| Dioscorea zingibernsis Wright, a native species in China, has the highest content of diosgenin in the rhizoma among all the species in Dioscorea. However, the diosgenin content and quantity in cultivated D. zingibernsis has been declined year and year. Microtubers, leaves, embryos and shoot-tips were used as initial explants. The effects of genotypes, explants, phytohormone combinations and light condition on calli induction of D. zingibernsis were studied by tissue culture. Calli which derived from leaves and microtubers were further differentiated into total plantlets and transplanted into fields, In order to improve rate of callus inducing and gain calli with higher ability of regeneration and total plantets with higher content of diosgenin. Simultaneously, young leaves, microtubers and calli were excised as material,The protoplast isolation and culture were also studied. It provided a new approach for breeding D. zingibernsis Wright with higer content and quality.The main results are as follows:The optimal media and culture condition of calli inducement were picked out, which were induced from four types of explants of same genotypes the rate of callus inducement of different genotypes of D. zingibernsis was compared; plant regeneration of the callus of leaves and microtubers was gained.The four different optimal culture mediums about callus inducement were obtained. Those are microtubers inducing medium:MS+2,4-D 2.5 mg L-1;leaves inducing medium:MS-NAA 0.1 mg L-1+6-BA 1.0 mg L-1; embyros inducing medium: MS+2,4-D 1.0 mg L-1 +6-BA 0.2 mg L-1 and stoop-tips inducing medium: MS+2,4-D 2.0 mg L-1'+6-BA0.2 mg L-1Four types of explants of same genotypes also appeared various ability in the rate and condition of callus formation. Embryos and mircrotubes exhibited high callus inducing percentage, shoot-tips followed by, while leaves were difficult to induce callus. Light culture condition provided different influence on callus inducement to different explants. Light condition enhanced callus formation in microtubes but this conditionshowed negative effect on callus formation in leaf segments, which were enhanced in dark cultured condition.To different explants, phytohormone combinations should be modified to get more effective callus inducement: Increasing of concentration of 2,4-D to some extent could promote callus inducement in microtubes, embryo, stoop-tip, but it was inefficient to callus inducing in leaf segments, which need addition of NAA for callus formation. The combination of 6-BA and auxin could remarkably enhance the callus inducement.The rate of calli inducement which was different genotypes microtubers of D. zingibernsis indicated remarkable difference.The whole plants have regenerated from calli of leaves and microtubers. Leaves-derived calli could do differentiation on inducing media, but the best differentiation culture medium is MS+NAA0.2+6-BA1.5;Microtubers-derived calli differentiated most plants on MS+NAA0.2+6-BA1.5.The best protoplast isolation system was established. We have obtained the cell aggrogate by protoplast culture.The best condition of isolation of leaves, microtubers and calli: Leaves which have grew for 5-10 days are cut up, then were put into enzyme consists of 0.6% cellulase, 0.5%pectinase and 0.8mol L~'mannitol and are sustaining for 21 hours in dark condition, itsprotoplast yield and energy is the highest; Microtubers which have grew 1 month are cut up and put into enzyme including 2.0% cellulase, 1.0% pectinase and0.7mol L-1 'mannitol ,then are sustaining for 10 hours in dark condition, its protoplastyield is the highest; its energy is better by phenosafranine staining. Calli which have suspended for 5 days are put into enzyme including 2.0% cellulase, 1.0% pectinase and0.7mol L -1 mannitol for 6-8 hours in dark condition, its protoplast yield and energy is the best.Protoplast is filtrated by nickel net and centrifuged 5 min in 100g min-1, we collectprotoplast deposition, then find a strap by grads...
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