METHOD AND COMPOSITION FOR THE PROMOTION OF LEGUMINOUS PLANT PRODUCTIVITY AND SEED YIELDS

Abstract: Method and composition for the promotion of leguminous plant growth and, more particularly, to increase the seed yields of leguminous plants by treatment of leguminous seeds, seedlings and maturing plants with a growth promoting composition comprising as active ingredient an effective amount of trigonelline.

Description: METHOD AND σ_*_POSITICN FOR THE PROMOTION OF I-EGRMENCϋS EEANT PBODO'l'±Vi'lY AND SEED YIELDS BACKGROUND OF THE INVENTION; This invention generally relates to a method for promoting and regulating the growth, seed yields and productiv¬ ity of leguminous crops by the controlled application of a growth promoting solution containing an effective concentration of trigonelline. Significant increases in seed yields and productivity of leguminous crops such as soybean, pinto bean, garden peas and others, are realized when the seeds or seedlings are treated with the growth regulating composition of the invention particularly during the first- ten days after germination. A dramatic increase in second generation seed -> yields has been found when providing foliar application of the growth promoting composition to first generation legumes. Trigonelline is a naturally occurring compound known to be present in a multitude of plants and animals and can be represented by the formula In. plants, trigonelline has been detected in Gymnospermae, Monocotyledoneae and numerous Dicotyledoneae. ( illeke, U. et al. Phytoche is ry 18, 105 (1979)). The presence of trigonelline has also been detected in animals including mammals which have been found to excrete trigonelline in the urine. It has also been found that trigonelline is typically the most abundant molecule of the pyridine nucleotide metabolic pathway for the production of nicotinamide adenine dinucleotide (NAD) indicating that trigonelline exhibits a regulatory function in NAD biosynthesis and in cell proliferation. (Godavari, H_ R. et " al. Can J. Botany 48, 2267 (1973) and Preiss, J. et al. Journal of American Chemical Society 79, 4246 (1957) ) . Other known components of the pyridine nucleotide metabolic pathway include nicotinic acid and nicotinamide. Although the use of nicotinamide and analogues of nicotinic acid as active ingredients in a plant growth regulator has been- generally disclosed, nicotinamide and nicotinic acid have been found to be relatively ineffective for the promotion of leguminous plant growth particularly when applied under nitro¬ gen-abundant conditions. Accordingly, a real need exists for an effective • and economic composition and method whereby the productivity of legumes is substantially increased. _ SUMMARY AND OBJECTS OF THE INVENTION It is accordingly an object of the present invention to provide an effective and economic composition and method effective for the promotion of growth and overall productivity of leguminous plants. It is a further object to provide a method which is " particularly effective for promoting the growth and productiv¬ ity of leguminous plants under nitrogen-abundant conditions. It is still a further object to provide a metho whereby the trigonelline concentration of leguminous plan seeds is increased. It is yet a further object to provide a metho whereby second generation seed yields are significantly in creased. By this invention a novel and effective method fo promoting leguminous plant growth is provided whereby a growt promoting composition containing an effective trigonelline concentration is controllably administered to leguminous plants and, more particularly, to the seeds, seedlings or maturing plants thereof. Although it has been known for some time that trigonelline is present in a plant body, a determination of its precise function in the biological metabolism of plants has not been sufficiently investigated and correlated. However, as the result of extensive research and development it has recently been discovered that when applying trigonelline in carefully controlled concentrations to leguminous plants the plant productivity and seed yields is unexpectedly and substantially increased. This very surprising discovery is the result of considerable qualitative and quantitative investigation and analysis of experiments conducted by us to determine the hormonal activity of trigonelline. In the course of our investigation it has been determined that trigonelline promotes preferential cell arrest in the G2 phase of the cell cycle for approximately 40% of the