As an alternative or supplement to field collections, a repository of 35 grapevine genotypes and 15 clones of cv. Optima was set up under minimal growth conditions. To prolong the storage period between subculturing to at least 12-18 months, some factors of long-term storage were optimized and the causes responsible for early senescence of sensitive genotypes were investigated  Besides indirect effects due to excision time, origin of the starting material and preculture, there is a direct influence of the light conditions on the survival rate of cultures. CCC application showed, in some cases, positive effects only after long-term storage.  An increase of carbon dioxide concentration during storage is considered to be responsible for the early death of some cultures

Alleweldt, G.

Harst-Langenbucher, M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

446 Section 5 Conservation of the genetic resources of Vitis M. HARST-LANGENBUCHER and G. ALLEWELDT Bundesforschungsanstalt filr Rebenziichtung Geilweilerhof, D-6741 Siebeldingen, F. R. Germany S u m m a r y : As an alternative or supplement to field collections, a repository of 35 grapevine genotypes and 15 clones of cv. Optima was set up under minimal growth conditions. To prolong the storage period between subculturing to at least 12-18 months, some factors of long-term storage were optimized and the causes responsible for early senescence of sensitive genotypes were investigated. Besides indirect effects due to excision time, origin of the staning material and preculture, there is a direct influence of the light conditions on the survival rate of cultures. CCC application showed, in some cases, positive effects only a ft e r long-term storage. An increase of carbon dioxide concentration during storage is considered to be responsible for the early death of some cultures. K e y w o r d s : gene resources, tissue culture, long-term storage, preculrure, light condition, growth regulator, carbon dioxide. Introduction For grapevines, as with most heterozygotic plants, maintainance of homologous material is normally carried out vegetatively in living collections. This form of preservation, however, is highly vulnerable by biotic and abiotic factors e. g. pests, pathogens, climatic stress or air pollution. An ideal alternative or supplement to field collections is preservation in vitro. Under normal culture conditions (NC), maintainance in vitro is laborious, time and space consuming. For storage purposes plants are maintained under reduced culture conditions (RC) to prolong the period between subculturing. In 1986 a repository of 3 5 grapevine genotypes and 15 clones of cv. Optima was set up under minimal growth conditions. A wide range of genotypical reactions was observed during a storage period of 12 months. To optimize the storage conditions and to determine the causes of early senescence, various examinations were carried out. Material and methods Plant material Rooted plants deriving from nodal cuttings were used as storage material. The plantlets were cultivated under NC up to a shoot length of5-7 cm and then transferred to RC. Culture conditions - NC: 25 °C, 16 h photoperiod, 50 µ E • m·2 • s· 1 - RC: 8 °C, lOhphotoperiod, lOµE• m·2 • s· 1 The medium ofL1Ns~AIER and SKooo (1965) supplemented with honnones (0.01 mg/I NAA and 0.03 mg/I BA), vitamins (0.40 mg/I thiamine), 30 g/I sucrose and 8 g/I agar, was used under NCandRC. CCC was applied to the nutrient medium before cultivation of the cuttings according to ALLEWELDT and HARST-LANGENBUCHER (1987). Tissue and cell culture 447 Carbon dioxide determination The detennination ofC02 was carried out with a special C02 detector Ultramat 22p Siemens modified for use with in virro cultures. Abbreviations used r>;A.A, 1-naphtaleneacetic acid; BA, 6-benzyladenine; CCC, chlorocholinechloride (2-chlorethyl-trimethyl-ammonium-chloride); KC, normal cullure conditions; RC, reduced culture conditions. Results and discussion Optimiz.ation of the long-term storage conditions For a successful in vitro cultivation and long-term storage of nodal cuttings the staning material is imponant. Best results were obtained when the explants were taken in the months of June and July. Cuttings from greenhouse-grown plants performed better than cuttings from the field (Table 1 ). Prior ·to long-tenn storage the plants have to be subcultured twice after excision and establishment in virro. Plants which were stored directly after sprouting of just excised cuttings were highly infected or died within a few weeks of storage (Table 2). Table 1: Survival rate(%) of in vitro cultures after 12 months oflong-term storage in dependence on excision time and origin of the plant material (n = 20) excision time JUNE JULY AUGUST origin FIELD 70 80 0 GREENHOUSE 90 100 50 Table 2: Survival rate(%) ofin l'irro cultures after 3, 6 and 12 months of long-term storage in dependence