In order to understand the hormonal factors affecting dormancy in grape seeds, studies were conducted on the endogenous auxins, inhibitcrs and gibberellin-like substances in the seeds stored moist at 5° C and at room temperature .The results show that moist after-ripening at 5° C resulted in an increase in the amount of growth promoting substances similar to auxins, in the extracts of chilled seeds. No correlation was observed between the content of inhibitor and dormancy in grape seeds. The extracts of neither chilled nor non-chilled seeds showed the presence of gibberellin-like substances when tested on D5 dwarf maize mutant plants.In the light of these findings, it is proposed that an increased synthesis of growth promoting substances during stratification may be one of the factors responsible for the termination of dormancy and resumption of germination of grape seeds

Chacko, E. K.

Kachru, R. B.

Singh, R. N.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 8, 12-18 (1969) Division of Horticulture, Indian Agricultural Research Institute, New Delhi, India Physiological studies on dormancy in grape seeds (Vitis Vinifera) 1. On the naturally occurring growth substances in grape seeds and their changes during low temperature after ripening by R. B. KAcHRu 1), E. K. CHACKo2) and R. N. S1NGH Introduction Grape seeds require a period of three months of moist after-ripening at 5° C for germination, (FLEMION 1937, SINGH 1961, RANDHAWA and NEG! 1964). Certain chemicals such as gibberellic acid and thiourea have also been found effective in partially substituting this after-ripening period there by hastening seed germination (Fox and TELLECHEA 1957, RANDHAWA and NEGI 1964, CHADHA 1965). But the factors respon­sible for the dormancy in grape seeds and the physiological changes taking place during low temperature stratification leading to germination have not been studied so far. However, recent studies on seeds and buds of a number of plants (LucKw1n 1952, HEMBERG 1965, VEGIS 1965, WAREING 1965, THOMAS et al. 1965) have demonstrated the involvement of certain growth inhibiting and growth promoting substances in the dormancy mechanism found in such organs. Investigations of this type would elucidate the factors involved in the physiology of dormancy in grape seeds. The results of our studies conducted during 1966-67 on the naturally occurring growth substances in dormancy of grape seeds, their changes during moist after-ripening at 5° C and dry storage at room temperature are dis­cussed in this report. Materials and Methods Grape seeds were collected from fully ripe berries of the variety Bharat Early, grown at the Experimental Orchard of Indian Agricultural Research Institute. The seeds, after cleaning, Were surface dried and were divided into two lots. One lot vJas mixed with moist sand in a petri dish and was stored in a refrigerator at 5° C. The other lot was stored dry at room temperature. The germination of the fresh seeds was tested immediately after extraction and that of chilled and non-chilled seeds later at monthly intervals. Germination tests were carried out by sowing seeds in sterilized sand and kept at a temperature of 25-28° C. When taking seed samples for germination tests, a duplicate lot was taken from both the treatments for extracting the endogenous growth substances. Extraction of growth substances Seeds were macerated in a chilled mortar with cold methanol. The macerated material was submerged in an excess of methanol and allowed to stand at -23° C ') Pool Officer, Indian Agricultural Research Inst., New Delhi 12, India. ') Junior Horticulturist, Inst. of Horticultural Research, Hessarghata, Bangalore 6, India. Dormancy in grape seeds 13 for 20 hours with occasional stirring. At the end of the extraction period, the methanolic extract was filtered and the residue was washed with fresh methanol. These washings were added to the original filtrate and the whole extract was eva­porated to dryness in a Buchler model flash evaporator under reduced pressure at 35-40° C. The dry residue was dissolved in a known volume of methanol for sub­sequent purification and bioassay.For determining the total auxins and