Pollen of five grape cultivars stored in liquid nitrogen ( -196 °C) showed no significant decrease in percentage germination after 64 weeks, whereas pollen stored under laboratory conditions lost viability within 4 weeks. Significant differences in pollen viability were recorded among the cultivars after the storage duration of 64 weeks. This technique of pollen cryopreservation will be advantageous for grape breeders and gene banks involved in pollen preservation for conservation of grape genetic resources

Ganeshan, S.

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Vitis 24, 169-173 (1985) Indian Institute of Horticultural Research, Bangalore, lndia Cryogenic preservation of grape (Vitis vinifera L.) pollen by S. GANESHAN Gefrierkonse1vierung von Pollen der Rebe (Vitis l'inifera L.) Zusammenfass u ng: Pollen von fünf Rebsorten, der in flüssigem Stickstoff ( - 196 °C) bis zu 64 Wochen lang gelagert worden war, zeigte nach dieser Zeit keine signifikante Verminde-rung seiner Keimfähigkeit in vitro. Dagegen verlor der bei Zimmertemperatur aufbewahrte Pollen se ine Lebensfähigkeit schon innerhalb von 4 Wochen. Zwischen den fünf Sorten wurden nach 64 Wochen dauernder Gefrie rl agerung s ignifikante Unterschiede der Keimfähigkeit beobachtet. Die beschriebene Technik der Gefrierkonservierung von Pollen kann sowohl für Rebenzüchter a ls auch für Genbanken von Nutzen sein. K e y wo r cl s : po llen, variety of vine, cold , storage, germination. Introduction Pollen of several grap e cultivars lose viability and fertilizing capacity after 2 weeks of collection and storage under ambient conditions (NAGARAJAN et al. 1965). Maximum longevity for pollen of Thompson Seedless, Mu scat of Alexandria and Monukka was reported as 4 years , wh en stored at -12 ° C and 28 % relative humidity (Ouv10 1942), resulting in a reduced germination profile. Pollen storage for a period of 4 months to over 1 year at low temperatures ( - 12 to 8 °C) within a humidity r ange of 0-50 % was a lso fo und suitable (NEBEL and RUTTLE 1937; NAGARAJAN et al. 1965; BAMZAI and RAN-DHAWA 1967), but not without a progressive reduction in viability after storage . Studies on th e effects of varying humidity for preserving viability (GüLLM ICK 1942; RANDHAWA et al. 1982) and storage in organic solvents (AGARWAL 1983) resulted in r educed germi-nation in vitro with increasing storage durations. Fluctuations in storage conditions alter the microclimate in which pollen is preserved, leading to an accelerated lass of viability (STANLEY and L! NSKENS 1974). Although successful pollinations were achieved with 4 years' stored pollen produc-ing germinat ion in vitro as low as 6 % (OLMO 1942), preserving pollen viability with negligible lass in terms of its germination potential and fe rtility would be most desira-b le. A 'pollen bank' containing stocks preserved with high viabi li ty profiles can be of prolific use to gr ape breeders. Tlrns, pollination between early and late flowering culti-vars could be carried out, irrespective of their geographic location and time of flower-ing. Recently, long-term pollen preservation for genetic resources conservation h as been given a serious consideration (HARRINGTON 1970; ROBERTS 1975 ; ÜMURA and AKI-HAMA 1980; ÜMURA et al. 1982). The possibility of using liquid nitrogen storage condi-t ions for preserving viability of grape pollen was demonstrated (PARFITT and ALMEHDI 1983). Pollen samples rapidly frozen and thawed after holding for 1 h at -196 °C retained viability in vitro equivalent to fresh pollen. Application of cryogenic techniques to preserve pollen viability for prolonged du-rations has been found suitable in grape (YAKIMOV 1977, 1979) and polle n of other eco-nomically important crops (FARMER and BARNET 1974; NATH and ANDERSON 1975 ; BARNA-170 S.GANESHAN BAS and RAJKI 1976, 1981; KOBAYASHI et al. 1978; WEATHERHEAD et al. 1978; AKIHAMA et al. 1979; FISCHER and WALTHER 1981; TOWILL 1981; FRANK et al. 1982; TISSERAT et al. 1983; PARFITT and