on the number of subculrures ( n = 20) number of months of storage subcultures after excision 3 6 10 0 100 100 55 1 100 95 37 2 100 90 80 448 Section 5 Table 3: Survival rate(%) of in vitro cultures from cv. Riesling and the wild species V. rupestris after 9 months of long-term storage in dependence on light intensity (n = 16) light intensity 1 µE· m- 2 • s- 1 genotype RIESLING 50 3 V.RUPESTRIS 88 81 Light conditions also seem to play an important role on survival rate. Within a few months of storage, the cultures stored under continuous light or in darkness died. Under short-day conditions and low light intensity (10µE·m· 2 ·s·1), most of the cultures survived a storage period of 15 months. A further reduction of the light intensity down to only 1 µE• m·2 • s· 1 caused rapid losses of sensitive cultivars within a few weeks under RC (Table 3). Fig. 1 (left) : Improvement of sprouting and shoot development after subculruring onto fresh medium in dependence on CCC treatment during long-term storage. a) Subculture of a CCC-treated plant, b) subculture of an untreated plant. Fig. 2 (right): Induction of senescence symptoms on in l'itro plantlets by Jong-term storage under RC (a) and by increasing the CO, concentration under NC (b ). Tissue and cell culture 449 Comparable results had been achieved by BARLAss and SKENE (1983) and FAUSTINI (1984). In a funher attempt to improve the survival rate, CCC was applied to in vitro cultures. Under field conditions an increase in cold colerance had been induced by treating grapevines with this growth inhibitor (BOURQUI~ and ALLEWELDT 1970). Table 4: Survival rate(%) of in l'irro culrures of V. riparia in dependence on CCC treaunent during-different storage periods ( n = 16) treatment storage period (months I 3 6 12 untreated CCC 100 100 71 100 36 64 Table 5: Comparison of the survival rates (%) from different genotypes of Vitis after a long-term storage of 12 months. Storage periods 1986/87and1987 /88 genotype storage period I storage period II 1986-1987 1987-1988 V.RUPESTRIS 31 88 V.RIPARIA 0 63 so 4 56 53 RIESLING 63 81 KERNER 81 100 FABERREBE 56 100 OPTIMA 56 100 ORION 6 100 VIDAL 50 81 mean 44 84 450 Section 5 Jn 1irro cultures of only a few cultivars showed a better survival rate in RC when treated with CCC (Table4). Some cultivars could not be stored longer than when untreated. However, after long-1enn storage these CCC-treated plants exhibited earlier sprouting and faster shoot development afcer subculturing onto fresh medium (Fig. 1 ). This result has to be confirmed in subsequent examinations. 3,0 • 2,5 V. rupestrls Richter 110 1,5 1.0 0,5 ""- ·------· 3,0 0 3 6 0 J 6 2,5 Bacchus r--· Optima ..,. 1,5 0 > c .9 1,0 -«I .. 0,5 - "'-c ID u c • 0 u I 3,0 0 1 3 6 0 1 3 6 ON u 2,5 Orion Sirius 1,5 1,0 / 0,5 "" • 0 3 6 0 3 6 months of long term Storage Fig. 3: CO 1 development during a storage period of 6 months in dependence on the genotype. Tissue and cell culture EXClSJON TRANS f"EH j SUBCULTURE ADAPTATION ! SUBCULTURE j~ GREENHOUSE ...,.___WECK® -JARS CULTURE•TUBES 451 passaJl,.t:> ( 1lur1tl ion I 0. l C8-l2 wc~ks) 0.2 to 0.3 : 12 -20 weeks) l. 0 (> 52 weeks) (4 weeks) l. l (B-12 weeks) l. 2 (6-10 weeks) Fig. 4: Long-tenn storage under reduced culture conditions during maimainance and propagation of Viris material by tissue culture 1echniques. 452 Section 5 Causes of senescence Besides these tests for optimizing the long-tenn storage conditions, the causes of early senescence exhibited by some genotypes were of great interest. Many authors have supposed that C02 becomes concentrated under in vitro conditions in the culture tubes (DONOVAN and MuRASHIGE 1979; DE PROFT et al. 1985; FouR:><mux and BEss1s 1986; ZoBEL 198 7). In a special examination, senescence symptoms similar to those observed during long-tenn storage could be induced by artificial rising the C02 concentration up to 3 % (v/v) under NC (Fig. 2). The development ofC02 concentrations during a storage period of6 months is demonstrated in Fig. 3. During the period under NC the CO, concentration increased to more than 0.5 % (v/v) of the environmental CO 2 content of the laboratory (start of storage). Within the first 4 weeks after transfer of the plantlets to RC, the C02 concentration declined rapidly (1 month of storage). During the following 5 months under RC the CO, concentration showed a characteristical rise dependent on genotype (6 months of storage). • Fig. 5: Resumption of growth vigour after 20 months of long-term storage when subcultured onto fresh medium. a) Plantier after 20 months of long-term storage, b) subcultivation onto fresh medium, c) sprouting 7 d after subcultivation, d) shoot development 21 dafter subcultivation. Tissue and cell culture 453 In the culture tubes of cvs Richter 110, Bacchus and Orion, which showed a very marked response to RC, a high C02 concentration of almost 3 % (v/v) was determined. To avoid high