V . . .. : . .t. . V .... D. . .. . . 1. , D . V D . .. , . V . . . . .... . . . A . .. .D. . . . I. . D A . V, . . , , . V , . . . _. . . .. , . .. . . . . . , ., . . . . , . , . ., . r . .r. p...‘ v i. <9 I .... :3...Ww:.% r “.41. A v v . 3/4 v . J fat. {IN/w 24.1....» ¢ :0?!" m' TOLEMNCE TO CHLORAMBEN ’M“ Tu fr DADD- AND mummif- .o y. y I . .5... “.1 gr Ivan/1v”. ”(fff! 5 NWT ’N STATE UNIVERSHY JUUAN CREIGHTGN HELLER, J! D. 5%. ,1: I 5.. CUCUMBER 19??! Thesis for me Degree of Ph. Di. m mm m D... m . . ..;r .. v .r . . Dr . y A D. 4 . .m . . ; .. 1.-.... . . . D. .. ._...V_.. .........3.r..>.......§ d 9- v.5. ,fi..r....a.. .5 .u . _. _. D DDDD bmmn ans “SW A 21 man ,... film This is to certify that the thesis entitled THE PHYSIOLOGICAL BASIS AND INHERITANCE OF TOLERANCE TO CHLORAMBEN METHYL ESTER IN CUCUMBER presented by Julian Creighton Miller, Jr. has been accepted towards fulfillment of the requirements for _____Ph- D. degree inmture D? W mic: mfm Date Maj! 4. 197L 0-7639 Deg-393:2. ABSTRACT THE PHYSIOLOGICAL BASIS AND INHERITANCE OF TOLERANCE TO CHLORAMBEN METHYL ESTER IN CUCUMBER BY Julian Creighton Miller, Jr. The physiological basis and inheritance of dif- ferential tolerance in cucumber (Cucumis sativus L.) to methyl 3-amino-2,S-dichlorobenzoic acid (chloramben methyl ester) was investigated. Two tolerant, MSU 3207 and MSU 0612, and two susceptible, MSU 3159 and MSU 0866, lines were selected for further study following screening of more than 200 inbred lines, plant introductions and cultivars. Intraspecific variability exhibited by these lines was related to higher concentrations of 3-amino-2,5- dichlorobenzoic acid (chloramben) in the roots of the susceptible lines, resulting from metabolic differences in both susceptible lines and rapid absorption of the herbicide by the roots of MSU 3159. Tolerance of MSU 3207 resulted primarily from low uptake and reduced transloca- tion of the herbicide, while tolerance of MSU 0612 appeared Julian Creighton Miller, Jr. to be related to a peculiar l4C-label distribution pattern in the leaves. Thin-layer chromatographic analysis of methanol-soluble extracts three days after l4C-chloramben methyl ester treatment separated six l4C-metabolites in the roots and five in the shoots. After a 4-hr treatment, l4C-chloramben methyl ester was absorbed and translocated more rapidly than l4C—chloramben by all four lines, although the extent of absorption among the lines was similar. Tolerance or susceptibility did not always cor- relate with the total concentration of radioactivity in methanol-soluble shoot and root extracts. Progeny from crosses among the four lines were evaluated for tolerance to chloramben methyl ester in the F1, F2, BCl and BC2 generations. Plant height, dry weight and visual appearance were evaluated following herbicide treatment. Partial dominance of genes conditioning tolerance was shown. The minimum number of effective factor pairs was variable depending on the trait evaluated; however, quantitative inheritance of tolerance was indi- cated. Transgressive segregation was observed in the F2 populations of the crosses tolerant x tolerant. Herita- bility estimates from crosses involving tolerant MSU 0612 were consistently higher than those involving tolerant MSU 3207, also indicating that the tolerance of these two lines may be conditioned by different genes. Estimates of heritability indicated that considerable progress can be Julian Creighton Miller, Jr. made in selection for tolerance when the criteria used are plant dry weight, apical inhibition, or epinasty. THE PHYSIOLOGICAL BASIS AND INHERITANCE OF TOLERANCE TO CHLORAMBEN METHYL ESTER IN CUCUMBER BY Julian Creighton Miller, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1972 ©C0pyright by JULIAN CREIGHTON MILLER, JR. 1972 ACKNOWLEDGMENTS The author wishes to express his appreciation to Drs. L. R. Baker and D. Penner for their help and guidance throughout this study and to Drs. M. W. Adams, M. J. Bukovac, D. D. Harpstead, S. Honma, and K. C. Sink for their valuable suggestions. Appreciation is also expressed to Roy Early for laboratory assistance, Darsan Wang for greenhouse assistance, and to John Barnard and Dr. Chung Lee for assistance in computer programming. ii Guidance Committee: Sections I and II are segments of related thesis research information condensed into formats suited and intended for publication in Weed Science (Section I) and the Journal of the American Society for Horticultural Science (Section II). iii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . . . LIST OF TABLES O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O O I O 0 SECTION I. II. THE BASIS FOR VARIABILITY IN CUCUMBER TOLERANCE TO CHLORAMBEN METHYL ESTER Abstract. . . . . . . . . . . . Introduction . . . . . . . . . . Materials and Methods . . . . . . . Results . . . . . . . . . . . . Discussion . . . . . . . . . . . Literature Cited . . . . . . . . . INHERITANCE OF TOLERANCE TO CHLORAMBEN METHYL ESTER IN CUCUMBER Abstract. . . . . . . . . . . . Introduction . . . . . . . . . . Materials and Methods . . . . . . . Results . . . . . . . . . . . . Discussion . . . . . . . . . . . Literature Cited . . . . . . . . . iv Page ii vi 29 3O 32 37 44 47 Table Section I 1. Section II 1. LIST OF TABLES Page Accumulation of 14C from l4C-chloramben and l4C-chloramben methyl ester (CME) in the roots and leaf blade of 16-day old cucumber seedlings treated for 4 hours. . ll Accumulation of 14C from l4C—chloramben methyl ester in the roots and second true leaves of four lines of 4-week-old cucumber plants treated for 3 days . . . . . . 12 The influence of rootstock and scion as shown by reciprocal grafts, on the accumu- lation of 14C in the second true leaf of four lines of 5-week-old Eucumber plants treated for 3 days with l C—chloramben methyl ester . . . . . . . . . . 14 R values of l4C-metabolites before and after acid hydrolysis for 1 hour with 1.0 N HCl at 70 C . . . . . . . . . . 18 Means and standard errors of parents, F1, F2, BCl, and BC2 populations from four cucumber crosses rated for Visual appearance, plant height, and dry weight 3 weeks following CME treatment . . . . 39 Estimated values of components of varia— tion and degree of dominance for visual appearance, plant height, and dry weight of four cucumber crosses 3 weeks follow- ing treatment with CME . . . . . . . 