Recombinant DNA technology comprises the removal of specific DNA fragments from one organism, the study of their functions and their subsequent incorporation into the existing genetic background of another organism, which may be a chosen plant species.
This comparatively new technology (the first transgenic plants were obtained in the labo-
GENETlCALL Y ENGINEERED GRAPEVINES 415 ratory in 1983, field tests with transgenic tobacco and tomatoes were first performed in 1987) has become an important part of plaI;lt breeding and it is even differentiated in the so-called "molecular breeding" (Logemann and Shell, 1993). This methodology enables new genes encoding new traits to be introduced without modifying the desired commer~
cial attributes of target crop plants.
Indeed genetic transformation has become an efficient tool to express alien genes into the crop plants. Along with the number of advantages: 1. The source of new genetic in- formation (the species not included in Planta, other plants, animal cells and etc.) that can be introduced into the plant genome is essentially unlimited; 2. The gene of interest (when a particular gene has been isolated and reconstructed) can be used in wide range of different crops; there are also a certain number of limitations. Because of that, genetic transformation cannot be assumed as a separate alternative of conventional plant breed- ing, but mostly as a complementary approach at a later stage.
There are also certain ethical and safety limitations (regulation of Genetic Modified Organisms - GMOs) that we are not going to discuss here. One essential limitation of pure methodological character is the number of transgenes that can be expressed in the same transgenic line, because of a process of co-suppression - "transgene silencing" that can be activated and to reduce the expression of trans gene (Galun and Breiman, 1997).
Much progress has been made in the last few years in the genetic manipulation of grape (see reviews by Gray and Meretith, 1992; Torregrosa, 1995; Perl, 1998). Agrobac- terium-mediated transformation has been the favored system for transformation of grape.
A combination of both methodologies: particle wounding by bombardment followed by Agrobacterium transformation has also been reported (Scorza et al., 1995, 1996).
The availability of an efficient adventitious regeneration procedure applicable to vari- ous cell and tissue culture system mainly determined the development of genetic trans- formation procedures in grape. The efficiency of regeneration is highly species depend- ent in Vilis (Gray and Meredith, 1992). Somatic embryogenesis is the favored regenera- tion protocol, suitable for genetic transformation of grape. Comparison of regeneration through embryogenesis with that achieved through organogenesis in grapes has demon- strated that the second one frequently leads to chimeras in transformed tissues (Mauro et al.,1995).
The efficiency of a transformation procedure depends on several factors: plasmid construct, optimal conditions for transformation, ability to regenerate plants from trans- formed tissue, proper selection and possibility of developing intact transgenic plants.
The crucial requirement in the process of transformation is th6 availability of cells hav- ing both the ability to be transformed and subsequently to regenerate into plants. Thus, embryogenic cells in suspension are very promising candidates for successful transfor- mation in grape (Fig. 16. 1).
On the basis of parameters for initiating and maintaining of embryogenic suspensions in seedless grapevine cultivars, reported by Lipsky et al. (1997), Colova-Tsolova et al. (2000) have reported two alternative protocols for the development of embryogenic cell suspen- sions from European grape Vitis vinifera L. cvs Velika and Prime. This study compared the
416 V. COLOVA-TSOLOVA et al
Figure 16.1. Proembryogenic mass (PEMs) in grape embryogenic suspension (cv Sugarone):
a) PEMs - light microscopy (faze contrast) XlOO; b) PEMs - scaning electron microscopy X 500.
development of somatic embryos from in vitro immature leaves and anthers. Embryo- genic cells were obtained after exposure of the explants to 2,4-D/BA (2,4- dichlorophenoxyacetic acidl6-benzyladenine) growth regulators. Both phytohormones
GENETICALLY ENGINEERED GRAPEVINES 417 were required to induce dedifferentiation, cell division and to detennine the embryogenic state. Both type of explants exhibited relatively high percentages of embryogenic callus initiation: 16.6% of leaves and 19.6% of anthers. A preliminary requirement for obtain- ing a high percentage of embryogenic calluses from leaves was two subsequent subcul- tures of the initial explants on a cytokinin-containing medium. Embryogenic lines were maintained both as clusters of differentiated somatic embryos through secondary em- bryogenesis and as proliferative PEMs (proembryogenic mass of cells) in embryogenic cell suspension culture. High frequency of conversion of PEMs to regenerate grape plantlets was achieved by combining dehydration of the culture with growth regulator treatment.