In vitro Long-term Storage and Regeneration of Bulgarian Grapevine Variety “Velika” via Repetitive Somatic Embryogenesis

Somatic embryogenesis being one of the in vitro techniques that produce high regeneration rates, serves for high frequency propagation, gene transfer and germplasm storage. The aim of this work is to study the possibilities for in vitro long-term storage of grapevine genetic resources via repetitive somatic embryogenesis. Thirteen years after induction from leaves explants embryogenic cultures of Bulgarian grapevine variety “Velika” are still propagating via repetitive somatic embryogenesis in frame of clusters keeping their embryogenic and morphogenic competence. Plantlents regeneration from cotyledonary stage somatic embryos showed a normal phenotype compared with shoot-tip propagated controls. The genetic fidelity of regenerated plants was assessed by DNA analysis using Inter Simple Sequence Repeat (ISSR) markers. The amplified loci showed no differences between mother and regenerated plants, confirming the genetic authenticity of in vitro stored and propagated plants.


Introduction
In vitro techniques are a convenient tool for vine breeding to overcome the difficulties in conventional breeding studies and obtaining new cultivars. One of these techniques is somatic embryogenesis. The term of somatic embryogenesis is described as embryo formation from somatic cell or tissue. These embryos are similar to zygotic embryos formed by fertilization. Somatic embryogenesis being one of the in vitro techniques that produce high regeneration rates, serves for high frequency propagation, gene transfer and germplasm storage (Tsvetkov et al., 2005 [1]). Additionally, genotype specificity within species mainly limited the application of somatic embryogenesis as an effective tool for plant regeneration in Vitis sp. (Tsvetkov et al., 2000 [2]). Different explants have been used for the induction of grapevine somatic embryogenesis, mainly anthers/ovules, but also tissues from vegetative organs (leaves, petioles, stems, internodes) (Martinelli and Gribaudo, 2001 [3]). Several strategies have been adopted in order to keep indefinitely the embryogenic competence of the cultures and thus obtain a long-term source of somatic embryos. The maintenance of embryogenic cultures has been reported to last even for several years, retaining their embryogenic competence (Martinelli and Gribaudo, 2009 [4]). In vitro culture techniques can lead to genetic variation in plants trough somaclonal variation (Neelakandan and Wang, 2011 [5]). Flow cytometry of cells of grapevine plants, regenerated via somatic embryogenesis has shown that polyploidy somaclonal variation is produced during in vitro culture (Prado et al., 2010 [6]; Acanda et al., 2013 [7]).

Plant Material and Somatic Embryogenesis
Embryogenic cultures from in vitro leaf tissues were established and multiplied on the base of the protocol described by Tsolova and Atanassov (1996 [8]). The 13 years storage of the repetitive somatic embryogenesis were done by periodic transfer (every 3 months) to fresh NN medium (Nitsch and Nitsch, 1969 [9]) supplemented with 2 mg/l 2,4 D (2,4 dichlorophenoxyacetic acid), 1 mg/l IAA (indole-3-acetic acid), 1 mg/l BAP (benzylaminopurine) and 0,6 mg/l GA 3 (gibberelic acid) for embryo maintenance and multiplication. In vitro plantlets were regenerated on hormone-free ½ MS medium (Murashige and Skoog, 1962 [10]) from selected somatic embryos in cotyledonary stage of their development.

Somatic Embryogenesis
Indirect somatic embryogenesis from in vitro leaf explants was obtained after 5 months induction with a rates of 78% of callus induction and 3,5% of embryo induction. New formedsomatic embryos at early stages were removed from primary callus (Fig.1) and placed on fresh embryo maintenance medium in groups of 10 as embryogenic clusters (Fig.2). The indefinite maintenance of embryogenic culture is a crucial point of somatic embryogenesis protocol. Different methods have been attempted in order to avoid the progressive loss via necrosis as well as culture aging during subcultures (Martinelli and Gribaudo, 2009 [4]).
Our strategy was to subculture embryogenic clusters as a complete unit. Thirteen years after induction from leaves explants embryogenic cultures are still propagating via repetitive somatic embryogenesis in frame of the clusters keeping their embryogenic and morphogenic competence. It is important to note that one of the major problems of plant tissue propagation is the occurrence of phenotypic variations between the original stock material and in vitro derivate plant material. These phenotypic differences are result of somaclonal variations (Larkin and Scowcroft, 1981 [12]). In present study, all plantlets regenerated from cotyledonary stage somatic embryos (Fig.3) showed normal phenotype features, which did not manifest any deviation in comparison with the shoot-tip propagated controls (Fig. 4). After the successful regeneration from somatic embryos the plantlets were micropropagated through typical in vitro techniques which kept the phenotypic characteristics of the cultivar. However, if changes and abnormalities in morphology are not observed, that does not exclude presence of genetic variation. Moreover, grapevines are woody perennial plants and possible variations can manifest in later development stages. To assess the genetic stability and fidelity of in vitro propagated plants, obtained through somatic embryogenesis, a DNA assay was conducted.

Genetic Analysis
To perform a genetic assay we chose ISSR markers, which are one of the favored DNA marker systems for assessment of the genome stability in regenerated plants. For present DNA assay we analyzed 7 randomly chosen in vitro propagated plantlets and the original stock material of cv. "Velika" with 10 ISSR primers. All primers amplified successfully and generated fingerprinting profiles for the analyzed samples based on distinct reproducible amplification products. Every ISSR-primer produced a fingerprint profile, which showed to be identical for all analyzed samples (Fig. 5).
So far, ISSR analysis has been successfully applied to study the genetic stability of micropropagated plantlets of different crops (Martins et al., 2004 [13]; Alizadeh et al., 2008Alizadeh et al., , 2009). Present results show that the ISSR-profile of all analyzed regenerants matches the profile of the original mother plant cv. "Velika". None of the primers showed polymorphism in the analyzed set of plants which confirmed the fidelity of regenerated plantlet population to the genotype of mother plant. Present study showed that the long term storage of somatic embryos and the in vitro propagation did not cause variation in phenotypic and genetic make-up of regenerated plantlets. Additionally, ploidy evaluation with a technique such as flow cytometry of somatic embryogenesis-regenerated plants is recommended to assure trueness-to-type.

Conclusions
The long term storage of grape somatic embryos under the described protocol did not affect the competence of embryos and the genetic fidelity of regenerated plantlets and therefore can be successfully applied for propagation, gene transfer and germplasm storage of grapes.