Agrobacterium – Mediated Genetic Transformation of the Resurrection Plant Haberlea Rhodopensis Friv.

 

G. PETROVA and D. DJILIANOV
AgroBioInstitute, BG – 1164 Sofia, Bulgaria

Abstract

PETROVA, G. and D. DJILIANOV, 2013. Agrobacterium – mediated genetic transformation of the resurrection plant Haberlea rhodopensis Friv. Bulg. J. Agric. Sci., Supplement 2, 19: 10–14

 

 Resurrection plants are widely used models for desiccation tolerance studies. Several genes have been successfully isolated from these species. In an attempt to study their role, these genes have been successfully transferred in other model plants. Since resurrection plants are as a rule, polyploids, they are pure targets for mutational studies. In this respect, the establishment of efficient and repeatable transformation system will contribute significantly for the elucidation of stress tolerance. In this study we describe for the first time, a procedure for Agrobacterium tumefaciens – mediated genetic transformation of the resurrection plant Haberlea rhodopensis Friv. For in vitro regeneration of Haberlea, we used liquid WPM media. It enables us to achieve direct regeneration and transformation system, which is an alternative to the callus-based transformation, used in other resurrection plants. The A. tumefaciens strain LBA4404 harbouring the plasmid pCAMBIA 1305.1 which contains the gus gene as a reporter gene and hpt II gene as a selectable marker gene was used. The initial experiments were conducted in order to establish the suitable concentration of cefotaxime for the elimination of Agrobacterium from cultures, as well as the optimal concentration of hygromycin for the selection of transformed plants. It was found that the highest concentration of cefotaxime that protocorms of H. rhodopensis could tolerate is 500 mg.l–1 and they are inhibited at 0.75 mg.l–1 hygromycin. Transformation was confirmed by histochemical GUS assay and 35S- / NOS- PCR analysis. The percentage of GUS activity was 3% and the optimal co-cultivation time was 60 minutes.

Key words: Agrobacterium, Haberlea rhodopensis, regeneration, transformation

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