ALMEHDI 1984 a, b). The present paper establishes the viability state of cryopreserved grape pollen in terms of germinability in vitro and provides a simple method for prolonging viability under cryogenic conditions. Materialsand methods Pol l en collection and processin g for cryopreservat i on Pollen was collected from plants in well established vineyards of Bangalore Blue, Bangalore Purple, Black Champa, Queen of Vineyards and Anab-e-Shahi using the method described by ÜLMO {1942). Pure pollen was transferred into gelatin capsules, which were individually packed in 5 ml Corning culture tubes with screw caps, contain-ing activated silica gel. Culture tubes were bound with adhesive tape and wrapped with aluminium foil after encasing them in small 5 cm lang PVC tubes. Storage at -196 °C was accomplished by direct immersion in liquid nitrogen after precooling to -20 °C. Culture tubes with pollen samples were stacked in canisters of a Mach-SM-43 cryobio-logical system {MVE, USA) and immersed in liquid nitrogen. Complete immersion of samples throughout the storage duration was ensured by frequent refilling of the cryo-flask with liquid nitrogen and capping the canisters with perforated lids, in order to prevent the stored samples from floating out into the Dewar. The cryoflask was main-tained in the laboratory at 22 ± 2 °C. Viability te sts Pollen viability was tested by of its ability to germinate in an artificial medium. Fresh and cryopreserved pollen samples were cultured in vitro following hanging drop techniq'..le, after 0 (fresh), 4, 26 and 64 weeks. Samples were kept for thawing at ambient temperature for 30 min before culture, after which the remaining pollen was refrozen to -196 ° C via -20 °C. The germination medium consisted of 20 % sucrose solution in which pollen germinated profusely. Hanging drop cultures were incubated for 5 h at 25 ± 2 ° C and stained with a drop of ALEXANDER's stain {1980). Slides with germinated pollen were scanned and scored using a Leitz Neo-Promar projection microscope. Pol-len whose tube lengths measuring more than the grain diameter were considered via-ble. More than 500 grains were scored for each replication. Three replicates, each of control and frozen pollen were assayed for viability after each storage duration. Data on germination were analysed for variance by completely randomised {factorial) design. Results and discussion Table 1 shows in vitro pollen germination percentages recorded after different durations of cryopreservation, as compared with fresh pollen. All five cultivars recorded germination rates equivalent to fresh pollen, after 4, 26 and 64 weeks of stor-age in liquid nitrogen. However, pollen samples from different cultivars showed signifi-cant variability in germination profiles after cryopreservation. Pollen could withstand direct freezing to - 196 °C via - 20 °C, r apid uncontrolled thawing to room temperature and refreezing to cryogenic temperatures (via - 20 °C) during viability assessment after each storage duration. On the contrary, pollen samples stored at room tempera-Cryogenic preservation of grape pollen 171 Table 1 Pollen germination in different grape cultivars after storage in liquid nitrogen (mean values as per cent) Pollenkeimung bei verschiedenen Rebsorten nach Lagerung in flüssigem Stickstoff (Mittelwerte in %) Cultivar Bangalore Blue Bangalore Purple Black Champa Queen of Vineyards Anab-e-Shahi Mean Standard error mean LSD (P0.05) LSD (P0.01) Duration of storage (weeks) 0 (fresh) 4 26 54.3 54.0 50.8 61.8 58.1 58.2 80.5 66.1 71.3 74.8 75.3 79.0 64.0 62.8 73 .2 67.l 63.3 66.5 Cultivar Duration 3.612 3.230 10.322 9.232 13.810 12.352 Mean 64 59.5 54.7 81.9 64.9 84.4 75.6 80.2 77.3 71.7 67.9 75.6 C x D Interaction 7.224 20 .643 27.620 ture (control) continuously decreased in their capacity to germinate in vitro, approach-ing less than 5 % by the end of 2 weeks, after which viability was completely lost within 4 weeks. Control pollen samples, cultured a long with cryopreserved