w.‘—v—w --c- -m— ‘ I‘M a ‘ ‘ k 3 (M \ | mm — — ~_-_ _ ; , — \ t ”I STUDYES ON ‘EHS Effié‘ff» £333 YEA?E&A?URES .iND MGM‘EC GHEMKAL fifi'fi‘ii‘ifififiéfi m 35%? #élefltTéGN {SF '3“Hi'1 DRY HES‘J.’ iphawaius .vuéflafia L. amfi P. scufiéasim inifiii‘i-Jx‘ {warts W um 3:16 Sages 5&9? {89 a wigs-is 3m} 3‘21“}; far “fie 8:33:35 a? fit. :3. é‘v‘uEHiGfiu‘é $33315 Wi‘iERSETY' Yea-than 3m: “£962 Studies on the Effects of Temperatures and Organic Chemical Supplements on Seed Production of the Dry Bean (Phaseolus vulgaris L. and E. acutifolius latifolius Freem) and Sugar Beet (Beta vulgaris L.) BY Teh-chien Shen A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Farm Crops 1962 ’ (\3 (A) _ - (\3, ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Drs. M. W. Adams, F. W. Snyder and S. T. Dexter for their helpful advice in conducting this investigation. The author deeply appreciates the financial support granted by the Agency for International Development, which enabled him to undertake this investigation. ii Studies on the Effects of Temperatures and Organic Chemical Supplements on Seed Production of the Dry Bean (Phaseolus vulgaris L. and E. acutifolius latifolius Freem) and Sugar Beet (Beta vulgaris L.) BY Teh-chien Shen AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Farm Crops 1962 Approved %7/>%W ABSTRACT Experiments we re conducted to determine the effects of high temperature on the seed yields of nine bean varieties and four sugar beet clones. Ten water-soluble vitamins and nucleic acid hydrolysate were applied as foliar sprays during the time of flowering and seed maturation. Yields of bean and sugar beet were suppressed by the high temperature. Its deleterious effects were enhanced by irregular air movement and competition between plants for light. No effect of the application of organic chemical supplements could be detected due to large experimental error in the data. iv TABLE OF CONTEN TS Page INTRODUCTION . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . 2 METHODS AND MATERIALS . . . . . . . . . . 6 RESULTS. . . . . . . . . . . . . . . . 13 DISCUSSION . . . . . . . . . . . . . . . 24 SUMMARY. . . . . . . . . . . . . . . . 30 LITERATURECITED . , . . . . . . . . . . 31 INTRODUCTION Adaptation of plants to an environment has been considered to depend upon an organism having no inherent deficiency of an essential enzyme or metabolite, or a deficiency induced by the environment (14). The flexibility of the biochemical system of a plant may enable it to adjust to the varying environment. The concept that decreased growth or even death of both higher and lower plants under unfavorable climatic conditions can sometimes be prevented by supplying certain specific metabolites has been supported by the findings of many workers in the past two decades. From the agronomic point of view, the chemical cure of climatic lesions (4) provides a new and prospective avenue of exploration aimed at solving the problem of insufficient food supply for the increasing world population through improving yield in the present cultivated areas or offering a chance to extend crop production to new lands. The main object of these experiments was to determine whether the poor seed yields of bean and sugar beet strains at high temperature could be improved with organic supplements applied as foliar sprays. REVIEW OF LITERAT URE The earliest demonstration of the chemical control of a tem- perature lesion was reported by Bonner (3) in 1943, who grew Cosmos plants at controlled temperatures of 80° and 68°F and applied thiamine in the nutrient solution at a rate of 0. 01 mg per liter. Pos- itive growth responses to added thiamine were obtained at a constant temperature of 68° but not at 80°F, nor at a day temperature of 80° combined with a night temperature of 68°F. It should be noted that temperature conditions favoring luxuriant growth appeared unfavor- able for demonstration of any apparent growth-promoting effect of thiamine. Earlier experiments conducted by the same author and others (1, 2) also showed that the application of vitamin B1 was some- times effective and sometimes not. It was suggested that it was effective only when the plant was low in vitamin B1 and that leaf vita- min content might be used to diagnose whether or not addition of vitamin B1 would promote the growth of a given crop or plant species. Galston and Hand (7) found that adenine stimulated the growth of epicotyl sections, leaf buds and roots of etiolated pea. The thermal inactivation of the growth of excised pea epicotyl-sections was largely prevented by the addition of adenine to the medium. I-lighkin (9) used two genetic strains of peas in studies of heat resistance and the inheritance of heat resistance. On the basis of work by Galston and others, he made measurements to determine whether there was a relationship between purine and pyrimidine -2- -3- composition of these pea strains and their ability to resist heat. The amount of adenine in the heat susceptible variety remained the same at low (14°C) and at high (26°) temperatures, but in the resistant var- iety, the adenine content at 26°C increased at least 100 per cent as compared to that in plants grown at 14°. Galston (8) reported the response of pea plants to adenine. All plants were kept at 23°C during