THE GROWTH AND PROTEIN CONTENT OF GRAIN :CROPS AFTER TREATMENT OF SEED WITH TRIAZINES PART I VACUUM INFILTRATION. OF SIMAZINE INTO BARLEY SEED. PART II SEED TREATMENT OF GRAINS WITH TRIAZINES Thesis for the Degree of M. S. MICHIGAN. STATE UNIVERSITY HARRY L. SEWARD 1972 "ii-DESI? 1-“ ‘ ; BINDING av HOAB & SONS‘ “ 800K emumv me. LIBRARY BINDERS ABSTRACT THE GROWTH AND PROTEIN CONTENT OF GRAIN CROPS AFTER TREATMENT OF SEED WITH TRIAZINES BY Harry L. Seward PART I VACUUM INFILTRATION OF SIMAZINE INTO BARLEY SEED ELarker' barley (Hordeum vulgare L.) was soaked and vacuum infiltrated in solutions of simazine (2-chloro—4,6— bis(ethylamino)-§rtriazine) to determine the feasibility of these methods for improving the protein content and/or yield. Absorption of water was closely correlated with uptake of simazine for both soaking and vacuum infiltration. More water and simazine were taken up by vacuum infiltration than by soaking. Uptake of simazine was positively corre— lated with the concentration of the treatment solution for both methods. The increase in uptake of simazine resulting from vacuum infiltration as compared to soaking was retained by seeds planted in soil, but released by seeds soaked in distilled water. The combustion procedure for recovery of 1"C simazine from seeds proved to be more effective than autoclaving after 6 hours of either soaking or vacuum Harry L. Seward infiltration. These studies indicate that a sufficient amount of simazine may be absorbed by the seed to make seed treatment a feasible alternative to spraying. PART II SEED TREATMENT OF GRAINS WITH TRIAZINES Studies were conducted to determine the effects of simazine (2-chloro-4,6-bis(ethylamino)—§ftriazine), OHEMT (2-chloro—4(2-hydroxyethylamino)-6-methylamino—§etriazine, and OHEET (2-chloro-4—ethylamino—6-(2-hydroxyethy1amino)- g—triazine), appIied as seed treatments, on protein content and yield of barley (HOrdeum vulgare L. 'Coho'), oats (Avena sativa L. 'Gary'), and rye (Secale cereale L. 'MSU Exp'I. Seed treatments included vacuum infiltration with aqueous solutions, and soaking in dichloromethane solutions. Solvent controls (water infiltration and soaking in DCM) increased yield, protein content, or total protein in most of these studies. In no instance did infiltration of triazines result in increases of yield or protein content greater than the solvent controls. THE GROWTH AND PROTEIN CONTENT OF GRAIN CROPS AFTER TREATMENT OF SEED WITH TRIAZINES PART I VACUUM INFILTRATION OF SIMAZINE INTO BARLEY SEED PART II SEED TREATMENT OF GRAINS WITH TRIAZINES BY Harry Df'Seward A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1972 ACKNOWLEDGEMENTS Recognition is extended to Drs. S. K. Ries, A. R. Putnam, and C. E. Cress for their assistance as members of my committee and their help in preparation of this thesis. The assistance of Mrs. Violet Wert is also grate— fully appreciated. ii TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . PART I VACUUM INFILTRATION OF SIMAZINE INTO BARLEY SEEDS IMRODUCTION. O O C O O . O O O O O O O O O 0 O C 0 MATERIALS AND METHODS . . . . . . . . . . . . . . RESULTS AND DISCUSSION. . . . . . . . . . . . . . LITERATURE CITED. . . . . . . . . . . . . . . . . PART II SEED TREATMENT OF GRAINS WITH TRIAZINES INTRODUCTION. . . . .'. . . . . . . . . . . . . . METHODS AND MATERIALS . . . . . . . . . . . . . . RESULTS AND DISCUSSION. . . . . . . . . . . . . . LITERATURE CITED. . . . . . . . . . . . . . . . . iii Page iv vi 24 26 28 31 4O LIST OF TABLES TABLE 1. 2. PART I A comparison of the rates of uptake of water and simazine by soaked and infiltrated seeds . . . . The relationship of uptake of 14C simazine with concentration of the treatment solution. . . . . A comparison between autoclaving and combustion procedures for extraction of 140 simazine from soaked and infiltrated seeds . . . . . . . . . . The rate of loss of 14C simazine with the time from barley seeds planted in soil; calculated from decreased retention of simazine by the seeds after different time intervals . . . . . . The rate of loss of 14C simazine with time from barley seeds soaked in distilled water . . . . . PART II The effect of vacuum infiltration of simazine and OHEMT on yield and protein content of barley grown at three locations . . . . . . . . . . . . The effect of seed protein (mg/seed) on seedling vigor of barley obtained from infiltration studies at East Lansing. . . . . . . . . . . . . The effect of vacuum infiltration of simazine and OHEMT on yield and protein content of oats grown at Entrican. . . . . . . . . . . . . . . . The effect of soaking oat seed in DCM and simazine solution of DCM on yield and protein content 0 O O O O I O O O O O O O O O O O O O O 9 iv Page 16 l7 l8 19 22 32 33 34 36 LIST OF TABLES--Continued TABLE 10. 11. Page The effect of soaking barley seed in DCM and simazine solutions of DCM on growth and protein content in the greenhouse (average of 1 and 24 hours). . . . . . . . . . . . . . . . . . . . . 37 The effect of vacuum infiltration of 3 tri— azines on the dry wt and protein content of rye grown in the greenhouse . . . . . . . . . . . . 38 LIST OF FIGURES FIGURE Page 1a. The uptake of water by soaked and infiltrated barley seeds during 6 hours. . . . . . . . . . 9 lb. The uptake of simazine by soaked and infil— trated barley seed during 6 hours. . . . . . . 