A modified method to prepare root tip squashes was used for the routine examination of chromosomes of grapevines developed from in vitro shoot cultures. The results established that adventitious buds obtained by culture of fragmented shoot apices were diploid, and that storage at 9.5 °C of multiple shoot cultures of eight Vitis genotypes for periods up to 12 months did not affect the ploidy level of plants regenerated from them

Barlass, M.

Goodwins, D. R.

Skene, K. G. M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 27, 41-46 (1988) CSIRO, Divis ion of Horticultura l Research, Adelaide, Austra lia Ploidy stability in grapevines following long term storage in vitro by K. G. M. SKENE, D. R. GooDWINS and M. BARLASS Stabilität des Ploidiegrades bei Reben nach Langzeitlagerung von in-vitro-Kulturen Zu sam menfa ss ung : Für die routinemäßige Überprüfung der Chromosomen von Reben aus in-vitro-Kultur wurde eine modifizie rte Methode zur Herstellung von Wurzelspitzen-Quetschpräparaten angewandt. Die Ergebnisse bewiesen, daß Adventivknospen, die durch Kultur zerstückelter Triebspitzen gewonnen wurden, diploid waren. Ferner wurde durch Lagerung multi-pler Sproßkulturen von acht Vitis-Genotypen - bis zu 12 Monate bei 9,5 °C - der Ploidiegrad der daraus regenerierten Pflanzen nicht beeinflußt. K e y wo r d s : tissue culture, chromosome, storage, temperature, Vitis, variety of vine . Introduction The culture of fragmented shoot apices of grapevine (FSAC) is a rapid method for producing !arge numbers of multiple shoots from adventitious buds (BARLi\SS and SKENE 1978, 1980 a, 1980 b). Inter alia, the technique is suitable for micropropagation (SKENE and BARLASS 1980), and shoot cultures from fragmented apices also have been used for in vitro germplasm storage (SKENE and BARLASS 1983 a) . Both of these applica-tions have a high requirement for genetic stability, one aspect being the strict mainte-nance of the chromosome number representative of the genotype (ScowCROFr 1984). Whilst it is weil known that the chromosome number of callus and other dediffer-entiated explants may be subject to change during in vitro culture (n'AMATO 1978; BAY-uss 1980), our earlier findings indicated that the ploidy of adventitious Cabernet Sau-vignon shoots (2n = 38) obtained through FSAC remained unchanged from the original material (BARLASS and SKENE 1978). The current study was initiated to investigate this point in greater detail, as weil as to observe the effects of culture on ploidy of a range of genotypes, both at normal temperatures and at the reduced temperatures used for germplasm storage. Routine observations on !arge numbers of mitotic figures were made possible by modifications to existing root squash techniques. Materials and methods Plant material Multiple shoot cultures were prepared from fragmented shoot apices of the geno-types listed in the table by methods described previously (BARLASS and SKENE 1978, 1980 b; SKENE and BARLASS 1980). Cytochimeric genotypes were avoided, as it has been shown that FSAC can separate the component cell types (SKENE and BARLASS 1983 b). Once cultures were weil established on a modified Murashige-Skoog medium, they were maintained for periods up to 12 months (0, 3, 6, 9, 12 months) at 9.5 °C in a 15 h 42 K. G. M. SKENE, D. R. GOODWJNS and M. BARLASS Grapevines from in l' itro storage: numbers sampled for chromosome counts Reben aus in-1,itro-Lagerung: Anzahl der Proben für die Chromosomenzählung Number examined Genotype Plants from Root tip Mitotic culture squashes figures Vitis vinifera cv. Cabernet Sauvignon 56 226 1 272 Cabernet Franc 12 47 220 Sultana 53 285 1 768 11. amurensis 19 134 684 11. berlandieri 15 98 476 Tf. champini cv. Ramsey 26 148 703 Tf. Jabrusca cv. Concord 15 68 343 Tf. berlandieri x Tf. rnpestris hybrid R 99 19 105 598 Total 215 1 111 6 064 light, 9 h dark photoperiod (white fluorescent tubes, 15-30 ~tE · m - 2 · s - 1). In som e cases, cultures were stored in constant darlrness at the same temperature. This work is outlined by SKENE and BARLASS (1983 a). At the completion of cold storage , material was transferred to fresh media and placed in a culture room with a 26 °C day a nd 20 °C night (BARLASS and SKENE 1978) until active shoot growth resumed. Shoots were transferred to rooting medium, ulti-mately hardened-off and placed in pots in an air-conditioned glasshouse for further growth prior to chromosome examination . For