the day but were divided into groups which were subjected to different night temperatures from 2° to 30°. Plants made most rapid growth in length at a night temperature of 17°, growth being inhibited about 35 per cent by a 10° deviation in tempera- ture, either upward or downward. Adenine had no effect at the optimum temperature but it promoted stem elongation slightly both at the higher and the lower temperatures. Similar results were obtained on the fresh weight of the pea stem. Langridge and Griffing (l6) grew different homozygous ecotypes of Arabidopsis thaliana aseptically in growth chambers. They found that the genotypic component of the measurable growth variation be- tween races was positively correlated with temperature. Eight out of 43 races showed a disproportionately large decrease in growth at 31. 5°C. Five of these 8 races had pronounced morphological symptoms of high temperature damage, and 3 gave significantly increased growth at 31. 5° with vitamins, yeast extract, or nucleic acids. The specific stimulating substance for the first two races was found to be biotin, which completely prevented the formation of the temperature lesion; in the third race, a partial alleviation of the temperature lesion resulted from the addition of cytidine. -4- In their recent work, Ketellaper and Bonner (11) grew plants at different temperatures including the optimal temperature, and found that they produced different amounts of dry matter at different temperatures. Further experiments showed that increases in dry weight up to 40 per cent were obtained by application of vitamin B or ribosides to pea plants grown-at 30°C day temperature and 23°C night temperature (30/230C). Application of the vitamin B mixture or thiamine to Cosmos inwcool temperature (17/10 or 20 /14°C) produced a 30 per cent increase in dry weight. Vitamin C was effective at 20/14 and 23/17°c. Much work has been done with lower plants and bacteria be- cause of the extensive background of biochemical studies and the ease of conducting experiments with these microorganisms under controlled conditions. Working with a mutant strain of Neurospora, Mitchell and Houlahan (19) showed that it synthesized riboflavin at a rate approach- ing that of a wild-type strain at temperatures below 25°C while an external source of this vitamin was necessary for normal growth above 28°C. In studies on the growth requirements of an Escherichia coli mutant, Maas and Davis (18) found that pantothenate was required by a certain mutant only at temperatures above 30°C. The mutant was derived from an ordinary auxotroph which requires pantothenate at any temperature. For both strains, pantoate and beta-alanine, the intermediates of pentothenate synthesis, were inactive as growth factors, indicating a block in the last stage of this biological reaction. The authors also found that the pantothenate-synthe sizing enzyme of the mutant was much more heat-labile than that of the wild types. -5- A similar result has been obtained by Horowitz and Fling (10) with Neurospora. The wild strain of Neurospora has a pair of alleles, TS and T1, governing the thermostability of the enzyme tyrosinase. Strains carrying TS produce a tyrosinase characterized by a half-life of at least 30 minutes at 59°C, whereas strains carrying T1 produce a tyrosinase with a half-life of 3 to 4 minutes at the same temperature. The template -modification and rate -modification hypotheses have been mentioned to explain the thermal stability of this enzyme. Campbell (5) has studied the thermal stability of highly purified alpha-amylase produced at 35° and 55°C by two facultative thermophilic bacteria, Bacillus coagulans and B. stearothermophilus. It was found that the enzymes produced at 55° we re more heat stable than those pro- duced at 35°, losing only 6 to 10 per cent of their activity in one hour at 90° whereas the latter preparations lost 90 to 92 per cent of their activity. From his experience on the nature of biological thermostability, Koffler (12) explained biological individuality on the molecular level. Two possibilities were suggested in regard to the existence of life at extremely high temperature. 1. The essential cell components of thermophiles are relatively more stable than those of mesophiles. 2. Rapid resynthe sis is the key to the problem of biological stability to heat. Koffler and his associates (13), using the flagella from E. coli as a model, found evidence supporting the first suggestion that might explain the remarkable heat stability of thermophile protein. METHODS AND MATERIALS Eight commercially grown bean varieties of Phaseolus vulgaris L. , one of E. acutifolius latifolius Freem and four sugar beet clones of different thermal sensitivities were used in the experiments. These experiments were conducted in the Plant Science Green- house of Michigan State University, at East Lansing, Michigan. Dur- ing the time of experimentation, the greenhouse temperatures were controlled by the automatic steam heating system coupled with electrical heaters and manual adjustments of the vents. The screening procedure for detecting a Specific deficiency, suggested by Dr. K. C. Atwood and used successfully by previous workers (17, 15), was adopted in these experiments. Four vitamin solutions having the