atsel. There 
 was evidence for thorough distribution in 
 the 1- and 4-day treatments; apparently 
 the supply to the roots became exhausted 
 in 14 days. 
 
 L-HISTIDINE If adjustment of exposure 
 time of the film to specific activity to 
 give a constant image density (see table 
 XIV, 1) is taken as a basis for compari- 
 son, histidine was the amino acid most 
 strongly absorbed by leaves. Movement 
 via phloem to developing leaves and roots 
 was very strong; there were indications 
 
 121 
 
of continued distribution in the 1-day and 
 4-day treatments and of lowering availa- 
 bility in the 14-day plants. The two bar- 
 ley varieties had similar responses. 
 
 Movement of histidine from roots to 
 tops was of medium intensity, with evi- 
 dence for complete distribution in the 
 1- and 4-day treatments. Evidently, roots 
 of barley accumulate and hold a large part 
 of the applied dosage within the first 4 
 days ; there seemed to be little more tra- 
 cer in the tops after 14 days. 
 
 DL-LYSINE Absorption and movement of ly- 
 sine was second in intensity to histidine, 
 with strong images of all leaves of both 
 barley varieties. Continuous movement is 
 evident in the 1- and 4-day trials; the 
 tracer supply was evidently exhausted by 
 the 14-day time period. Accumulation of 
 translocated lysine in roots was high in 
 the growing regions of the 1- and 4-day 
 plants, and only medium in roots of the 
 14-day plants. 
 
 Movement of lysine from roots to tops 
 gave only light labeling; accumulation in 
 roots was high - evidently release from 
 symplast to apoplast was not free in the 
 
 case of this amino acid. There is evi- 
 dence for secondary movement via phloem 
 in the 1- and 4-day plants. 
 
 DL-PI-ENYLALANINE This amino acid resem- 
 bles glycine in its distribution in bar- 
 ley; absorption and movement are strong; 
 prolonged movement is evident but not pro- 
 minent; apoplastic movement in the treated 
 leaves varied from medium to strong; 
 availability as shown by the young leaves 
 of the 14-day plants was limited by this 
 time. 
 
 Accumulation of phenylalanine by bar- 
 ley roots was strong throughout the 14- 
 day period; transport to the tops was 
 medium to light; redistribution was not 
 prominent in the short treatment periods 
 and was lacking in the 14-day plants. 
 The veins of the leaves of the plants 
 treated via roots were prominently la- 
 beled, indicating strong accumulation by 
 phloem and border parenchyma cells. 
 
 DL-TRYPTOPHAN Phloem movement of trypto- 
 phan from treated leaves to tops and 
 roots of barley was in the medium range; 
 mature roots were lightly labeled; con- 
 
 1/ 
 
 2° 
 
 {) <iP \f 
 
 2b 
 
 W 
 
 Fig. XIV, 2a, 2b. Distribution of tryptophan it Atlas (left) and Atsel barley 
 (right) treated via the leaves (2a) and via the roots(2b). Dosage was 0.1 yumole 
 per plant; treatment times were (left to right) 1 day, 4" days, and 14 days. 
 
 122 
 
tinued movement occurred but was not pro- 
 minent; and availability was low after 
 the 4-day period. Apoplastic movement in 
 treated leaves was medium to low (fig. XIV, 
 2a). 
 
 Movement from roots to tops from root 
 application was lowest for tryptophan 
 among the eight compounds tested; images 
 were light in all cases; evidence for 
 phloem movement was present only in the 
 1-day plant of Atlas barley. Accumula- 
 tion in the roots themselves was high for 
 all treatment times (fig. XIV, 2b) ; these 
 plants resembled the 2,4,5-T treated plants 
 described in chapter 8. 
 
 L- VALINE This amino acid shewed the most 
 similarity between leaf and root uptake 
 (fig. XIV, 3a, b) . Absorption and phloem 
 transport via the leaves were medium; 
 movement continued throughout the 1- and 
 4-day treatments; apoplastic movement in 
 treated leaves was medium to heavy. 
 
 Movement of L-valine to the tops from 
 root treatment was greatest of all the 
 amino acids tested, and equal to or greater 
 than was distribution from leaf treatment. 
 Accumulation remained high in all roots 
 throughout the 14-day period; redistribu- 
 tion from old to young leaves was promin- 
 
 ent in the 1- and 4-day treatment periods; 
 the tracer supply became exhausted toward 
 the end of the 14-day treatment, but pri- 
 mary accumulation in the tips of the num- 
 ber two leaves was prominent in plants of 
 both varieties. 
 
 PL- VALINE Phloem transport from leaf ap- 
 plication was weakest in DL-valine-treated 
 plants. Concentrations of tracer in 
 untreated leaves was medium to light, and 
 medium in roots. Apoplastic movement in 
 treated leaves averaged medium; evidence 
 for redistribution via phloem was lacking. 
 
 Evidence for xylem movement of DL- 
 valine from roots to tops indicated medium 
 transport in Atlas barley and low trans- 
 port in Atsel. Accumulations in roots 
 remained high, particularly in young roots 
 and in growing root tips. Evidence for 
 redistribution from old to young leaves 
 was lacking. 
 
 Table XIV, 2, gives a summary of ob- 
 servations on the 4-day treatments of the 
 Atlas and Atsel varieties. It is obvious 
 that there are distinct differences in the 
 amounts of amino acids moved, even though 
 amino acids generally translocate from 
 leaves via the phloem by symplastic move- 
 
 i 3b V U 
 
 
 \ 
 
 Fig. XIV, 3a, 3b. Distribution of L-valine- in Atlas and Atsel barleys treated 
 via leaves (3a) and via roots (3b). Dosage was 0.1 /umole per plant; treatment 
 times were (left to right) 1 day, 4 days, 14 days. 
 
 123 
 
< 
 
 o 
 
 c 
 
 E 
 < 
 
 0) 
 
 — l/> 
 
 1> 1) 
 
 jD — 
 
 TJ *-> 
 
 -I V 
 
 CM w- 
 
 *- L. 
 
 - -C (0 
 > Cn=> 
 •— l •»- 
 
 XU X 
 
 01 
 
 V <*- — 
 
 — O L. 
 
 m i/i si 
 
 I- c 
 
 L 2 
 
 
 ■P 
 
 C 
 
 t 
 
 a 
 
 & 
 
 xi 
 
 S 
 
 •5 
 6 
 
 •H 
 
 2 
 
 C 
 
 8 
 
 ■a 
 
 w 
 
 CM O 0> ID J" 
 
 rH CM rH rH rH 
 
 + + + + + 
 
 +■ + + + + 
 
 + + + + + 
 
 8 
 
 0) 
 
 rH 
 
 -q 
 
 rH 
 
 O 
 
 
 0) 
 
 0) .Q 
 ITJ 4-1 
 
 £3 
 
 S'S 
 
 I 
 
 U 
 
 •I 
 
 •H 
 (0 
 
 i 
 
 0) 
 
 .5 
 
 W 
 
 J. 
 
 Q 
 
 rH 
 
 J. 
 