11 2a. The uptake of water by soaked and infiltrated barley seeds during 24 hours . . . . . . . . . 13 2b. The uptake of simazine by soaked and infil— trated barley seeds during 24 hours. . . . . . l4 3. A comparison of the retention of simazine by soaked and infiltrated barley seed in soil and the release of simazine by soaked and infil- trated barley seed in distilled water. . . . . 21 vi PART I VACUUM INFILTRATION OF SIMAZINE INTO BARLEY SEED INTRODUCTION Increases in both protein content and yield of agronomic crOps have been attained with spray applications of nontoxic levels of simazine (2—chloro-4,6-bis(ethylamino)v§rtriazine) (1,5,11). Further investigations have indicated that the factors necessary for obtaining a consistent maximum response are not understood (7,10,14,16,17). It is possible that a seed treatment prior to planting would assure a more accurate and consistent amount of chemical being made available to the plant. -Absorption of herbicides by seeds has been investigated to determine if sensitivity or tolerance may be related to the rate and amount of herbicides absorbed. Hansen and Buchholtz (3) reported that the amount of 2,4-dichlorophenoxy- acetic acid (2,4—D) absorbed by corn (Zea mays L.) and pea (Pisum sativum L.) seeds was influenced by, but not closely correlated with, water uptake. Uptake of 2,4—D by various seeds has been reported by other investigators (2,6). Swan and Slife (15) found that soybeans (Glycine max L.) soaked in 3—amino-2,5-dichlorobenzoic acid (amiben) contained large. amounts of the herbicide in the cotyledons, but translocated relatively small amounts to the developing seedling. Distribution of labelled 4—iod0phenoxyacetic acid in develop- ing seedlings varied.with seed type (8). Rieder, Buchholtz, and Kust (9), working with soybeans, concluded that various herbicides were absorbed at different rates, and that uptake was directly related to temperature, but not correlated with water uptake. Scott and Phillips (13) reported that seed size influenced diffusion of isoprOpyl m-chlorocarbanilate (chlorOprOpham) into soybean seed. Vacuum infiltration of growth regulators into seeds is a relatively undeve10ped technique. The only reported use of vacuum infiltration on seeds was made by Schwiezer (12). Oat (Avena sativa L.) and wheat (Triticum aestivum L.) seeds were vacuum infiltrated with amino acids; however, no data is available concerning uptake of the amino acids by the seed during infiltration or translocation of the amino acids into the germinated seedlings. The objective of this research was to investigate some of the parameters of soaking and vacuum infiltration as methods of treating grain seed with nontoxic levels of simazine to increase growth and protein content. MATERIALS AND METHODS (Graded barley (Hordeum vulgare L. 'Larker') and aqueous f 14C ring-labelled simazine (2-chloro—4,6-bis- solutions 0 (ethylamino)-§rtriazine) (4.9 uC/mg) were used in these studies. The procedure for vacuum infiltration (11) con- sisted of placing test tubes containing the seeds and solution in a desiccator and applying a vacuum (35 mm Hg of absolute pressure). All infiltrations were done at 20 C and the vacuum was momentarily released every 30 minutes. Solutions were immediately decanted upon removal from the desiccator, and the seeds were rinsed twice with distilled water. Excess water was removed by blotting with paper towels, and the seeds were dried for 24 hours at 43 C. The procedure for soaking was identical except that the seeds were not placed in a partial vacuum. Uptake of water and labelled simazine by both soaked and vacuum infiltrated seeds was measured after i, 1, 3, and 6 hours in a short term study, and after 3, 6, 12, and 24 hours in a second study. Lots composed of 40 seeds were weighed and placed in distilled water. Following treatment, the solutions were decanted and the seeds were centrifuged for 2 minutes at 200 x G in Filterfuge tubes (LaPINE Scien— tific Co., Chicago, Illinois, 60629). These tubes permit excess water to pass through a screen into a lower reservoir during centrifugation. The seeds were weighed and water uptake determined. Uptake of simazine during treatment was measured by placing seeds in a 9.0 wM 14C simazine solution. Seeds were treated at a rate of 10 seeds per 2 ml in the short term study, and 10 seeds per 5 ml in the second study due to the amount of evaporation during the 24 hour infiltra- tion. Labelled simazine was extracted from treated seeds by placing 5 seeds in'2 ml of distilled water and autoclaving for 10 minutes at 18 psi and 121 C. A 1.0 m1 aliquot was then counted. The vaIidity of this extraction method was tested by combusting duplicate samples from the 24 hour study. Samples composed of 5 seeds were weighed and ground to pass through a 40 mesh screen. A 50 mg aliquot was com- busted and the 14CO; was trapped in 15 ml of ethanol- ethanolamine (2:1). Three ml of this solution was counted, and the percent recovery determined by spiking a 50 mg sample of untreated seed meal with 10 ul of 9.0 uM 14C simazine. All solutions containing 14C simazine were added to 15 m1 scintillation solution (4 g BBOT : 1000 ml toluene : 400 ml Triton X—lOO). Cpms were corrected for chemical quenching by preparing quench curves. -After correcting for aliquots used, dpms were converted to