Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain)

Abstract

An efficient protocol for plant regeneration through somatic embryogenesis was developed for the first time in five autochthonous grapevine cultivars (Treixadura, Torrontés, Mencía, Merenzao and Brancellao) from Galicia (north-western Spain). Improvements of the induction protocol for the cv. Albariño in respect to previously reported data were also made. Media containing NN salts and MS vitamins supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) were effective in inducing somatic embryogenesis. The addition of casein hydrolysate produced the best results for up to four cultivars (Albariño, Treixadura, Merenzao and Brancellao). Somatic embryogenesis was also induced in explants collected during the binucleate pollen microsporogenesis stage (R3 flower stage) of all cultivars with the exception of Treixadura, suggesting that under appropriate conditions explants can display longer windows of competence. Transfer of embryogenic callus to differentiation medium produced callus proliferation and somatic embryo development proliferation by secondary embryogenesis. However, an extensive process of precocious embryo germination was observed reducing the efficiency of secondary embryo proliferation. This situation was overcame by the use of differentiation medium lacking growth regulators (DM1 medium), which allowed reducing precocious germination by half on average and improving embryo proliferation by secondary embryogenesis. Transfer of normally developed, ungerminated isolated embryos to germination medium allowed obtaining very high percentages of embryo germination (up to 97% in Mencía, more than 87% averaging all cultivars). A comparison of plant conversion between precociously and normally germinated embryos showed that precocious germination in differentiation medium reduced plant conversion, even at high rates depending on the cultivar (from 93% to 39% in Brancellao, from 86% to 61% averaging all cultivars).