Alleweldt, G.

Harst, Margit

Reustle, G.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 33, 173-174 (1994) Research Note Regeneration of grapevine (Vitis sp.) protoplasts G. REUSTLE1), MARGIT HARs-r2) and G. ÄLLEWELD-r2) The availability of a protoplast-to-plant system would offer new perspectives for breeding of disease resistant grapevines. Grapevine, known to be recalcitrant for tissue culture experiments as many other perennial species, has been successfully regenerated from nodes and meristems from a wide range of varieties, whereas the regeneration of an-thers, ovules, or leaf and petiole tissue (STAMP and MEREDITH 1988, CHENG and REISCH 1989, MARTINELLI et al. 1993) is restricted to few genotypes. Although intensive efforts have been made during the last decade to develop a regenera-tion protocol for grapevine protoplasts, only cell division and formation of microcalli could be achieved (LEE and WETZSTEIN 1988, Mn et al. 1991, REusTLE and ALLEWELDT 1990, U1 et al. 1990). In our recent experiments, we could now demonstrate that grapevine protoplast regeneration is possible. . Materials and methods: For protoplast isolation, embryogenic tissue (somatic embryos in different stages of development), which has been induced on leaf disks of Vitis vinifera L. cv. Seyval blanc (HARsT and ÄLLEWELDT 1994) was used as donor tissue. The isolation protocol was carried out according to REUSTLE and NATTER (1994). After over-night digestion and subsequent purification, the de-rived protoplasts were resuspended in 0.65 M mannitol (pH 5.8). Protoplast density was adjusted to 0.5 x 106 cells per ml mannitol. For cultivation, protoplasts were immo-bilized in Na-alginate (2 %) applying a modified method of KARESCH et al. (1991). Protoplasts, embedded in the alginate layers ( density of 0.25 x 106 per ml) were trans-ferred into petri dishes (5 cm 0) with 2 ml of several media and cultivated at 24-26 oc in the dark. As culture media a modified CPW-13 medium (FREARSON et al. 1973) with 0.54 M glucose and NN-69 medium (NITSCH and NITSCH 1969) with 0.6 M glucose, were used for the initial cultivation step. Each medium was supplemented with 1.0 % PVP-40. During the initial-izing period, two hormone treatments i) 1 ppm 2,4-D and 0.5 ppm BAP or ii) 4.0 ppm NOA and 0.9 ppm TDZ were tested. As control treatment, a hormone-free variant was used. After 4 weeks, alginate layers were transferred to hor-mone-free NN-69 medium (for 4 weeks) containing 0.4 M glucose and 1 % PVP-40, followed by a further subculti-vation in the same medium but with reduced glucose con-centration (0.2 M). ') Universität Hohenheim, Institut für Obst- , Gemüse- und Weinbau, D-70593 Stuttgart, Germany 2) Bundesanstalt für Züchtu?gsf?rschung an Kult_urpfl~nzen , Institut für Rebenzüchtung Geilweilerhof, D-76833 Siebeldmgen, Germany Fragments of the alginate layers (1.5-2.0 mm in size) containing microcalli of different types and sizes were then transferred in petri dishes (10 cm 0) onto solidified (0.3 % gelrite) NN-69 medium with 0.2 M glucose. Results and discussion: I s o 1 a t i o n : With the applied isolation method, yields of 0.8 to 4.7 x 106 proto-plasts per gram material were achieved. The protoplasts varied in size from 10 to 50 t-tm and most of them were rich in cytoplasm. C e 1 1 d i v i s i o n : Beginning and rate of cell division depended on the media used and on the applied hormones. When CPW-13 medium was used, protoplasts started to divide during the 2nd week of cultivation inde-pendent of hormone treatment. Using the NN-69 medium, cell division occurred only after NOA{TDZ application. In contrast to CPW-13 medium, first cell division could be observed at the earliest 4-5 weeks after culture initiation. Furthermore, plating efficiency in CPW-13 medium was higher. Without hormones, protoplasts formed new cell walls within 2 weeks, however, cell division could not be observed. Mi c r o c a 11 u s f o r m a t i o n : Best development of divided protoplasts to microcallus arose in CPW-13 me-dium with NOA{TDZ as hormone treatment. 6-8 weeks after culture initiation, friable microcalli, consisting of large cells, developed and the microcalli reached sizes of 0.5-1.5 mm. In case of 2,4-D/BAP application, growth of the divided protoplasts was low. Using NN-69 medium with NOA{TDZ as initializing variant, the developed microcalli consisted of smaller cells and the texture remained com-pact. The positive effect of CPW-13 medium on protoplast division and microcallus formation was already found in earlier studies with grapevine protoplasts (REusTLE and NATTER 1994). The reduced growth of divided protoplasts in the presence of 2,4-D seems to be due to a genotype-specific sensitivity of Seyval to this hormone. S o m a t i c e m b r y o g e n e s i s : Small embryogenic cell aggregates (100 - 200 t-tm) could be ob-served in the alginate layers at the earliest 8 weeks after cultivation when NN-69 medium with NOA!TDZ was used in the initial cultivation step (Fig. 1). Ten weeks after cul-ture initiation, somatic embryos and embryoides were vis-ible on the alginate layers with the naked eyes (Fig. 2). Fig.l: Freshly isolated protoplasts from embryogenic tissue of grapevine 1 7 4  G .  R E U S T L E ,  M A R G I T  H A R s T  a n d  G .  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Regeneration of grapevine (Vitis sp.) protoplasts

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Regeneration of grapevine (Vitis sp.) protoplasts

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