components shown in Table I and a nucleic acid hydrolysate solution were applied as foliar sprays in all experiments. Stock solutions of ten water-soluble vitamins were prepared at con- Table I. Components of Four Vitamin Solutions Solutions Components Vl riboflavin, nicotinic acid, pantothenic acid, pyridoxine V2 nicotinic acid, biotin, inositol, folic acid V3 pantothenic acid, inositol, ascorbic acid, choline V4 pyridoxine, folic acid, choline, p-aminobenzoic acid centrations as shown in Table 11. Each vitamin solution was prepared by mixing the four component stock solutions in equal volume before applications. Nucleic acid hydrolysate (NA) was prepared from sodium -6... -7- ribonucleate by the method proposed by Lang ridge (15). Five grams of sodium ribonucleate were hydrolysed and made up to one liter of solution. All of these solutions were made weekly and stored in refrigerators. Table II. Concentration of the Stock Solutions of Ten Vitamins Vitamins Concentrations Vitamins Concentrations riboflavin 10 mg /l i-inositol 2000 mg /1 nicotinic acid 50 folic acid 10 calcium pantothenate 10 ascorbic acid 200 pyridoxine hydro- choline chloride 10 chloride 10 p-aminobenzoic acid 10 biotin 0. 5 Plants were grown in a mixture of sand and peat or vermiculite. Complete nutrient solution containing the following salts was supplied two or three times a week. Ca(NO3)2° 4HzO 0.4723 ng/l H3BO3 0.0015 gms/l KHzPO4 0. 068 l MnSO4° 2HzO 0. 00086 KNO3 9. 4044 ZnSO4o 7H20 0. 000 l 1 KCl 0. 2982 CuSO4o 5HzO 0. 00004 NH4H2PO4 0. 2302 (NI-I4)6Mo7Oz4° H20 0. 00006 MgSO4° 7H20 0. 3697 FeSO4o 7H20 0. 0 143 NH4NO3 0. 080 l Sequestrene 0. 02 10 Experiment 1. On October 16, 1961, seeds of seven bean varieties, namely Columbia Pinto (CP), Michelite #62 (M62), California Dark Red Kidney (CDRK), Idaho Sanilac (IS), Red Mexican #35 (RM35), Great Northern #59 (GN59) and #123 (GN123) of P. vulgaris and Tepary Bean (TB) of P. acutifolius latifolius we re sown in ten inch pots filled with the mix- ture of 1:1 builders "sand and peat. Six seeds were sown in each pot. -8... There were eighteen pots for each variety placed adjacent to each other on the floor of the greenhouse. After the development of foliage leaves, seedlings were thinned to four uniform plants per pot. All plants we re kept in a room in the greenhouse at a day temperature varying from 70 to 80°F and a night temperature at approximately 60°F. When a variety started to differentiate flower buds, fourteen very uniform pot cultures were moved into a high temperature room having a day temper- ature kept above 90° and night temperature around 80°F; the other 4 pots were retained in the lower temperature room and served as controls. The fourteen pots of each variety in the high temperature room were divided into six groups. Five groups, each consisting of two pots, received the five spray treatments respectively (Tables I and 11). One of these two pots was sprayed once a week, the other three times a week. The sixth group consisted of four pots and was used as the high temperature control. Pots receiving same treatment were arranged adjacent to each other in plots, but the plots themselves were separated by 18-inch alleyways on two sides, and by 36-inch alleyways on the other two sides. In the high temperature room, a turbulator (Model T24D, ACME Engineering and Manufacturing Corp.) operated by a 1/6 HP electric motor was used for air circulation during the experiment. The date of macroscopic flower bud appearance and the starting date of treatment for the eight varieties are given in Table III. For each spray treatment, 28 ml of vitamin solution or nucleic acid hydrolysate solution were sprayed on the four plants in each pot. All treatments were stopped on January 5, 1962, when the flowering -9- period was over. Plants in the high temperature room were harvested on January 27, 1962. The low temperature controls were harvested on February 17, 1962. Numbers and weights of pods and seeds produced in each pot were recorded. Table 111. Date of Flower Bud Appearance and the Starting Date of Treatment with Sprays for Eight Bean Varieties Varieties Date of flower bud Starting date of spray appearance treatment California Dark Red Kidney Nov. 14, 1961 Nov. 21, 1961 Columbia Pinto Nov. 15 " Great Northern #123 n it Red Mexican #35 H n Great Northern #59 H n Idaho Sanilac Nov. 19 " Michelite #62 " " Tepary Bean Nov. 26 Nov. 28 Experiment 2. A further experiment on bean varieties was started on February 18, 1962, based on the differential varietal responses observed in the first experiment. Three varieties, namely California Small White (CSW), Idaho Sanilac (IS) and Tepary Bean (TB) were used. Two rooms in the greenhouse were kept at the same day temperature (80-90°F) and night temperature (70-75°F). Seeds of each variety were sown in 36 pots, initially spaced as in Experiment 1, in each room. Seedlings were thinned in the same manner as in Experiment 1. The earliest variety, Idaho Sanilac, started to initiate flower buds on March 20, 1962. From that date, the temperature in one room was raised to a day temperature above 90°F and night temperature from 80° to 85°F. In the other room, -10 - the day temperature was reduced to 75° to 80°F and the night temper- ature to approximately 65°F. When spray