ug simazine. Allowances were also made for evaporation of treatment solutions which re— sulted in concentration of these solutions with time. Germination of infiltrated and soaked seeds was deter- mined on seeds treated for i, 3, and 6 hours. Each treatment was replicated 4 times with 50 seeds per replicate. The seeds were placed in petri dishes between filter papers sup- ported on a layer of cotton saturated with distilled water. Germination was recorded after 36 and 48 hours at 24 C. Release of simazine to the soil was studied by measuring the amount of labelled simazine retained by seeds over time. Seeds soaked and infiltrated in 14C simazine were planted in an unsterilized silt loam. Each seed was planted in an in- dividual plastic container (with drainage), and the soil was watered to field capacity. Seeds of both soaked and infil— trated treatments were removed after 6, 18, 30, 42, 66, and 90 hours. The soil was brushed off prior to drying at 43 C for 24 hours, and the 14C simazine was extracted by auto- claving. Each replicate consisted of 3 seeds. The release of 14C simazine from treated seeds was also assayed in distilled water. Treated seeds were placed in 2 ml of distilled water for each of the following times: 6, 18, 30, 42, 66, and 90 hours. A 1.0 ml aliquot of the dis- tilled water was then removed and counted. Each replicate consisted of 5 seeds. The effect of concentration on uptake by the seed was tested by soaking and infiltrating seeds in 2.5, 5.0, and 10.0 uM 14C simazine solutions. Seeds were treated at a rate of 10 seeds per 5 ml of solution. Following rinsing and drying of the seeds, the 14C simazine was extracted by auto- claving. f 14C simazine by barley plants grown in 10"7 Uptake o and 10"8 M simazine solutions for 3 days was studied. Plants were grown as described by Wert (18). A 1.0 ml aliquot of the simazine solution was counted before and after the 3 day growth period in these solutions. Duplicate containers with solutions, but no plants, were also monitored. After 3 days, solutions were brought back up to volume with distilled water, and a 1.0 ml aliquot was counted. Loss of simazine from solutions was calculated and uptake of simazine ex- pressed in ng per plant. .All experiments except for the germination study were done with 3 replications in completely randomized designs. Analyses of variance were computed, and F values calculated for single degrees of freedom. Linear correlations were calculated between relevant parameters and the following code indicates levels of significance : *P = .05 and **P = .01. Individual observations were correlated except in the case of rate studies where means were correlated. RESULTS AND DISCUSSION The uptake of water was increased after 30 minutes of vacuum infiltration as compared to soaking (Figure 1a). This initial increase was maintained with little further change in rate. The uptake of 14C simazine was increased after 1 hour of vacuum infiltration compared with soaking, and the rate of uptake continued to increase up to 6 hours (Figure 1b). Uptake of water in the 24 hour study indi— cated that the same increase observed in the 6 hour study was maintained even after 24 hours of vacuum infiltration (Figure 2a). Uptake of 14C simazine was similarly increased by vacuum infiltration, and this increase was maintained after 24 hours (Figure 2b). This study indicated a maximum increase in rate of simazine uptake due to vacuum infiltra— tion after 3 hours. After 30 minutes of treatment, the rate of simazine up- take per hour was greater for infiltration than for soaking. This may be accounted for by the effect of vacuum on the structural components of the seed resulting in increased water uptake. rApparently, vacuum infiltration predisposes some portion of the seed to more water absorption than does soaking seed (Figure la). This may be related to the Figure la. The uptake of water by soaked and infiltrated barley seeds during 6 hours. The F value for soaking vs. infiltration is significant at the .01 level. UPTAKE (pl/g seen) WATER 300 INFILTRATED .. " SOAKED TOO 3 T'ME (hr) Figure 1a 10 Figure lb. The uptake of simazine by soaked and infiltrated barley seed during 6 hours. The F value for the interaction of time and treatment method is significant at the .01 level. SIMAZINE (pg/g SEED) UPTAKE OF 1.00 .80 .60 11 INFILTRATED I 3 TIME (hr) Figure lb " SOAKED 12 Figure 2a. The uptake of water by soaked and infiltrated . barley seeds during 24 hours. The F value for soaking vs. infiltration is significant at the .01 level. (pl/g SEED) UPTAKE WATER 500 400 200 100 13 INFILTRATED a " SOAKED 12 TIME (hr ) Figure 2a 24 14 Figure 2b. The uptake of simazine by soaked and infiltrated barley seeds during 24 hours. The F value for soaking vs. infiltration is significant at the .01 level. 15 INFILTRATED 2.50 ‘ 3 I.“ I.” U) o: 2.00 } v‘soIIIKED a. I.