Somatic embryogenesis from immature anther explants: Toward the development of an efficient protocol production of grapevine

Abstract

This paper reports on successful establishment of friable embryogenic callus (FEc) initiated from immature anther explants collected from the vineyard within 1 month. The anther collection, filament attachment, filament length, stages, anther size, color, and maturation are the multiple factors involved in the establishment of embryogenic callus. Anthers with immature filaments having a translucent white color, a length that was between 0.02 mm and 0.05 mm, and at either stage II or stage III gave a high frequency of embryogenic callus. In contrast, mature anthers with green translucent or yellow color filaments attached with a length between 0.08 mm and 0.2 mm and at either stage V or stage VI produced non-embryogenic callus (NEc), with browning of the anther in the medium. Inflorescences collected from bud initiation onward could be cultured as immature anthers, and a good response was recorded; FEc and NFc were separated from initiation in the form of a callus as early as 9 weeks. The initiation of proembryogenic masses (PEMs) and the subsequent stages of development were analyzed; the maximum percentage of embryogenic callus obtained was 56% for Sultana, 48% for Red Globe, and 39% for Merlot at stage III. In the modified medium as described in Franks et al. (Mol Breed 4:321-333, 1998), subculturing was carried out on NN medium containing 2,4-D (1.5 mg dm-3), BA (1.5 mg dm-3), sucrose (60 g dm-3), and PVP (25 mg dm-3). The excretion of polyphenols into the subculture was reduced by culturing the callus in the same medium in the presence of 25 mg dm-3 PVP, and this reduced the browning of the culture medium. In conclusion, many factors are involved in determining embryogenic callus initiation, and these are, in turn, linked to subsequent embryo initiation, development, and regeneration during somatic embryogenesis.