cell population in root meristems of Pisuiri sativu . Trigonelline which is present in ungerminated • ORE- seeds is transported to the roots and shoots from cotyledons during early seedling development. Although concentrations of other components of the pyridine nucleotide metabolic pathway for NAD production such as nicotinic acid and nicotinamide have been found to promote cell arrest in G2 when added to a suit¬ able culture medium, it has been discovered that trigonelline exhibits the greatest positive effect on 'cell arrest. It is presently believed that nicotinic acid and nicotinamide are converted to trigonelline thereby producing an effective concentration for promotion of cell arrest. Accordingly, trigonelline has been found to be the first chemically iden¬ tified plant hormone which controls the cell cycle in plants or animals. (Tramontano et al. "Relationship Between Trigonelline Concentration and Promotion of Cell Arrest in G2 in Cultured Roots of Pisum sativum", Phytochemistry, Vol. 21, No. 6, pp 1201-1206 (1982); Evans, L.S. and Tramontano, .A. , "Is Trigonelline a Plant Hormone", American Journal of Botany, 68 (9) , 1282-1289 (1981)) . It has further been determined that the concentration of trigonelline in germinated seeds varies among different plant species. In an effort to determine the ubiquity of trigonelline in ungerminated seeds of different plant species to ascertain which particular species exhibit a positive response to trigonelline as a plant hormone, ungerminated seeds of various plant species were ground and extracted with ethanol in known manner and the trigonelline concentration measured. By conventional procedures, trigonelline was extracted and samples were injected into a high pressure liquid chromatograph (HPLC) T with appropriate trigonelline standards. The results set forth in Table 1 indicate that high trigonelline concen¬ trations, i.e., greater than 70 μg trigonelline per gram of tissue, are present in ungerminated seeds of all legumes tested except peanut. Correspondingly, relatively low trigonelline concentrations were detected in the cereal grain crops tested. The data of Table 1 indicates that legumes contain significant concentrations of trigonelline in their seeds demonstrating a propensity of leguminous crops to respond positively to exogenous applications of trigonelline. -6- TABLE 1 Concentration of trigonelline in ungerminated.seeds of several plant species Plant Species Concentration of Trigonelline (μg g -1 tissue) Legumes Garden peas (Pisum sativum) 227 Broad bean (Viσia faba) 294 Soybean (Glycine max) 71.8 Pinto bean (Phaseolus vulgaris) 344 White bean (Phaseolus vulgaris) 554 Peanut (Arachis hypogea) 8.7 Wing bean (Psophocarpus tetragonolobus) 384 Lentils (Vicia augustifolia) 363 Non-legumes - - Dicotyledonous plants Radish (Raphanus sativus) 7.6 Cereal crops Barley (Hordeum vulgaris) 8.9 Corn (Zea mays) 4.0 Rye (Serale cereale) 52 Sorghum (Sorgum vulgare) 14.9 Wheat (Triticum aestivum) not detectable Concentration levels of trigonelline, nicotinic acid and nicotinamide in plant tissues from ungerminated seeds to 10 day old- seedlings have also been investigated for Pisum sativum. It was found that the total quantity of trigonelline from ungerminated seeds to 7 day old seedlings was in the range of 70 to 81 μg with an increase in trigonelline to 101 μg in 10 day old seedlings. By comparison, nicotinic acid concen¬ trations were approximately 10% of the trigonelline -7- concentration and the concentration of nicotinamide was less than 1% of the trigonelline concentration. On the basis of these findings, it was determined that application of trigonelline to the seeds or seedlings of legumes particularly during the first ten days after germination resulted in unexpectedly and significantly in¬ creased plant productivity. Specific experiments demonstrating the unexpected effectiveness of trigonelline on the growth and productivity of legumes are presented below. It has further been found that foliar applications to maturing plants of effective amounts of trigonelline produces seeds, which upon planting will result in greater plant produc¬ tivity. Particularly effective results have been realized when the trigonelline composition is applied during the pod filling period or that period from inception of pod elongation until seed enlargement is substantially complete. DESCRIPTION OF PREFERRED EMBODIMENTS The preparation and application of effective growth regulating compositions containing trigonelline to leguminous plants in