comparison , potted plants obtained by conventional propagation of hardwood cuttings were also available . Examination of c hromo so me s Pr e t r e a t m e n t : 2-3 d before tips were required, each plant was tapped from its pot, and then gently returned to the same pot. This disturbance created air spaces into which actively growing roots proliferated. Root tips were collected in the morning, rinsed and placed in a 10 mg/1 solution of o-isopropyl-N-phenylcarbamate (IPC) for 2.5-3.0 h at room temperature. IPC is very effective in con t racting and sepa-rating chromosomes of a wide range of plant species (STOREY and MANN 1967). Fixation : Tips were rinscd in at least two changes of distilled water at 10 min intervals, before immersing in freshly prepared Carnoy fixative (3 parts abs. eth anol : 1 part glacial ace tic acid) for 2-16 h. Tips were then transferred to 70 % ethanol and stored at 4 ° C. In general, observations were made the fo llowing day. Ti s s u e s oft e n i n g : The tips were taken from 70 % ethanol to 40 % eth anol for 10 min , followed by two 5 min changes of water , then left in water for a fur-ther 15- 20 min whilst fresh enzyme solution was being prepared. Tissue was softened in 2 % w/v Macerozyme R-10 (Yakult Biochemical Co. Ltd., Nishinomiya, Japan) at pH 6.0 for 2.5--:3 .0 h at ambient temperature. Approximately 1.5 ml solution was used Ploidy stability following long term storage in vitro 43 for each five root tips. After maceration, tips were rinsed once in water for 5 min before hydrolysing in 5N HCl for 30 min at ambient temperature. This served to further soften the tissue, clear the cytoplasm and allowed easy separation of cells into a single layer when squashed. Care was taken not to overhydrolyse the tissues, as this caused the cells to burst easily. Tips were again rinsed in two 5 min changes of water, and left in water prior to preparation of squashes. S q u a s h p r e p a r a t i o n : Each root tip was transferred to a drop of 45 % acetic acid on a precleaned microsope slide, and the root-cap, epidermis and outer cor-tex were removed with dissecting needles, leaving a 0.5-1.0 mm tip. After further dis-section into small pieces, a coverslip was added, and the preparation squashed between filter papers until the desired flatness was achieved . When required, squashes were made permanent by freezing the slide from below with either a stream of compressed C02 or a block of dry ice to facilitate removal of the cover slip, followed by washing in two 5 min changes of absolute ethanol and immediately mounting in Euparal. M i c r o s c o p i c ob s e r v a t i o n : Preparations were usually not stained prior to examination by phase contrast microscopy. Whenever possible, squashes were prepared from five roots per plant, and at least five mitotic figures were examined in each squash, preferably from cells in metaphase. Chromosomes were counted, and cells scored for ploidy (2n = 38). In several instances, squashes were also prepared from shoot apices or immature tendrils by techniques essentially the same as described . Results In general, chromosomes were contracted, weil separated, and confined within the boundaries of the cell (Figs. 1 and 2). If the cell had burst, cell separation in good pre-parations also was sufficient to avoid confusion in scoring chromosomes (Figs." 3 and 4). Contrast in density between chromosomes and background cytoplasm was good (Fig. 1) to excellent (Figs. 2-6), and occasionally chromosome detail was better than usual, with satellites being visible (Fig. 4). The examples illustrated represent cells of Vitis 11inifera root tips taken from conventional cuttings (Fig. 1) and from tissue-cultured plants (Figs. 2-5). Squashes of similar quality were prepared from other genotypes (Fig. 6) . With the exception of Fig. 5 (a tetraploid cell from a mixoploid root with both diploid and tetraploid cells present), all examples are diploid. The majority of squashes were very flat, with chromosomes at the same plane of focus for photography and ease of counting. Roots from 215 plants (8 genotypes) derived from tissue cultures were examined. This represented a total of 6064 mitotic figures from 1111 root squashes (Table). The diploid number of chromosomes was observed in roots from all plants. No aneuploid cells were detected in any of the preparations examined. Tetraploid cells (4n = 76) as weil as diploid cells were present in