| The regeneration system was established on Saposhnikovia divaricata （Turcz.） Schischk.through inducement and differentiation of callus with different explants. The results can besummarized as follows:1.Explants: Callus could be induced by different explants including seed, radicle, stem,cotyledon and leaf, but induce ratio and growth form of callus were different from each other.It was proved that stem was the best explant for inducing callus.2.Culture medium and hormone ratio: The effects of different hormone combinations oncallus formation were greatly, and auxin2,4-D is a key factor in callus formation. The bestculture medium was MS+2,4-D1.5mg.L-1+6-BA1.0mg.L-1+KT0.5mg.L-1, on which thehighest induce ratio was96.7%. Callus under different hormone combinations were bothproliferated, but the best subculture medium was MS+6-BA1.0mg.L-1+NAA1.0mg.L-1, onwhich the callus multiplication times could reach to5.2. This kind of subculture callus couldbe differentiated with a high degree in the later differentiation process.3.Differentiation of callus: The callus turned green and formatted many buds aftertransferred into the culture medium, and then clustered shoot was formatted. The best mediumfor differentiation was MS+6-BA1.0mg.L-1+NAA0.5mg.L-1,on which the highestdifferentiation could reach to75.0%4.Root regeneration: Roots began to grow after the callus were transferred into therooting medium15days later. On the1/2MS medium with NAA0.5mg.L-1and6-BA0.2mg.L-1, the highest rooting rate could reach to65.0%, on which the number of rootswas16.3.5.Refine and transplant the seedings: The regeneration plants were hardened2d to3d inclosed bottles natural light. Then opend the bottle caps and hardened1d to2d. The mediummust be sterilized before transplanting. The best transplanting matrix was rotten quality soil,vermiculite and river sand by1to1to1and92.0%seedlings could survive.6.The RAPD result of tissue culture seedlings:15tissue culture seedlings differentiatedby the same stem callus and CK were analyzed by RAPD, and only one primer amplified onestably polymorphic segment of640bp from the6th regenerated plant. The result told that tissue culture could keep genetic characteristics very well, but at the same time it could leadto the change of DNA and produce the genetic variation.7.Culture conditions and vitrification: Hormones, agar concentration and light intensityhad apparent effect on the occurrence of vitreous buds during tissue culture. The resultshowed that too high6-BA concentration, too low agar and faint light intensity easily inducedthe occurrence of vitreous buds.8.The analysis of peroxidase isozyme of green and vitreous plantlets: The result showedthat the vitreous plantlets lost three bands such as Rf0.150, Rf0.179and Rf0.300of highactivity, but added four bands such as Rf0.140, Rf0.147, Rf0.156and Rf0.182of high activityand two secondary bands. The possible reasons were that a structure gene might be regulatedby multiple promoter genes, and vitrification caused structure gene expression unprofessional;vitrification might also promote other genes which changed the normal metabolism andproduced more secondary bands. |
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