 Q 
 
 
 >) 
 
 > 
 
 > 
 
 Q 
 
 
 CM 
 
 o> 
 
 CO 
 
 o 
 
 l£> 
 
 .3- 
 
 d- 
 
 CO 
 
 
 rH 
 
 rH 
 
 rH 
 
 CM 
 
 r-t 
 
 rH 
 
 rH 
 
 rH 
 
 
 
 ♦ 
 
 
 4- 
 
 
 + 
 
 
 
 
 
 + 
 
 ♦ 
 
 H- 
 
 4- 
 
 + 
 
 + 
 
 ■r 
 
 
 + 
 
 H- 
 
 + 
 
 H- 
 
 4- 
 
 H- 
 
 + 
 
 + 
 
 
 + 
 
 4- 
 
 H- 
 
 + 
 
 4- 
 
 ♦ 
 
 + 
 
 + 
 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 
 
 h 
 
 
 
 
 + 
 
 + 
 
 ■► 
 
 + 
 
 + 
 
 +J 
 
 + 
 
 + 
 
 ■M 
 
 
 
 
 
 
 
 
 
 C 
 
 8 
 
 
 
 
 
 
 
 
 
 + 
 
 ■4- 
 
 + 
 
 4- 
 
 
 
 + 
 
 
 T) 
 
 + 
 
 + 
 
 H- 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 e 
 
 
 
 
 
 
 
 
 
 ■M 
 
 
 
 
 
 
 
 
 
 I 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 J 
 
 
 H- 
 
 + 
 
 + 
 
 
 
 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 H- 
 
 + 
 
 *» 
 
 
 
 
 
 
 
 
 
 >> 
 
 
 
 
 
 
 
 
 
 ii 
 
 
 
 
 
 
 
 
 
 i— 1 
 
 1 
 
 
 
 
 
 
 
 
 
 -H 
 
 
 
 
 
 
 
 
 
 0) 
 
 
 
 
 
 
 
 
 
 W 
 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 +-> 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 + 
 
 <. 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 
 + 
 
 + 
 
 4- 
 
 4- 
 
 
 
 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 4- 
 
 4- 
 
 
 
 
 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 
 
 
 + 
 
 + 
 
 + 
 
 + 
 
 4- 
 
 + 
 
 + 
 
 + 
 
 
 
 
 
 
 <U 
 
 
 
 
 
 
 
 
 
 .5 
 
 
 
 
 
 
 
 
 
 i 
 
 § 
 
 
 
 
 0) 
 
 
 1 
 
 0) 
 
 rH 
 
 x: 
 
 
 0) 
 
 
 1 
 
 l 
 
 ■H 
 
 4-> 
 W 
 
 a 
 
 3 
 5 
 
 >> 
 
 i 
 
 4-> 
 
 0) 
 
 I 
 
 ■3 
 
 Ifl 
 
 > 
 
 
 J. 
 
 I 1 
 
 j. 
 
 s 
 
 J. 
 
 > 
 
 J, 
 
 
 a 
 
 o 
 
 Q 
 
 a 
 
 Q 
 
 Q 
 
 u 
 
 It) 
 T3 
 
 10 
 
 lb 
 
 b0 
 •H 
 
 O C 
 
 a) 
 
 M 
 
 t, 
 
 •H 
 
 <-• 
 
 w 
 
 n 
 
 10 
 
 
 o 
 
 ^ 
 
 ^H 
 
 ■M 
 
 a- 
 
 
 i^. 
 
 • • 
 
 o 
 
 (0 
 
 
 -H 
 
 c 
 
 o 
 
 o 
 
 1 
 
 •H 
 ■M 
 
 >. 
 
 10 
 
 10 
 
 H 
 
 
 e 
 
 o 
 
 3 
 
 *-> 
 
 CO 
 
 >, 
 
 ^_ 
 
 <u 
 
 
 ^ 
 
 
 124 
 
ment and from roots via xylem by apoplas- 
 tic movement. 
 
 If the numbers of plus signs for 
 each amino acid in table XIV, 2, are 
 totaled as a rough measure of uptake and 
 distribution for these 4-day treatments, 
 the amino acids assume the following ser- 
 ies in the two barley varieties: 
 
 Atlas 
 
 Glycine 
 
 L-histidine 
 
 Lr-valine 
 
 DL- lysine 
 
 DL-tryotophan 
 
 DL-pheny lalanine 
 
 DL-arginine 
 DL- valine 
 
 Atsel 
 
 DL- lysine 
 Glycine 
 L-histidine 
 DL-pheny lalanine 
 DL- tryptophan 
 L-valine 
 DL-valine 
 DL-arginine 
 
 Some amino acids are apparently more 
 mobile than others in the phloem, some 
 excel in xylem mobility, and there are 
 over-all differences in mobility between 
 the two varieties of barley. In general, 
 greater quantities of these acids move 
 about the plant following leaf application 
 than they do following root application. 
 Some amino acids act like 2,4-D inasmuch 
 as they accumulate in roots and fail to 
 move to tops in appreciable quantity; 
 DL-valine and tryptophan are two such 
 acids (fig. XIV, 2b), Others, such as 
 L-valine, apparently move about as freely 
 in the transpiration stream as they do via 
 the phloem (fig. XJV, 3a, b) , 
 
 Amino acids are normal products of 
 plant metabolism and may be present in 
 
 the assimilate stream in appreciable quan- 
 tities (Mittler, 19572?). Probably a small 
 fraction of the mobile assimilates at all 
 times, they are known to move out of se- 
 nile leaves in autumn in relatively large 
 quantities, and thus it is not unexpected 
 to find that they are quite mobile in the 
 phloem. In xylem, however, simple nitro- 
 gen compounds are more often found in the 
 amine and amide forms, and the rather 
 free movement of L-valine from roots to 
 tops of barley comes as a surprise. Be- 
 cause of the rather wide variation in 
 xylem mobility between L-valine and tryp- 
 tophan, these molecules should be examined 
 carefully in hopes of finding some clue to 
 the molecular configuration responsible 
 for free xylem mobility in plants. 
 
 In regard to the xylem mobility shown 
 in the dosage series of root treatments 
 with tryptophan, it is interesting to note 
 that the 0.05 /umole dosage in Atlas barley 
 failed to enter the xylem and move up to 
 the leaves , and that root accumulation was 
 high. Since the movement at the higher 
 dosages was normal it seems that there may 
 be a threshold effect in this situation. 
 This type of response is found with 2,4-D 
 at injurious levels. However, there were 
 no symptoms of injury in the case of the 
 0.1 Aimole dosage shown in figure XIV, 2£>, 
 nor in the 0.2 /umole dosage shown in fig- 
 ure XIV, 12?. The avidity with which 
 plant roots absorb, accumulate, and retain 
 various compounds is an important pheno- 
 menon and one of great interest to people 
 engaged in application of pesticides 
 through the soil. 
 
 125 
 
Chapter XV. Role of Formulation 
 Additives in Absorption and 
 Translocation of Herbicides 
 
 L 
 
 .n the formulation of pesticides, 
 carriers - such as water and oils - may 
 largely determine the type of herbicidal 
 action of a chemical. For example, if 
 dinitro-ortho-cresol is dissolved in oil 
 and applied to vegatation it has a general 
 contact action, but when dissolved in 
 water its sodium salt is highly selective. 
 Surfactants also have important effects on 
 spray action: sulfuric acid, a highly 
 selective spray, may be made more general 
 in its action by addition of Vatsol OT. 
 
