Abstract
BackgroundSomatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type).ResultsSomatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV.ConclusionsCut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.
Highlights
Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L
The percentage of embryogenic explants ranged from 18.50%, in Airén, to 65.46%, in Verdil and it was mainly achieved via direct embryogenesis which was observed in at least 90.0% of the explants of the varieties Cabernet Franc, Mencía, Petit Verdot, Pinot Blanc, Pinot Gris, Pinot Meunier, Pinot Noir, and Verdil (Table 1; Fig. 2a)
After three (T3) and 4 months of culture (T4), the somatic embryogenesis percentage (E) had increased, decreased, or been maintained at similar rates - with respect to the previous period - depending on the cultivar (Table 1)
Summary
Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. Pathogenic agents, including 65 viruses, five viroids and eight phytoplasmas have been detected in a large number of infected grapevines [1]. Somatic embryogenesis consists in the induction of somatic embryos from cells of the explants cultured in vitro and is the preferred method for cell to plant regeneration in Vitis vinifera L. and its intraspecific or interspecific hybrids. It has been used for micropropagation [13, 14], generation of transgenic plants [15] and virus sanitation [3, 12]. The time required to recover plants was shortened with respect to other protocols
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