Effect of sucrose on shoot regeneration in Agrobacterium transformed Hypericum perforatum L. roots

Abstract

The aim of the present study was to investigate the effect of sucrose on shoot regeneration potential in Hypericum perforatum L. roots obtained by Agrobacterium rhizogenes transformation. The morphological evaluation of transgenic roots grown on media supplemented with sucrose (0.5, 1, 2, 4, 6 and 8 \%) indicated that both genotype and sucrose concentration significantly affected root elongation and branching, as well as shoot regeneration. For two of five analyzed clones, lower sucrose concentrations (up to 2 \%) led to intensive shoot regeneration, while the other three clones intensified shoot development only at elevated sucrose concentrations (4 \%). For all clones, concentrations above 4 \% had a deleterious effect on both root and shoot development. Genetic characterization of regenerated shoots revealed that all tested clones were diploid with an average of 0.670 +/- 0.002 pg of DNA per nucleus, with no significant differences between transgenic and non-transformed plants and, according to PCR, with integrated A. rhizogenes rolA, -B, -C and -D genes. Real-time RT-PCR confirmed the expression of rolA, -B and -C, while expression of the rolD gene was not detected. Differences were detected in the absolute amounts of transcripts between analyzed clones, with the highest levels of expression for all three analyzed rol genes in a clone previously defined as having high root differentiation and less effective shoot regeneration potential. The observed variations in morphogenesis potential could be attributed to different levels of expression of integrated rolA, -B and -Cgenes; while sucrose additionally pointed out these trends.