accordance with the present invention are set forth below to clearly demonstrate the unexpectedly superior results obtainable by the method of our invention. It is to be clearly understood that the scope of the invention is not intended to be limited by these specific embodiments. The biological activity of trigonelline, as a plant growth hormone, has been investigated with respect to a number of representative legumes. In all cases the plants were carefully maintained under environmental conditions condusive -8- for good plant growth. For purposes of experimentation, seeds of garden pea (Pisum sativum) , Soybean (Glycine max) and bean (Phaseolous vulgaris) were planted in a commercial soil mix¬ ture. Four seeds were placed in four inch diameter plastic pots with only one seedling from each pot allowed to survive after the second week. The soils were watered with a water solution either with or without test compounds as necessary from initial soil wetting to final harvest at maturity. The plants were inoculated with an effective concentration of Rhizobium inoculum and 0.25 g of a suitable peat-bacterium mixture was applied to each pot. The plants were watered with the test solutions as needed and, on average, each pot received 100 ml. during each routine watering. The plants were harvest¬ ed when shoots, leaves and pods were completely senescent. The numbers of mature pods and seeds and both fresh and dry masses of mature seeds were determined on an analytic balance accurate to 0.01 g for each plant. > EXPERIMENT 1 The plant productivity of soybean and pinto bean was measured as compared to a suitable control with the results being presented in Table 2. The basic water solution used was a 25% strength standard Hoagland solution which omitted all sources of nitrogen. The soybean (Glycine max) and pinto beans (Phaseolus vulgaris) were exposed to an aqueous solution containing a 10 molar trigonelline concentration as a soil drench. After exposure to these two solutions throughout the entire growing season the seed yields of the plants were measuredr -9- TABLE 2 Seed yields of soybean (Glycine max) and pinto beans (Phaseolus vulgaris) Plant Yield (g plant "1 ) Control 10~ M trigonelline Soybean 0.93 1.32 Pinto bean 1.49 1.81 The results of Table 2 demonstrate that application of a trigonelline solution of 10~ molar concentration (0.39 g. per plant over the entire growing season) as a soil drench to soybeans and pinto beans significantly increases plant produc¬ tivity, as measured by seed yield, approximately 21% for pinto beans and 42% for soybeans as compared to the control. EXPERIMENT 2 In this experiment the productivity of garden peas (Pisum sativum) was determined when exposed to an aqueous solution containing a 10 " molar concentration (0.11 g. per plant over the entire growing season) of trigonelline as a soil drench. The basic watering solution was a 25% strength stan¬ dard Hoagland solution which omitted all sources of nitrogen. The plants were exposed to the solutions either with or without trigonelline throughout the entire growing season. The results of this experiment are shown in Table 3. -10- TABLE 3 Seed yields of garden peas (Pisum sativum) Mean number Mean number Mean number Mean seed -Mean seed of seeds of pods of seeds (fresh) (fresh) per plant per plant per pod biαnass biαπass per plant per seed First experiment without trigonelline 4.65 2.25 2.06 1. 843 0.396 with trigonelline 5.0 2.28 2.19 2.248 0.448 Second experiment without trigonelline 3.15 1.45 2.17 1.28 0 .366 with trigonelline 4.95 2.05 2.41 1.81 0. 406 EXPERIMENT 3 In this experiment the comparative seed yields of garden peas (Pisum sativum) when exposed for 1 week of watering 3 times at 100 ml per application with 10 * molar concen¬ trations of trigonelline (0.03 g. per plant), nicotinic acid (0.02 g. per plant) and nicotinamide (0.02 g. per plant), in the presence of nitrogen, was determined. The basic watering solution was 25% strength standard Hoagland solution which included nitrogen (+N) at an amount of approximately 1.56 grams nitrogen per pot. In each case the solutions were applied as a soil drench. The results of this experiment are shown in Table 4. QMPI -11- TABLE 4 Seed yields of garden peas (Pisum sativum) Substance Yield (g seed plant "* ) +N Water only 0.35 10 Trigonelline 0.55 10~ Nicotinic acid 0.46 -3 10 Nicotinamide 0.32 Analysis of the data set forth in Table 4 shows that the plant productivity in the case of application of trigonelline, as measured in seed yield, increased