samples after 26 and 64 weeks, did not germinate. Pollen stored in liquid nitrogen continued to germinate as successfully as fresh pollen. Analysis of variance between cultivars, stor-age durations and their interaction is given in Table 2. Differential staining with ALEXANDER's stain was observed for fresh and cryopre-served germinated pollen, until the 64th week of viability assay. Germinated grains stained in two colours, the light pink grains becoming more greenish as the tubes elon-gated. The non-germinated grains stained dark pink. Pollen samples of all cultivars stored in liquid nitrogen maintained total viability after 64 weeks, indicating that a rapid cooling rate (from -20 to -196 °C) had no significant effect on the biological integrity of pollen under cryogenic conditions. Pol-len preserved under these conditions could remain viable for 12 years (YAKIMOV 1979) w ith negligible loss of its potential to germinate in vitro, retaining high fertility rates by producing normal fruit and seed set when used in field pollinations. This method of pollen preservation is considered superior to storage under low temperature and hu-midity, where viability was gradually reduced after prolonged durations of storage. Field pollinations with stored pollen germinating as low as 6 % in 1ritro was reported to be sufficient for effecting a normal berry set (OLMO 1942). In the present investigation, pollen of a ll five cultivars recorded very high germination profiles after 64 weeks of cryopreservation, which is 10-14 times more than the stipulated require-ment for a normal berry set. Therefore, the major implication of this study from a breeding view point is that storage in liquid nitrogen over a prolonged duration allows grape breeders to pollinate early flowering cultivars with pollen from late flowering cultivars taken during previous years. 172 Source Cultivar Duration C x D lnteraction Error S. GANESHAN Table 2 Analysis of variance of pollen ge rmination percentages Varianzanalyse der prozentualen Pollenkeimung df MS F 4 995.16 6.357 3 413.04 2.638 12 83.28 0.532 40 156.55 Significance NS NS As a simple technique, storing fresh grape pollen (without the need for desiccation or freeze-drying) for long-term preservation of plant germplasm is possible by cryo-preservation in liquid nitrogen at -196 °C. This can )Je used as a routine method by grape breeders and gene banks involved in conservation in iritro. Summa1·y Pollen of five grape cultivars stored in liquid nitrogen ( -196 °C) showed no signifi-cant decrease in percentage germination after 64 weeks, whereas pollen stored under laboratory conditions lost viability within 4 weeks. Significant differences in pollen viability were recorded among the cultivars after the storage duration of 64 weeks. This technique of pollen cryopreservation will be advantageous for grape breeders and gene banks involved in pollen preservation for conservation of grape genetic resources. Acknowledgement The author thanks Dr. K. L. CHADHA, Director, Indian Institute of Horticultural Research, Ban-galore , and Dr. M. P . ALEXANDER, Head, Division of Plant Genetic Resources, for facilities and encouragement. Literature cited AGARWAL, P. K. ; 1983 : Effect of storage in organic solvents on the germination of grapevine pollen. J. Hort. Sei. 58, 389-392. AKIHA~IA, T .; O~IURA, M.; KozAKI, I.; 1979: Long-term storage of fruit tree pollen and its application in breeding. J ARQ 13, 238-241. ALEXANDER, M. P.; 1980 : A ve rsati le sta i11 for pollen, fungi, yeast and bacteria. Stain Techno!. 55, 13-18. BAMZAI, R. D.; RANDHAWA, G. S.; 1967: Effect of certain growth substances on germination, tube growth and s torage of grape pollen grains. Vitis 6, 269-277. BARNABAS, B.; RAJK I, E.; 1976: Storage of maize (Zea mays L.) pollen at -196 °C in liquid nitrogen. Euphytica 25, 747-752. - - ; - -; 1981: Fertility of deep-frozen maize (Zea mays L .) pollen. Ann. Bot. 48, 861-864 . FARMER, R. E„ JR.; BARNET, P. E.; 1974: Low-temperature storage of black walnut pollen. Cryobiology 11, 366-367. FISCHER, M.; WALTHER, I.; 1981: Erste Versuche zur Überlagerung von Süßkirschenpollen, Prunus avium L. Arch. Züchtungsforsch. 