40 Estimated values of heritability and probable number of effective factors for tolerance to CME of 4 cucumber crosses evaluated 3 weeks after herbicide treatment . . . . . . . . . 41 Figure Section 1. Section 1. I II LIST OF FIGURES Distribution of radioactivity in grafted and ungrafted cucumber lines treated for 3 days with l4C-chloramben methyl ester . . . . l4C—metabolites in the methanol-soluble extracts of root tissue from cucumber plants treated for 3 days with l4C-chloramben methyl ester . . . . . . . . . . . l4C-metabolites in the methanol-soluble extracts of leaf tissue from cucumber plants treated for 3 days with l4C-chloramben methyl ester . . . . . . . . . . . Reactions of tolerant MSU 0612 and suseCpti- ble MSU 3159 twenty-one days after treatment with 3.12 ppm amiben methyl ester (chloramben methyl ester). . . . . . . . . . . vi Page 15 21 34 SECTION I THE BASIS FOR VARIABILITY IN CUCUMBER TOLERANCE TO CHLORAMBEN METHYL ESTER ABSTRACT The physiological basis for tolerance and sus- ceptibility of four lines selected from inbred lines, plant introductions and cultivars of cucumber (Cucumis sativus L.) to methyl 3-amino-2,S-dichlorobenzoic acid (chloramben methyl ester) was investigated. MSU 3207 and MSU 0612 were tolerant (T), whereas MSU 3159 and MSU 0866 were susceptible (S). Intraspecific variability exhibited by these lines was related to higher concentrations of 3-amino- 2,5-dichlorobenzoic acid (chloramben) in the roots of the susceptible lines, resulting from metabolic differences in both lines and rapid absorption of the herbicide by the roots of S-MSU 3159. Tolerance of T-MSU 3207 resulted primarily from low uptake and reduced translocation of the herbicide, while tolerance of T-MSU 0612 was related to a peculiar l4C-label distribution pattern in the leaves. Thin-layer chromatographic analysis of methanol-soluble extracts three days after 14C-chloramben methyl ester treatment separated six l4C—metabolites in the roots and five in the shoots. After a 4-hr treatment, l4C-chloramben methyl ester was absorbed and translocated more rapidly 14 than C-chloramben by all four lines, although the extent of absorption among the lines was similar. Tolerance 1 or susceptibility did not always correlate with the total concentration of radioactivity in methanol-soluble shoot and root extracts. INTRODUCTION Interspecific variability in tolerance of various crop and weed species to chloramben and its analogs has been associated with several physiological mechanisms. Chloramben is readily absorbed by roots of tolerant and susceptible species (2, 3, 7, 20, 21, 22, 23, 24). Absorption is concentration dependent (20, 21) and does not appear to be related to species sensitivity (3, 21, 22). Conversely, differential translocation is an important factor in determining species selectivity (3, 7, 9, 21, 23) and appears to be closely associated with metabolism particularly in the roots (3, 21, 24). The metabolism of chloramben methyl ester is con- sidered to proceed by hydrolysis to chloramben in the soil (6). The fate of chloramben in most plants is the formation of complexes or conjugates with natural plant products (5, 8, 24). These conjugates, upon either base or acid hydrolysis release free chloramben (5, 6, 8, 21, 25). Two conjugates, N-(3-carboxy-2,5-dichlorophenyl)-g1ucosyl- amhma(N-glucosy1 chloramben) and an unidentified chloramben conjugate (chloramben-X) have been found (5, 6, 10, 22, 25) and are considered to be detoxication products (5, 19, 20, 24). In several species, tolerance to chloramben has been related to rate of chloramben metabolism (20, 21, 24) and composition or distribution of metabolites (5, 21, 22); however, no such relationship was observed in cucumber and squash (3). The metabolic basis for the selective action of chloramben remains unclear. The occurrence of intraspecific variability in the response of weed and crop plants to specific herbicides has recently been reviewed (11). Weed control in cucumber with preemergence application of chloramben methyl ester has resulted in variable tolerance dependent on environmental conditions1 and cultivars (14). The purpose of this investigation was to determine the physiological basis for intraspecific variability in cucumber tolerance to chloramben methyl ester by studying absorption, translocation, distribution and metabolism in two tolerant and two susceptible cucumber lines. lAmchem Products Inc. 1971. Chloramben methyl ester (Vegiben 2-E) for weed control in snap beans, cucumbers and cantaloupes. Technical Service Data Sheet No. 3/71. 5p. MATERIALS AND METHODS Herbicide. Carboxy-labeled l4C-chloramben methyl ester was prepared by reacting carboxy-labeled l4C- chloramben (specific activity 1.19 mc/mmole) dissolved in a small volume of methanol with diazomethane diethyl ether solution (1, 4). This reaction yielded 100 percent l4C- chloramben methyl ester as shown by thin-layer chromato- graphy. Non-labeled herbicide was added to the radioactive form to obtain the desired concentration of 1.8 x 10'.5 M in the nutrient treatment solution. Plant material and treatment. Preliminary experi- ments under both greenhouse and field conditions revealed three to seven+fold differences among approximately 200 cucumber lines tested for their response to chloramben methyl ester. From these lines, two susceptible, S-MSU 3159 and S-MSU 0866, and two relatively tolerant lines, T-MSU 0612 and T-MSU 3207, were selected for this study. Seeds of the four inbred lines were germinated in moist vermiculite. After ten days, when the first true leaf had expanded to about 0.5 cm, seedlings were trans- planted to an aerated Hoagland's no. 1 nutrient solution (12). Three days after transplant, reciprocal hypocotyl approach grafts were made in all combinations utilizing the techniques of de Stigter (18). Sealtex Latex Bandage was used to secure graft unions. Seven days later the unwanted rootstock and scion were removed. Self grafts and nongrafted plants of each line were also included in the study. Five days after excision, when two to three true leaves were evident, plants were transferred to calibrated plastic cups containing 80 ml of nutrient solution including 1.8 x 