treatments were started on March 26, 1962, in both high and low temperature rooms, plant cul- tures were very uniform in size in each variety. In each room, the experiment consisted of a split plot experiment. Six treatments (five spray treatments and a control sprayed with distilled water) were applied as main plots distributed at random in 2 replications. Each main plot consisted of 3 varieu’es in a latin square of 3 replications. Individual pots in plots were 5 inches apart in one direction and 8 inches in the other. Main plots were 18 inches apart in one dire'c1ion and 36 inches apart in the other. Plants were sprayed twice a week. From March 26 to April 8, 1962, 250 ml of solution were applied to each main plot. After April 11, the volume was increased to 500 ml per plot, and after April 25, the volume was increased to 800 ml per plot, because the plants had grown so large that the original volume could not be sprayed evenly over all leaves. Spray treatments were stopped on May 16, 1962. In the high temperature room, the Sanilac plants were harvested on May 12; in the low temperature room on May 25, 1962. Plants of the other variefies, bearing mature pods at that time, still continued vegetative growth at both temperatures and started secondary flowering at the lower temperature. Nutrient supply was reduced to once a week after May 16, 1962; watering was stOpped on May 29, 1962. Plants were harvested on May 31 in both temperature rooms. -11- Experiment 3. Sugar beet cuttings of four clones, namely 59-2, 30-1, 137-11 and 137-8, were made on September 29, 1961. On December 2, 1961, 30 cuttings of each clone were transplanted into ten-inch pots filled with about two inches of sand at the bottom and vermiculite on top. One plant was planted in each pot. These plants were photothermally induced at 48°F under continuous light from December 26, 1961 to March 6, 1962. During this treatment, many plants which were small and weak died. The surviving plants were then grown in a cool greenhouse temperature until bolting. After bolting, plants were paired according to their size and then separated into two groups. One group was moved into the high tem- perature room of Experiment 2; another group was left in the cooler room. Five or six treatments of vitamin and nucleic acid hydrolysate solutions were applied on the sugar beet plants in both temperatures with one or two replications according to the number of available plants of each clone. The different treatments and number of plants used are shown in Table IV. All treatments were started on April 8, 1962, except the second replication of 59-2 which was started on April 22, 1962. A volume of vitamin solutions or nucleic acid hydrolysate sufficient to cover the leaf surface and inflorescences of the beet plants was applied twice a week as a foliar spray. All treatments ended on May 23, 1962. Plants were harvested on June 27, 1962. Fresh weight of tops and seed yields were recorded. -12- Table IV. Treatments, Replications, and Number of Sugar Beet Plants Used in Experiment 3. Clones Temperature Treatments Replica1ions No. of Plants high and 4 vitamin solutions 2 24 59-2 low nucleic acid and control high and 4 vitamin solutions 2 20 30-1 low and control high and 4 vitamin solutions, 1 12 137-11 low nucleic acid and control RESULTS Experiment 1. Temperatures during the time of treatment and seed maturation are shown in Figure 1. In the first half of this period, air temperature of the high temperature room could not be raised using the steam heating system to above 90°F except on sunny days. After December 29, 1961, two electric heaters were used as a reinforcement. The day tem- peratures could then be raised to above 90°F, however, ,daily fluctuations were still caused by the external weather. After January 13, 1962, daily maxima were recorded as 100° to 110°F. The exposure of the thermo- graph to direct sunshine made the recorded temperatures several degrees higher than the true air temperatures on sunny days. Seed yields of plants in all treatments and at both temperatures are shown in Table V. All of these varieties, except Tepary Bean, performed better at the lower temperature. The differences between yields at these two temperatures were Significant at the l per cent level. Most of the spray treatments tended to increase the bean yields in California Dark Red Kidney, Great Northern #123, Idaho Sanilac, and Tepary Bean, but for the rest, all spray treatments tended to suppress seed production. No trend can be found from the differences between effects of one and three applications per week. At the lower temperature, bean plants of all eight varieties grew Slower and matured later than those at high temperature. Plants at -13- Table V. Total Yields of Seeds (in gms) of Eight Bean Varieties for Diffe rent T reatments . Sprays Varieties Treatments per week CDRK CP GN123 RM35 GN59 IS M62 TB High temp. control# 0 4. 87 16. 37 8. 50 16. 62 6. 87 3. 62 17. 75 12. 12 Low temp. control# 0 17. 13 23. 12 27. 62 32. 37 21. 13 17. 13 26. 37 1. 00 Differences 12.26** 6.75* 19. 12** 15.75% 14.26* 13.51% 8.62** 11. 12* High temp. plus v1 1 5.5 12.5 9.5 12.5 3.5 6.0 13.0 15.0 3 3. 5 13 0 10.0 13 0 5. 0 2. 5 12. 5 13 0 V2 1 4.5 12.0 11.0 11.5 2.0 4.5 9.5 12.5 3 4.0 11 5 9.0 18 0 5.5 5.0 11.5 18 5 v3 1 5. 