Genetic transformation of african violet (Saintpaulia ionantha Wendl.) and bud regeneration from transformed roots

Abstract:

Genetic transformation of African violet (Saintpaulia ionantha Wendl.) was performed using leaves isolated from in vitro-cultivated plants. Agrobacterium rhizogenes strain A4GUS was used for inoculation. The explants have been co-cultivated with bacteria on Murashige and Skoog (MS) medium with or without 50 mM acetosyringone (AS) for 3 days, and thereafter further cultivated on MS plant growth regulator (PGR)-free medium. A few days later, necrosis of inoculated explants was observed and, following 3 weeks of inoculation, it was recorded in 53 % and 71 % of explants co-cultivated on medium with or without AS, respectively, whereas the frequency of necrosis of uninoculated explants, treated and maintained in the same way as the inoculated explants, was about 3 % on both media. Following 6 weeks of inoculation, adventitious roots appeared on 19.3 % and 14.9 % of inoculated explants co-cultivated on medium with or without AS, respectively. By the time, 25 adventitious roots were isolated from the explants co-cultivated with AS, while 14 adventitious roots were isolated from the explants co-cultivated on AS-free medium, all obtained from independent transformation events. The roots were further grown as separate putative transgenic lines. After 16 weeks, only one hairy root line survived (marked as Si5). Genetic transformation of this line has been confirmed by the presence of the rolA, -B and -C genes and the absence of the virD1 gene in its genomic DNA. Roots of line Si5 were difficult‑to‑regenerate, and efficient shoot regeneration was achieved only in calli induced from the roots grown on medium with thidiazuron, and subsequently subcultivated on medium with benzyladenine. Five phenotypically most distinguishing lines, out of 20 lines of regenerants obtained from independent regeneration events, were subjected to molecular analysis. PCR analysis confirmed the presence of the rolA, -B, -C, and -D genes in 3 lines, while 2 lines, as well as the negative control, showed only a weak signal for all 4 rol genes. The presence of weak signal in these 2 lines of regenerants and the negative control might be due to the deletion of the rol genes, and could be explained by the presence of cT‑DNA within the genome of African violet. However, this phenomenon should be further investigated by hybridization with specific probes. The expression of all 4 rol genes has been shown in 3 lines of regenerants with positive PCR amplification. The rolC gene had the higest level of expression compared to the rolB and rolA, while the expression of the rolD gene was very low. In these 3 lines spontaneous occurrence of adventitious roots on the leaves cultivated on PGR-free medium was observed, but other symptoms typical for hairy root syndrome were absent. In negative control and 2 lines displaying only weak amplification of the rol genes by PCR, the expression of rol genes was absent, as well as any morphological disorders.

Key words: African violet, Gesneriaceae, hairy roots, caulogenesis, Saintpaulia ionantha, tissue culture, transgenic plants

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