10—5n414C-chloramben methyl ester. The cups were protected from light with aluminum foil. Plants were grown in the greenhouse under a 27 C day and 21 C night regime, with supplemental incandescent light provided during the l4-hr day. Sufficient Hoagland's solution was added daily to maintain the 80 m1 volume. At the end of the 3-day treatment period, roots were rinsed three times with distilled water and blotted dry, then the plants were immediately placed on dry ice and freeze dried. In a separate 4-hr absorption and translocation study with l4C-chloramben methyl ester and l4C-chloramben, l4—day-old plants were transferred at the first true leaf stage to vials containing 10 m1 of treatment solution (0.5 uc radioactivity per 100 ml Hoagland's solution) and 'placed in a growth chamber at 30 C with a light intensity of 21,500 lux. At the end of the treatment period the seedlings were immediately placed on dry ice, freeze—dried, mounted, radioautographed, removed from the mount and weighed. Roots and leaf blade of each plant were com— busted using the Scthiger method of Wang and Willis (26), and radioactivity was determined using the scintil- lation solution of Lui gt_§1. (13). Values reported for each study are the means from two separate experiments consisting of at least two replications. Determination of absorption, translocation and metabolism. Following freeze-drying, plants were mounted and radioautographed according to the methods of Crafts and Yamaguchi (9). The mounted plants were exposed to x-ray film for two weeks. After radioautography, the second true leaf including its petiole, and the root system including the hypocotyl, of each mounted plant were removed from the blotter paper and weighed. Methanol- soluble metabolites were extracted from the leaf and root tissue according to the procedures described by Swanson g£_§1. (25). The tissue was homogenized in warm, absolute methanol, the homogenate filtered through Whatman no. 2 filter paper and concentrated in vacuo at less than 50 C. The concentrated extracts were adsorbed on 12 by 150 mm Florisil columns, eluted with methanol and reduced to dryness in vacuo. The extract was made to 100 01 with methanol. Fifty ul was chromatographed on 20-cm-sq glass thin-layer plates coated with 0.25 mm of silica gel HP or GF. The chromatographs were developed ascendingly for 15 cm using a solvent system consisting of n-butanol:ethanol:ammonium hydroxide (2:1:1; V/v/v). Radioautographs of the thin— layer plates were used for detecting the precise location of the l4C-components. Rf values of the five to seven l4C-labeled spots obtained were compared with those of Stoller and Wax (22). Chloramben and chloramben methyl ester standards were localized with UV light, Ehrlichs reagent, and by radioautographic technique. The 14C- labeled spots were scraped from the plates and radio- assayed by liquid scintillation spectrometry. The scintillation solution contained 5.0 g/L PPO plus 0.3 g/L POPOP in toluene. The l4C-metabolites were eluted from thin—layer plates and hydrolized with 1.0 N HCl for 1 hr at 70 C. The hydrolysates were then re—chromatographed. Thin-layer chromatographic plates were also sprayed with 1.0 N HCl and then heated to 70 C for hydrolysis of metabolites. Reducing sugars were detected with silver nitrate spray reagent. Membrane stud . The fatty acid composition of membranes from each of the four lines was investigated to determine whether it contributed to variation among lines tested. Membranes were isolated by differential centrifugation using the general procedure of Raison and Lyons (17). Lipids were extracted from the membranes with chloroform and methanol (2:1, v/v). Fatty acids were extracted from the lipid fraction, methylated, and separated by gas-liquid chromatography (l6). RESULTS Absorption and translocation. All lines readily absorbed 14C-chloramben and 14C-chloramben methyl ester during the 4-hr treatment period, the latter being absorbed to a greater extent (Table 1). A comparison between T-MSU 3207 and S-MSU 3159 showed a significantly lower concentration of 14C activity in the root tissue of T—MSU 3207 following 14C-chloramben methyl ester treat- ment. The other tolerant line, T-MSU 0612, absorbed as much l4C-chloramben methyl ester after 4 hr as did both susceptible lines (Table 1). After 3 days, the concen- tration of l4C-chloramben methyl ester was greater in this line than in S-MSU 0866, but less than in S-MSU 3159 (Table 2). Concentration of 14C in the leaves of the four lines did not differ significantly following 4-hr treatment with either l4C-chloramben or l4C-chloramben methyl ester (Table 1). Comparison of T—MSU 3207 and S-MSU 3159 following the 3-day treatment period revealed the trans- location of a 3-fold higher concentration of 14C to the leaf tissue of the susceptible line (Table 2). The con- centration of 14C in the roots of S-MSU 3159 was twice 10 11 TABLE l.--Accumulation of 14C from l4C—chloramben and 14C- chloramben methyl ester (CME) in the roots and leaf blade of 16-day old cucumber seedlings treated for 4 hours. ========r 14C in roots 14C in leaf Cucumber line no. (dpm/mg dry wt) (dpm/mg dry wt) Chloramben CME Chloramben CME T-MSU 3207 393 aa 523 a 19 a 48 a S-MSU 3159 452 a 778 b 30 a 73 a S—MSU 0866 407 a 677 ab 28 a 79 a T—MSU 0612 446 a 712 b 25 a 56 a aMeans within columns with common letters did not differ significantly at the 5% level by Duncan's Multiple Range Test. 12 TABLE 2.