5 11. 0 13. 5 17.0 6.0 5. 5 10.0 19.0 3 6.5 13 5 12.5 18 0 1. 5 6.0 14.0 17.0 V4 1 5.5 13.0 6.5 14.5 5.0 6.5 11.0 17.0 3 5. 0 14. 0 6. 5 19. 5 7. o 4. 0 12. 5 19 5 NA 1 7. 5 9. 5 12. 5 15. 5 4. 5 2. 5 14. 5 13. 5 3 3.0 11.0 10.0 8.0 4.0 4. 5 4.0 8.0 # Average of 4 pots *, ** Significant at 0. 05, 0. 01 level, respectively .Eoou ousuduoafiou :mE 5 Shawn magma >30 555635 .0 33.1.530on oh .500.” ouBMuomEo» 30.“ can smE “I own." o>m :13 .m can .4. A “:05? 096. mo «03qu mcfiuom poo» pad «sesame: waist ousuduomEou mo acoflmmuflr 3me 4 ouwmrm -15.. zoo . «3. .8. : 232...... .u : ..r2..§. 5 : 7.3.568 5.85252 - q A q T A q q 1 oo 1 Os. 1 8 a. on 1 oo. o p P b p h b P b o.— 5. um 0»de ~16- low temperature had a healthier appearance, and darker green leaves, while those at the high temperature were somewhat elongated, and possessed somewhat yellowish-green leaves. The low yields of the seven bean varieties at high temperature may be attributed to the fact that flower buds of most of these varieties aborted before anthesis. Generally, the bean plants grown at high temperature had fewer pods per pot (four plants), fewer and smaller seeds per pod, and set pods at the higher nodes in comparison with the plants at low temperatures (Table VI). The only exception was the Tepary Bean. It set pods at low temperature but had fewer and poorly developed seeds. Experiment 2. Temperatures for the period of treatment and seed maturation are Shown in Figure 2. Air temperatures were recorded under shade in this experiment. Growth of plants and seed yields were highly variable between replications at high temperature. In each main block, 9 pots of'three varieties were arranged as a 3 x 3 latin square. Plants located along the border usually were larger, leafier and had better yields than those located inside the square. Tepary Bean was more sensitive to the border effect than others. In extreme cases, contrast between replica- tions of 5. 5 to 96.0 gms (Control) and 2. 5 to 80.0 gms (V3) were recorded. -17 - Table VI. Temperature Effects on Yield Constituent of Bean Varieties*. Varieties Character Temperature CDRK CP GN123 RM35 GN59 15 M62 TB No. of pods high 11. 2 20. 0 ll. 5 24. 7 11. 0 17. 7 23. 2 22. 7 low 10. 5 2 l. 2 22. 3 28. 5 l7. 8 3 l. 3 35. 5 18. 5 Weight of high 6. 75 20. 25 10. 50 19. 87 8. 75 4. 87 22. 37 14. 00 pods (gms) low 22.75 27.62 32.75 40.75 24. 50 23. 35 37.75 4.25 No. of seeds high 13. 7 47.0 31.4 58.0 25.2 26.0 90.0 88.0 low 23. 0 48. 2 7 l. 3 92. 7 55. 0 96. 3 136. 5 38. 2 No. of seed high 1. 22 2. 35 2. 73 2. 35 2. 29 l. 47 3. 88 3. 88 per pod low 2. 19 2.27 3. 20 3. 25 3.09 3. 08 3. 84 2.06 Average weight high 0. 36 0. 35 0. 27 0. 29 0. 27 0. 14 0. 20 0. 14 of seed (gms) low 0. 74 0. 48 0. 39 0. 35 0. 38 0. 18 0. l9 0. 02 Node where lst high 4. 7 8. 1 9. l 10. 6 8. 9 7. 0 10. 4 8. l pod was set low 5.4 3. 6 5.4 6.9 6.0 6.0 9.4 8. 7 * Data are averages of 4 pots. .500.” span-mundane» awE a: mug—damage» hep 555-me .U 33 103309." on .500.“ ousumuomF-ou 30- find 5m? 5 owmuo>d 336 .m pad 4 .N acme-pong mo powuom wafluomupoom pad ado-human» war-5p ousuduomfiou mo mac-33.19, 33G .N thaw-h 260 «we. .82. .N : :6: a : :34 .853: 4 — T d d a u 8 1 on I I. 8 . m 8 1 - 'Z‘ 1 < x 1 cm - ~ - ~ 0 I I , A _ _ p . . _ e o: J. amuuadwal -19 .. In determining the effects of temperatures, spray treatments and varietal responses, analysis of variance of the data (Table VII) showed highly significant differences between varieties and their inter- action with temperatures (Table VIII). The varietal means in Table IX Show that, only at low temperatures are the differences in seed yields Significant. The yields of these three varieties were very low at high temperature. No significant difference was obtained. Table VII. Seed Yield (in gms) for Different Bean Varieties and Treatments at High and Low Temperatures. High temperature Low temperature Treatments CSW IS TB Total CSW IS TB Total V 1 34. 5 24. 5 6. 5 65. 5 407. 5 86. 5 413. 5 907. 5 V2 31. 5 36. 5 12.5 80. 5 437.0 61. 5 364.5 863.0 V3 34. 0 37. 0 82. 5 153. 5 408. 5 110. 5 37 l. 0 890. 0 V4 46.0 35.0 13.0 94.0 427.0 87. 5 368.0 882. 5 NA 55.5 34.0 49.5 139.0 450.0 66.0 415.0 931.0 Control 38. 5 46. 0 10 l. 5 186. 0 500. 5 95. 5 403. 0 999. 0 Total 240.0 213.0 265.5 718. 5 2630. 5 507. 5 2335.0 5473.0 The yield of Tepary Bean at the lower temperature was much better than in Experiment 1 (see Tables V and IX, 1. 0 gm per pot in Expt. 1 vs. 194. 58 gms per plot or 64. 86 per pot in Expt. 2). It is believed that the high day temperature after May 14, 1962 (Figure 2) favored the development of seeds of this variety. -20- Table VIII. Analysis of Variance of the Total Seed Yields of the Experiments at Both High and Low Temperatures. Sources DF SS MS F Temperatures 1 3 13962. 09 3 13962. 09 415. 420* Replications 2 3748. 75 1874. 38 2. 480 Treatments 5 328 1. 56 656. 3 1 0. 868 Temp. X Treat. 5 510. 23 102. 05 0. 135 Error (a) 10 7557. 73 755. 77 Varieties 2 1 14302. 78 57 15 l. 39 190. 963** Var. X Treat. 10 2 185. 97 2 18. 60 0. 730 Var. X Temp 2 106206. 69 53 103. 34 177. 437** Var. X Treat. X Temp. 10 3683. 30 368. 33 1. 231 Error (b) 24 7182. 65 299.28 Total 71 562621. 75 * Significant at 0. 05 level ** Significant at 0. 