 Since the introduction of 2,4-D and 
 other systemic herbicides it has been ob- 
 served that inclusion of surfactants ap- 
 preciably increases the effectiveness of 
 these materials. Figure XV, 1, shows the 
 penetration of a fluorescent dye into the 
 upper halves of two pear leaves having 
 open stomata when Vatsol OT was included 
 in the solution (upper leaves); where 
 stomata were closed, no penetration took 
 place (lower leaves). Recently, addition 
 of solublizing compounds of a surfactant 
 nature has been found to greatly increase 
 the herbicidal activity of the substituted 
 ureas. Because of these effects the 
 authors made a study of surfactants in an 
 effort to throw some light on the ways in 
 which these compounds may alter the uptake, 
 distribution, and toxic action of herbi- 
 cides. 
 
 ROLE OF SURFACTANTS IN TRANSLOCATION 
 
 In many of the early studies on the 
 absorption and translocation of tracer 
 molecules it was observed that inclusion 
 of a surfactant in the treatment solution 
 enhanced the uptake of the tracer (Mitch- 
 ell and Linder, 1950a, b) . Although 
 early thinking about this effect was al- 
 most entirely in terms of increased wet- 
 ting, the concept gradually evolved that 
 
 in some way penetration of the cuticle was 
 being increased. Studies on the lowering 
 of surface tension of water by surfactants 
 proved that a limiting value was reached 
 around 30 dynes per square centimeter, 
 and that increasing surfactant concentra- 
 tion more than about 0.1 per cent by 
 weight had little further effect on low- 
 ering surface tension. Results of field 
 trials with 2,4-D, dalapon, and other 
 herbicides indicated that concentration 
 
 Fig. XV, 1. Penetration of fluores- 
 cent dye through the stomata of pear 
 leaves. Dye solution included the surfac- 
 tant Vatsol OT at 0.5 per cent. Upper 
 pair of leaves had open stomata; the low- 
 er pair had closed stomata. Treatment 
 time, 2 minutes; apical halves of leaves 
 immersed . 
 
 126 
 
increases to approximately 1.0 per cent 
 gave definite increases in herbicidal ac- 
 tivity, particularly on perennial plants 
 where both penetration and translocation 
 were involved. 
 
 TWEEN 20 Jansen, Gentner and Shaw (1961) 
 have made a comprehensive study of the 
 effects of surfactants on herbicidal ac- 
 tivity. Working with 63 surfactants, in- 
 cluding cationic, anionic, nonionic, am- 
 pholytic and blended materials, and using 
 these in conjunction with 2,4-0, dalapon, 
 DNBP and amitrole on corn and soybeans , 
 they found three characteristic types of 
 action. Many surfactants caused progres- 
 sive increase of herbicidal activity with 
 logarithmic increases in concentration. 
 Some surfactants caused a progressive su- 
 pression of herbicidal activity with in- 
 crease in concentration; others had no 
 effect. Many surfactants showed marked 
 phytotoxicity at high concentrations, and 
 some were stimulating in the absence of 
 herbicide at low concentration. Surfac- 
 tant effects varied between the two spe- 
 cies of test plants used and there were no 
 obvious correlations between surfactant 
 effects and surfactant structure. Jansen 
 and his colleagues (1961) used Tween 20 
 (S-145), an anionic surfactant of very 
 low phytotoxicity, as a standard. 
 
 Webster (1962) has studied the effect 
 of Tween 20 on the entry of 2,4-D into 
 leaves of Kalmia augusti folia. He found 
 that entry rate of 2,4-D into young leaves 
 decreased with an increase in Tween 20 of 
 two- fold to eleven- fold for a given 2,4-D 
 dosage; with a two- fold increase entry of 
 2,4-D into old leaves was increased. 
 
 In our early studies with labeled 
 tracers we observed the increased wetting 
 and penetration when 0.1 per cent Tween 
 20 was incorporated into the formulation 
 of the treatment solution. In order to 
 rule out wetting as a factor in our trans- 
 location studies, we used 1/10 per cent 
 Tween 20 in all of our aqueous and 50 per 
 cent alcohol treatment solutions (Wein- 
 traub et al. t 1950; Brown and Weintraub, 
 1950; Weintraub et al. t 1952a, b; Wein- 
 traub et al. t 1954). 
 
 Since we had used Tween 20 in most 
 of our treatment solutions, it seemed ad- 
 visable to study the effects of varying 
 the surfactant concentration on the up- 
 
 take of certain herbicides. Our first 
 series of experiments used 2,4-D*; the 
 second involved 2,4-D* and amitrole*. 
 
 The first experiment used 2,4-D* at 
 dosages of 1/10 /jmole, 4/10 jumole, and 
 8/10 ;umole each, in 20 ul of solution. 
 Tween 20 concentrations were 1/10 per 
 cent and 4.0 per cent. Treatment times 
 were 1 day and 4 days; plants were beans 
 and barley. The only factors resulting 
 in differences were concentration of 
 2,4-D, and time; intensity of labeling 
 with 2,4-D* increased regularly through 
 the concentration series and formative 
 effects increased with both concentration 
 and time. These responses were more ap- 
 parent on bean than on barley, but the 
 difference in Tween 20 concentration pro- 
 duced no effect visible in the autoradio- 
 graphs of either plant. 
 
 A second experiment compared 2,4-D* 
 and amitrole* at the same micromolar dos- 
 ages as in the preceding experiment. 
 Treatment time was 4 days; the plant was 
 nasturtium. Again, there were no differ- 
 ences between the Tween 20 concentrations; 
 the usual differences between 2,4-D* and 
 amitrole* were expressed (fig. VIII, 2). 
 Amitrole* was transported both symplas- 
 tically and apoplastically ; 2,4-D* dis- 
 tribution was entirely symplastic. Treat- 
 ment in this experiment was to under- 
 surfaces of leaves where there are numer- 
 ous stomates. 2,4-D* caused the treated 
 nasturtium leaf to turn yellow; amitrole* 
 had no visible effect on the treated leaf 
 within the 4-day treatment period. 
 
 A third experiment examined the role 
 of Tween 20 on uptake of 2,4-D* and ami- 
 trole* by coleus plants (fig. XI, 3). 
 Forty /ul volumes of 2,4-D* and amitrole* 
 treatment solutions of 0.005M concentra- 
 tion were applied to opposite leaves in 
 the form of about twenty 2-/ul droplets; 
 the whole group of plants was covered by 
 a polyethylene bag to maintain high humi- 
 dity and was left for 4 days. Tween 20 
 concentrations were 1/10, 1.0 and 4.0 per 
 cent. One leaf in each pair was treated 
 on the upper surface, which was free of 
 stomates, the other was treated on the 
 lower surface where stomates were numer- 
 ous. 
 
 Again there were no apparent differ- 
 ences between responses to different Tween 
 
 127 
 
20 concentrations; with 2,4-D* there were 
 narked differences between applications 
 to upper and lower surfaces, but such dif- 
 ferences were not apparent in the case of 
 amitrole*. It is apparent that with 2,4-D* 
 much more tracer entered through the lower 
 leaf surface, as is shown by the strong 
 labeling of a side shoot above the treated 
 leaf and by similar labeling of the stem 
 on the side of lower surface treatment. 
 Translocation was strictly symplastic when 
 2,4-D* was used. 
 
 Amitrole* apparently moved through 
 upper and lower leaf surfaces in about 
 equal quantities; after entry there was 
 considerable apoplastic movement, as in- 
 dicated by the labeling of the untreated 
 portions of the treated leaves and the 
 intermediate-sized untreated leaves above 
 the treated ones. But symplastic movement 
 was also strong, as was accumulation in 
 active sinks; this is apparent in the 
 intense labeling of the young expanding 
 leaves and of the buds at the upper end 
 of and along the stem, and by the labeling 
 of young growing roots. Old roots and 
 stem tissue held an intermediate concen- 
 tration of amitrole* - an indication of a 
 lower accumulative avidity for this com- 
 pound. 
 