--Accumulation of 14C from l4C-chloramben methyl ester in the roots and second true leaves of four lines of 4-week-old cucumber plants treated for 3 days. l4 l4 Cucumber line no. C in roots C in leaf (dpm/mg dry wt) (dpm/mg dry wt) T-MSU 3207 20,518 aa 542 a S-MSU 3159 40,626 c 1,712 b S-MSU 0866 20,360 a. 714 a T-MSU 0612 ' 30,011 b 1,700 b aMeans within columns with common letters did not differ significantly at the 5% level by Duncan's Multiple Range Test. 13 as great as in T-MSU 3207. However, S-MSU 0866 absorbed and translocated no more 14C-label to the leaf than did T-MSU 3207 (Table 2). T-MSU 0612, the other tolerant line, was intermediate between the two susceptible lines in absorption of 14C, but it was equal to S-MSU 3159 in translocation of 14C to the leaves (Table 2). The role of roots and shoots in imparting tolerance or susceptibility to these four lines is shown in Table 3. Plants with T-MSU 3207 scions had signifi- cantly less 14C in the leaf tissue than plants with scions of the other three lines, implying reduced trans- location of 14C to the leaves. Plants with S-MSU 3159 rootstocks accumulated more 14C in the leaf tissue than plants with other rootstocks. Radioautographs of plants treated with 14C- chloramben methyl ester showed that movement of radio- activity was principally into the older leaves (Figure l). The intensity of l4C-labeling in S-MSU 3159 was greater than in T-MSU 3207. The grafted plant T-MSU 3207/T-MSU 3207 did not differ appreciably from the ungrafted T-MSU 3207 plants. However, when T-MSU 3207 was grafted onto S-MSU 3159 rootstocks more 14C was translocated to the T-MSU 3207 shoot than when it was on its own rootstock (Figure l-A and B and Table 3). Though tolerant, T-MSU 0612 absorbed and translocated as much 14C as S-MSU 3159 (Figure l-C and D and Table 3). 'T—MSU 0612 exhibited a unique l4 TABLE 3.--The influence of rootstock and scion as shownl4 by reciprocal grafts, on the accumulation of C in the second true leaf of four lines of 5-week- old cucumber plants treated for 3 days with 14C- chloramben methyl ester. 14C in leaf of scion (dpm/mg dry wt) Rootstock Scion T-MSU 3207 S-MSU 3159 ‘s-Msu 0866 T-MSU 0612 Meana T-MSU 3207 558 1812 1483 1518 1343 S-MSU 3159 1136 1824 2292 2060 1828 S-MSU 0866 541 1308 850 1830 1133 T—MSU 0612 1264 1034 1116 1732 1287 Meana 875 a 1495 b 1436 b 1785 b aMeans followed by common letters did not differ significantly at the 5% level by Duncan's Multiple Range Test. 15 Figure l.--Distribution of radioactivity in grafted and ungrafted cucumber lines treated for 3 days with l4C-chloramben methyl ester. Plants in Figure l-A and B from left to right are S-MSU 3159, T-MSU 3207, T-MSU 3207/T-MSU 3207, and T-MSU 3207/S-MSU 3159. Figure l-A: Plants; Figure l-B: Radioautographs. Plants in Figure l-C and D from left to right are S-MSU 3159/T-MSU 0612, T-MSU 0612/S-MSU 3159, S-MSU 3159, and T-MSU 0612. Figure l-C: Plants; Figure 1-D: Radioautographs. 17 distribution of l4C-label in the leaves, with an accumula- tion of l4C—label in and adjacent to the vascular tissue. This distribution of l4C-label was not observed in the other lines studied. T-MSU 0612 retained this character— istic l4C distribution pattern even when grafted onto other rootstocks. Thus, control of this pattern was due to factors in the leaf rather than in the roots of T-MSU 0612. Radioautographs of S-MSU 3159/T-MSU 0612 revealed a l4C-labeling intensity and distribution similar to the ungrafted S-MSU 3159 plant (Figure 1-C and D). The rootstock of T~MSU 0612 did not alter the distribution of l4C-label in susceptible S-MSU 3159, as there was no accumulation of l4C-label along the veins as was seen in T-MSU 0612. Metabolism. Examination of radioautographs of chromatographic plates spotted with methanol-soluble root extracts showed the presence of the following metabolites proceeding from the origin: N-glucosyl chloramben, chloramben conjugate, nonhydrolyzable metabolite, chloramben methyl ester conjugate, free chloramben, chloramben-X (traceS) and the parent compound, chloramben methyl ester (Table 4). The percentage dis- tribution among l4C-metabolites found in root tissue after 14 C-chloramben methyl ester treatment for three days was determined (Figure 2). T-MSU 0612 contained the least 18 TABLE 4.--Rf values of l4C-metabolites before and after acid hydrolysis for 1 hour with 1.0 N HCl at 70 C. Metabolites Original Hydrolysate R R f f N-glucosyl chloramben .14 - .17 .41 Chloramben conjugate .21 - .24 .41 Nonhydrolyzable metabolite .27 - .29 .22 Chloramben methyl ester conjugate .38 - .41 .78 Chloramben standard .41 — .45 .41 Chloramben methyl ester standard .78 - .84 .78 T- MSU 0612 19 Percentage Distribution Cucumber 9,2? , 4? . 6? T8? .1 13° 35.6 13.2 6.6 22.4 14.5 7.7 fD-MSU 3207 ' ' 7 28.1 16.2 6.1 20.4 20.5 8.7 37.0 11.6 8.7 11.0 25.3 6.4 S‘MSU 0866 20.2 10.0 7.9 32.0 16.6 13.4 Metabolites LSD (0.05) N—glucosyl chloramben E] 13.7 Chloramben conjugate MB 6.8 Nonhydrolyzable metabolite E 6 . 5 Chloramben methyl ester conjugate CE 7.7 Chloramben E3 6.2 Chloramben—x (trace) * Chloramben methyl ester fig 4.6 FIGURE 2.--l4C-metabolites in the methanol-soluble extracts of root tissui from cucumber plants treated for 3 days with l C-chloramben methyl ester. Meta- bolites were separated on silica gel GF TLC plates with a n-butanol:ethanol : ammonium hydroxide (2:1:l,v/v/v) solvent system. 20 N-glucosyl chloramben, while containing the most chloramben methyl ester conjugate and chloramben methyl ester. S-MSU 3159 and S-MSU 0866 retained more chloramben in the roots than either of the tolerant lines, possibly contributing to their susceptibility. S-MSU 0866 contained significantly less chloramben methyl ester conjugate in the roots than any of the other three lines. The percentage distribution among 14C-metabolites in methanol-soluble extracts of leaf tissue following three day treatment with l4C—chloramben methyl ester was also determined (Figure 3). Of the six metabolites found in the root tissue, the nonhydrolyzable metabolite as well as chloramben methyl ester, were not detected in this tissue. Again, T-MSU 0612 contained less N-glucosyl chloramben and more chloramben and chloramben methyl ester conjugate than did the other lines. Among the other three lines there was no significant difference in the dis- tribution of metabolites in the leaf tissue. Following HCl hydrolysis, three of the hydrolysates (N—glucosyl chloramben, chloramben conjugate, chloramben) moved to an Rf corresponding to that of the l4C-chloramben standard; two others (chloramben methyl ester, chloramben methyl ester conjugate) to an Rf corresponding to that of the chloramben methyl ester standard. One 14C-metabolite proved to be nonhydrolyzable under these conditions. 21 Cucumber Percentage Distribution Line 0 20 40 60 80 100 l I F I a u I I l I ‘i 45.9 22. 9 7.8 23.4 — ': “1.11:2:315'2'" "‘2” // T MSU 3207 s 4 %W* 49.4 19.0 8.5 23.1 s— MSU 3159 ,.13:111.”..'I.'."..':,'2.",‘131.3'....‘z.’ AW'I: 51.7 21.2 8.9 18.1 s-msu 0866 2"'4:«:4.::4.::251.1.42».V/mir 37.5 14. 