inhibitors, an aliquot of the above methanolic extract was evaporated to dryness and redissolved in acetonitrile which was shaken with an equal volume of hexane (N1TscH 1956). The acetonitrile phase, after evaporating to dryness, was dissolved in a known volume of methanol for chromatography. The remaining part of the original concentrated methanolic extract correspond­ing to a known weight of seeds was evaporated to dryness. The residue was dissolved in distilled water. The acidic gibberellin-like substances were extracted from this aqueous solution using ethylacetate by the" method suggested by KATO (1964). Paper Chromatography of growth substances Descending paper chromatography in darkness at a temperature of 25° C ± 2 using Whatman No. 1 chromatographic filter paper was do:1e throughout this study. An aliquot of the final methanolic solution containing growth substances cor­responding to a known weight of the seed sample to be tested was applied along a five cm starting line of the paper. The chromatograms were equilibrated for 6-8 hours in the solvent vapour and then developed in isopropanol/ammonia/water (10/1/1 : v/v/v) to a distance of 30 cm. Occasionally, chromatograms of seed ex­tracts were developed in distilled water and 70% ethyl alcohol along with synthetic IAA. The developed chromatograms were sprayed with Ehrlich and Salkowski reagents (KEFFORo 1955) for locating the growth substances and their possible identi­fication. Bi o a s s a y: The auxin activity in the developed chromatograms was tested using Avena (var. Algerian) first internode test as suggested by NnscH and N1TSCH (1956). All tests were conducted in a temperature controlled dark room (25° C ± 2) with subdued green light. Final measurements of the sections were made using a photo­graphic enlarger and recording the magnified (X5) images with a mm-scale. Sta­tistical analysis was done to show significant differences. The response of first internode sections of the variety Algerian to synthetic IAA (0.0001-1.0 µg) and GA3 (0.001-100 µg) was also tested along with the seed extracts. In hib i t o r s  : The presence and the activity of the inhibitors present in the extracts of grape seeds were studied by cress (Lepidium sativum) seed germination test. The chromatograms of seed extracts were cut into l O equal pieces and were placed in 5 cm petri-dishes and moistened with two ml of double distilled water. As control, a blank filter paper strip was used in the petri-dish. In each of these petri­dishes, 50 cress seeds were allowed to germinate in darkness at a temperature of 25° C ± 2. The number of seeds germinated was counted after 48 hours and the percentage inhibition of germination was calculated from duplicate tests. The chromatograms presumably containing gibberellin-like substances were dried and cut into equal parts, shredded and eluted with 10 ml of 80% alcohol. The resulting eluates were dried under a current of air and redissolved in a known volume of 0.05 per cent Tween 80 solution. Aliquots of this were tested for bio­lo,�ical activity using the dwarf maize assay, (D5 dwarf mutant maize plants; PHINNEY 1961). 14 R. B. KACHRU, E. K. CHACKO and R. N. SINGH Results The germination percentage of fresh seeds as well as those stored at 5° C and room temperature for different periods are given in Table 1. Table 1 Germination percentage of grape seeds (var. Bharat Early) stored at 50 C and room temperature for different periods Period of storage Fresh 1 month 2 months 3 months 10 26 60 Germination percentage 50 C Room temperature 6 18 It is evident from the above data that freshly harvested grape seeds were in a state of dormancy and did not germinate. This state of dormancy continued in the seeds stored at room temperature during the first month and even after the second and third month of storage, only a meager percentage of germination such as 6 and 18 respectively was recorded. This indicates that dormancy in grape seeds is not broken during dry storage at room temperature. On the contrary, seeds stored in moist sand at 5° C germinated to the extent of 26 per cent after two months and 60 per cent after 3 months storage, indicating a gradual breakage