root tips of 29 plants, and five other plants also bore both diploid roots and roots with tips appearing to be exclusively tetraploid. Shoot apices and tendrils were examined in all these latter plants and in selected plants with mixoploid roots; only diploid cells were observed. For comparison, it should be noted that one Sultana vine in a series of glass-house-grown plants derived from conventional hardwood cuttings also bore both diploid and exclusively tetraploid roots (thirteen 2n tips and five 4n tips, each from independent adventitious roots). As in the case of the tissue-cultured plants, only diploid cells were observed in the shoot apex. 44 K. G . M. SKENE, D. R. GooDWINS and M. B ARLASS \ . •• .......... _.. . • ,...--:-·· . „, • ' I' .. . .... ~ •• •• ••• • • • 't ' \, ., ~·· -·„····" " • • „ • \ . , -•,. . \ I ~. ... . • , -·. ,, . •. •. „ • . • • ',, . . . ... -· .. „ •• . _, • \ . . • • • • • • • .. . -• • , • . • ./ 2 1 "· . ~· . • • ~ '· . 1. . • " •• „. • • "' "' „ . " • , 1' , „ ' • ~ „ •• „ „ „ • ., • .t, • •• • „ ~ -· . . •• • • • - . • •• • •• • " • ~ • . •• '„ 4 3 ~ - ~ - ---, ' • •• • • • .. • • . • • • . ,,,. • • „ • / . • • •• • • • • .. „ „ • • . ~ „ ...... .... - • „ • ' • • . . ·'··\•.,.. • •• •• • • • . -.; '·· .. • • • • „ .... „ • • • • • •• • •• . ··~.. . . • 1 • • • • -. 5 • 6 • Figs. 1-6: Metaphase plates of unstained grapevine root tip squashes observed by phase contrast microscopy. All at the same magnification. Bar (Fig . 6) = 5 µm. Fig. 1: Vitis vinifera cv. Sultana, from conventional hardwood cutting. Diploid (2n = 38). Figs . 2-4: V. vinifera cv. Sultana, ex tissue culture. Diploid. Arrow (Fig. 4) indicates chromosome with satellites . Fig. 5: V: vinifera cv. Sultana, extissue culture. Tetraploid (4n = 76). Fig. 6: V. berlandieri x v: rupestris hybrid R 99, ex tissue culture. Diploid. Abb. 1-6: Metaphaseplatten in ungefärbten Quetschpräparaten von Rebwurzelspitzen, Phasen-kontrastmikroskopie. Alle Abbildungen in gleicher Vergrößerung, Maßstab (Abb. 6) = 5µm . Abb. 1: Vitis vinifera cv. Sultana, konventionell vermehrter Schnittholzsteckling. Diploid (2n = 38). Abb. 2-4: V. vinifera cv. Sultana, aus in-vitro-Kultur. Diploid. Der Pfeil (Abb. 4) weis t auf ein Satel-litenchromosom hin. Abb. 5: V. vinifera cv. Sultana, aus in-vitro Kultur. Tetraploid (4n = 76). Abb. 6: Kreuzung R 99 (V: berlandieri x V: rupestris), aus in-vitro-Kultur. Diploid. Ploidy stability following long term storage in vitro 45 No attempt has been made to provide detailed results for individual experiments, as all plants coming from culture were considered diploid (as confirmed by examina-tion of shoot apices where necessary), and the occurrence of mixoploid or tetraploid roots was independent of either culture temperature or the length of time stored at reduced temperature in light or darkness. Discussion Grapevine chromosomes are difficult to count due to their number and small size, a situation exacerbated by dense cytoplasm, which does not permit easy separation of chromosomes during preparation of squashes. Nevertheless, for routine counting of chromosomes in !arge numbers, reliable methods for their visualisation are needed. There are several reports of the examination of grape chromosomes (e. g., THOMPSON and ÜLMO 1963; RAJ and SEETHAIAH 1969; BOUQUET 1978), and some have produced good results. During this study, however, results with published methods were inconsistent, especially with respect to flatness of the figures, the degree of staining and amount of contrast between chromosomes and background cytoplasm. The method of squash preparation finally adopted permitted the routine examina-tion of !arge numbers of root tips. IPC proved to be particularly effective in contracting grape chromosomes, and combined with a careful balance between Macerozyme and HCI, the tissue was softened sufficiently to obtain consistently flat figures with good chromosome separation. High contrast between chromosomes and background was achieved by the clearing effects of fixative and HCI on cytoplasm, and especially by the simple expedient of examining unstained material with phase contrast microscopy. In this way the task of counting the small grape chromosomes was relatively simple . The examination of root squashes from in 1;itro-cultured plants in this study has presented a great deal more evidence in support of previous conclusions based on Cabernet Sauvignon, that plants