6 14.3 33.6 T- MSU 0612 (TifffW‘IHlf'IEWZ/Qm* Metabolites LSD (0.05) N—glucosyl chloramben E] 9.2 Chloramben conjugate EB 12.1 Chloramben methyl ester conjugate IE 3.8 Chloramben ES 8.7 Chloramben-x (trace) FIGURE .3.--14 C-metabolites in the methanol-soluble extracts of leaf tissue from cucumber plants treated for 3 days with 4C-chloramben methyl ester. Metabolites were separated on silica gel GF TLC plates with a n-butanol:ethanol : ammonium hydroxide (2:1:l,v/v/v) solvent system. 22 Chloramben-X was present in such trace quantities that no attempt was made to hydrolyze it, and its tentative identification was based on similarity to an earlier study (22). Chloramben, chloramben—X, and chloramben methyl ester had Rf values similar to, but slightly lower than, those previously reported for these metabolites (7, 22, 24, 25). Membrane study. The results indicated that the fatty acid fraction of cucumber membranes was composed primarily of palmitic, stearic, oleic, linoleic, and linolinic acids. There were significant differences in the fatty acid composition between roots and shoots of all lines; however, there were essentially no significant differences among root or leaf tissue of the four lines which could account for the observed differential sensitivity to chloramben methyl ester. DISCUSSION Cucumber lines which absorbed the most l4C— chloramben methyl ester also transported the most 14C to the leaves during the 3—day treatment period (Table 2). After treatment for four hours, there was greater 14 absorption and translocation of C-chloramben methyl ester than l4C-chloramben by all four lines (Table 1). This indicated that l4C-chloramben methyl ester can be absorbed directly by cucumber without first being hydrolyzed to l4c-chloramben. The tolerance of'T-MSU 3207 and susceptibility of S-MSU 3159 was apparently due to inherent differences in their ability to absorb and translocate 14C—chloramben methyl ester or its metabolites, resulting in higher l4C accumulation in the roots and leaves of S-MSU 3159. The divergent phytotoxic reactions exhibited by S—MSU 3159 and T-MSU 3207 may be partially due to differences in metabolism, as S-MSU 3159 contained more phytotoxic chloramben and less non-phytotoxic N-glucosyl chloramben in the roots than did T-MSU 3207. 23 all... . fag I 24 The susceptibility of S-MSU 0866 could not be attributed to absorption or translocation; however, the concentration of chloramben found in the roots of this line and S-MSU 3159 may partially explain their suscepti- bility. It may be that the availability of the moiety which conjugates with chloramben methyl ester or chloramben is the limiting factor in the metabolism of these two toxic compounds. The tolerance exhibited by T-MSU 0612 cannot be attributed to low root absorption or reduced translocation to the shoot. It is also difficult to attribute the tolerance of this line solely to metabolic differences, although such differences were observed. The unique 14C- labeling pattern in the leaves of T-MSU 0612 may play an important role in determining tolerance in this line. Chloramben or other metabolites may not enter the cyto- plasm of this line to the same extent as in the other three lines, although the fatty acid fraction from mem- branes of this line did not differ in composition from that found in the other lines. The distribution of metabolites observed in T-MSU 0612 may be confounded by the mechanism responsible for the unusual labeling pattern, rendering the metabolic data an inaccurate basis on which to explain tolerance. 25 Three l4C-chloramben methyl ester metabolites not previously reported were observed in this study and designated as chloramben conjugate (Rf .21 - .24), non- hydrolyzable metabolite (Rf .27 - .29) and chloramben methyl ester conjugate (Rf .38 - .41). The phytotoxicity and importance of these metabolites in determining selectivity was not studied. The tolerance of these four cucumber lines to chloramben methyl ester did not always correlate with total concentration of radioactivity in the methanol- soluble shoot and root extracts. This agrees with the observations of Stoller and Wax (22) who studied six plant species with a wide range of tolerance to chloramben. Widely divergent responses of cucumber to chloramben methyl ester have been observed (14) and tolerance is highly heritable (15). Data obtained in this study indicates that the physiological basis for tolerance of T-MSU 3207 differs from that of T-MSU 0612. Likewise, the susceptibility of S-MSU 3159 has a different physio- logical basis than that of S—MSU 0866. The presence of intraspecific variability in cucumber with respect to tolerance to chloramben methyl ester and of more than one physiological basis for this tolerance offers the possi- bility of breeding cucumber cultivars with a greater tolerance level than those presently available. 10. 11. LITERATURE CITED Aldrich Chemical Catalog 14, 1969-70 directory of organic chemicals. 1968. Aldrich Chemical Company, Inc., Milwaukee, Wis. p. 484. Ashton, F. M. 1966. Fate of amiben-Cl4 in carrots. Weeds 14:55-57. Baker, R. S. and G. F. Warren. 1962. Selective herbicidal action of amiben on cucumber and squash. Weeds 10:219-224. de Boer, T. J. and H. J. Backer. 1956. Diazomethane. Organic Synthesis 36:16-19. Colby, S. R. 1965. Herbicide metabolism: N-glycoside of amiben isolated from soybean plants. Science 150:619-620. Colby, S. R. 1966. Fate of the amide and methyl ester of amiben in soybean plants and soil. Proc. No. East. Weed Contr. Conf. 20:619-626. Colby, S. R. 1966. The mechanism of selectivity of amiben. Weeds 14:197-201. Colby, S. R., G. F. Warren and R. S. Baker. 1964. Fate of amiben in tomato plants. J. Agr. Food Chem. 12:320-321. Crafts, A. S. and S. Yamaguchi. 1964. The auto- radiography of plant materials. California Agr. Exp. Sta. Man. 35. 143 p. Frear, D. S., C. R. Swanson and R. E. Kadunce. 1967. The biosynthesis of N-(3-carboxy-2,5-dichloropheny1)- glucosylamine in plant tissue sections. Weeds 15:101-104. Hammerton, J. L. 1967. Intra-specific variations in susceptibility to herbicides. Ghent. Rijksfac. Landbouwwetensch. Meded. 1967:999-1012. 26 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 27 Hoagland, D. R. and D. I. Arnon. 1950. The water- culture method for growing plants without soil. California Agr. Exp. Sta. Circ. 347. 