approxi¬ mately 57% when compared with the control. Nicotinic acid exhibited a considerably smaller effect on productivity while nicotinamide showed no increase at all. EXPERIMENT 4 This experiment was conducted to determine the growth regulating effect of trigonelline, nicotinic acid and nicotinamide on soybeans (Glycine max) . In this experiment the plant productivity, as measured by seed yield, was determined upon treatment with trigonelline (1.04 g. per plant), nicotinic acid (0.73 g. per plant) and nicotinamide (0.73 g. per plant) at a molar concentration of 10 " applied throughout the entire growing season. The basic watering solution was a 25% strength standard Hoagland solution which included nitrogen (+N) at an -12- amount of approximately 3.12 grams nitrogen per pot over 80 days. The results are set forth in Table 5. TABLE 5 Seed yields of soybeans (Glycine max) Treatment g seed/plant ' pod #/plant Control 7.8 38 Trigonelline ιo- 3 M 16.4 57 Nicotinic acid ID "3 M 10.5 42 Nicotinamide ID "3 M 13.6 43 The data of Table 5 clearly shows that trigonelline exhibits the greatest positive effect on plant growth. The seed yield increased more than 100% as compared to the control. Correspondingly, the number of pods per plant increased approx¬ imately 66%. Our investigations have shown that treatment of leguminous plants with a growth promoting composition contain¬ ing at least a 10 molar concentration of trigonelline is preferred and results in significantly increased plant produc¬ tivity and, more particularly, dramatically increased seed yield. However, lower concentrations of trigonelline such as -4 10 molar also result in positive increases in plant produc¬ tivity. It has further been discovered that particularly effective results, in accordance with the present invention. OMPI . -13- are achieved by treating leguminous plants with the inventive growth promoting solution containing an effective concentration of trigonelline during the pod filling period which can be defined as the period from inception of pod elongation until seed enlargement is substantially complete. It has been found that after harvesting the mature seeds from plants which have been treated with a trigonelline growth promoting solution as described above the seed yields of the plants grown from such pre-conditioned seeds are unexpectedly and significantly enhanced. The trigonelline containing solution is administered in the form of a foliar , spray to the maturing plants and contains, preferably, a suitable surfactant additive. Experiments were conducted to determine the growth regulating effect on soybeans and garden peas from one gen¬ eration to the next when applying trigonelline to the first generation. Specifically, trigonelline was applied to the foliage (i.e., leaves, stems and pods) of maturing garden pea and soybean plants during the pod filling period and the seeds were then harvested. It has been found as shown in Tables 6 and 7 below that trigonelline is synthesized in the foliage of maturing garden pea and soybean plants and that the trigonelline so formed is transported to the seeds as plant senescence occurs. The trigonelline concentration in plant tissues of garden peas and soybeans is shown in Tables 6 and 7, respectively. -14- TABLE 6 Trigonelline concentration in tissues of Pisum Satvium during ontogeny. Trigonelline Concentration (μg g "* fresh mass) Plant age Roots Shoots Leaf Pods Seeds (days) 20 6.3+0.2 24.2+5.3 88.2+6.0 30 6.743.0 19.8+1.0 28.0±0.0 40 0.7+0.2 3.5±0.2 11.1+3.0 50 • * h J 1.2+0.5 20.2±0.0 37.7+7.0 127.6+2.2 60 1.0+0.6 * 8.8±4.6 5.8±4.8 166.4+16.0 Immature seeds were not separated from the pods at this point because seeds were very small and would have an insignificant mass compared with the mass of the pods. Values represent mean and standard error, respectively. Asterisks denote samples below 0.5 μg g~ tissue which is the lowest limit of detection of trigonelline with the procedures used. -15- TABLE 7 Trigonelline concentration in fresh tissues of. Glycine max during ontogeny. Plant cage Triejonelline concentration (μg g~ fresh mass) (days) Roots Stems Leaf Pods Seeds 20 *b/ * 63.2±4.4 - < • - 30 * * 40.3+5.6 - - * 40 * . * 48.8+2.2 - - 50 * * 27.8+2.3 - - 60 * * 39.4+2.0 16.2+3.2 a - 70 * 7.6±1.0 19.5±1.9 14.8±1.4 a - 80 1, .1+0.05 1.5+1.0 18.3+11.6 5.3+0.4 a/ - 90 * 2.0+1.0 10.8+0.4 4.5+0.5 19, .7+2.6 100 * 3.0+0.5 10.5±4.5 3.712.0 21. .7±3.3 Immature seeds were not separated from the pods at this point because seeds were very small and would have an insignificant