 A test on fair-sized cotton plants, 
 in which Tween 20 was incorporated at con- 
 centrations of 1/10 per cent and 4 per 
 cent into 2,4-D* solutions of 2/10, 4/10, 
 and 8/10 jumole dosages, likewise snowed 
 no observable differences in uptake or 
 movement due to the surfactant. 
 
 A final test on the possible effect 
 of Tween 20 on 2,4-D* absorption by soy- 
 bean roots from the culture medium showed, 
 if anything, a reduction in absorption be- 
 tween 1/10 per cent and 4 per cent Tween 
 20 concentration. The 2,4-D* was present 
 at 1.10 jumole in 100 ml of Hoagland's sol- 
 ution. 
 
 These tests with unlabeled Tween 20 
 and labeled 2,4-D and amitrole apparently 
 indicate that this surfactant has no role 
 in the uptake and distribution of systemic 
 herbicides. However, a large amount of 
 observational data from the field indi- 
 cates an enhancement of uptake by surfac- 
 tants. Possibly, the growing of the 
 plants in the greenhouse, the single- 
 droplet application within a lanolin ring, 
 or some other part of our experimental 
 
 procedure was responsible for our nega- 
 tive results. 
 
 SODIUM LAURYL SULFATE One of the common- 
 est of surfactants is sodium lauryl sul- 
 fate, an anionic surfactant which has long 
 been readily available. When it was fi- 
 nally produced in the S35_iabeled form, 
 we used some in our standard test. Ap- 
 plied to bean leaves at dosages of 1/10 
 umole, 4/10 pmole and 8/10 jumole of treat- 
 ment solution of 0.01 M concentration, 
 with and without Tween 20 in the formula- 
 tion, it showed no tendency to penetrate 
 (fig. XV, 2, left). In bean, cucumber, 
 and cotton it showed no apoplastic move- 
 ment in the treated leaf; in soybean and 
 barley there was slight apoplastic move- 
 ment in an acropetal direction from the 
 treated spot. Applied to roots via the 
 culture medium at 1/10 jumole, 4/10 wmole 
 and 8/10 jumole per 100 ml of solution, 
 it moved slightly into bean in one day; 
 labeling increased through the above dos- 
 age series. In cucumber and barley it 
 was also moved in 1 day; in cotton and 
 soybean it entered the roots but did not 
 translocate to tops. In 4 days all the 
 above species had faint to heavy labeling 
 of the tops from root treatment (fig. XV, 
 2, 3, 4). In bean, where dosages were 
 1/10, 4/10, and 8/10 jumole per 100 ml, 
 labeling increased in proportion to dosage 
 (fig. XV, 2, right). 
 
 In leaf treatments, incorporation of 
 Tween 20 at 1/10 per cent concentrations 
 had no effect on penetration of sodium 
 lauryl sulfate , nor did incorporation- of 
 unlabeled 2,4-D, When sodium lauryl sul- 
 fate-S 35- treated plants were extracted 
 and the extracts chromatographed there 
 was no evidence for movement of the S35 
 from the position of treatment on the 
 leaf; extracts from roots and stems of 
 root-treated plants produced spots indi- 
 cating that the S^ remained near the ori- 
 gin. These spots correspond in Rf value 
 to SOi^ion. S35 in sodium lauryl sulfate 
 moved freely to the solvent front. 
 
 C. L. Foy, using S35-iabeled sodium 
 lauryl sulfate alone and in conjunction 
 with dalapon on leaves of cotton plants, 
 found that this surfactant penetrated the 
 cuticle and moved toward the leaf tip 
 (apoplastic movement) but not out of the 
 leaf via the phloem. After 7 days the 
 S35 label was found throughout the plant; 
 Dr. Foy considered it probable that this 
 
 128 
 
Fig. XV, 2. Leaf treatment (left) and root treatment (right) of bean plants with 
 s35_i aDe i ec | sodium lauryl sulfate at 0.4jumo1e dosage for k days. The formulation 
 was made up with 0.1 per cent Tween 20 in 50 per cent ethyl alcohol. 
 
 129 
 
V 
 
 
 
 
 Fig. XV, 3- Movement of s35_i a b e i ec | sodium lauryl sulfate in cotton (left) and 
 barley (right) from 4-day treatment via roots. Dosage was 0.1 /umole; the solution 
 contained 0.1 per cent Tween 20. 
 
 130 
 

 
 • -, A 
 
 -' 
 
 r 
 
 
 Fig. XV, 4. Movement of S35-1abe1ed sodium lauryl sulfate in soybean and cucumber 
 
 131 
 
was metabolized sodium lauryl sulfate, 
 possibly sulfate^ ion. 
 
 Dr. Foy also studied uptake of 
 T-1947-Cl 1 *, a surfactant of low phototox- 
 icity. This compound penetrated very 
 slowly and moved only acropetally to the 
 leaf tips; no Cl 1 * moved out of the leaf 
 even in 7 days. Sodium lauryl sulfate- 
 S 35 1 which was found to be intact in the 
 leaf after 24 hours, moved predominantly 
 in the veins; T-1947-C14 moved mostly in 
 the interveinal regions. Moreover, 
 T-1947-Cl 1 * accumulated in the lysigenous 
 glands of the cotton leaf, as do the 
 chloro- and methoxy-triazines ; sodium 
 lauryl sulfate-S35 did not do this. 
 
 These tests seem to indicate that 
 sodium lauryl sulfate penetrated the leaf 
 and was strongly retained in living cells 
 
 in an altered form not mobile on chroma- 
 tograph paper. To have enhancing action 
 on a pesticide, such a material could act 
 only in two ways - by increasing wetting, 
 and by increasing penetration. Since the 
 surfactant molecules did not move within 
 the plant, it is difficult to see how 
 they could affect the action of a chemi- 
 cal at a site removed from the point of 
 application. Thus the action of the two 
 surfactants so far tested must consist of 
 an enhancement of uptake through increased 
 penetration. The lack of effect noted in 
 these tests, using droplet application to 
 leaves of greenhouse-grown plants, must 
 result from the conditions of the experi- 
 ment. Droplet application in effect is 
 a very different treatment from spraying, 
 which may cover the total foliage surface 
 with a liquid film. 
 