of dormancy. The results of the bioassay for auxins in the extracts of fresh, non-chilled and chilled seeds (corresponding to 0.5 g of seed) during different months of storage are presented in Figure 1. It can be seen from the histograms in Figure 1 that in the chromatograms of fresh seed extracts, growth activity was located in two zones at Rf 0.3--0.5 and Rf 0.8. The peak activity was at 0.3-0.5. In the chromatograms of seed extracts taken after one, two and three months of storag� at 5° C, the activity of this zone was diffused over Rf 0.0-0.6 which may be due to the high amount of growth active substance present in such extracts. The amount of growth active substances present at Rf 0.0-0.6 in each chromatogram was calculated in terms of IAA equivalents and is presented in Table 2. Table 2 Auxin level in the extracts of chilled and non-chilled grape seeds var. Bharat Early. Results expressed as !AA equivalents in µg at Rf 0.0 - 0.6 of the chromatograms representing 0.5 g of seeds Storage Chilled Non-chilled period Fresh 0.004 1 month 0.126 0.0035 2 months 0.469 0.0028 3 months 0.569 0.189 It is evident from Figure 1 and Table 2 that the extracts of chilled and non­chilled seeds showed a marked difference in the levels of growth promoting sub­stances during storage. The extracts of fresh seeds contained only 0.004 µg IAA equivalent, whereas a gradual and steady increase in the amount of such growth 160 ..c., --c r� 140 _., �1120 inu.. "iii 100 :� 200 0 0 180 � --c t� 160 c,..c a,'f -g g 140 EZ 120 100 200 180 -c 160 o 140 120 100 1 Month 0,5 Dormancy in grape seeds 2 Months 1,0 0,5 1,0 15 3 Months 0,5 1,0Rf Fig. 1: Acetonitrile soluble growth promoting substances in the extracts corresponding to 0.5 g of chilled and non-chilled grape seeds (variety Bharat-Early). Darkened areas represent significant growth at 5 percent level. promoting substances was noticed in the extracts of chilled seeds during storage. In the extracts of seeds stored at room temperature, a slight increase in the amount of growth promoting substances was noticed only after 3 months of storage which was however insignificant when compared to that of chilled seeds. The increase in the germination percentage of seeds stored at 5° C was thus correlated with an increase in the amount of growth active substances in the seeds. When the developed chromatogram of extracts of seeds stored for three months at 5° C was sprayed with Ehrlich reagent, a bright pink colour turning blue after heating was developed at Rf 0.2-0.4. The intensity of this colour development, even though observed in the chromatograms of seed extracts stored at 5° C and room temperature for one and two months, was much less in the latter case than of seed extracts stored at 5° C for three months. Synthetic IAA located at Rf 0.4-0.5 when developed, also gave a pink colour turning blue after heating. However, colour development with Salkowski reagent, which is a more specific test for indole auxins, was not observed in the chromatograms of chilled or non-chilled grape seed extracts. Also, chromatograms of seed extracts developed in 70 per cent ethanol and distilled water did not give any conclusive proof to establish the identity of the growth ac­tive substance as IAA. The chromatograms of fresh, chilled and non-chilled seed extracts taken during different periods of storage although inhibiting the germination of cress seeds at 16 R. B. KAcHRu, E. K. CHACKO and R. N. SINGH Rf 0.7-0.8, indicating the presence of inhibitors, no difference was noticed in the degree of inhibition of cress seed germination by different seed extracts. Bioassay of the acidic ethylacetate fraction of the seed extracts on D5 dwarf maize mutant plants did not show any growth response in the first and second leaf sheath elongation. Discussion A close correlation between an increase in the amount of growth promoting activity in the extracts of grape seeds during stratification and the removal of dormancy was noticed in the present study. The extracts of freshly harvested seeds as well as those stored dry at room temperature for three months contained much smaller amounts of such growth promoting substances as compared to