0 1 level Table IX. Average Yields (in gms) of Seeds of Three Bean Varieties at High and Low Temperatures. Temperatures Varieties High Low CSW ' 20. 00 , 219.21 IS 17. 75 42. 29 TB 22. 12 194. 58 LSD at 0.05 16. 13 14. 63 LSD at 0.01 22.64 20. 53 The effects of vitamin and nucleic acid treatments were not significant (Table VIII). Further effort was directed toward detecting the effect of the vitamin solutions and nucleic acid hydrolysate. When -21.. the yields of CSW in the low temperature room were analysed separ- ' ately, the effects of treatments were significant at the 5 per cent level (Table X). Statistically significant differences were obtained between mean yields (Table XI), however, the degrees of freedom are too small, in this case, for reliable conclusion. Table X. Analysis of Variance of the Yield of CSW Bean at Low Temperature. Source DF SS MS F Replications 1 6. 02 6. 02 Treatments 5 33 1. 9 1 66. 38 5. 96* Error 5 55. 70 11. 14 Total 11 393. 63 * Significant at 0. 05 level In comparing the data presented in Tables VII or IX, the differ- ences between yields in the high and low temperature rooms are very striking, however, the effects cannot be analysed statistically because no replication was possible for the temperature factor. Table XI. Average Yields (in gms) of CSW Bean Seed from Plants receiving Different Spray Treatments at Low Temperature. Treatments Yields V1 67. 92 V2 72. 84 V3 68. 08 V4 7 1. 16 NA 75. 00 Control 83. 42 LSD at 0. 05 8. 74 LSD at 0.01 13.70 -22- Experiment 3. The seedball yields from the beets are expressed in the form of yield in grams per gram of fresh top, as shown in Table XII, because the different plant sizes at harvesting time made the data variable and interpretation difficult. Table XII. Seedball Yields (gms/gm of fresh top) for Three Sugar Beet Clones and Different Treatments at High and Low Temperatures. Clones Temperature Treatments 59-2* 137-1 1** 30-1* V1 0.000 0.000 0.023 V2 0.000 0.000 0.029 High V3 0.000 0.000 0.019 V4 0.001 0.000 0.011 NA 0.001 0.000 --- Control 0.001 0.000 0.008 V1 0.018 0.024 0. 147 V2 0.011 0.052 0. 158 Low V3 0.034 0.018 0. 161 V4 0.021 0.035 0. 123 NA 0.014 0.037 --- Control 0.015 0.033 0. 139 * Average of two replications ** Data of one replication The most apparent fact is that all three clones produced more seeds per gram of fresh top at the lower temperature. Clone s 59 -2 and 137 -l 1, which have been categorized as poor seed producers at high temperature (22), produced only a few seeds or none at high tem- perature. Vitamin solutions and nucleic acid hydrolysate treatments could not overcome the pronounced effects of environmental factors, -23- but it seems that the yields of clone 30 -1 at high temperature have been improved by the first three vitamin solutions. DISC USSION The hypothesis, first presented by Bonner (4) that the temper- ature-induced deficiencies may be overcome by the application of appropriate chemicals offers new opportunities for agronomic researchers. However, these experiments failed to show whether or not it is applica- ble in the cases of bean and sugar beet seed production. The yields of most bean varieties and sugar beet clones used in these experiments were suppressed by the high temperature. In the first experiment, the spray treatments of vitamin solutions and nucleic acid hydrolysate appeared to improve seed setting in some varieties at high temperature, but it could not be verified in the more detailed studies of Experiment 2. Table XIII. Average Yields (in gms) of Seeds of Different Treatments at High and Low Temperatures. Temperatures Treatments High Low V l 10. 92 15 1. 25 V3 25. 58 148. 33 V4 15. 67 147. 08 NA 23. 17 155. 17 Control 3 l. 00 166. 50 LSD at 0.05 46. 18 34. 57 LSD at 0. 0 l 72. 42 54. 20 In comparing the mean yields between different chemical treat- ments the least significant difference at l per cent level was as high as 234 per cent of the mean yield of the control at high temperature (Table XIII). (It should be pointed out that the LSD in Table XIII was calculated from a generalized error over all treatments and varieties; -24- -25- but since the treatment variation in TB far exceeds the treatment var- iation of the other strains, there is a marked lack of homogeneity of errors, and the LSD as given is not critically valid for any comparisons involving treatments.) The increases of fresh or dry weight in other higher plants induced by similar chemical sprays have been reported to fall at levels below 50 per cent of the weight of control plants (Table XIV). Therefore, it would have been impossible to detect any effect of the supplements from data of this large variability except in the presence of an unusual increase in yield. It is believed that in- adequate environmental control and the resulting high error components masked all the effects that these supplements might have had. Table XIV. Increase of Fresh or Dry Weight in Higher Plants Induced by Organic Supplements. Plants Supplements Effect in increasing References fresh or dry weight of begetative organs Cosmos Vitamin B1 20-48% Bonner (1943) Vitamin B mix- ture 30% Ketellapper and Bonner (1961) thiamine 30% ” Pea adenine 1-13% Galston (1957) Vitamin B 40% Ketellapper and Bonner (1961) ribosides 40% " Arabidop sis vitamins 44% Langridge and thaliana Griffing (1959 Experiments