| Indigoblue woad(/sato indigotica Fort.) is not only a largely used medicinal plant, but also an important industrial material, used as dyer's-weed in China. It is widely cultured in our country. In this paper, genetic transformation systems by Agrobacterhim rhizogems was established and optimized and high quality regenerating system was selected. The products of indigotin and indirubin in regenerating plantlets had been detected by HPLC. The main studies and results were reported as follows: 1 Establishment and selection of hairy root clonesTaken cotyledons of autotetraploid indigoblue woad as explants and hairy roots induced successfully from them by Agrobaterium rhizogenes strains A4 and R1000. A4 had better root-inducing capability than R1000. The percentage of root induction by A4 was 100% and average root number per cotyledon was 30. Both IAA and light had significant effects on root-inducing efficiency. Percentage of root induction could be enhanced with MS medium supplemented with 0.5mg/l IAA. No root occurred without light. DNA of hairy roots for polymerase chain reaction (PCR) analysis was extracted. The presence of the rolB and rolC genes in the hairy roots was confirmed by PCR analysis. The positive result obtained in the amplification of the rolB and rolC in hairy roots could be attributed to successful transformation rather than to residual Agrobacterhim. These hairy roots grew well on MS agar medium or liquid medium withour growth regulator for over one year. They grew vigorously and produced numerous lateral branches. Four hairy root clones, namely TRA1, TRA2,TRA3 and TRA4, were established. Compared to hairy root system HRA3 which came from diploidindigoblue woad(2n=14), hairy roots from autotetraploid indigoblue woad(2n=28) grew faster. It was shown that hairy root of autotetraploid indigoblue woad was better than that of diploid indigoblue woad. 2 Establishment and optimization of regenerating plantlet system from hairy roots.(1) Differentiation of bud and root directly from hairy root. Hairy roots were subcultured every month. Twenty one adventitious buds from 30 root segment cultures were obtained after subculturing on medium lacking growth regulator for 90 days. The effects of hormones 6-BA and NAA on the adventitious bud differentiation were investigated and optimized. The results were showed that 6-BA had significant effect on bud differentiation and the optimized medium for adventitious bud differentiation was MS + l.0mg/l NAA + 0.5mg/L 6-BA. There were 67 adventitious buds occurred from average 30 hairy root segment cultures.(2) Regenerating plantlets were obtained from calli, which derived of hairy roots. The results of bud induction showed that 2,4-D had the significant effects on callus formation and followed bud differentiation. No callus occurred from hairy roots on medium lacking 2,4-D. High concentration (l.0mg/1) of 2,4-D could induce much more calli, but it obstructed the growth of calli and followed buds differentiation. The result of callus induction showed that the optimum medium for callus induction was MS + 0.5mg/l 2,4-D + 0.5mg/l 6-BA and MS + 0. 1mg/1 2,4-D + 0. 1mg/l 6-BA for callus growth. Adventitious buds could be spontaneously formed from calli on MS medium lacking hormones. Despite of it, the bud-inducing frequency was low. High frequency of bud induction was found on MS medium supplemented with 6-BA The optimum medium for adventitious bud induction was MS + l.0mg/1 NAA + 0.5mg/l 6-BA, and MS + 1.5mg/l NAA + 0.5mg/l IAA for bud rooting.(3) Regenerating plantlets were obtained via somatic embryo, which derived from hairy root. The effects on the induction of somatic embryos from hairy root were investigated using orthogonal test. The best somatic embryo induction was obtained on MS + 0.5mg/l NAA + 0.5mg/l KT + 30g/l sucrose. Among the three factors tested in our experiments, KT had the significant effect on the somatic embryo induction and its development, followed by NAA Sucrose had smallest effect on somatic embryo induction, but had the obviou...
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