the seeds stratified for three months. LucKwILL (1952), in apple seeds, also found that low tem­perature moist after-ripening resulted in an increase in the growth promoting ac­tivity of seed extracts. Such stratification treatment also reduced the amount of an inhibitor present in apple seeds. Thus, LucKWILL concluded that the synthesis of growth promoting substances coupled with the leaching cut of the inhibitor from the seeds during low temperature after-ripening resulted in the germination of apple seeds. The presence of growth inhibiting substances in grape seeds has been reported by LucKWILL (1953). In the present study, no correlation between dormancy and the level of such inhibitor was observed, although inhibition of cress seed germination was noticed in the chromatogram of seed extracts at Rf 0.7-0.8. WAREING (1965) has given evidence to show that growth inhibitors are involved in the regulation of dormancy in many seeds, although in seeds like pea nut, GoRDEN­SHARIR and WAREING (1964) could not isolate any inhibitor from dormant seeds. Thus, the presence of inhibitors alone cannot be taken as the sole factor controlling dormancy in seeds. P1LLAY and EDGERTON (1965) reported an increased level of growth promoting substances in the extracts of after-ripened cherry seeds with and without gibberellic acid pre-treatment. The present findings also show that stratification resulted in increased synthesis of growth promoting substances which may be necessary for the germination of grape seeds. The importance of growth promoting and gibberellin-like substances in the dormancy regulation of seeds has been stressed by a number of workers especially uvring the onset of dormancy, (VILLERS and WAREING 1960, FRANKLAND and WAREING 1962, WAREING 1965). In our experiments, the presence of gibberellin-like substances could not be detected in D5 dwarf maize mutant bioassay. Since oat first internodes from the variety Algerian do not respond to gibberellin-like substances significantly, it is to be concluded that this large amount of growth promotion obtained at Rf 0.0-0.6 in the chromatograms of chilled seed extracts is due to substances similar to auxins. However, none of the chromogenic or chromatographic tests indicated that this substance is identical with IAA. Also, when the chromatograms of chilled seed extracts were developed in distilled water and 70 per cent ethanol, growth activity was located at Rf 0.4-0.5, 0.9-1.0; and 0.7-0.8 respectively. Hence it is to be assumed that a complex of substances with growth promoting activity are syn­thesised in the grape seeds during low temperature storage. Since GA3 has been found effective in hastening the breakage of dormancy in grape seeds (RANDHAWA and NEGI, 1964), it is reasonable to assume that low tem­perature after-ripening results in an increased synthesis of endogenous gibberellins in the seeds. However, our results do not show any evidence to substantiate this. Dormancy in grape seeds 17 Hence, further experiments are under way to see how GA3 treatment brings about enhanced germination of partially chilled grape seeds. The results of our studies in regard to the effect of GA3 on the metabolism of auxin-like growth promoting substances present in grape seeds and their exact biological and chemical properties will be communicated at a later date. Summary In order to understand the hormonal factors affecting dormancy in grape seeds, studies were conducted on the endogenous auxins, inhibitcrs and gibberellin-like substances in the seeds stored moist at 5° C and at room temperature .The results show that moist after-ripening at 5° C resulted in an increase in the amount of growth promoting substances similar to auxins, in the extracts of chilled seeds. No correlation was observed between the content of inhibitor and dormancy in grape seeds. The extracts of neither chilled nor non-chilled seeds showed the presence of gibberellin-like substances when tested on D5 dwarf maize mutant plants. In the light of these findings, it is proposed that an increased synthesis of growth promoting substances during stratification may be one of the factors responsible for the termination of dormancy