“ z 1.50 I. < E U) ‘6 1.0. IIJ x < p— A. :3 .50 6 I2 24 TIME (hr) Figure 2b 16 observation of rapid release of gases from seeds during the first 30 minutes of vacuum infiltration. While water uptake associated with vacuum infiltration attains its maximum rate after 30 minutes, simazine uptake does not attain a maximum rate of increase until some time after 3 hours. This may be explained through the possible influence of simazine on absorptive sites under conditions of vacuum infiltration, causing structural changes in some seed food reserves which further alters simazine uptake (9). Although there was an initial lag of herbicide uptake com— pared to water uptake, water uptake and simazine uptake were closely correlated over 24 hours for both soaked and infiltrated seeds (Table l). Haskell and Rogers (4) also found a close correlation between water uptake and herbicide penetration of simazine into various seeds. Table l. A comparison of the rates of uptake of water and simazine by soaked and infiltrated seeds. Rate of Uptake Time Water Simazine interval Soaked Infiltrated Soaked Infiltrated (hr) 0-3 64.3 78.0 21.7 27.0 3-6 27.3 27.3 14.0 13.7 6-12 16.5 16.2 10.3 12.7 12-24 9.3 10.2~ 7.0 6.0 Correlations between water and simazine uptake r = .986* .980* 17 Uptake of simazine was found to be directly correlated to the concentration of the treatment solution for both soaking and vacuum infiltration (Table 2). This indicates two methods for controlling the amount of simazine absorbed by the seed : time and concentration of the treatment solu- tion. Table 2. The relationship of uptake of 14C simazine with concentration of the treatment solution. Simazine Uptake by Seeds Concentration Soaked Infiltrated (PM) (ng/g seed) 2.5 130 133 5.0 239 248 10.0 430 513 Correlation with simazine concentration r = .997** .995** A comparison of extraction methods indicated that both f 14C simazine due to vacuum in- show an increase in uptake 0 filtration (Table 3). Recovery was the same for seed treat- ments of 3 and 6 hours with both methods of extraction, but was reduced for seed treatments of 12 and 24 hours for ex- tractions done by autoclaving. Increases of vacuum infiltra- tion over soaking are not affected by this reduction in recovery. Neither method indicated a significant interaction 18 .meu no mmmaoummmn mooaume coauomuuxm Avon How Hm>ma Ho. mnu um ucmuwmwsmam ma coaumnuaflwsw cam msflxmom smm3wmn mocmummwao mnp How msam> m mass 0.0H m.h X sowumflum> mo ucmfiowmmmoo ha. am. on.~ mm.a UmumupaflmsH mm.m wo.a pmxmom em mm. - ma. mm.H m¢.a omumuuaflmsH mm.a 5N.H cmxmom NH ma. 0H. NN.a 0H.H woumuuawmsH no.H oo.H Umxmom m . . Hm. mm. smumuuaemcH EH OH me. me. cmxmom m Apmmm m\mjv Apmmm.m\m1v Aunv coaumoneoo om>maoou94 coaumonsou om>mauouo¢ useEummHa mEflB ImGMXmom Hm>o svmuomnuxm ucsosd Ummm sowumuuafimcH mo mmmmuusH . .mvmmm Umumnuaflmsw can UmMm0m EDHM mewNmEHm Des mo newuumunxm Mom umuovmooum coaumsneoo paw mcw>mauousm com3umn comflHmmEOU 4 .m wanna 19 of time with method of treatment (soaking vs. infiltration) and the coefficients of variation were similar. The rate of release of simazine by seeds, as measured by decreased retention of simazine by seeds in soil, was the same for both soaked and infiltrated seeds (Table 4). Table 4. The rate of loss of 14C simazine with the time from barley seeds planted in soil; calculated from decreased retention of simazine by the seeds after different time intervals. Time Seed Treatment Intervals Soaked -Infi1trated (hr) ' (ng simazine/g seed/hr) 0-6 700.0 700.0 6-18 3.0 5.0 18—30 5.0 4.0 30-42 2.5 2.5 42-66 1.6 1.2 66-90 0.4 1.2 Since the rate of release was the same, and infiltrated seeds contained a larger amount of simazine initially, more sima— zine was retained by infiltrated seeds than by soaked seedA in soil (Figure 3). Reledse of simazine by seeds in dis- tilled water indicated a different phenomenon. Infiltrated seeds released more simazine than soaked seeds in distilled water (Figure 3). This was accounted for by an increased rate of release during the first 6 hours (Table 5). Figure 3. 20 A comparison of the retention of simazine by soaked and infiltrated barley seed in soil and the release of simazine by soaked and infil- trated barley seed in distilled water. The F value for soak vs. infiltration is sig- nificant at the .01 level for both studies. .70 '0 o ed) 0 U! E (us/9 Se SIMAZINE O N b o .10 21 /* Infiltrated/t /—* Soaked ‘ Released by Seed (water) p Retained by Seed (soil) 'fi \ Infiltrated . *\ Soaked \ T' ——43 IO 20 30 40 50 60 70 80 90 TIME (hr) Figure 3 22 Table 5. The rate of loss of 14C simazine with time from barley seeds soaked in distilled water. Time Seed Treatment Interval Soaked Infiltrated (hr) (ng simazine/g seed/hr) 0-6 400.0 470.0 e-ia 3.0 5.0 18-30 0.0 4.0 30-42 1.6 0.0 42-66 3.7 1.7 66-90 0.8 1.7 The conflicting results in the release of simazine in dis- tilled water compared to soil may indicate that the presence of solutes affects release to the soil solution. Both studies revealed however, that simazine was readily released by both soaked and vacuum infiltrated seeds. .Germination was not affected by soaking or vacuum infil— tration. The average germination over a period of 6 hours of treatment was 97% for soaked seed and 96% for infiltrated seed. Wert (18) has observed increased plant growth of barley grown in nutrient cultures after 3 days in 10'7 and 10‘9 M simazine solutions. 