In vitro regeneration and transformation of rye (Secale cereale L.)
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In vitro cultures are an integral part of plant transformation. Genetic manipulation can be performed only on a single cell level. Therefore in vitro culture and regeneration of plants from a single cell are very important for successful transformation In vitro culture of rye is more difficult to conduct than of others cereals. Difficulties with in vitro regeneration of rye seems to be the main factor limiting the development of rye transformation systems. Genetic transformation process includes three main steps, single cell transformation, selection of transgenic cells and regeneration of plants from single cells. Efficiency of each of these steps can influence the result of the transformation process. Therefore optimisation of those steps is very important.Transformation has been performed using the microprojecticle method and scutellum of rye embryos as a target. Two constructs have been used in cotransformation, pDB1 containing a marker bar gene (phosphinotricine acetylotransferase) for Basta herbicide resistance and a repoter uidA gen (beta-glucuronidase) and pAwact-Sec containing 196 bp fragment of a sec-1 gene in anti-sense orientation. Using pAWact-Sec construct we tried to block the expression of the endogenous gene sec-1 coding omega-secalin which is storage protein of rye grain. We were able to regenerate transgenic rye plants containing all introduced genes. The efficiency of sec-1 expression blocking was analysed by SDS-PAGE method. Among 50 analysed kernels of T1 generation we found 5 with lower omega-secalin level. Complete blocking of omega-secalin was not observed.
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S. Sowa, Instytut Hodowli i Aklimatyzacji Rosin, 05-870 Radzikow, Poland, e-mail: email@example.com