and resumption of germination of grape seeds. Acknowledgements The authors are thankful to Dr. S. K. MUKHERJEE, Head of the Division of Horticulture for providing facilities and encouragement. Literature Cited CHADHA, K. L., 1965: Studies on phenotypic variability and seed germination in some grape cultivars. Unpublished thesis submitted to the Post-graduate School, Indian Agricult. Res. Inst., New Delhi. FLEMION, F., 1937: After-ripening at 5' C favours germination of grape seeds. Contr. Boyce Thompson Inst. Plant Res. 9, 7-15. Fox, J. M. and TELLECHEA, H., 1950: Accion delatio-urea y tio cianato de amonie como estimulants de la germination de la semilla de vid. Rev. Fae. Cienc. Agrar. (Mendoza) 2, 53-57. FRANKLAND, B. and WA REING, P. F., 1962: Changes in endogenous gibberellins in relation to chilling of dormant seeds. Nature 194, 313. GoRoON-SHARIR, A. and WAREING, P. F., 1964: Nature 204, 1308. Quoted from: WAREING P. F.: Dormancy in plants. Sci. Progr. (London) 53, 529-537. HEMBERG, T., 1949: Significance of growth inhibiting substances and auxins for the rest period of the potato tuber. Physiol. Plant. (Lund) 2, 24--37. KATO, J., 1964: Isolation of a gibberellic-like substance from bamboo shoots and its biological' activity. In: ANoNn<: Regulateurs naturels et croissance vegetable. Edit. Cent. Natl. Rech. Scientif. (Paris) 123, 275-285. LucKWILL, L. C., 1952: Growth inhibiting and growth promoting substances in relation to dormancy in apple seeds. J. Hort. Sci. 27, 53-57. - - , 1953: Studies on fruit development in relation to plant hormones. 1. Hormone production by the developing apple seed in relation to fruit drop. J. Hort. Sci. 28, 14-24. NITSCH, J. P., 1956: Methods for the investigation of natural auxins and growth inhibitors. In: WAIN, R. L. and WIGHTMAN, F. (Edit.): Chemistry and mode of action:·of plant growth substances. Butterworths Scientif. Pub!. - - and Nnsctt, C., 1956: Studies on the growth of coleoptile and first internode sections. A new ,sensitive straight growth test for auxins. Plant Physiol. 31, 94-111. 18 R. B. KACHRu, E. K. CHACKO and R. N. SINGH PHINNEY, B. 0., 1961: Dwarfing genes in Zea mays and their relaticn to gibberellins. In: KLEIN, R. H. (Edit.): Plant growth regulation. 4. Intern. Conf. on Plant Growth Regulation. Iowa State Univ. Press. Ames, Iowa, p. 489-503. P1LLAY, D. T. N. and EDGERTON, L. J., 1965: Relationship of growth substance, to rest period and germination in mazzard cherry seeds. Proc. Amer. Soc. Hort. Sci., 86, 108-114. RANDHAWA, G. S. and NEG1, S. S., 1964: Preliminary studies on seed germination and subsequent seedlings growth on grapes. Indian J. Hort. 21, 186-196. S1NGH, S. N., 1961: Germination of grape (Vitis vinifera) hybrid seeds by chilling. Curr. Sci. (Bangalore) 30, 62. THOMAS, T. H., WAREING, P. F. and Roe1NsoN, P. M., 1965: Action of the sycamore 'dormin' as a gibberellin antagonist. Nature 205, 1270-1272. VEG1s, A., 1965: Die Bedeutung von physikalischen und chemischen AuLlenfaktoren bei der In­duktion und Beendigung von Ruhezustiinden bei Organen u:1d Geweben hoherer Pflan­zen. In: RUHLAND, W. (Hrsg.): Handb. d. Pflanzenphysiol. Ver!. Springer, Berlin, 15, 534-668. Viu.Ens, T. A. and WAREING, P. F., 1960: Interaction of growth inhibitor and a natural ger­mination stimulator in the dormancy of Fraxinus exe!sior L. Nature 185, 112. WAREING, P. F., 1965: Dormancy in plants. Sci. Progr. (London) 53, 529-537. - - , 1965: Endogenous growth inhibitors in seed germination and dormancy. In: Ru11LAND, W. (Hrsg.): Handb. d. Pflanzenphys. Ver!. Springer, Berlin, 15, 909-924. Eingegangen am 4. 11. 1968 Prof. Dr. R. N. SINGH Division of Horticulture Indian Agricult. Res. Inst. New Delhi 12 India 

Physiological studies on dormancy in grape seeds (Vitis vinifera) 1. On the naturally occurring growth substances in grape seeds and their changes during low temperature after ripening

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Physiological studies on dormancy in grape seeds (Vitis vinifera) 1. On the naturally occurring growth substances in grape seeds and their changes during low temperature after ripening

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