'Barley plants, similarly grown, removed 8 ng of 14C simazine per plant from 10‘8 M simazine solutions and 56 ng of 14C simazine per plant from 10‘7 M simazine solu— tions. Uptake was not measured in 10"9 M solutions because of insufficient activity. Vacuum infiltration or soaking in 23 a 9.0 qusimazine solution for 3 hours results in uptake of approximately 30 ng of simazine per seed. This would indicate that seed treatment may provide sufficient chemical to affect the plant under field conditions and thus serve as an alternative to spraying. These studies indicated that vacuum infiltration of barley seeds resulted in increased simazine uptake, but give no indication of the distribution of this increase in the seed. Further work should be directed toward investi— gating the effect of infiltration on distribution of chemicals in the seed; specifically on whether there are structural components of the seed which are absorbing water and simazine under conditions of vacuum infiltration. 10. LITERATURE CITED Allinson, D. W., and R. A. Peters. 1970. Influence of simazine on crude protein and cellulose content and yield of forage grasses. Agron. J. 62:246-250. Everson, L. 1950. Further studies on the effect of 2,4-D on seeds. Proc. Assoc. of Seed Anal. 40:84—87. Hansen, J. R., and K. P. Buchholtz. 1952. Absorption of 2,4-D by corn and pea seeds. Agron. J. 44:493— 496. Haskell, D. A. and B. J. Rogers. 1960. The entry of herbicides into seeds. Proc. North Centr. Weed Contr. Conf. 17:39. Kay, Burgess L. 1971. Atrazine and simazine increase yield and quality of range forage. Weed Sci. 19:370-371. Mitchell, J..W., and J. W. Brown. 1947. Relative sensi— tivity of dormant and germinating seeds to 2,4-D. Science 106:266-267. Monson, W. G., G. W. Burton, W. S. Wilkinson, and S. W. Dumford. 1971. Effect of N fertilization and simazine on yield, protein, amino—acid content, and carotenoid pigments of coastal bermudagrass. Agron. J. 63:928—930. Rakitin, Y., and A. K. POtapova. 1959. (Penetration of herbicides into plants and their influence on phos- phorus uptake). Fiziologiya restenil. 6(5):614-616. Eng. translation Soviet Plant Physiol. 6(5):621-623. Rieder, G., K. P. Buchholtz, and C. A. Kust. 1970. Uptake of herbicides by soybean seed. Weed Sci. 18(1):101-105. Ries, S. K., O. Moreno, W. F. Meggitt, C. J. Schweizer, and S. A. Ashkar. 1970. Wheat seed protein : Chemical influence on the relationship to subsequent growth and yield in Michigan and Mexico. Agron. J. 62:746-748. 24 11. 12. 13. 14. 15. l6. 17. 18. 25 Ries, S. K., C. J. Schweizer, and H. Chmiel. 1968. The increase of protein content and yield of simazine-treated‘cr0ps in Michigan and Costa Rica. Bio Sci. 18:205—208. Schweizer, C. J. 1970. Seed protein content and amino acid infiltration of grain: Relation to the effect on subsequent seedling growth and yield. Ph. D. Thesis, Michigan State University. Scott, H. D., and R. E. Phillips. 1971. Diffusion and herbicides to seed. Weed Sci. 19(2):128-132. Smith, Dudley T. 1971. Cotton yield and quality follow- ing sublethal applications of simazine and terbacil. Agron. J. 63:945—947. Swan, D. G., and F. W. Slife. 1965. The absorption, translocation, and fate of amiben in soybeans. Weeds. 13:133—138. Tweedy, J. A., A. D. Kern, G. Kaputsa, and D. E. Millis. 1971. Yield and nitrogen content of wheat and sorghum treated with different rates of nitrogen fertilizer and herbicides. Agron. J. 62:216-218. Vergara, B. S., M. Miller, and E. Avelino. 1970° Effect of simazine on protein content of rice grain (Oryza sativa L.). Agron. J. 62:269-272. Wert, V. Unpublished data. PART II SEED TREATMENT OF GRAINS WITH TRIAZINES INTRODUCTION Variable results have been obtained from applications of (nontoxic levels of) g-triazines to agronomic crops in an effort to increase protein content and/or yield. Ries, Schweizer, and Chmiel (11) reported increases in both yield and protein content of various agronomic crOps with spray applications of simazine (2-chloro—4,6-bis(ethylamino)—§— triazine). Increases in crude protein with no accompanying decrease in yield resulting from spray applications of simazine on forage grasses was reported by Allinson and Peters (1). Tweedy et a1. (15) failed to increase protein content or yield of wheat (Triticum aestivum L.) and sorghum (Sorghum vulgare L. Moench) sprayed with simazine, atrazine (2-chloro-4-ethylamino-6-iSOpropylamino-setriazine), and terbacil (3-tertbuty1-5-chloro—6-methyluracil). Monson et a1. (9) reported that spray applications of simazine on coastal bermudagrass (Cynoden dactylon L. Pers.) had no effect on dry matter yield, protein yield, or protein percentage. Vergara, Miller, and Avelino (16) increased percent protein in rice grain (Oryza sativa L.) with simazine applied in the irriga— tion water but also reduced yield. Foliar application and soil incorporation of terbacil and simazine produced no increase in growth or quality of cotton (Gossypium hirsutum L.) (14). Ries et a1. (10) reported inconsistent results 26 27 following application of simazine and terbacil on wheat in Michigan. These studies indicate that the factors necessary for obtaining a consistent maximum response are not understood. It is possible that seed treatment prior to planting would assure that a more accurate and consistent amount of chemical would be available to the plant. Treatment of seeds with biologically active compounds has been investigated by several workers (2,4,5,12). Schweizer (12) reported the use of vacuum infiltration as a method of treating grain seeds with amino acids for improve— ment of growth and yield. Meyer and Mayer (8) introduced the use of dichloromethane (DCM) as a solvent for chemical treat- ment of seeds. It was reported as having no effect on germi— nation or respiration of lettuce (Lactuca sativa L. 