 132 
 
Chapter XVI. Conclusions 
 
 a 
 
 "ver 10 years of work with labeled 
 tracers, using autoradiography as the 
 principal tool, has resulted in pertinent 
 contributions to man's understanding of 
 fundamental plant physiology and of herbi- 
 cidal action. It now seems certain that 
 systemic distribution of foliage-applied 
 compounds follows a source-to-sink pat- 
 tern indicative of movement en masse of 
 the assimilate stream via the phloem. 
 The consistent bypassing of mature leaves, 
 the high concentrations in young growing 
 shoot tips, root tips, and intercalary 
 meristems, and the reversibility of flow 
 brought about by proper manipulation, all 
 indicate a mass-flow type of mechanism. 
 To best utilize such a mechanism the fol- 
 lowing conditions should be met: 
 
 a) For critical situations where sys- 
 temic distribution of herbicides in per- 
 ennial plants is desired, formulation 
 of the phenoxy compounds in the emulsi- 
 fiable acid or alkoxy aliphatic ester 
 forms is essential. 
 
 b) Formulations and dosages should 
 be chosen to avoid rapid contact toxi- 
 city to foliage, and to facilitate slow, 
 ordered uptake. The dosage optimum for 
 2,4-D is lowest with light aliphatic 
 esters, intermediate with amine salts, 
 and higher with alkoxy esters and emul- 
 sifiable acid formulations. 
 
 c) Timing of application should be 
 done on a physiological basis - that 
 is, when leaf maturity, root growth and 
 photosynthetic activity are all favor- 
 able. 
 
 d) Application procedures should 
 meet the specific requirements of the 
 weed being controlled. If there is a 
 mixture of species, even the wisest com- 
 
 promise on procedures might not give 
 satisfactory results, and repeated 
 treatments may be necessary. 
 
 e) Activity of sink as well as source 
 is essential; insufficient soil moisture 
 for root growth may cause the most fa- 
 vorable formulation of phenoxy compounds 
 to fail. 
 
 f) The use of amitrole, which is more 
 mobile than 2,4-D and which does not re- 
 quire active root growth for response, 
 may be the answer to many of the prob- 
 lems encountered with the phenoxy com- 
 pounds. With amitrole, selectivity may 
 constitute a problem, however, as some 
 weed species are not susceptible to this 
 herbicide. 
 
 Autoradiographic studies have shown that 
 2,4-D brings about a disruption of the 
 vascular tissues of treated plants. 
 
 a) Where 2,4-D is being used, the 
 dosage should be optimum; too high a 
 dosage injures the source and too low a 
 dosage will not kill the plant; too low 
 a dosage may also block the phloem so 
 that a repeat treatment may fail. 
 
 b) Where two or more herbicides are 
 being used in combination, 2,H-D should 
 be applied at the same time or later 
 than the other material. If applied 
 before, it stops movement of all mater- 
 ials into roots. 
 
 Comparative mobility studies on many la- 
 beled herbicides indicate that they move 
 in the assimilate stream, and that entry 
 into and removal from this stream are the 
 principal factors affecting the amounts 
 moved. 
 
 133 
 
a) Requirements for translocation of 
 the phloem mobile compounds are very 
 much alike for all. Limitation of move- 
 ment of the phenoxy compounds by accumu- 
 lation or binding is substantial and can 
 best be met by proper timing of appli- 
 cation with respect to the physiologi- 
 cal status of the plants. Plants reach- 
 ing the optimum condition at different 
 times should not be expected to respond 
 favorably to a single common treatment. 
 
 b) Because of differing requirements 
 for different herbicides, application 
 times and methods should fit the chemi- 
 cal being used, the plants being spray- 
 ed, and the relevant physiological fac- 
 tors. 
 
 c) The need for understanding the 
 physical nature of the translocation 
 process, the chemical nature of the 
 compounds translocated, and the spe- 
 cial requirements for effective dis- 
 tribution, precludes the discovery by 
 crude testing methods of new and revo- 
 lutionary formulations or application 
 procedures for using the well-known 
 herbicides. 
 
 Different herbicide molecules move into 
 plant roots at different rates. Some, 
 such as the substituted ureas and sym- 
 metrical triazines, move through roots 
 and into the tops quite rapidly; others, 
 such as 2,4-D and maleic hydrazide, may 
 move slowly. 
 
 a) Where 2,4-D is used as a pre- 
 emergence treatment, it must be applied 
 so as to contact the roots of germina- 
 ting seedlings. Urea and triazine 
 herbicides may successfully be applied 
 to young seedling growth. 
 
 b) Formulation of 2,4-D for pre- 
 emergence application in soil should 
 allow for soil type and rainfall. The 
 acid, low in solubility, is most per*- 
 sistent; the aliphatic esters are inter- 
 mediate; the amine or sodium salts are 
 least persistent. 
 
 c) The herbicide should come in con- 
 tact with the emerging roots of the 
 seedling weeds. In the absence of rain- 
 fall, soil incorporation is necessary. 
 
 The great differences in species suscep- 
 
 tibility, observed in the use of herbi- 
 cides in the field and confirmed by auto- 
 radiography, result mainly from differen- 
 ces in the reaction of living cells to 
 herbicide toxicity at specific sites with- 
 in the plant, not to differences in 
 penetration or translocation. 
 
 a) Differences in formulation will 
 bring about differences mainly in tox- 
 icity but not in selectivity. Selecti- 
 vity of the newer foliar sprays, and of 
 all soil-applied herbicides, is little 
 affected by formulation. 
 
 b) If dosage is varied through a 
 sufficient range, most herbicides are 
 selective. Broad spectrum selectivity 
 is most desirable; compounds having 
 narrow selectivities are hazardous to 
 use. 
 
 Autoradiographic studies have been carried 
 out with labeled forms of the following 
 herbicides : 
 
 2,H-D Propazine 
 
 2,4,5-T Prometone 
 
 Amitrole Amiben 
 
 Maleic hydrazide 2,3,6-TBA 
 
 Dalapon EPTC 
 
 Monuron Barban 
 
 Simazine Dacthal 
 
 2,4-DB Alanap 
 
 NH4SCN Arsenate 
 
 Atrazine PCP 
 
 Trietazine Ammonium thiocyanate 
 
 2 ,4 ,5-T-butoxy-ethanol ester 
 
 Sodium trichloropropionate 
 
 Applied to a monocotyledonous test 
 plant (barley) and a dicotyledonous test 
 plant (kidney bean) through leaves and 
 roots and given 1-, 4-, and 16-day test 
 periods, these twenty- four compounds have 
 provided a broad picture of the uptake, 
 transport and redistribution of herbicides 
 by plants. These studies give invaluable 
 information for designing formulations 
 and application methods for herbicides 
 and coupled with studies on herbicidal 
 breakdown in plants and soils they pro- 
 vide guidelines for the intelligent inter- 
 pretation of residue data. 
 
 Autoradiography of whole treated 
 plants will undoubtedly become more wide- 
 ly used as its advantages are increasing- 
 ly recognized. The fact that treatment 
 
 134 
 
normally consists of the application of 
 microcurie amounts of a compound to an 
 intact leaf, and that no further manipu- 
 lation is needed during the normal treat- 
 ment period, indicates the value of the 
 method. Ready control of light, temper- 
 ature, and humidity enables the researcher 
 to study the effects of innumerable exper- 
 imental conditions on the processes of 
 absorption and translocation in plants. 
 From our results to date we can see no 
 effects of radiation per se on these pro- 
 cesses; we conclude therefore that the 
 labeled compounds used in our studies can 
 be considered as nontoxic tracers so far 
 as radiation effects are concerned. 
 
 With such a convenient method avail- 
 able there should be a bright future for 
 studies using these tracer techniques. 
 Additionally, a wide range of physiologi- 
 cal investigations are indicated. Further 
 studies on the division of labor between 
 various plant organs (Bernard Forde, 1963) 
 will undoubtedly challenge plant physio- 
 logists. More work on the basic aspects 
 
 of solute absorption by roots and foliage 
 should be carried on using autoradiogra- 
 phy. And translocation studies aimed at 
 a final resolution of the 100-year-old 
 controversy over mechanism should be pur- 
 sued. 
 
 When these studies have been made 
 and the true mechanisms of transport pro- 
 cesses have been elucidated, there remains 
 the task of studying distribution patterns 
 of inorganic nutrients, plant foods and 
 agricultural chemicals in all of the major 
 groups of plants. Only when knowledge 
 from such studies is at hand will the 
 plant physiologist and the agricultural 
 consultant be able to prescribe fertilizer 
 use, irrigation practices, hormone treat- 
 ments and pesticide application with a 
 satisfactory degree of certainty. Auto- 
 radiography will be one of the tools that 
 will eventually enable the agriculturalist 
 to bring on those increases in production 
 of food, feeds, and fiber needed to pro- 
 vide for the world's expanding population. 
 
