Somatic Embryogenesis in Iberian Grapevine (Vitis vinifera) Cultivars Using Carpels as Initial Explants: Protocol Establishment and Histological Evaluation

Abstract

Trincadeira and Aragonez are two important grapevine cultivars in the Iberian Peninsula, used for high quality red wines production. Both cultivars are strongly affected by fungal diseases, with consequent high loses on plant productivity and fruit quality. A successful protocol for plant regeneration via somatic embryogenesis (SE) was established for both cultivars allowing further plant improvement based on gene transfer technology. Several factors were evaluated during the three different phases which characterize an SE plant regeneration protocol. The culture room temperature during the induction phase, a parameter usually accepted as standard by most researchers, proved in these trials to significantly affect the embryo induction rates. Concerning embryo conversion, it was specially affected by the embryo developmental stage, by the intensity and duration of the chilling treatment and by the supplementation of conversion culture medium with activated charcoal. The responses obtained, both for induction and conversion, proved to be highly genotype dependent. Calli structure, as well as embryo integrity, was histologically observed, allowing to characterize embryonic and non-embryonic masses and to identify abnormalities on embryo development.

Somatic Embryogenesis from Mature Embryos of Olea europaea L. cv. ‘Galega Vulgar’ and Long-Term Management of Calli Morphogenic Capacity

“…Embryos at different developmental stages, from globular to cotyledonous, were simultaneously identified on the same callus ( Figure 5A). This asynchronous development has been commonly found in SE across plant species [41,63,64]. Higher homogeneity on embryo development can only be achieved with the selection of embryogenic cell lines, as proposed by Frederico et al [65].…”

“…The efficiency of SE induction depends on several factors with a complex interaction among them. The main factors affecting SE efficiency are the genotype of the donor plant, the type of explant taken for the establishment of in vitro cultures, the development and physiological stage, the growth conditions (mainly photoperiod and temperature), and the chemical composition of the culture media (including type and concentration of growth regulators) [40,41]. The genotype strongly affects the ability of a tissue to dedifferentiate and de novo acquire meristematic competence and to differentiate embryogenic structures [39,[42][43][44].…”

“…The genotype strongly affects the ability of a tissue to dedifferentiate and de novo acquire meristematic competence and to differentiate embryogenic structures [39,[42][43][44]. Individual genotypes within the same species vary greatly in embryogenic capacity, reflecting substantial differences in the ability to activate key elements for the achievement of embryogenic competence [41] which shows the need to develop improved methodologies for each genotype. Results reported by Narváez and co-authors [39] emphasize this dependence, showing SE induction efficiency in two olive wild genotypes from the four initially considered, and embryos conversion from a single one.…”

“…Besides the genotype, the developmental stage and age of the explants used as start material, are key factors determining the success of SE induction. Several reports on woody plant species, Vitis vinifera [40,41] Picea abies [45], Prunus incisa [46], Pinus radiata [47], and Eucalyptus globulus [48], emphasize the effect of explant developmental stage on embryogenesis efficiency.…”