32 p. Lui, T. Y., A. Oppenheim and P. Castelfranco. 1965. Ethyl alcohol metabolism in leguminous seedlings. Plant Physiol. 40:1261-1268. Miller, J. C., Jr. and L. R. Baker. 1971. Dif- ferential phytotoxicity of amiben methyl ester to Cucumis sativus lines. (Abstr.) Hort Sci. 6:276. Miller, J. C., Jr., L. R. Baker and D. Penner. Inheritance of tolerance to chloramben methyl ester in cucumber. (In preparation). Penner, D. and W. F. Meggitt. 1970. Herbicide effects on soybean (Glycine max L. Merrill) seed lipids. Crop Sci. 10:553-555. Raison, J. K. and J. M. Lyons. 1970. The influence of mitochondrial concentration and storage on the respiratory control of isolated plant mitochondria. Plant Physiol. 45:382-385. de Stigter, H. C. M. 1956. Studies on the nature of the incompatibility in a cucurbitaceous graft. Mededelingen van de Wandbouwhogschool te Wageningen, Nederland 56(8):1-51. Stoller, E. W. 1968. Differential phytotoxicity of an amiben metabolite. Weed Sci. 16:384-386. Stoller, E. W. 1969. The kinetics of amiben absorption and metabolism as related to species sensitivity. Plant Physiol. 44:854-860. Stoller, E. W. 1970. Mechanism for the differential translocation of amiben in plants. Plant Physiol. 46:732-737. Stoller, E. W. and L. M. Wax. 1968. Amiben metabolism and selectivity. Weed Sci. 16:283- 288. Swan, D. G. and F. W. Slife. 1965. The absorption, translocation, and fate of amiben in soybeans. Weeds 13:133-138. 28 24. Swanson, C. R., R. H. Hodgson, R. E. Kadunce and H. R. Swanson. 1966. Amiben metabolism in plants. II. Physiological factors in N-glucosyl amiben formation. Weeds 14:323-327. 25. Swanson, C. R., R. E. Kadunce, R. H. Hodgson and D. S. Frear. 1966. Amiben metabolism in plants. I. Isolation and identification of an N-glucosyl complex. Weeds 14:319-323. 26. Wang, C. H. and D. L. Willis. 1965. Radiotracer Methodology in Biological Science. Prentice- Hall, Inc., Englewood Cliffs, N.J. 363 p. SECTION II INHERITANCE OF TOLERANCE TO CHLORAMBEN METHYL ESTER IN CUCUMBER ABSTRACT Progeny from crosses between two tolerant and two susceptible lines of cucumber (Cucumis sativus L.) were evaluated for tolerance to methyl 3-amino—2,5-dichloro- benzoic acid (chloramben methyl ester) in the F1' F2, BCl and BC2 generations. Plant height, dry weight and visual appearance were evaluated following herbicide treatment. Partial dominance of genes controlling tolerance was noted. The minimum number of effective factor pairs was variable depending on the trait evaluated; however, quantitative inheritance of tolerance was indicated. Transgressive segregation was observed in the F2 popula- tions of the crosses tolerant x tolerant. Heritability estimates from crosses involving tolerant MSU 0612 were consistently higher than those involving tolerant MSU 3207, also suggesting that the tolerance of these two lines may be conditioned by different genes. Estimates of heritability indicated that considerable progress can be made in selection for tolerance when the criteria used are plant dry weight, apical inhibition, or epinasty. 29 INTRODUCTION The occurrence of intraspecific variability in the response of weed and crop plants to specific herbicides has recently been reviewed (7). Methyl 3—amino-2,5- dichlorobenzoic acid (chloramben methyl ester or CME) is an effectiveajpreemergence herbicide for cucumber (Cucumis sativus L.); however, use under certain conditions has resulted in varying degrees of plant injury and occasional yield reductions (14). This variability in tolerance has been related to environmental conditions (14) and cultivars grown (12). Although many examples of intraspecific variability within crop and weed species in their response to agri- cultural chemicals have been reported, relatively few investigators have studied the genetic basis for the observed variability. Resistance to insecticide damage by DDT in barley (8, 18) and trichlorfon in sorghum (15) was found to be conditioned by the recessive genes ddt and dtp_respective1y. Methyl parathion damage to sorghum (3) and toxaphene damage to oats (5) are determined by single dominant genes. Resistance to barban chlorosis in barley was shown to be conditioned by a single recessive gene, 23, while resistance to apical inhibition appeared to be 30 31 quantitatively inherited (9). Hayes et a1. (9) reported that resistance to barban and DDT in barley is indepen- dently inherited. Most maize cultivars are resistant to injury by simazine and atrazine. However, Grogan et a1. (6) isolated a susceptible inbred line and found that susceptibility was controlled by a single recessive gene. Tolerance to atrazine (4) and MCPA (17) in flax was polygenic, exhibiting relatively low heritability. Schooler et a1. (16) showed that siduron tolerance in foxtail barley was controlled by three complementary factors. Cultivar differences in growth response to CME are evidence for genetic control of this characteristic. Control of environmental conditions during and following herbicide application is often impossible; however, the development of cucumber cultivars with a relatively high degree of tolerance to CME may be possible. Knowledge of the inheritance of CME tolerance would facilitate the development of highly tolerant cultivars. The purpose of this investigation was to identify tolerant lines, and to determine the inheritance of tolerance to CME and the feasibility of incorporating this tolerance into new and existing cultivars of cucumber. MATERIALS AND METHODS Plant material and treatment. More than 200 inbred lines, plant introductions, and cultivars of cucumber were examined for tolerance to CME in preliminary experiments. An emulsifiable concentrate containing 0.9 kg/liter of chloramben as the methyl ester was used throughout this investigation. A treatment concentration of 3.12 ppm herbicide in Hoagland's no. 1 (10) aerated nutrient solu- tion culture was used in the greenhouse studies, while rates of 0, 2.2, 4.5, and 6.7 kg/ha were applied in the field during 3 growing seasons. Seeds were germinated in moist vermiculite for all greenhouse experiments. After 10 days, when the first true leaf had expanded to about 0.5 cm, seedlings were transplanted to the nutrient solution culture. Herbicide was added 1 day after transplanting. Plants