The effect of genotype on grapevine embryogenic culture was studied on eight Vitis spp. Cultures were obtained from excised anthers, isolated from flower buds. Somatic embryogenesis was achieved from 6 of the 8 varieties at various frequencies. Embryos were germinated either on solid or in liquid medium in the dark before being exposed to light. The regeneration rate ranged from 8 to 81 %. For induction of recurrent somatic embryogenesis, torpedoes and early cotyledonary stage somatic embryos were employed applying two plant growth regulator treatments. Results demonstrate that grape cultivars are highly variable in their response to induce somatic embryogenesis, recurrent somatic embryogenesis and germination of embryos

Mozsár, J.

Viczián, O.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 35 (4), 155-157 (1996) Genotype effect on somatic embryogenesis and plant regeneration of Vitis spp. by J. MozsAR and 0. VICZIAN Plant Protection Institute, Hungarian Academy of Sciences, Dept. of Biotechnology, Budapest, Hungary S u m m a r y : The effect of genotype on grapevine embryogenic culture was studied on eight Vitis spp. Cultures were obtained from excised anthers, isolated from flower buds. Somatic embryogenesis was achieved from 6 of the 8 varieties at various frequen-cies. Embryos were germinated either on solid or in liquid medium in the dark before being exposed to light. The regeneration rate ranged from 8 to 81 %. For induction of recurrent somatic embryogenesis, torpedoes and early cotyledonary stage somatic embryos were employed applying two plant growth regulator treatments. Results demonstrate that grape cultivars are highly variable in their response to induce somatic embryogenesis, recurrent somatic embryogenesis and germination of embryos. K e y w o r d s : somatic embryogenesis, grapevine, genotype effect, germination. Introduction Although it has been relatively easy to achieve somatic embryogenesis from grapevine, the frequency of plant re-generation from these embryos usually is low. Several protocols have been developed to increase the regenera-tion rate, including chilling of embryos at 4 °C (RAJASEKARAN and MULLINS 1979; MARTINELLI et al. 1993), daily subculturing of embryos in a fresh medium (CouTos-THEVENOT et al. 1992), removal of cotyledons from em-bryos (MAuRo et al. 1986), application of plant growth regu-lators and dehydration of mature embryos (GRAY 1989), induction of adventitious buds on isolated hypocotyls (VILAPLANA and MULLINS 1989) and germination in liquid medium (MozsAR and SDLE 1994). Grape somatic embryos were employed in Agrobacterium-mediated gene transfer (MARTINELLI and MANDOLINO 1994, ScoRZA et al. 1995). In these experiments regeneration of transgenic plants oc-curred via recurrent embryogenesis. According to these results in vitro embryogenic culture with a high rate of plant recovery and recurrent somatic embryogenesis seems to be a prerequisite to successful genetic manipulation. In this work 8 Vitis spp. were used to induce somatic embryogenesis and to obtain somatic embryos which were germinated or were subjected to the induction of recurrent somatic embryogenesis. Materials and methods Five Vitis vinifera L. cultivars, Chasselas, Sztirkebanit, Ezerj6, Szlanka and EME (Ezereves Magyarorszag Emleke, Chasselas Queen Victorian White x Calabrian White) and Vitis amurensis Rupr. (clone No. 66), Vitis riparia Michx. cv. Gloire de Montpellier and the inter-specific rootstock G 28 (V. berlandieri x V. riparia x V. vinifera) were used in this study. The initiation of embryogenic cultures from excised anthers has been described previously (MozsAR and SDLE 1994). In the germination experiments globular and heart-shaped embryos were placed in 100 ml Erlenmeyer flasks in 25 mlliquid MS medium (MuRASHIGE and SKooG 1962) containing. 20 g r' sucrose and incubated in an orbital shaker (150 rpm at 28 oc in the dark). The medium was replenished weekly. In parallel with this experiment em-bryos were germinated on solid MS (0.8 % Difco Noble-agar) at the same temperature in darkness. After 3 weeks embryos with cotyledons and primary roots were placed individually in test tubes, which were filled with 10 ml induction medium (half-strength MS plus 10 g r' sucrose) and incubated at 25-27 oc in permanent light. Two months later plantlets with well-defined root and shoot systems were registered. For induction of repetitive somatic embryogenesis torpedos (ea. 2-3 mm long) and early cotyledonary stage somatic embryos (ea. 5-8 mm long) were selected. They were cultured either on MSE medium containing MS plus 20 g r'sucrose supplemented with 5 j.lM 2,4-dichloro-phenoxyacetic acid (2,4D) and 0.9 j.lM 6-benzylamino-purine (BA) or MSE/2 medium containing