were conducted in the greenhouse. The first diffi- culty encountered in these experiments was the control of temperature. -26- At the beginning of Experiment 1, the temperature in the high temper- ature room could not be raised effectively to a level around 90°F with the steam-heating system. During the time of Experiment 2, the high temperature could me maintained somewhat constant with a new thermo- stat, but in the latter part of this experiment, the hot spring weather after May 14, 1962 made the temperature in the low temperature room impossible to maintain at the low level initially eStabli shed. During the second half of May, the day temperatures usually were above 90°F. Tepary Bean grew well at high temperature and it was the only bean variety which yielded poorly at the lower temperature of Experiment 1. This variety set pods satisfactorily at low temperature (day temperature 70°-80°, night temperature 60°F) but the seeds did not develop well (See Table VI). In Experiment 2, this variety set only some empty pods at low temperature as was expected, but after the middle of May, all seeds developed rapidly as the day temperature increased. Finally, it had a seed yield only somewhat lower than that of California Small White at the same temperature. If there were some effects of the applied supplements on seed setting at low temperature, they may have been masked by the subsequent rapid growth of seeds at the increased day temperature. It is evident that a well regulated greenhouse or growth chamber is necessary for conducting critical experiments of this nature. The second difficulty which was encountered was the limitation of space in the greenhouse in Experiment 2. At the beginning, a split latin square design with six treatments as main plots and 3 varieties as subplots was adopted for balancing the effects of light variation but it was found impracticable to spray the chemical solutions on a plot without contaminating others. The change of the design reduced the degrees of freedom for treatments; and the intra-plot competition was found to be very serious. Idaho Sanilac was the only bush type variety used in Experiment 2. When it was located between plants of the two Vining varieties according to the latin square arrangement in plots, it grew well during the early stage but was finally completely shaded by its neighboring plants which became vegetatively dominant in the latter half of the experimental period. It has been pointed out that, in Experiment 2, the location of plants greatly affected the yields of bean plants grown at high temperature. Although the plants were very uniform in size when spraying with organic supplements was initiated, crowding in subsequent growth led to excessive variation in yield between replicates. Significant differences occurred between yields of plants along the bor- ders and in the inner part of plots in all three varieties (Table XV). The border effects were not appreciable at low temperature. In Experiment 2, bean plants showed luxuriant vegetative growth at high temperature. Plants of the two 1Vining varieties had spindly growth, long inte modes and small yellowish leaves like those of Experiment 1, but they grew more rapidly. The mean temperature at the time of treatment and seed maturation in the high temperature. room we re 84. 53° and 85. 30°F for Experiments 1 and 2, respectively. This difference did not seem to have much effect on the growth habit and yields of bean plants. However, during Experiment 2 the high temperature room was more crowded with bean plants than in Experiment 1. In order to raise the temperature of that room, door and vents were usually closed. The turbulator used for improving ventilation in the high temperature room -23- of Experiment 1 was not available for Experiment 2. Therefore, the possible effects of high relative humidity of stagnant air between plants on transpiration and on preventing the loss of heat of leaves by radiation in the infra-red (6, 21) are worth noting. The prevention of heat transfer between plants and their environment and the re sult- ing high leaf temperature might have enhanced the temperature effect in the high temperature room. Table XV. Border Effect on the Seed Yield (in gms) of Three Bean Varietie s. Location Mean yields Difference between Varieties Temperature of plants per pot mean yields border 8.86 high inside 5. 27 3. 59* CSW border 73.64 low inside 72. 70 0. 94 border 8.33 high inside 3. 7 l 3. 98** IS border 14.61 low inside 13. 77 0. 32 border 16.07 high inside 1. l7 14. 90* TB border 67.73 low inside 62. 8 l 4. 92 * significant at 0. 05 level ** significant at 0. 