were grown for 3 weeks under a day and night regime of approximately 27°C and 21°C respectively, with supplemental fluorescent light provided during the l4-hr day. Fe was added to the nutrient solution every 3 days, and distilled water was added to maintain the desired level. Screening tests and all genetic studies were conducted in 6 polyethylene 32 33 trays, 74 x 117 x 11 cm, covered with stainless steel lids containing 84 holes each. Studies using carboxy-labeled l4C-CME revealed that sufficient herbicide was available at the concentration used to eliminate differential com- petition among lines for the available herbicide. Low and high scoring lines from the greenhouse water culture test reacted similarly in the replicated field test. Injury was assessed 3 weeks after herbicide treatment and was indexed by degree of epinasty, apical inhibition, callus formation, root inhibition and discolora- tion, and size (weight) reduction. There were 3 to 7- fold differences, dependent upon the trait measured, among lines tested, as related to performance of the control (untreated). Two susceptible, MSU 3159 and MSU 0866, and 2 tolerant, MSU 0612 and MSU 3207, lines were selected for use as parental material (Figure 1). Inheritance study. Selfed single plant progeny of the 4 parental lines were increased asexually to obtain enough plants of each genotype to make a complete 4 x 4 diallel with reciprocals. For each possible cross, 10 populations were used to obtain genetic data: P1’ P2, Fl, Fl', BCl' BCl', BCZ' BC2', F2, F2'. To test each family, herbicide was added to 5 trays. A 6th tray without herbicide served as control. Each population was sampled 8 to 10 times per tank. The experimental design for 34 Figure l.--Reactions of tolerant MSU 0612 and susceptible MSU 3159 twenty-one days after treatment with 3.12 ppm amiben methyl ester (chloramben methyl ester). Plants from left to right are control, treated, control, and treated. $5 32 1 ma am: m> H. noH.Hmm.| mmo.wmm.| mmo.Hmm.| eHo.HHm.I mmo.HmH.H| mao.wvv.u memo sz x Naeo sz mmo.wov.u mmo.fimo.l mmo.HmN.I mHo.Hmm.l mmo.wme.l mao.fimo. memo sz x bomm sz mmo.Hme.| oao.wea.| mmo.wvm.| moo.wmo.| hHo.Hmm.l moo.wvo.u mmam sz x NHeo sz emo.wmm.u mvo.wao. mmo.Hmo.I «Ho.weo.n mmo.wem.n mao.flam. mmam 3m: x homm sz munmflmz mun mmo.u~m. omo.Hmm. mmo.won. mao.Hmo. mmo.wmm. oao.wmo.a memo 3m: x Naeo sz mmo.wom. mmo.wmo.a emo.wwm. emo.wmm. mmo.wem. HHo.HHN.H memo sz x nomm sz Nmo.fimm. Nvo.wmo.a hmo.wmo.a ooo.wom.a mmo.fiav. ooo.wvm.a mmam sz x maeo sz avo.wom. Hmo.wno.a mmo.wom. mmo.wmm. mmo.wmm. moo.HHN.H mmam sz x nomm sz mummems unwed mvo.woa. mao.wnm. mmo.wov. mao.u>m. nmo.wma.| moo.Hme. memo sz x mHmo sz Hmo.umm. mmo.anm. nmo.fimv. moo.wmv. vmo.nao. moo.wom. memo sz x homm sz mmo.w~m. mHo.Hmm. mmo.wev. Hao.wvm. «Ho.Hmo. moo.wom. mmam 0m: x mHmo sz mao.wve. mHo.Hmm. mHo.me. hoo.wmm. mao.wbm. moo.HHe. mmam sz x homm sz HHMSmH> mom Hum ah ah mm Hm peepsum mmmmouu mcofiumasmom .ucwsumwuu mzo mcfl3oHH0m mxmwB m usmflmz who pom .unmwwn Danae .mocmummmem HmSmH> new cmbmu mmmmouo umbesoso “sow Eoum mcoflumasmom mum tam .Hom .mm .Hm .mucmumm mo muouum pumpcmum pan mammZDu.H mqmae 40 TABLE 2.--Estimated values of components of variation and degree of dominance for visual appearance, plant height, and dry weight of four cucumber crosses 3 weeks following treatment with CME. Trait Evaluated Crosses Studied Visual ,Height Dry Weight Environmental variance (E) MSU 3207 x MSU 3159 0.0038 0.0206 0.0156 MSU 0612 x MSU 3159 0.0045 0.0091 0.0060 MSU 3207 x MSU 0866 0.0088 0.0186 0.0120 MSU 0612 x MSU 0866 0.0203 0.0120 0.0140 Dominance variance (H) MSU 3207 x MSU 3159 0.0104 0.0127 0.0325 MSU 0612 x MSU 3159 0.0348 0.1396 0.0353 MSU 3207 x MSU 0866 0.0423 0.0520 0.0522 MSU 0612 x MSU 0866 0.0220 0.0363 0.0347 Additive variance (D) MSU 3207 x MSU 3159 0.0039 0.0604 0.0331 MSU 0612 x MSU 3159 0.0283 0.1390 0.0268 MSU 3207 x MSU 0866 0.0338 0.0662 0.0278 MSU 0612 x MSU 0866 0.0708 0.0826 0.0308 Dominance ratio MSU 3207 x MSU 3159 1.6397 0.9183 0.9895 MSU 0612 x MSU 3159 1.1100 1.0022 1.1482 MSU 3207 x MSU 0866 1.1183 0.8864 1.3702 MSU 0612 x MSU 0866 0.5572 0.6627 1.0623 41 TABLE 3.-—Estimated values of heritability and probable number of effective factors for tolerance to CME of 4 cucumber crosses evaluated 3 weeks after herbicide treatment. Trait Evaluated Crosses Studied Visual Height Dry Weight MSU MSU MSU MSU MSU MSU MSU MSU 3207 0612 3207 0612 3207 0612 3207 0612 x MSU 3159 broad sense narrow sense x MSU 3159 broad sense narrow sense x MSU 0866 broad sense narrow sense x MSU 0866 broad sense narrow sense X MSU 3159 Castle Mather Wright Burton 1921 1971 1968 1951 x MSU 3159 Castle Mather Wright Burton 1921 1971 1968 1951 x MSU 0866 Castle Mather Wright Burton 1921 1971 1968 1951 x MSU 0866 Castle Mather Wright Burton 1921 1971 1968 1951 Heritability (hz) for F 42 equal in the other crosses, as reflected by the correspond- ing dominance ratios (Table 2). All crosses exhibited relatively high heritability (Table 3). The minimum number of effective factor pairs involved in plant height as an expression of tolerance ranged from 1 to 2, depending on the particular cross involved and the method of calculation employed. Gene estimates for MSU 3207 x MSU 3159 were higher than those of the other crosses. Dry weight. Partial dominance of genes controlling plant weight following herbicide treatment was evident from the F mean values in all 4 crosses being higher 1 than their correspondimgndd-parentvalues, and closer to the mean of the tolerant parents (Table 1). Variances of the BC2 populations were generally larger than those of the BCl populations. The dominance variances were higher than additive variances in all crosses, excepting MSU 3207 x MSU 3159 (Table 2). The estimate of narrow sense heritability in the cross MSU 3207 x MSU 0866 was low (Table 3). The probable number of genes controlling the expression of tolerance as measured by plant dry weight ranged from 1 to 5. In general, more genes appeared to be segregating in the expression of dry weight than in either of the other traits measured. Transgressive combination and other observations. The F1 plants from crosses of the 2 susceptible lines, 43 MSU 3159 x MSU 0866, were no more or less susceptible than either