developed from adventitious buds following FSAC retained the diploid number of chromosomes (BARLASS and SKENE 1978). In particular, chromosome numbers of eight genotypes remained unchanged during extended cul-ture at normal temperatures (26/20 °C) and were unaffected by storage at 9.5 °C for periods up to 12 months. The presence of mixoploid meristems with diploid and tetraploid mitotic figures in roots from some of the plants is not unusual, as it is possible for polyploid mitoses to occur within the older parts of a predominantly diploid meristem (NAGL 1978). The occurrence of five plants with both tetraploid and diploid root tips is puzzling. They are unlikely to be cytochimeras, as shoot apices were exclusively diploid. In view of the plant propagated from hardwood cuttings (also with a diploid apex) that bore several tetraploid roots, it is suggested that these changes in root ploidy were a consequence of events occurring at the level of the root or its initials during growth in the glasshouse, and were unrelated to in vitro culture. lt is concluded that from the point of view of stability of chromosome number, FSAC is a suitable method of preparing multiple shoot cultures from non-chimeric Vitis species, and these may be stored at reduced temperature without change in ploidy level. Summary A modified method to prepare root tip squashes was used for the routine examina-tion of chromosomes of grapevines developed from in vitro shoot cultures. The results 46 K. G. M. SKENE, D. R. GOODWJNS'and M. BARLASS established that adventitious buds obtained by culture of fragmented shoot apices were diploid, and that storage at 9.5 °C of multiple shoot cultures of eight Vitis genotypes for periods up to 12 months did not affect the ploidy level of plants regenerated from them. Acknowledgements This work was carried out with the support of a grant from the International Board of Plant Genetic Resources. The competent technical assistance of Ms. LYNN KRAMM is acknowledged. Literature cited o'AMATO, F.; 1978: Chromosome number .variation in cultured cells and regenerated plants. In: THORPE, T. A. (Ed.}: Frontiers of Plant Tissue Culture, 287- 295. Int. Ass . P lant Tissue Culture, Calgary. BARLAss, M.; SKENE, K. G. M.; 1978: In 1ri tr o propagation of grapevine ( llitis \finifera L.) from frag-mented shoot apices. Vitis 17, 335-340. - - ; - - ; 1980 a: Studies on the fragmented shoot apex of grapevine I. The regenerative capacity of leaf primordial fragments in \fitm. J. Exp. Bot. 31, 483-488. - - ; --; 1980 b: Studies on the fragmented shoot apex of grapevine II . Factors affecting growth and differentiation in \fitm. J . Exp. Bot. 31, 489-495. BAY LISS, M. W.; 1980: Chromosomal variation in plant tissue culture. Int . Rev. Cytol., Suppl. 11 A, 113-144 . BouQUET, A.; 1978: Methode de denombrement chromosomique dans Je genre llitis. Ann . Amelior. Plantes 28, 251- 255. NAGL, W.; 1978: Endopolyploidy and Polyteny in Differentiation and Evolution. North Holland Biomedical Press, Amsterdam. RAJ, A. S.; SEETHA!AH, L. ; 1969: Karyotype analysis and meiotic studies in three varieties of grape ( llitis \finitem L.). Cytologia 34, 475-483. ScowcROIT, W. R.; 1984: Genetic Variability in Tissue Culture: Impact on Germplasm Conservation and Utilization. IBPGR, Rome. SKENE, K. G. M. ; BARLASS , M.; 1980 : IV!icropropagation of grapevine. Proc. Int. Plant Prop. Soc. 30, 564- 570. - - ; - - ; 1983 a: Long term storage of grape in l'itm. Plant Gen. Res. Newsletter No. 53, 19-21. - - ; - - ; 1983 b : Studies on the fragmented shoot apex of grapevine IV. Separation of pheno-types in a periclinal chimera in l'itl'O. J. Exp. Bot. 34, 1271-1280. STOREY, W. B.; MANN , J. D. ; 1967: Chromosome contraction by o-isopropyl-N-phenylcarbamate (IPC). Stain Techno!. 42, 15-18. THOMPSON, M. M.; ÜL:110, H. P .; 1963: Cytohistological studies of cytochimeric and tetraploid grapes . Amer . J. Bot. 50, 901-906. Eingegangen am 28. 7. 1987 Dr. K. G. M. SKENE D. R. Goonw1Ns CSIRO Division of Horticultural Research GPO Box 350, Adelaide South Australia, 5001 Australia Dr. M. BARLASS CSIRO Division of Horticultural Research Private Mail Bag, Merbein Victoria, 3505 Australi a 

Ploidy stability in grapevines following long term storage in vitro

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Ploidy stability in grapevines following long term storage in vitro

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