MS plus 20 g r' sucrose with 2.5 j.lM 2,4D and 0.45 j.lM BA. A solidifying agent (0.8 % Difco Noble-agar) was added. Plates were incubated at 28 oc in the dark. After 2 months explants that had produced somatic embryos or yellow embryogenic calli were registered. Calli were transferred to the induc-tion medium to determine their embryogenic capacity. Results Induction of somatic embryogenesis: The embryogenic callus had a light-yellow color and was composed of an undifferentiated mass of cells and clusters of proembryo and globular, embryo-like structures. Pri-mary somatic embryos appeared on these calli after 2 weeks of incubation on the induction medium. We were not able to induce somatic embryogenesis on explants ofEzerj6 and V. amurensis. Of the tested cultivars, Correspondence to: J. MozsAR, Plant Protection Institute, Hungarian Academy of Sciences, Herman Ott6 ut 15 Pf. 102, H-1525 Budapest, Hungary. Fax: (36) 156 3698. 156 J. Mozs..\R and 0. VICZIAN Chasselas was superior in somatic embryogenesis induc-tion (5.5 %). Although its anthers yielded relatively few calli, almost all (91 %) were embryogenic. The efficiency of somatic embryogenesis induction of the other cultivars was quite low (less than I % ), because only a low portion of calli were embryogenic (6-18 % ). Embryogenic calli formed numerous somatic embryos asynchronously on the induction medium, except for Sziirkebanit. Somatic embryos of Sziirkebanit were there-fore exclusively employed in germination experiments. Embryo germ in at ion : In previous experi-ments we found that the liquid MS medium with weekly replenishment greatly stimulated plant regeneration of V. riparia somatic embryos (Mozs..\R and SOLE 1994) as compared to solid MS. Therefore we tested this method on embryos of other Vitis spp. Significant differences between the two germination methods were observed only with 3 out of the 5 cultivars (Tab. 1). Germination of somatic embryos of V. riparia and Chasselas increased in liquid medium while those of Szlanka decreased. Although the percentage of regenerated plants from Sziirkebanit embryos was nearly doubled due to the liquid medium, statistically significant differences could not be demonstrated. Table 1 Effects of different treatments of germination on plant regeneration Embryos germinated Embryos germinated on solid medium I on liquid medium I regenerated plantlets regenerated plantlets 62/24 46/26 Chasselas* 38% a 52 % a 46/14 78/22 EME 30%a 28 %b 30/14 39/4 Szlanka* 46% a 10% c 16/2 46/10 Sziirkebanit 12 % b 21% bed 69/24 104/32 G 28 35 %a 30%bd 45/4 52/32 Vitis riparia* 8%b 61 % a Values with the same letter are not significantly different at P = 0.05 according to the Student's t test (by columns). * Significant difference between the two germination methods. Repetitive somatic embryogenesis: In most cases responsive embryos produced both somatic embryos and yellow embryogenic calli. Some explants became swollen, turned entirely or partly dark-brown and necrotic. In many cases from this kind of necrotic tissue an induction of secondary somatic embryogenesis or embryogenic calli could be observed. The results shown in Tab. 2 indicate that repetitive somatic embryogenesis was affected by genotype, devel-opmental stage of the somatic embryos and concentration of plant growth regulators. The best results were obtained with G 28 embryos, regardless of developmental stage and medium while Chasselas had the poorest capability for re-current somatic embryogenesis. The cotyledonary stage em-bryos gave a higher frequency of embryogenesis than tor-pedo stage embryos except for V. riparia on MSE medium. In 4 cases significant differences were observed between the MSE and the MSE/2 medium. G 28 cotyledonary and V. riparia torpedo and cotyledonary stage embryos pro-duced a significantly higher level of embryogenesis on MSE/2 than on MSE. However, 19 % of the Chasselas cotyledonary embryos showed a secondary embryo induc-tion on MSE whereas only 2.4 % on MSE/2. T ab le 2 Effect of developmental stages and different media (MSE, MSEI2) on the induction of recurrent somatic embryogenesis MSE MSE/2 Number of Number of embryos I recurrent embryos I recurrent embryogenesis embryogenesis Chasselas 76/1 93/2 torpedo (1.3 %) a (2.1 %) a Chasselas 56111 41/1 cotyledonary* (19 %) bf (2.4%)a EME 91121 94/49 torpedo (23 %) bcf (52%) bd EME 65/36 38/25 coty ledonary (55%) d (65 %) bd Szlanka 40/27 54/23 torpedo (67 %) d (42 %) bd Szlanka 49/37 40/32 cotyledonary (75 %) d (80 %) cd G28 67/61 53150 torpedo (91 %) e (94 %) c G28 38/37 42/42 cotyledonary* (97 %) e (100 %) Riparia 35/11 37/18 torpedo* (36 %) bed (48 %) b Riparia 39/5 23/30 coty ledonary* (13 %) f (76 %) d Values with the same letter are not significantly