Vitis 47 (4), 251–252 (2008) Research Note An efficient buds culture method for the regeneration via somatic embryo- genesis of table grapes 'Red Globe' and 'Flame Seedless' S. ARAYA, H. PRIETO and P. HINRICHSEN Laboratorio de Biotecnología, Centro de Investigación La Pla- tina, Instituto de Investigaciones Agropecuarias, Santiago, Chile K e y w o r d s : T able grapes, somatic embryo- genesis, growth regulators, genetic transformation. A b b r e v i a t i o n s : NOA = 2-naphthoxiacetic acid; NAA = α-naphthalene acetic acid; BA = 6-benzyladenine; 2,4-D = 2,4-dichlorphenoxyacetic acid; IBA = indolbutyric acid; SE, somatic embryogenesis. Introduction: Chile is the leading table grape ex- porter from the Southern Hemisphere and this industry is mainly based on 'Thompson Seedless', 'Red Globe' and 'Flame Seedless' ('Flame'), covering more than 75 % of the table grape planted area in the country. With the aim of im- proving the productivity and quality of these and other cul- tivars, a genetic transformation program was initiated by 1999, mainly focused in the introduction of genes related to biotic stress tolerance (HINRICHSEN et al., 2005). With this goal in mind, somatic embryogenesis (SE) was settled for these cultivars, as this has become the most efficient procedure for the generation of in vitro cultures prone to genetic transformation (STAMP and MEREDITH 1988, SCORZA et al. 1996, MARTINELLI et al. 2001, IOCCO et al. 2001, TOR- REGROSA et al. 2002). However, grapevine SE is not a rou- tine procedure that can be easily and efficiently reproduced in different cultivars (MARTINELLI et al. 2001). There exists a genotype-specific response even when using the same culture medium in the induction process (SRINIVASAN and MULLINS 1980, STAMP and MEREDITH 1988, TORREGROSA et al. 2002). In this regard, most of the advances published on SE has been focused on wine cultivars (STAMP and MEREDITH 1988, IOCCO et al. 2001) as well as in 'Thompson Seedless' (SCORZA et al. 1996). Very few additional work has been directed to optimize SE in other table grape cultivars such as 'Red Globe' (PERL et al. 1996) and 'Flame Seedless'. In the case of 'Flame', there are no reports specifically related to the optimization of the production of somatic embryos. Taking these antecedents into consideration, the purpose of this note is to describe the best combination of growth regulators for these two table grape cultivars, in order to optimize the induction of somatic embryos. Material and Methods: Apical and axial buds with 2 to 4 leaves were cut from in vitro grown plants of 'Red Globe' and 'Flame'. Plants were grown in C2D medium (CHEÉ and POOL 1987) supplemented with 4 μM 6-benzy- ladenine (BA) to induce multiple budding, in a period of about 30 d. Buds were excised using a stereoscopic lens and later incubated for callus induction in a modified basal medium [NB® (PhytoTechnology Labs., Shawnee Mis- sion, KS) plus 3 % sucrose, 1 g·l -1 myoinositol and 0.7 % (p/v) TC-agar® (PhytoTechnology) and 5 µM 2,4-dichlo- rphenoxyacetic acid (2,4-D)] plus a range of concentra- tions of α-naphthalene acetic acid (NAA) and BA (see Ta- ble for specific conditions). Media were adjusted to pH 5.8 and sterilized by autoclaving at 121°C for 20 min before pouring in 90 x 15 mm Petri dishes. Five explants per plate were incubated for 30 d in darkness at 24 ± 2 °C. After this time, calli formed were kept at 16 h light/8 h darkness, at the same temperature for 150 d. Correspondence to: Dr. P. HINRICHSEN, Laboratorio de Biotecno- logía, La Platina Research Centre, Instituto de Investigaciones Agropecuarias, P.O. Box 439-3, Santiago, Chile. Fax: +56-2-757-5139. E-mail: phinrichsen@inia.cl T a b l e Concentrations and combinations of growth regulators added to the modified NN medium for the induction of somatic embryo- genesis from apical and axilar buds of the 'Red Globe' and 'Flame Seedless' cultivars Medium Concentration of growth regulators (µM) 2,4 – D BA NAA NB1 5 1 --- NB2 5 5 --- NB3 5 2.5 2.5 NB4 5 2.5 5 NB5 5 5 5 Somatic embryo induction and devel- opment: Pro-embryogenic masses were transferred to X6 medium (LI et al. 2001) and kept at 24 ± 2 °C under the same photoperiod for 90 d. Somatic embryos were picked up from masses according to their developmental stage and maintained on the same medium. To induce their develop- ment, embryos at cotyledonary stage were transferred to C2D medium supplemented with 0.4 μM BA, 0.25 μM in- dolbutyric acid (IBA), 2 g·l -1 glycine and 0.5 g·l -1 activated charcoal and kept under the same conditions of tempera- ture and photoperiod for 20 d. The total time involved in the whole induction and development of embryos was at least 110 d. S t a t i s t i c a l a n a l y s i s : A completely randomized design with sub sampling was used. The experimental unit was conformed by five bud explants maintained in a single Petri dish, and each treat- ment consisted of 20 replicates. To stabilize the variance, data were transformed according to √(x + 0.5). Means were separated using the Duncan multiple test (p = 0.01). Results and Discussion: Most commonly used explant and growth regulator combinations for the induction of SE on grapes include the use of leaves and anthers and low doses of cytokinins plus high doses of auxins, such as me- dium NB1 (Table) (SRINIVASAN and MULLINS 1980, STAMP