'Grand Rapids') or peas (Pisum sativum L. 'Alaska'). The objective of this research was to evaluate the use of vacuum infiltration of seeds, in aqueous solutions of triazines, and soaking of seeds, in dichloromethane solutions of triazines, on the improvement of yield and protein content of several grain crops. METHODS AND MATERIALS Barley (Hordeum vulgare L. 'Coho'), oats (Avena Sativa L. 'Gary'), and rye (Secale cereale L. 'MSU Exp') were treated with simazine (2—chloro-4,6—bis(ethylamino)-§r triazine), OHEMT (2-chloro-4(2-hydroxyethylamino)-6-methyl- amino-g-trazine), and OHEET (2-chloro-4-ethylamino-6(2—hy- droxyethylamino)-§-triazine). The procedure for vacuum infiltration consisted of plac- ing 60 g of seed in 250 m1 of aqueous treatment solutions for 3 hours at 35 mm of Hg of absolute pressure with six periodic releases (12). The solutions were decanted, and the seeds rinsed twice with distilled water. Excess water was removed by blotting with paper towels, and the seeds were dryed for 12 hours at 43 C. Controls for experiments involving vacuum infiltration consisted of untreated seed, dry seed held under vacuum, and water infiltrated seed. Another seed treatment involved soaking seeds in dichloro- methane (DCM) solutions of simazine (60 g/250 ml). After soaking, the seeds were rinsed twice with DCM, blotted with paper towels, and dryed at room temperature for 24 hours. Controls included untreated seed and DCM soaked seeds (no simazine). 28 29 Field tests were initiated in the Spring of 1971 at three experiment stations located at Sodus, Entrican, and East Lansing, Michigan. Coho barley infiltrated with 20 EM simazine and OHEMT at 20, 100, 200, 400, 800, and 1200 EM concentrations was planted at all three locations. Gary oats, similarly treated, were planted at Entrican, and in addition, Gary oats soaked in 50, 100, 250, and 500 ”M simazine DCM solutions with treatment times of 4 and 24 hours, were also planted. Field plots were arranged in randomized complete block designs with 4 replications. A plot consisted of two rows 7 m long and 25 cm apart. Seed was sown at a rate of 85 kg/ha. Applications of 337 kg/ha of 12-12-12 fertilizer were made at East Lansing and Sodus. At Entrican lupines had been grown in 1970 followed by a rye cover crop: thus no supple— mental fertilizer was applied. After harvesting, the seed was cleaned, air dried, and weighed. Samples of each plot were ground to pass through a 40 mesh screen, and total nitrogen was determined by an automatic micro—kjeldahl procedure (3). Rye (MSU Exp), infiltrated with simazine, OHEMT, and OHEET at concentrations of .05, .1, .5, l, and 5 EM, and Coho barley, soaked in 20 and 100 wM simazine solutions with treat— ment times of 4 and 24 hours, were grown under greenhouse conditions. Seed samples of the 3 controls from the harvested barley field experiment at East Lansing were grown in the 3O greenhouse to determine if the increases in mg of protein per seed among these treatments would result in increased seedling vigor as previously reported for wheat (9). The rye experiment was grown in an unsterilized silt loam and the barley experiment in vermiculite. Seeds were sown at a rate of 10 seeds per pot and thinned to 5 seedlings. Application of 150 ml of 3 mM (N) 50% Hoaglands solution (6) per pot was made to the seedling vigor study after thinning. Weekly applications were made to the DCM barley experiment. The rye plants were harvested after 6 weeks and the barley DCM study was terminated after 5 weeks. The seedling vigor study was harvested after 21 days. After drying for 72 hours at 43 C, plants were weighed, ground, and total N was determined. The greenhouse rye study and the seedling vigor study were randomized complete block designs with 4 replications. The greenhouse barley study (DCM) was a split plot design with 5 replicates. The main split was for time of treatment and the minor split was for chemical treatment. The analysis of variance was computed according to the experimental design. Where a significant F value for treat— ments was obtained, Duncan's Multiple Range Test was used to determine the difference between means. Where applicable nonorthogonal comparisons were made, the linear correlations were calculated between relevant parameters. The following code indicates levels of significance: *P = °05, and **P = .01. RESULTS AND DISCUSSION Field tests at 3 locations resulted in no effect of vacuum infiltration of 'Coho' barley with simazine and OHEMT upon yield (Table 6). Levels of OHEMT up to 1200 HM resulted in no reduction of yield. Protein content (mg/g) was increased over the untreated control by 20 and 800 nM OHEMT, and by the water infiltrated control. A set of non— orthogonal comparisons indicated that total protein (kg/ha) was increased over the untreated control by the water in— filtration alone. Vacuum infiltration of simazine and OHEMT did not increase total protein per hectare. A subsequent greenhouse study indicated that the seedling vigor of seeds obtained from the 3 control treat- ments at East Lansing was closely correlated with the protein content of the seed (Table 7). This is the first reported evidence that seedling vigor in barley is related to seed protein. The yield was reduced by