mass compared with the mass of the pods. Values represent mean and standard error, respectively. •___. • . Asterisks denote samples below 0.5 μg g~ tissue which is the lowest limit of detection of trigonelline with the procedures used. Very importantly it has further been found that the foliar application of trigonelline to the maturing plants during the pod filling period significantly increases the trigonelline concentration in the seeds above natural levels of trigonelline as shown in Table 8. Each foliar application contained approximately 500 ml of a 10~ 3 M trigonelline concentration (.09 g. trigonelline) and approximately 3 g. of a -16- suitable surfactant and was applied to fifty garden pea plants. In the case of soybeans, each foliar application contained approximately 2000 ml of a 10 M trigonelline concentration {.35 g. trigonelline) and a suitable surfactant was applied to thirty soybean plants. TABLE 8 Concentration of trigonelline in seeds of Pisum sativum or Glycine max exposed to foliar sprays of trigonelline during pod filling Pisum Sativum # of sprays trig. cone, (μg g -1 tissue) 0 227.0 2 667.8 3 533.3 4 475.3 6 629.5 Glycine Max 0 71.8 2 165 4 145 6 370 By foliar application as described above of trigonelline to the maturing pea plants during the first generation it was found that the concentration of trigonelline in seedling roots was significantly increased. The results of -17- these experiments are set forth in Table 9. The data presented in Table 9 shows that the trigonelline concentrations in seedling roots were increased by a factor of from 2 to 3 times the normal trigonelline concentrations. TABLE 9 Trigonelline concentrations in seeds, cotyledons and roots of resultant seedlings from pea plants of the previous generation that received foliar application of trigonelline during pod filling. Trigonelline cone, (μg g~ tissue) Planted seeds Control 227 Three foliar applications 533 Four foliar applications 475 Seedling cotyledons (3-day-old _seedlingsj_ Control 217 Three foliar applications 200 Four foliar applications 197 Seedling roots (3-day-old ' seedlingsj Control 131 Three foliar applications 304 . Four foliar applications 447 -18- By foliar application as described above of trigonelline to the maturing soybean plant during the first generation it was found that the seed yields of field grown soybeans in the next generation were unexpectedly and dramat¬ ically increased. The results of these experiments are set forth in Table 10. Table 10 Seed yield of second generation soybean plants in which foliage of the first generation was exposed to trigonelline applica¬ tions Plant population Seed mass Seed mass density (grams per (kg per (plants per plant) hectare) hectare) Control plants 5.90xl0 5 8.81 5,198 Trigonelline exposed ς plants* 6.4x10 s 9.53 6,099 * Six lθ " ~ M trigonelline applications (2.1 g. trigonelline) were provided to thirty first generation plants over a 10 day period during the pod filling period. The data presented in Table 10 shows that the seed yield of soybeans in the second generation increased 17%, calculated as seed mass per hectare, when treating the foliage of the first generation with trigonelline. More particularly, the trigonelline foliar application increased the plant popu¬ lation density 8.5% demonstrating that trigonelline promotes seedling survival and increased the seed yields of individual plants 8.2%. -19- In the case of garden peas, trigonelline applications to the foliage of the first generation of garden peas increased seed yields of field grown plants in the second generation as shown in Table 11. TABLE 11 Seed yields of second generation pea plants in which foliage of the first generation was exposed to trigonelline application Number of plants Total seed mass per Treatment per 5 meters of row 5 meters of row (grams) Control 58 268 #1 treated* 124 450 #2 treated* 70 193 #3 treated* 88 381 * #1 treated - Two 10 *" M trigonelline foliar applications (0.174 g. trigonelline total) were provided to fifty plants 12 and 13 days prior to harvest. #2 treated - Two 10~ M trigonelline foliar applications (0.174 g. trigonelline total) were provided to fifty plants 9 and 10 days prior to harvest. #3 treated - Six 10 M trigonelline foliar applications (0.52 g. trigonelline total) were provided to fifty plants 18 and 26 days prior to harvest. When plants were exposed to trigonelline just 9 and 10 days prior to harvest (#2 treated) no increases in seed yields were obtained. However, when two trigonelline applica¬ tions