C02 concentration, the N2 content in the atmosphere of the culture tubes could be risen to keep the C02 content on a non-toxic level. The long-term storage of ornamentals is carried out in this way (PREIL 1989, personal communication). Due to these tests for optimizing the long-term storage conditions and the examination of the development ofC02 concentration during the storage period, some general recommendations for carrying-out conservation of in vitro cultures of grapevine can be given (Fig. 4): After excising the starting material from field- or greenhouse-grown grapes, the cultures have to be propagated twice under NC before they can be transferred to RC. If more than 50 % of each cultivar have died, the surviving cultures should be re-transferred into NC and adapted for about 4 weeks before subculturing onto fresh medium. After being subcultured twice, the plants can be stored again or, if necessary, adapted to soil. By following this procedure the survival rate was almost doubled in a further storage period, as demonstrated on some cultivars (Table 5). The stored material did not loose vitality even when the plants were subcultured onto fresh medium after a storage period of more than 20 mo.nths under RC (Fig. 5). After adaptation to soil and acclimatization to field, the long-term stored plant materialis true to type (Fig. 6). So far, some important factors influencing the survival rate under RC have been described. Other factors, like an adaptation to short-day conditions before and an adaptation to long photoperiod after the storage, have to be examined. A further perspective for optimization of the long-tenn storage can be the examination of the composition of the nutrient medium (full- or half. strength salt concentration, NH4N0 3 concentration, reduction of sucrose concentration, activated charcoal, etc.) . For almost all of the stored genotypes a minimum storage period of 12 months was realized. There are no problems in subculturing and in the acclimatization to field conditions after long-term storage. By using differenciated explants like shoot-tips or nodal cultures, somaclonal variation can beexcluded(D'AMATO 1978). Fig. 6: Plants are true to type when adapted to field conditions. a ) Leaves from an in l'itro long· term stored plant after adaptation to field conditions, b) leaves from the same cultivar derived from the field collection. 454 Section 5 At the present situation of plant cell and tissue culture research of the grapevine this conservation method of in vi1ro plantlets seems to be well-suited for a safe maintainance of healthy plant material. Thus, the establishment of a gene repository and the long-term storage of clonal material for breeding and/or propagation has been initiated. Conclusion A simple and safe maintainance procedure for rooted plantlets of in vi1ro cultures of grapevine has been established by improving the storage conditions (starting material, preculture, light conditions, C02 development). The period between two subcultivations could therefore be prolonged to 12·18 months. For those genotypes which show yet an unsatisf)'1ng survival rate, optimization of the long·tenn storage has co be continued. References ALLEWELDT, G.; HARST·LANGENBFCHER, M.; 1987: Der EinfluB von Wachstumsinhibitoren auf die Langzeitlagerung von in-1•itro-Kulruren der Rebe. Vitis 26, 57-64. BARLASS, M.; SKENE, K. G. M.; 1983: Long·terrn storage of grape in vitro. FAO/IBPGR Plant Genetic Resources Newsletter 53, 19-21. BouRQUIK, H. D.; ALLEWELDT, G.; 1970: Der Einflul3 von CCC und B-995 aufdas Triebwachstum der Reben. Vitis9, 105-120. D' AMATO, F.; 1978: Chromosome number variation in cultured cells and regenerated plants. In: THORPE, T. A (Ed.): Frontiers of Plant Tissue Culture, 287-295. International Association of Plant Tissue Culture, Calgary. DONOVAK, S. T.; MlJR.'\SHIGE, T.; 1979: Volatile emissions ofplam tissue culrures. I. ldentif1Cation of the nuijor components. In Vitro 15, 654-658. F AUSTINJ., R.; 1982: Mecodi alternativi di conservazione de! materiale genetiro della vite: "colrure in vitro". Vlgnevini9(9),11-13. FouRNIOUX, 1. C.; BES SIS, R.; 1986: Influence de la teneur en gaz carbonique sur la morphogenese de la vigne en culture in »itro. Can 1. Bot. 64, 2608-2616. LINS MAIER, E. M.; SKOOG, F.; 1965: Organic growth factor requirement of tobacco tissue culture. Physic!. Plant. 18, lOCJ.127. PROFT, M. P. DE; MAENE, L. J.; DEBERGH, P. C.; 1985: Carbon dioxide and etllylene t>Volution in the culture atmosphere of Magnolia culrured in vi1ro. Physic!. Plant. 65, 37 5-3 79. ZOBEL, R. W., 1987: Gaseous compounds of soybean tissue cultures: Carbon dioxide and ethylene evolution. Environ Exp. Bot. 27, 223-226. 

Conservation of the genetic resources of Vitis

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Conservation of the genetic resources of Vitis

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