levels of 800 and 1200 EM OHEMT infiltrated into Gary oats planted at Entrican (Table 8). There were no increases in yield over the untreated control, and it is possible that these levels were generally too high as indicated by the toxicity of the higher levels of OHEMT 31 32 Table 6. The effect of vacuum infiltration of simazine and OHEMT on yield and protein content of barley grown at three locations. Treatment Yield (kg/hectare) _ Chemical Infiltration Sodus Entrican East Lansing X (IIM) Control 987 1577 2883 1816 Air 1133 1470 3300 1968 Water 1107 1623 3180 1970 Simazine 20 1013 1500 2720 1744 OHEMT 20 933 1320 2937 1730 OHEMT 100 1040 1397 2813 1750 OHEMT 200 967 1437 3467 1957 OHEMT 400 1073 1630 3147 1950 ~OHEMT 800 960 1343 2483 1595 OHEMT 1200 1000 1373 3183 1852 Protein content (mg/g) 1 Control 161 158 122 147 a' Air 173 161 121 ' 152 ab Water 168 #119 #140 159 bc Simazine 20 178 165 121 155 abc OHEMT 20 177 172 127 159 be OHEMT 100 171 161 132 155 abc OHEMT 200 179 163 129 157 abc OHEMT 400 176 165 119 153 abc OHEMT 800 178 174 134 162 c OHEMT 1200 175 164 121 153 abc Total Protein (kg/hectare) Control 159 249 353 254 2 Air 202 235 379 272 Water 182 276 450 303 Simazine 20 179 247 328 251 OHEMT 20 165 229 376 257 OHEMT 100 175 225 369 256 OHEMT 200 172. 237 456 288 OHEMT 400 188 267 371 275 OHEMT 800 168 234 346 249 OHEMT 1200 174 226 386 262 1Values in a column followed by the same letter are not signif— icantly different at the .01 level using Duncan's Multiple Range Test. 2F value for comparison of control vs. water infiltration significant at the .01 level. 33 Table 7. The effect of seed protein (mg/seed) on seedling vigor of barley obtained from infiltration studies at East Lansing. Seed Seed Seed Seedling 1 Seed Repli— Weight Protein Protein Weight Treatment cate (mg/seed) (mg/g) (mg/seed) (mg/plant) Control I 42.9 105 4.40 28 II 42.1 105 4.42 29 III 43.5 141 6.13 36 a IV 43.9 135 5.93 37 Air I 43.3 115 4.98 33 II 44.1 127 5.60 30 III 43.5 113 4.91 32 a IV 43.3 121 5.24 33 Water I 42.7 123 5.25 34 II 44.8 118 5.29 34 III 46.0 152 6.99 41 b IV 42.8 157 6.80 39 1Values in a column followed by the same letter are not sig— nificantly different at the .05 level using Duncan's Multiple Range Test. 2Correlation of seed protein and seedling wt significant at the .01 level r = .915. 34 Table 8. The effect of vacuum infiltration of simazine and OHEMT on yield and protein content of oats grown at Entrican. Treatment Chemical Infiltration Yield Protein (HM) (hg/ha) (mg/9) (kg/ha) Control 1180 ab 2 148 1 174 ab Air 1264 a 153 190 ab Water 1142 ab 159 181 ab Simazine 20 1316 a 159 209 a OHEMT 20 1198 ab 159 191 ab OHEMT 100 1166 ab 158 184 ab OHEMT 200 1111 ab 159 176 ab OHEMT 400 961 bc 166 157 b OHEMT 800 860 cd 163 142 be OHEMT 1200 623 d 163 100 c 1Values in a column fOllowed by the same letter are not nificantly different at the .05 level using Duncan's Multiple Range Test. sig- 2F value for nonorthogonal comparison of 400, 800, and 1200 EM OHEMT vs. control significant at the .05 level. 35 in this experiment. This reduction in yield was accompanied by increased protein content as indicated by comparison of the untreated control versus 400, 800, and 1200 wM OHEMT. A reduction in total protein per hectare was produced by 1200 nM OHEMT. Vacuum infiltration of simazine and OHEMT did not increase total protein in this experiment. The re- duction in yield and total protein per hectare associated with high levels of OHEMT indicate that vacuum infiltration in this experiment results in sufficient levels of chemical availability to affect the plant: in this case causing injury. There was no effect of water infiltration (no triazines) on yield or protein content in this experiment. Gary oats soaked in simazine solutions (DMC) and planted at Entrican yielded the same as the DCM soaked control (Table 9). Nonorthogonal comparisons indicated that the 4 hour DCM control resulted in an increased yield over the untreated control, and the 24 hour DCM control increased protein content over the untreated contfol. The,4 hour DCM control increased total protein per hectare as a result of the increased yield. Seeds soaked in simazine solutions produced no increases in total protein over the DCM controls. It should be noted that no toxicity occurred even at treatment rates of 250 and 500 UM.of simazine. Barley seed soaked in simazine solutions (DCM) and grown in the greenhouse in scil, were no larger than the control (Table 10). However, soaking seeds in DCM without simazine 36 Table 9. The effect of soaking oat seed in DCM and simazine solution of DCM on yield and protein content. Treatment Simazine Time Yield Protein (UM) (hr) (kg/ha) (mg/g) (kg/ha) o — 1269 1 132 1 167 1 0 4 1728 146 255 50 4 1647 140 230 100 4 1604 142 225 250 4 1334 137 185 500 4 1571 139 225 0 24 1375 154 218 50 24 1426 143 203 100 24 1510 151 228 250 24 1596 156 248 500 24 1402 142 202 lF value for comparison of control vs. all DCM treatments is significant at the .05 level. 37 Table 10. The effect of soaking barley seed in DCM and simazine solutions of DCM on growth and protein content in the greenhouse (average of 1 and 24 hours). Treatment Solvent Simazine Dry wt Protein (UM) (mg/plant) (mg/g) (mg/plant) - — 79 b 1 183 14.6 DCM 0 92 a 186 17.0 DCM 20 . 