 135 
 
Glossary of Terms 
 
 u = micro mm = millimeter 
 
 Aig = microgram cm = centimeter 
 
 ul = microliter oz = ounce 
 
 mc = mi lli curie MW = molecular weight 
 ml = milliliter 
 
 Rf = a term used in chromatography to designate the ratio of the distance 
 moved by a solute to that moved by the solvent. 
 
 As 77 , Cl 1 *, Ca 1 ^, C136, Fe59, p32, s35, Zn65 = radioactive isotopes of the designated 
 
 chemical elements. 
 
 * = radioactive. For example, 2,4-D* = radioactive 2, 4-dichlorophenoxy acetic acid. 
 
 -Cl 4 = compounds labeled with carbon-14. For example, Cl^C^ = carbon- 14- labeled carbon 
 dioxide; 2,4-D-Cl4 = carbon- 14- labeled 2,4-D. Carboxy-carbon labeling was used 
 in this work. 
 
 Cold 2,4-D = unlabeled 2,4-D 
 
 ADP = adenosine diphosphate 
 
 Alanap = N-1-naphylphthalamic acid 
 
 Amiben = 3-amino-2,5-dichlorobenzoic acid 
 
 Amitrole = 3-amino-l,2,4-triazole 
 
 ATP = adenosine triphosphate 
 
 Atrazine = 2-chlorK^4-ethylamincH-6-isoprcpylamino-s-triazine 
 
 Barban = 4-chloro-2-butynyl tf-(3-chlorophenyl) carbamate 
 
 Dalapon = 2,2-dichloropropionic acid 
 
 Dacthal = 2,3,5,6-tetrachloroterephthalic acid, dimethyl ester 
 
 DNP = dinitrophenol 
 
 Duraset = N-meta-tolyl phthalamic acid 
 
 136 
 
EPTC = ethyl N, N-di-n-propylthiol carbamate, Eptam 
 
 IAA = indole- 3-acetic acid 
 
 MH = maleic hydrazide 
 
 Monuron = 3-(p-chlorophenyl)-l,l-dimethyl urea 
 
 Simazine = 2-chloro-4,6-bis (diethylamino)-s-triazine 
 
 TBA = 2,3,6-trichlorobenzoic acid 
 
 2,4-D = 2, 4-dichlorophenoxy acetic acid 
 
 2,4-DB = 4-(2,4-dichlorophenoxy) butyric acid 
 
 2,4,5-T = 2,4,5-dichlorophenoxyacetic acid 
 
 2060 = ethyl, ethyl-n-butylthiol carbamate 
 1607 = n-propyl, di-n-propylthiol carbamate 
 
 2061 = propyl, ethyl-n-butylthiol carbamate 
 
 T-1947 = polyoxyethylene polyol, a nonionic surfactant 
 
 TWeen 20 = polyoxyethylene sorbitan monolaurate, a nonionic surfactant of 
 low phytotoxLcity. 
 
 137 
 
Literature Cited 
 
 ANDERSON, OTTO 
 
 1958. Studies on the absorption and translocation of Amitrol (3-amino-l,2,4-triazole) 
 by nutgrass (Cyperus rotundus L. ). Weeds 6:370-85. 
 
 ARNON, D. I., P. R. STOUT, and F. SIPOS 
 
 19 40. Radioactive phosphorus as an indicator of phosphorus absorption of tomato 
 fruits at various stages of development. Amer. Jour. Bot. 27:791-98. 
 
 ASHTON, F. M. , G. ZWEIG, and G. W. MASON 
 
 1960. The effect of certain triazines on 01^02 fixation in red kidney beans. 
 Weeds 8:448-51. 
 
 ASHTON, F. M. , E. G. URIBE, and G. ZWEIG 
 
 1961. Effect of monuron on Q\^02 fixation by red kidney bean leaves. 
 Weeds 9:575-79. 
 
 BIDDULPH, 0., S. BIDDULPH, R. CORY, and H. K00NTZ 
 
 1958. Circulation patterns for phosphorus, sulfur, and calcium in the bean plant. 
 Plant Physiol. 33:293-300. 
 
 BLACKMAN, G. E. 
 
 1955. Interrelationships between the uptake of 2,4-dichlorophenoxyacetic acid, 
 
 growth, and ion absorption. In: The Chemistry and Mode of Action of Plant 
 Growth Substances. Wye College Symposium. Edited by Wain and Wightman. 
 Published by Butterworths Scientific Publications, London. 
 
 1957. Selective toxicity in relation to specific differences in retention, penetration, 
 and uptake. Proc. Intern. Congr. of Crop Protection 4th Meeting (Hamburg, 
 Germany, Sept. 8-15, 1957). 
 
 BROWN, J. W. , and R. L. WEINTRAUB 
 
 1950. A leaf-repression method for evaluation of formative activity of plant growth- 
 regulating chemicals. Bot. Gaz. 111:448-56. 
 
 CLOR, M. A. 
 
 1959. Comparative studies on translocation of Cl^-labeled 2,4-D, urea, and amino 
 triazole in cotton and oaks. Doctoral dissertation. University of 
 California, Davis, Calif. 
 
 CLOR, M. A., A. S. CRAFTS, and S. YAMAGUCHI 
 
 1962. Effects of high humidity on translocation of foliar-applied labeled compounds 
 in plants. Part I. Plant Physiol. 37:609-17. 
 
 COLWELL, R. N. 
 
 1942. The use of radioactive phosphorus in translocation studies. 
 Amer. Jour. Bot. 29:798-807. 
 
 138 
 
COOKE, A. R. 
 
 1956. A possible mechanism of action of the urea type herbicides. Weeds 4:397-98. 
 
 CRAFTS, A. S. 
 
 1933. Sieve-tube structure and translocation in the potato. Plant Physiol. 8:81-104. 
 
 1948. A theory of herbicidal action. Sci. 108:85-86. 
 
 1956a. Translocation of herbicides. I. The mechanism of translocation. Methods 
 of study with Cl^-labeled 2,4-D. Hilgardia 26(6) :287-334. 
 
 19562?. Translocation of herbicides. II. Absorption and translocation of 2,4-D by wild 
 morning-glory. Hilgardia 26(6) : 335-65. 
 
 1959a. Evidence for hydrolysis of esters of 2,4-D during absorption by plants. 
 Weeds 8:19-25. 
 
 1959b. Further studies on comparative mobility of labeled herbicides. 
 Plant Physiol. 34:613-20. 
 
 1961a. The chemistry and mode of action of herbicides. Interscience Publishers. 
 New York, N. Y. 269 pp. 
 
 19612?. Translocation in plants. Holt, Rinehart and Winston. New York, N.Y. 182 pp. 
 
 1961a. Improvement of growth regulator formulation. In: Plant Growth Regulation. 
 Iowa State Univ. Press. Ames, Iowa. 850 pp. 
 
 1963. Herbicide behavior in the plant. In: The Physiology and Biochemistry of 
 Herbicides. Academic Press, London. 
 
 CRAFTS, A. S., and T. C. BROYER 
 1938. Migration of salts and water into xylem of the roots of higher plants. 
 Amer. Jour. Bot. 25:529-35. 
 
