ISHS Acta Horticulturae 459: International Symposium Brassica 97, Xth Crucifer Genetics Workshop
HIGH PLANT REGENERATION, GENETIC STABILITY OF REGENERANTS, AND GENETIC TRANSFORMATION OF HERBICIDE RESISTANCE GENE (BAR) IN CHINESE CABBAGE (BRASSICA CAMPESTRIS SSP. PEKINENSIS) |
| Authors: | H.T. Lim, Y.S. You, E.J. Park, Y.N. Song, H.K. Park |
| Keywords: | Brassica campestris ssp. pekinensis, Chinese cabbage, bar, regeneration, genetic transformation, hygromycin, kanamycin, zeatin, silver nitrate, tobacco feeder cells, genetic stability |
| Abstract:
Ten breeding lines including two CMS (Cytoplasmic Male Sterility) lines and one cultivar, Seoul, of Chinese cabbage (Brassica campestris ssp. pekinensis), known to be one of the most difficult vegetable crops to be regenerated from in vitro tissue culture, were used to establish the plant regeneration system. Two different types of explants, cotyledonary petiole and hypocotyl segments were cultured on MS basal medium supplemented with several combinations of plant growth regulators (PGRs). The optimum level of AgNO3 for shoot regeneration ranged from 0.5 to 1.0 mg/l. An addition of AgNO3 at higher than 1.0 mg/l caused the high vitrification rate of regenerated shoots. There were great differences in the abilities in shoot regeneration among genotypes and media types. In all genotypes, MS basal medium supplemented with higher levels of PGRs (NAA 1.0 mg/l, BAP 3.0 mg/l, AgNO3 0.5 mg/l) was far more effective in shoot regeneration than that containing lower level of PGRs(NAA 0.5 mg/l, BAP 2.0 mg/l, AgNO3 0.5 mg/l). The best shoot regeneration (68%), however, took place when hypocotyl explants were cultured on MS basal medium in conjunction with NAA 1.0 mg/l, BAP 2.0 mg/l, zeatin 2.0 mg/l, AgNO3 0.5 mg/l. RAPD (Randomly Amplified Polymorphic DNA) analysis of regenerants using several arbitrary decamer oligonuclotides showed that there were no major detectable somaclonal variations except for one regenerant, indicating that most of the regenerants were genetically stable. For genetic transformation experiments, hypocotyl segments of selected genotypes were precultured in the medium (NAA 1.0 mg/l, BAP 2.0 mg/l, zeatin 2.0 mg/l) for two days and cocultivated with Agrobacterium tumefaciens strains containing NPTII and bar (bialaphos resistance) genes for two days. Tobacco feeder cells were included in the medium from preculture to cocultivation procedure. Shoot regenerated directly from the explants in the selection medium (NAA 1.0 mg/l, BAP 2.0 mg/l, zeatin 2.0 mg/l, AgNO3 0.5 mg/l) containing kanamycin 10 mg/l in a few weeks. Putatively transgenic plants were acclimatized to green house condition and applied to PCR analysis using bar primer. Ten out of 14 plants regenerated and survived from selection medium were found to be transgenic plants. | |
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