different at P = 0.05 according to the Student's t test (by columns). * Significant difference between the two plant growth regulator treatments. Somatic embryogenesis and plant regeneration 157 Discussion During induction of somatic embryogenesis, differ-ences were observed among cultivars not only in the effi-ciency of embryogenesis but also in the behavior of embryogenic calli, e.g., calli of Szi.irkebanit produced few embryos. Replenishment of liquid medium is supposed to re-move inhibitory compounds (CouTos-THEVENOT et al. 1992) and helps to promote normal shoot meristem. However, this technique was not clearly beneficial for all varieties tested. It promoted the conversion of Chasselas and V. riparia embryos to plants but reduced that of Szlanka and did not change significantly the conversion rate of EME, Szi.irkebanit and G 28. This indicates that the previ-ously described method (MozSAR and SOLE 1994) is appro-priate only for certain cultivars. Other procedures published to improve regeneration frequency of grapevine somatic embryos had the same limitation (GRAY 1989, VJLAPLANA and MuLu Ns 1989, CouTos-THEVENOT et al. 1992). For grape transformation recurrent cycles of embryo-genesis seem to be the most promising way for selection of genetically transformed clones (MARTJNELLJ and MANDOUNO 1994). In our experiments its efficiency de-pended on genotype, developmental stage and growth regu-lator treatment. For the induction of repetitive embryogenesis early cotyledonary stage embryos gener-ally were more efficient than embryos in the torpedo stage and the MSE/2 medium was more efficient than MSE. Of the investigated genotypes, G 28 appears to be the best variety for a successful genetic transformation. Its an-thers are adequate explants for the induction of somatic embryogenesis, it has an acceptable regeneration rate and an extremely good inclination for recurrent somatic embryogenesis. Suitable results were achieved with V. riparia, Szlanka and EME and Chasselas, if the opti-mum treatment was applied in each step. E.g., 76% of the V. riparia cotyledonary embryos produced adventitious em-bryos on MSE/2, while only 13 % on MSE, 46 % of Szlanka embryos converted to plants when germinated on solid MS whereas only l 0 % when germinated in liquid medium. This study demonstrated that the behavior of grape-vines in an in vitro embryogenic culture is highly influ-enced by the genotype. No correlation was be observed between the degree of relatedness and the responses of grapevines. Closely related genotypes often gave diver-gent responses, while genetically different grapes behaved similarly. Acknowledgements This work was supported by OTKA F017358. We thank Dr. K. B AKONYI and Dr. I. KoLEDA for plant material. References COUTOS-THEVENOT, P.; GOEBEL-TOU RAND, !. ; MAURO, M. C.; 10UANNEAU, 1. P.; BoYLAY, M.; DELOIRE, A.; GuERN,.J.; 1992: Somatic embryogenesis from grapevine cells. I. Improvement of development by changes in culture conditions. Plant Cell Tissue Organ Cult. 29, 125-133. GRAY, D. J.; 1989: Effect of dehydration and exogenous growth regulators on dormancy, quiescence and germination of grape somatic embryos. In Vitro Cell. Dev. Bioi. 25, 1173-1178. MARTJNELLI, L.; BRAGAGNA, P.; POLETT, V.; Sc!ENZA, A.; 1993: Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris. Plant Cell Rep. 12, 207-210. - -; MANDOLJNO, G.; 1994: Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.). Theorel. Appl. Genel. 88, 621-628. MAURO, M. C.; NEF, C.; FALLOTH, J.; 1986: Stimulation of somatic embryo-genesis and plant regeneration from anther culture of Vitis vinifera cv. Cabernet Sauvignon. Plant Cell Rep. 5, 377-380. MozsAR, 1. ; SOLE, S.; 1994: Rapid method for induction of somatic embryogenesis from Vitis riparia. Vitis 33, 245-246. MURASHIGE, T.; SKOOG, F.; 1962: A revised medium for rapid growth and bioassay with tobacco tissues. Physiol. Plant. 15,473-497. RAJASEKARAN, K.; MuLLJNS, M. G.; 1979: Embryos and plantlets from cul-tured anthers of hybrid grapevines. J. Exp. Bot. 30, 399-407. SCORZA, R.; CoRDTS, 1. M.; RAMMING, D. W.; EMERSHAD, R. L.; 1995: Trans-formation of grapevine (Vitis vinifera L.) zygotic-derived somatic embryos and plant regeneration of transgenic plants. Plant Cell Rep. 14, 589-592. VJLAPLANA, M. ; MuLLJNS, M. G.; 1989: Regeneration of grapevines (Vitis spp.) in vitro: formation of adventitious buds on hypocotyls and coty-ledons of somatic embryos. 1. Plant Physiol. 134,413-419. Received June 17, 1996 

Genotype effect on somatic embryogenesis and plant regeneration of Vitis spp.

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Genotype effect on somatic embryogenesis and plant regeneration of Vitis spp.

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