11, 359-362. FRANK, J.; BARNABAS, B.; GAL, E.; FARKAS, J.; 1982: Storage of sunflower pollen. Z. Pflanzenzücht. 89, 341-343. Cryogenic preservation of grape pollen 173 GOLLMICK, F.; 1942: Über die Lebensdauer des Rebenpollens . Angew. Bot. 24, 221-232. H ARRINGTON, J. F.; 1970: Seed and pollen storage for conservation of plant gene resources. In: FRAN-KEL, 0. H; BENNET, E . (Eds.): Genetic Resources in Plants - their Exploration and Conserva-tion, 501-521. Blackwell, Oxford, Edinburgh. KoBAYASHl, S.; IKEDA, I.; NAKATANl, M.; 1978: Long-term storage of citrus pollen. In: AKIHA~IA , T.; NAKAJIMA, K. (Eds .): Long-term Preservation of Favourable Germplasm in Arboreal Crops, 8-12. The Fruit Tree Research Station· of the Ministry of Agriculture and Forestry, Fujimoto, Yatabe-Cho, Tsukuba-gun, lbaraki-ken, Japan. NAGARAJAN, C. R.; KRISHNAMURTHY, S.; MADHAVA RAO, V. N.; 1965: Storage studies with grape pollen. S. Indian Hort. 13, 1-14. NATH, J.; ANDERSON, J. O.; 1975: Effect of freezing and freeze-drying on the viability and storage of Lilium longiflorum L. and Zea mays L. pollen. Cryobiology 12, 81-88. NEBEL, B. R.; RuTILE, M. L.; 1937: Storage experiments w ith pollen of cultivated fruit trees. J. Pomol. Hort. Sei. 14, 347-359. ÜL~IO, H. P.; 1942: Storage of grape pollen. Proc. Amer. Soc. Hort. Sei. 41, 219-224. ÜMURA, M.; AKIHMIA, T.; 1980: Pollen preservation of fru it trees for gene banks in Japan. FAO/ IBPGR Plant Genet. Resources Newsletter 43, 28-31. - - ; MATSUTA, N.; AKIHMIA, T.; Yos HIDA, M.; 1982: Pollen preservation of Japanese apricot and mume. FAO/ IBPGR P lant Genet. Resources Newsletter 49, 19-21. PARFITT, D. E.; ALMEHDI, A. A.; 1983: Cryogenic storage of grape pollen. Amer. J. Enol. Viticult. 34, 227-228. - - ; - -; 1984 a: L iquid nitrogen storage of pollen from 5 cultivated Pnwus species. Hort. Sei. 19, 69-70. - - ; - -; 1984 b: Cryogenic storage of o live pollen. Fruit Varieties J. 38, 14-16. RANDHAWA, G. S.; AGARWAL, P. K.; S1NGH, R.; 1982: Pollen Storage studies in grape. I. Effect of differ-ent humidity regimes on viabili ty. Indian J. Hort. 39, 24-28. ROBERTS, E. H.; 1975: Problems of long-term storage of seed a nd pollen for genetic resources conser-vation. In: FRANKEL, 0 . H.; HAWKES, S. G. (Eds .): Crop Genetic Resources for Today and Tomor-row, 269- 296. Cambridge University Press, Cambridge, London, New York, Melbourne. STANLEY, R. G.; LINSKENS, H. F.; 1974: Pollen: B iology, Biochemistry, Management, 56-86. Springer Verlag, Berlin , Heidelberg , New York. TISSERAT, B.; ULRICH, J. M.; F lNKLE, B. J.; 1983: Survival of Phoenix pollen grains under cryogenic conditions. Crop Sei. 23, 254- 255. Tow1LL, L. E.; 1981: Liquid nitrogen preservation of pollen from tuber-bearing Solanum species. HortSci.16, 177-179. WEATHERHEAD, M. A.; GROUT, B. W. W.; HENSHAW, G G.; 1978: Advantages of storage of potato pollen in liquid nitrogen. Potato Res. 21, 331-334. YA1<1~1ov, L. iVl. ; 1977: Method for the prolonged storage of plant pollen at very low temperatures (Russ.). Molekul. i prikl. biofiz. s .-kh. rastenil i primenenie novel s h. fiz.-tekhn. metodov v s . kh. Ki shinev, Moldavian SSR, 108. IAbstr.: P lant Breeding Abstr. 49, 8730.J - - ; 1979: The prolonged s torage of plant pollen at very low temperatures (Russ.). Bio!., ekol. i fiziol. kul't. i !es. rast. Kishinev, Moldavian SSR, 14-18. IAbstr.: Plant Breeding Abstr. 51, 61.J Eingegangen am 18. 1. 1985 S. GANESHAN Pollen Storage Laboratory Division of Plant Genetic Resources Indian Institute of Horticultural Research Hessarghatta Lake P.O. Bangalore - 560 089 India 

Cryogenic preservation of grape (Vitis vinifera L.) pollen

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Cryogenic preservation of grape (Vitis vinifera L.) pollen

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