 CRAFTS, A. S., H. B. CURRIER, and H. R. DREVER 
 
 1958. Some studies on the herbicidal properties of maleic hydrazide. 
 Hilgardia 27(22) :723-57. 
 
 CRAFTS, A. S., and C. L. FOY 
 
 1959. Autoradiography of labeled dalapon. Down-to-Earth 14:2-6. Dow Chemical Co. 
 
 1962. The chemical and physical nature of plant surfaces in relation to the use of 
 pesticides. In: Residue Rev. 1:112-39. 
 
 CRAFTS, A. S., and H. G. REIBER 
 
 1945 Studies on the activation of herbicides. Hilgardia 16(10) :487-500. 
 
 CRAFTS, A. S., and S. YAMAGUCHI 
 
 1958. Comparative tests on the uptake and distribution of labeled herbicides 
 
 by Zebrina pendula and Tradescantia fluminensis. Hilgardia 27(16) :421-54. 
 
 1960a. Absorption of herbicides by roots. Amer. Jour. Bot. 47:248-55. 
 
 19602?. Gross autoradiography of solute translocation and distribution in plants. 
 Med. and Biol. Illustration 10:103-09. London. 
 
 CURRIER, H. B., and A. S. CRAFTS 
 
 1950. Maleic hydrazide, a selective herbicide. Sci. 111:152-53. 
 
 139 
 
DAY, B. E. 
 
 1952. The absorption and translocation of 2,4-dichlorophenoxyacetic acid by bean 
 plants. Plant Physiol. 27:143-52. 
 
 DE STTGTER, H. C. M. 
 
 1961. Translocation of Cl^-photosynthates in the graft muskme Ion/ Cuourbita fieifolia. 
 Acta Bot. Neerl. 10:466-73. 
 
 DIXON, H. H. 
 
 1914. Transpiration and the ascent of sap in plants. Macmillan and Co. 
 London. 216 pp. 
 
 EAMES, A. J. 
 
 1949. Comparative effect of spray treatments with growth regulating substances on 
 nutgrass (Cyperus rotundas L. ) and anatomical modifications following treat- 
 ment with butyl 2, 4-dichlorophenoxy acetate. Amer. Jour. Bot. 36:571-84. 
 
 1950. Destruction of phloem in young bean plants after treatment with 2,4-D. 
 Amer. Jour. Bot. 37:840-47. 
 
 ESAU, K. 
 
 1953. Plant anatomy. John Wiley and Sons, Inc. New York, N. Y. 735 pp. 
 
 ESAU, K. , and V. I. CHEADLE 
 
 1961. An evaluation of studies on ultrastructure of sieve plates. 
 Natl. Acad. Sci. Proc. 47:1716-26. 
 
 EXER, B. 
 
 1958. Uber Pflanzenwachstumregulatoren. Der Einfluss von Simazin auf den Planzen- 
 stoffwechsel. Experimentia 14:135-37. 
 
 FORDE, B. J. 
 
 1959. Some studies on growth and nutrition of perennial ryegrass (Lolium perenne L. ) 
 and cocksfoot (Dactylis glomerata L. ). Dissertation for M.S. Degree. Victoria 
 University of Wellington, Wellington, N. Z. 
 
 1963. Some aspects of phloem translocation in three grass species. Dissertation for 
 the Ph.D. Degree. University of California, Davis, California. 
 
 FOY, C. L. 
 
 1958. Studies on the absorption, distribution and metabolism of 2,2-dichloropro- 
 
 pionic acid in relation to phototoxicity. Doctoral dissertation, University 
 of California, Davis, California. 
 
 1960. The adaptation of qualitative and quantitative techniques for determination 
 of radioactive dalapon in plant tissues. Hilgardia 30(5) :153-73. 
 
 FOY, C. L. , and L. W. SMITH 
 
 1963. Tracer studies with two radio- labeled surfactants and dalapon. Res. Prog. 
 Report. Western Weed Control Conf. pp. 88-89. 
 
 GYSIN, H. and E. KNUSLI 
 
 1958. Activity and mode of action of triazine herbicides. British Weed Control 
 Conf. Proc. 4th Meeting. 
 
 1960. Chemistry and herbicidal properties of triazine derivatives. In: Advances 
 in Pest Control Research. Interscience Publ. New York, N. Y. 
 
 HAUN, J. R. , and J. H. PETERSON 
 
 1954. Translocation of 3-(p-chlorophenyl)-l,l-dimethylurea in plants. Weeds 3:177-87. 
 
 140 
 
JACKSON, D. I. 
 
 1962. The response of Oxalis lati folia to hormone herbicides. Weeds 10:23-25. 
 
 JAGENDORF, A. T. 
 
 1958. The relationship between electron transport and phosphorylation of spinach 
 chlorop lasts . Brookhaven Symposia in Biology 11:236-57. 
 
 JANSEN, L. L. , W. A. GENTNER and W. C. SHAW 
 
 1961. Effects of surfactants on the herbicidal activity of several herbicides in 
 aqueous spray systems. Weeds 9:381-405. 
 
 KEY, J. L. 
 
 1962. Changes in ascorbic acid metabolism associated with auxin-induced growth. 
 Plant Physiol. 37:349-56. 
 
 LEONARD, 0. A., and A. S. CRAFTS 
 
 1956. Translocation of herbicides. III. Uptake and distribution of radioactive 
 2,4-D by brush species. Hilgardia 26(6): 36 6-415. 
 
 MASON, G. 
 
 1960. The absorption, translocation and metabolism of 2,3,6-trichlorobenzoic acid 
 
 in plants. Doctoral dissertation, University of California, Davis, California. 
 
 MASON, J. G., and E. PHILLIS 
 
 1937. The migration of solutes. Bot. Rev. 3:47-71. 
 
 MITCHELL, J. W. , and J. W. BROWN 
 
 1946. Movement of 2, 4-dichlorophenoxy acetic acid stimulus and its relation to the 
 translocation of organic food materials in plants. Bot. Gaz. 107:393-407. 
 
 MITCHELL, J. W. , and P. J. LINDER 
 
 1950a. Absorption and translocation of radioactive 2,4-DI by bean plants as affected 
 by cosolvents and surface agents. Science 112:54-55. 
 
 19502?. Absorption of radioactive 2,4-DI as affected by wetting agents. N. E. Weed 
 Control Conf. 4th Annual Proc. 21-25. 
 
 MTTTLER, T. E. 
 
 1957a. Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin). 
 I. The uptake of phloem sap. Jour. Exptl. Biol. 34:334-41. 
 
 19572?. Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin). 
 
 II. The nitrogen and sugar composition of ingested phloem sap and excreted 
 honey dew. Jour. Exptl. Biol. 35:74-84. 
 
 MORELAND, D. E. , W. A. GENTNER, J. L. HILTON, and K. L. HILL 
 
 1959. Studies on the mechanism of herbicidal action of 2-chloro-4,6-bis 
 (ethylamino)-s-triazine. Plant Physiol. 34:432-35. 
 
 MUNCH, E. 
 
 1930. Die Stoffbewegungen in der Pflanze. Gustav Fischer, Jena. 234 pp. 
 
 MUNI, A. P. 
 
 1959. Investigations on the anatomy of weeds and selective weed control. 
 Doctoral dissertation. Bose Institute, University of Calcutta. 
 