were made 12 and 13 days prior to harvest (#1 treated) , a yield increase of 68% was obtained. Further, when six trigonelline applications were made between 18 and 26 days prior to harvest (#3 treated) , a yield increase of 42% was found. These data demonstrate that foliar trigonelline appli¬ cations during the pod filling period (i.e., #1 treated and #3 treated) of the first generation result in dramatically in¬ creased seed yields of garden pea plants in the second gen¬ eration. As is clearly demonstrated by the above experiments, foliar application of trigonelline on soybeans and garden peas increase the trigonelline concentration in harvested seeds of the first generation. Particularly effective results have been obtained with foliar applications of trigonelline during the pod filling period for both soybeans and garden peas. Such . exogenous trigonelline applications increase the endogenous trigonelline concentrations in seeds of the first generation. When seeds of the first generation are germinated higher than normal trigonelline concentrations are present in seedling roots. When such seeds are cultivated under standard agronomic conditions, the increased trigonelline concentration in these seeds and seedling roots increases both seedling survival and seed yields of plants in the second generation. In this manner foliar application of trigonelline in the first generation increases the seed yields of treated soybean and garden pea plants in the second generation. It is of course to be understood that the foregoing description is intended to be illustrative only in that the embodiments described can be modified in various ways within the scope of the invention. For example, the growth promoter comprising an effective concentration of trigonelline can be applied either continuously or by pulse addition to the leguminous plants, seeds or seedlings to promote and regulate their growth.

Claims: WE CLAIM: 1. A method for the promotion of leguminous plant growth which comprises administering to a' first generation leguminous plant a growth promoting amount of a composition containing an effective amount of trigonelline as active growth promoter, harvesting seeds from said first generation leguminous plant and cultivating said harvested seeds to produce a second generation leguminous plant having increased seed yield. 2. The method of claim 1 wherein the active growth promoter is administered by foliar application to said first generation leguminous plant. 3. The method of claim 2 wherein said growth promot¬ er is administered to the first generation leguminous plant during the pod filling period. 4. The method of claim 1 wherein the active growth promoter is administered at a molar concentration in the range of 10 ""4 to lθ "3 . 5. The method of claim 1 wherein the active growth promoter is administered at a molar concentration of about 10~ 3 . 6. The method of claim 1 wherein said leguminous -plant- is—selected from the group consisting of Pisum sativum, -Vicia faba, Glycine max, Phaseolus vulgaris, Arachis hypogea, Psophocarpus tetragonolobus and Vicia augustifolia. 7. The method of claim 3 wherein said leguminous plant is Pisum sativum or Glycine max. 8. The method of claim 1 wherein said growth promoter is applied under nitrogen abundant conditions. 9. A method for the promotion of leguminous plant growth which comprises treating leguminous plant seeds and seedlings for the first ten days following seed germination with a growth promoting amount of a composition containing an effective amount of trigonelline as active growth promoter. 10. The method of claim 8 wherein the active growth promoter is employed at a molar concentration of at least 10 -4 11. The method of claim 8 wherein the active • growth promoter is employed at a molar concentration in the range of 12. The method of claim 8 wherein the active growth promoter is employed at a molar concentration of about 10 "" . 13. The method of claim 8 wherein said leguminous plant seeds are selected from the group consisting of Pisum sativum, Vicia faba, Glycine max, Phaseolus vulgaris, Arachis hypogea, Psophocarpus tetragonolobus and .Vicia augustifolia. 14. The method of claim 8 wherein said trigonelline is applied as a soil drench. 15. The method of claim 9 wherein said growth promoter is applied under nitrogen abundant conditions.

Inventors: EVANS LANCE S, TRAMONTANO WILLIAM A, EVANS, LANCE, S., TRAMONTANO, WILLIAM, A.
Applicants: MANHATTAN COLLEGE
Application year: 1983
Main classification: A01N43
IPC codes: A01N43