of the parents. The pOpulations from crosses of the 2 tolerant lines, MSU 0612 x MSU 3207, were tested under conditions of environmental stress (low temperature and light intensity) and under the conditions employed throughout this investigation. Under conditions of stress the F1 population means exceeded those of the parental means, thus exhibiting physiological homeostasis. When grown under normal conditions, the F1 individuals did not exceed either of the tolerant parents. Approximately 4% of the F population gave indication of transgressive 2 recombination for both tolerance and susceptibility. DISCUSSION Several assumptions are made when the additive- dominance model (11) and the various methods (1, 2, 11, 19) of estimating the minimum number of effective factors are employed. If not all of these assumptions are met, the results obtained may be confounded. BC variances are used in estimating the number of genetic factors in the Mather (11) and Wright (19) formulas. Where BC data is available, these methods tend to be more reliable than the other 2 methods employed in this investigation (1, 2). Those estimates including BC variances generally suggested polygenic control of CME tolerance, and were consistently higher than those not including these variances. The results illustrate one of the problems in evaluating a collection of plant material. It is difficult to determine which trait is the most reliable measure of tolerance, as each may be reflecting a different response. Plants of one line exhibited only slight apical inhibi- tion, yet showed severe epinasty in the few leaves present. Conversely, other lines were stunted and bushy with little or no epinasty, also suggesting a complex pattern of inheritance. It must be considered that plant height and weight may reflect genes segregating for these traits 44 45 irrespective of gene segregation for CME tolerance. Also, heterosis was observed in control Fl populations from all 4 crosses. The visual rating system was possibly the most accurate, since it is a type of discriminate function which includes primarily epinasty but also general plant appearance. The various responses observed may not have been pleiotropic per s3. Significant differences in tolerance were observed among some reciprocal crosses; however, it was difficult to attribute these differences conclusively to cytoplasmic effects. These differences may have been confounded by seed of low viability obtained from plants taken from cuttings. Studies to determine the physiological basis for tolerance of cucumber to CME with these same lines showed that the tolerance of MSU 3207 and MSU 0612 resulted from 2 distinct physiological mechanisms (13). Regardless of susceptible parent used or trait studied, crosses involving MSU 0612 consistently exhibited higher narrow sense heritability than those involving MSU 3207. Results obtained in these studies would suggest that generaliza- tions regarding the entire cultivar spectrum of a species can be misleading when based on the reactions of a single cultivar. 46 The results showed that both dominance and additive gene action play a role in the inheritance of tolerance to CME. The relatively high narrow sense heritabilities, particularly in crosses involving MSU 0612, indicate that considerable progress can be made by selection for CME tolerance when the criteria are plant dry weight, plant height (apical inhibition), or epinasty. These results support the idea of Weibe and Hayes (18) that plant breeding and genetics can play an important role in developing agricultural chemicals for use on crop plants. 10. 11. LITERATURE CITED Burton, G. W. 1951. Quantitative inheritance in pearl millet (Pennisetum glaucum). Agron. J. 43:409-417. Castle, W. E. 1921. An improved method of estimating the number of genetic factors concerned in cases of blending inheritance. Science 54:223. Coleman, 0. H. and J. L. Dean. 1964. Inheritance of resistance to methyl parathion in sorgo. Crop Sci. 4:371-372. Comstock, V. E. and R. N. Andersen. 1968. An inheritance study of tolerance to atrazine in a cross of flax (Linum usitatissimum L.) Crop Sci. 8:508-509. Gardenhire, J. H. and M. E. Daniel. 1970. Inheritance of reaction to toxaphene in oats. Crop Sci. 10:299-300. Grogan, C. O., E. F. Eastin, and R. D. Palmer. 1963. Inheritance of susceptibility of a line of maize to simazine and atrazine. Crop Sci. 3:451. Hammerton, J. L. 1967. Intra-specific variations in susceptibility to herbicides. Ghent. Rijksfac. Landbouwwetensch. Meded. 1967:999-1012. Hayes, J. D. 1959. Varietal resistance to spray damage in barley. Nature 183:551—552. , R. K. Pfeiffer, and M. S. Rana. 1965. The genetic response of barley to DDT and barban and its significance in crop protection. Weed Res. 5:191-206. Hoagland, D. R. and D. I. Arnon. 1950. The water- culture method for growing plants without soil. Calif. Agr. Expt. Sta. Circ. 347. 32 pp. Mather, K. and J. L. Jinks. 11971. Biometrical Genetics. Cornell University Press, Ithaca, N.Y. 382 pp. 47 12. 13. 14. 15. 16. 17. 18. 19. 48 Miller, Jr., J. C. and L. R. Baker. 1971. Differen- tial phytotoxicity of amiben methyl ester to Cucumis sativus lines. (Abstr.) Hort Sci. 6:276. , D. Penner, and L. R. Baker. The basis for variability in cucumber tolerance to chloramben methyl ester. (In preparation). Putnam, A. R. and F. D. Hess. 1971. Techniques for improved weed control efficiency in cucumbers. Mich. Agr. Expt. Sta. Research Report 140. 7 pp. Ricelli-Mattei, M. 1971. Differential phytotoxic reaction of sorghum cultivars to insecticides. I. Genetic resistance to trichlorfon. Crop Sci. 11:923-926. Schooler, A. B., A. R. Bell, and J. D. Nalewaja. 1972. Inheritance of siduron tolerance in foxtail barley. Weed Sci. 20:167-169. Stafford, R. E., V. E. Comstock, and J. H. Ford. 1968. Inheritance of tolerance in flax (Linum usitatissimum L.) treated with MCPA. Crop Sci. 8:423-426. Wiebe, G. A. and J. D. Hayes. 1960. The role of genetics in the use of agricultural chemicals. Agron. J. 52:685-686. ‘ Wright, S. 1968. Genetic and Biometric Foundations. Vol. 1, Evolution and Genetics of Populations, a treatise in three volumes. University of Chicago Press, Chicago, I11. 469 pp. ..cl‘ l. hll I! I: I I i] ll. 1 I l l I l l l I 'II I l ill-l 1'!“ I l.1|lvlu|li I