 PALLAS, J. E. Jr., and A. S. CRAFTS 
 
 1957. A critical preparation of plant material for autoradiography. Sci. 625:192-93. 
 
 141 
 
PEREIRA, J. F. , A. S. CRAFTS, and S. YAMAGUCHI 
 
 1963. Translocation in coffee plants. Turrialba 13:64-79. 
 
 PETERSEN, H. I. 
 
 1958. Translocation of Cl^-labeled 2,4-dichlorophenoxyacetic acid in barley and oats. 
 Nature, London. 182:1685-86. 
 
 1962. Nogle ukrudtsbekaempelsesmidlers translokation i forskellige plantearter. 
 Saertryk. Ugeskrift for Landmaend 25, 26. 
 
 PRIESTLEY, J. H. 
 
 1920. The mechanism of root pressure. New Phytol. 19:189-200. 
 
 QUINLAN, J. D. , and G. R. SAGAR 
 
 1962. An autoradiographic study of the movement of Cl^-labeled assimilates in the 
 developing wheat plant. Weed Res. 2:264-73. 
 
 ROHRBAUGH, L. M. , and E. L. RICE 
 
 1949. The effect of application of sugar on the translocation of sodium 2,4-dichloro- 
 phenoxy acetate by bean plants in the dark. Bot. Gaz. 110:85-89. 
 
 ROTH, W. 
 
 1958. Substances regulatrices de la crossance vegetale. Etude de l 1 action de la 
 Simazine sur la physiologie d'Elodea. Experimentia 14:137-38. 
 
 RUBEN, S., and M. D. KAMEN 
 
 1940. Radioactive carbon at long half- life. Phys. Rev. 57:549. 
 
 1941. Long-lived radioactive: Cl4. Phys. Rev. 59:349. 
 
 SMITH, A. E., J. W. ZUKEL, G. M. STONE, and J. A. RIDDELL 
 
 1959. Factors affecting the performance of maleic hydrazide. 
 Agric. and Food Chem. 7:341-44. 
 
 STOUT, P. R. and D. R. HOAGLAND 
 
 1939. Upward and lateral movement of salt in certain plants as indicated by radio- 
 active isotopes of potassium, sodium, and phosphorus absorbed by roots. 
 Amer. Jour. Bot. 26:320-24. 
 
 STRUGGER, S. 
 1938. Fluoreszenzmikroskopische untersuchungen uber die Speicherung und Wanderung des 
 Fluoreszeinkaliums in pflanzlichen Geweben. Flora 132:253-304. 
 
 SWEETSER, P. B. , and C. W. TODD 
 
 1961. The effect of monuron on oxygen liberation in photosynthesis. Biochim. 
 et Biophys. Acta 51:504-08. 
 
 SWEETSER, P. B. , C. W. TODD, and R. T. HERSH, 
 
 1961. Effect of photosynthesis inhibitors on light re-emission in photosynthesis. 
 Biochim. et Biophys. Acta 51:509-18. 
 
 SZABO, S. S. 
 
 1963. The hydrolysis of certain 2,4-D esters by bean and corn plants. Weeds 11:292-94. 
 
 TODD, C. W. 
 
 1960. The effect of urea herbicides on photosynthesis. Mimeo. report, E. I. du Pont 
 de Nemours and Co., Wilmington, Del. 
 
 VAN DER ZWEEP, W. 
 
 1961. The movement of labeled 2,4-D in young barley plants. Weed Res. 1:258-66. 
 
 142 
 
WEBSTER, D. H. 
 
 1962. Entry of 2 , 4-dichlorophenoxy acetic acid into lambkill leaves at varied 2,4-D/ 
 Tween 20 ratios. Weeds 10:250-51. 
 
 WEINTRAUB, R. L. , and J. W. BROWN 
 
 1950. Translocation of exogenous growth-regulators in the bean seedling. Plant 
 Physiol. 25:140-49. 
 
 WEINTRAUB, R. L. , J. W. BROWN, and J. N. YEATMAN 
 
 1950. Recovery of growth regulator from plants treated with 2,4-dichlorophenoxy- 
 acetic acid. Science 111:493-94. 
 
 WEINTRAUB, R. L. , J. W. BROWN, J. C. NICKERSON, and K. N. TAYLOR 
 
 1952a. Studies on the relation between molecular structure and physiological activity 
 of plant growth regulators. I. Abscission- inducing activity. 
 Bot. Gaz. 113:348-62. 
 
 WEINTRAUB, R. L. , J. W. BROWN, M. FIELDS, and J. ROHAN 
 
 19522?. Metabolism of 2, 4-dichlorophenoxy acetic acid. I. Cl 4 02 production by 
 bean plants treated with labeled 2, 4-dichlorophenoxy acetic acids. 
 Plant Physiol. 27:293-301. 
 
 WEINTRAUB, R. L. , J. N. YEATMAN, J. W. BROWN, J. A. THRONE, J. D. SKOSS, and J. R. CONOVER 
 1954. Studies on entry of 2,4-D into leaves. N. E. Weed Control Conf. 8th Proc: 5-10. 
 
 WESSELS, J. S. C. , and R. VAN DER VEEN 
 
 1956. The action of some derivatives of phenylurethan and 3-phenyl-l,l-dimethyl 
 urea on the Hill reaction. Biochim. et Biophys. Acta 19:548-49. 
 
 WIESE, A. F. , and H. E. REA 
 
 1962. Factors affecting the toxicity of phenoxy herbicides to field bindweed. 
 Weeds 10:58-61. 
 
 WIJEWANTHA, R. T. , and G. L. STEBBINS 
 
 1963. Private communication. 
 
 YAMAGUCHI, S. , and A. S. CRAFTS 
 
 1958. Autoradiographic method for studying absorption and translocation of herbi- 
 cides using Cl^- labeled compounds. Hilgardia 28(6) :161-91. 
 
 1959. Comparative studies with labeled herbicides on woody plants. 
 Hilgardia 29(4) :171-204. 
 
 ZIMMERMAN, M. H. 
 
 1961. Movement of organic substances in trees. Sci. 133(3446) :73-79. 
 
 ZWEIG, G., and F. M. ASHTON 
 
 1962. The effect of 2-chlor\D-4-ethyl-ajidno-6-isopropyl-amino-s-triazine (Atrazine) 
 on distribution of Cl^-compounds following C1402 fixation in excised kidney 
 bean leaves. Jour. Exptl. Bot. 13:5-11. 
 
 143 
 
To simplify this information it is sometimes necessary to use 
 trade names of products or equipment. No endorsement of named pro- 
 ducts is intended nor is criticism implied of similar products 
 which are not mentioned. 
 
 THIS MANUAL is one of a series published by the University of California 
 College of Agriculture and sold for a charge which is based upon return- 
 ing only a portion of the production cost. By this means it is possible 
 to make available publications which, due to relatively high cost of pro- 
 duction, or limited audience, would otherwise be beyond the scope of the 
 College publishing program. 
 
▼HIS BOOK IS DUE ON THE LAST DATE 
 
 im 
 
 To obtain additional copies of this manual or a catalog listing other 
 manuals and free publications available, see your University of California 
 Farm Advisor (offices located in most California counties), or write to: 
 
 Agricultural Publications 
 207 University Hall 
 2200 University Avenue 
 Berkeley 4, California 
 
 Orders of 10 or more copies of any one manual take a 20 per cent discount 
 off the list price. All manuals are shipped prepaid. 
 
 When ordering manuals, send orders and payment to the above address. 
 Make checks or money orders payable to The Regents of the University 
 of California. 
 
 1