88 ab 184 16.2 DCM 100 78 b 195 15.2 1Values in a column followed by the same letter are not sig- nificantly different at the .05 level using Duncan's Multiple Range Test. did produce larger plants than the control. There was no effect on either protein content or total protein by either simazine treatments or DCM. There was no difference between treatment times of 1 hour and 24 hours. Vacuum infiltration of 3 getriazines into rye seed increased the dry weight of plants at levels of 10"7 and 10-5 M compared to the untreated control and air infiltration control (Table 11). Protein content was increased by 10"7 and 10'6 M concentrations of simazine compared to the un- treated and air infiltrated controls. OHEMT and OHEET did not increase protein content. The total protein per plant was increased by all 3 triazines at 10‘6 and 10"7 M concen- trations. These increases can not however, be considered as 38 Table 11. The effect of vacuum infiltration of 3 triazines on the dry wt and protein content of rye grown in the greenhouse. Treatment Chemical ‘_ (HM) Simazine OHEMT OHEET x Dry wt (mg/plant) Control 178 1 178 1 178 2 178 Air 165 165 165 165 Water 195 195 195 195 0.05 223 169 189 194 0.10 194 191 215 200 0.50 189 210 217 205 1.00 234 218 218 223 5.00 189 209 202 200 Protein (mg/g) Control 131 1 131 131 131 Air 141 141 141 141 Water 141 141 141 141 0.05 138 131 142 137 0.10 142 135 137 138 0.50 151 145 131 142 1.00 138 135 123 132 5.00 151 135 144 143 Protein (mg/plant) Control 23.4 2 23.4 1 23.4 1 23.4 Air 23.6 23.6 23.6 23.6 Water 27.6 27.6 27.6 27.6 0.05 30.7 22.9 26.8 26.8 0.10 27.4 25.8 29.6 27.6 0.50 28.6 30.6 28.8 29.3 1.00 31.8 29.2 26.9 29.3 5.00 28.6 28.6 29.3 28.8 1F value for comparison of control and air versus all vacuum infiltrations significant at .05 level. 2F value for comparison of control and air versus all vacuum infiltrations significant at .01 level. 39 resulting from infiltration of triazines since none of these chemicals significantly increased yield, protein content, or total protein over the water infiltrated control. Perhaps the most significant finding in these studies was that water infiltration and soaking seeds in DCM often increased yield, protein content, or total protein. A hy- pothesis for these findings is offered by May, Milthorpe, and Milthorpe (7). Grain seed soaked in water for lengthy intervals (2 to 3 cycles of 24 hours) and air dryed produced significantly increased above-ground growth and yield. A greater response occurred when the plants were subjected to water stress. No workers however, have determined whether protein content or total protein was increased by this treat- ment. It must be noted however, that early emergence was associated with at least some of these results. This was not the case with water infiltration of DCM soaking. 10. LITERATURE CITED Allinson, D. W., and R. A. Peters. 1970. Influence of simazine on crude protein and cellulose content and yield of forage grasses. Agron. J. 62:246-250. Brown, R. T. 1967. The influence of naturally occurring compounds on germination and growth of Jack Pine. Ecology. 48:542-546. Ferrari, A. 1960. Nitrogen determination by a continu- ous digestion and analysis system. N. Y. Acad. Sci. 87:792—800. Grace, N. H. 1939. Treatment of plant seed. Canada Patent 383,345- Aug. 15 (Chem. Abstr. 33:8350). Heyl, G. E. 1937. Treating seeds with fertilizing and fungicidal substances. U. S. Patent 2,081,667. May 25, and U. S. Patent 2,083,065 June 5. (Chem. Abstr. 31:5096). Hoagland, D. R. and D. I. Arnon. 1938. The water culture method for growing plants without soil. Calif. Agri. Exp. Sta. Circ. 347. May, H. L., E. J. Milthorpe, and F. L. Milthorpe. 1962. Pre-sowing hardening of plants to drought. Field CrOp Abstr. 115(2):93-98. Meyer H. and A. M. Mayer. 1971. Permeation of dry seeds with chemicals: Use of dichloromethane. Science. 171:583-584. Monson, W. G., G. W. Burton, W. S. Wilkinson, and S. W. Dumford. 1971. Effect of N fertilization and sima— zine on yield, protein, amino acid content, and carotenoid pigments of coastal bermudagrass. Agron° J. 63:928-930. Ries, S. K., 0. Moreno, W. F. Meggit, C. J. Schweizer, and S. A. Ashkar. 1970. Wheat seed protein: Chemical influence on and relationship to subsequent growth and yield in Michigan and Mexico. Agron. J. 62:746—748. 40 ll. 12. 13. 14. 15. 16. 41 Ries, S. K., C. J. Schweizer, and H. Chmiel. 1968. The increase of protein content and yield of simazine treated cr0ps in Michigan and Costa Rica. Bio Sci. 18:205-208. Schweizer, C. J. 1970. Seed protein content and amino acid infiltration of grain: relation to and effect on subsequent seedling growth and yield. Ph. D. Thesis. Michigan State University. Sinha, R. N. 1969. Effect of presoaking seeds with plant— growth regulators and nutrient solution on dry matter production of rice. Madras Agr. J. 56:16-19. (Chem. Abstr. 71:48512). Smith, Dudley T. 1971. Cotton yield and quality following sublethal applications of simazine and terbacil. Agron. J. 63:945-947. Tweedy, J. A., A. D. Kern, G. Kaputsa, and D. E. Millis. 1971. Yield and nitrogen content of wheat and sorghum treated with different rates of nitrogen fertilizer and herbicides. Agron. J. 63:216-218. Vergara, B. S., M. Miller, and E. Avelino. 1970. Effect of simazine on protein content of rice grain (Oryza sativa L.). Agron. J. 62:269-272. "71111117111111Milt/1117111511111 1117'“