0 1 level -29- The effects of light can be considered separately according to its effects on different physiological activities. As pointed out by Neale (20), in the glasshouse, there was a correlation between the transpiration rate of tomato, lettuce and carnation plants and the total light radiation. It is possible that the greater amount of light which was available for bean plants along the border would also help in aeration and water relations. It has been reported (23) that under high temperature, bean plants needed a relatively large amount of light energy for photosynthesis. For normal growth, flowering and fruit development, light of 1200-2000 f. c. from above should be supplemented by light of equal intensity from the sides of the plants. Experiment 2 was conducted in winter and early spring under the natural short day and low light intensity. Six 500 W tungsten lamps, at a level about two feet above the top of bean plants, were used in each temperature room as supplementary light from 7 a. m. to 7 p. m. , but these do little to improve the photosynthesis of bean plants. During the course of this experiment, most plants in the high temperature room lost their leaves on the lower portion of stems, except for the plants located along the borders. In the latter case, there is no doubt that those plants had larger photosynthetic area for synthesizing carbohydrates to meet the necessities of rapid vegetative growth and the consumption of a large amount of organic food in the greater respira- tion caused by high temperature. In Experiment 3, no significant effect of treatments was obtained. The experimental plants were propagated asexually. The limited number of plants, their different initial size and deteriorated growth vigor made the data insufficient for a concrete conclusion. SUMMARY Bean varieties and sugar beet clones were grown under high temperature (day temperature 90°-100°, night temperature 80°-85°F) and lower temperature (day temperature 700-800, night temperature 60°-65°F) in the greenhouse during anthesis and seed maturation. Ten water-soluble vitamins and nucleic acid hydrolysate were applied as foliar sprays during anthesis and seed development. 1. Seed yields of bean varieties and sugar beet clones were greater at the lower temperature except for the Tepary Bean which needs a moderately high day temperature (around 90°F) for good seed development. 2. Variations between replications were so great as to obviate the possibility of reliability of observation of effects of chemical supplements. 3. High temperature effects were enhanced by poor ventila- tion and compe1ition between plants for light. -30- 10. ll. 12. 13. LITERATURE CITED Bonner, J., and J. Greene. Experiments on the relation of vitamin B1 to the growth of green plants. Bot. Gaz. 100: 226- 237. 1938. Bonner, J. . and J. Greene. Further experiment on the relation of vitamin B1 to the growth of green plants. Bot. Gaz. 101: 491- 500. 1939. Bonner, J. Effects of application of thiamine to Cosmos. Bot. Gaz. 104: 475‘4790 19430 Bonner. J. The chemical cure of climatic lesions. Engng. Sci. Mag. Campbell, L. L. Crystallization of alpha-amylase from a thermo- philic bacterium. J. Amer. Chem. Soc. 76: 5256. 1954. Curtis, 0. F. Leaf temperature and the cooling of leaves by radiation. Plant Physiol. 11: 343-364. 1936. Galston, A. W. , and M.- E. Hand. Adenine as a growth factor for etiolated peas and its relation to the thermal inactivation of growth. Arch. Biochem. 22: 434-443. 1949. Galston, A. W. In ”The experimental control of plant growth" (Ed. F. W. Went) pp. 313-317. (Chronica Botanica Co. :Waltham, Mass.) 1957. Highkin, H. R. The relationship between temperature resistance and purine and pyrimidine composition in peas. Plant Physiol. Suppl. 32: L. 1957. Horowitz, N. H., and M. Fling. Genetic determination of tyrosinase thermostability in Neurospora. Genetics 38: 360-374. 1953. Ketellapper, H. J., and J. Bonner. The chemical basis of temper- ature responses in plants. Plant Physiol. Suppl. 36: 21. 1961. Koffler, H. Protoplasmic differences between mesophiles and thermophiles. Bact. Revs. 21: 227-240. 1957. Koffler, H. G. E. Mallett, and J. Adye. Molecular basis of biological stability to high temperatures. Proc. Natl. Acad. Sci. 43: 464- 477. 1957. -31- 14. 15. 16. 17, 18. 19. 20. 21. 22. 23. -32- Kurtz, C. B. Biochemistry of Adaptation in Plants to Environ- ment. Amer. Nat. 94: 237-242. 1960. Langridge, J. A hypothesis of developmental selection exemplified by lethal and semi-lethal mutants of Arabidopsis. Aust. J. Biol. Sci. 11: 58-68. 1958. Langridge, J. , and B. Griffing. A study of high temperature lesions in Arabidopsis thaliana. Aust. J. Biol. Sci. 12: 117- 135. 1959. Lindegren, C. C., and G. Lindegren. Linkage relations in Saccharomyces of genes controlling the formation of carbohydrates and the synthesis of vitamins, amino-acids and nucleic acid components. Indian Phyt0path. 4: 11-20. 1951. Maas, W. K., and B. D. Davis. Production of an altered pantothenate- synthesizing enzyme by a temperature-sensitive mutant of Escherichia coli. Proc. Natl. Acad. Sci. 38: 785-797. 1952. Mitchell, H. K., and M. B. Houlahan. Neurospora. IV. A temper- ature sensitive riboflavinless mutant. Amer. J. Bot. 33: 31-35. 1946. Neale, F. E. Transpiration of glasshouse tomatoes, lettuce and carnations. Neth. J. Agr. Sci. 4: 48:56. 1956. Raschke, K. Heat transfer between the plant and the environment. , Ann. Rev. Plant Physiol. 11: 111-126. 1960. Snyder, F. W. and G. J. Hogaboam. Effect of temperature during anthesis and seed maturation on yield and germinability of sugar beet seed. Journal article No. 2188, Michigan Agricultural Experiment Station. (in press). Viglierchio. D. R., and F. W. Went. Plant growth under controlled conditions. IX. Growth and fruiting of the Kentucky Wonder Bean (Phaseolus vulgaris). Amer. Jour. Bot. 44: 449-453. 1957. 7%..“ in i‘ ' RIES H M'111111/111111'MIMI/(1111111!