| A sound regeneration system was established using a combination of different culturemediums/explants and hormone and different spacing intervals of the subculture. And then,the optimum concentration of EMS was studied for mutagenesis for both callus and leaves ofCodonopsis lanceolata.The roots which were induced from callus were determined thecontent of saponins. The roots were also analyzed by RAPD and clustering analysis to screenout variant plants. The main results can be summarized as follows:1. Improvement of regeneration system: The highest induce ratio and average ratio onMS were100%and70%, respectively, which was significantly higher than that of N6.Calluscould be induced by different explants including leaf, stem and seed, of which the callusinduced by leaves grew fast with close texture, low browning rate. And the highest induceratio on leaf was100%. Concentration of hormone contributed to browning rate, it wasproved that both high level of6-BA and KT could promote browning rate. So the bestculture medium was MS+2,4-D0.5mg.L L-1+6-BA1.0mg.L L-1+KT0.5mg.L L-1, on whichthe browning rate was the lowest（3.3%）, the induce rate was the second largest one（86.7%）.2. Influence of spacing intervals on browning rate: The lowest browning rate was3.3%,with3days as spacing interval, and the callus grew fast and texture closely. When thespacing interval exceeded15days, some calluses grew slowly with yellowish-brown orbrown. When it was over30days, most calluses browned. So shrinking time was aneffective way to reduce browning rate.3. EMS treatments and material survival: Both the survival rate and regeneration rate ofmutagenized leaves or calluses by EMS were under control, and reduced along with theEMS concentration increased. The results showed that the fatal combination of EMSconcentration and treatments time for leaf was （0.4%+4h）; for callus was （0.3%+4h）,respectively and the median lethal concentration of EMS for leaf was （0.4%+2h）; for calluswas （0.3%+2h）, respectively, the survival rate of the former was46.7%, the latter was50%and the differentiation rate of the former was14.3%, the latter was13.3%.4. Roots regeneration: Roots began to grow after the calluses were transferred into the rooting medium.45days later the growth of roots was stable. The rooting rate wasdramatically reduced by EMS. The roots from both leaves and calluses were successfullyinduced under half lethal EMS concentrations. It proved that the rooting rate of treatedleaves and calluses with median lethal concentration of EMS were9.8%and12.3%respectively, which were significantly below controls（78.3%and79.2%,respectively）. Theroots from both leaves and calluses were successfully induced under half lethal EMSconcentrations, namely,0.3%+2.0h and0.2%+2h could be regarded as the ideal treatmentof mutagenesis. And the best EMS concentration of mutagenizing Codonopsis lanceolatawas （0.2%+2h） on callus.5. Gain of variant: Ginsenosides determination was made on10mutagenesis seedlingswhich had external shape differences. It turned out that two of them had relatively highdiosgenin content, of which No.6contained5.061mg/g diosgenin,5.39%higher than that ofaverage, and No.6contained4.926mg/g diosgenin,2.58%higher than that of average. Thevariation of diosgenin content of treatments was0.372mg/g.6. Genetic analysis of mutagenesis regeneration group：The RAPD and clusteringanalysis on seedlings which were mutagenized by callus. It turned out that there were59strips amplificed by8special primers on10materials, of which44of the strips werepolymorphic which took up74.6%. Genetic similarity coefficients within the differentseedings were among0.454-0.774.the similarity coefficient between No.6and others was0.5. The high diosgenin content and larger genetic distance showed that there was aninevitable relationship between the two. So, No.6was the variation. |
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