Rapid propagation technology of Dioscorea bulbifera virus-free plantlets / 中草药

Objective Effects of different factors on proliferation and rooting of stems with a bud were studied by using Dioscorea bulbifera as test material to optimize the rapid propagation system of D.bulbifera virus-free plantlets.Methods Plant tissue culture method was used in shoot tip culture and rapid propagation study,and RT-PCR method was used in virus detection of virus-free plantlets.Results The best proliferation medium of D.bulbifera stems with a bud was MS+KT 2 mg/L+6-BA 1 mg/L+NAA 0.5 mg/L;The best sucrose and agar concentration of D.bulbifera stems with a bud was 30 g/L and 0 g/L,respectively;The best rooting medium of D.bulbifera stems with a bud was 1/2 MS+IBA 0.1 mg/L+NAA 0.5 mg/L+PP_(333) 1 mg/L;The best transplanting matrix of regeneration plantlets from D. bulbifera stems with a bud was perlite-vermiculite(2:1);The best PP_(333) concentration of D.bulbifera regeneration plantlets for transplanting was 50 mg/L.Conclusion The rapid propagation system of D. bulbifera virus-free plantlets is established successfully for the first time,which provides a technological basis for factory production of D.bulbifera virus-free plantlets.

Shoot tip culture and virus detection of Dioscorea bulbifera / 中草药

Objective Effects of different factors on shoot tip culture in vitro were studied by using Dioscorea bulbifera as test material to find the media suitable to the shoot tip differentiation of D.bulbifera and the method of virus delection.Methods Plant tissue culture method was used in shoot tip culture and the symptom observation method,instruction plant method,and RT-PCR method were used in virus detection of virus-free plantlets.Results The best disinfection method for D.bulbifera shoot tips was firstly disinfected for 30 s with 70% alcohol and then disinfected for 12 min with 0.1% HgCl2;It is better for D.bulbifera to cut shoot tips in 0.5 mm length after 37 ℃ heat treatment for 7 d;The best proliferation medium of D.bulbifera shoot tips was MS+KT 2 mg/L+NAA 0.5 mg/L;The best rooting medium of regeneration buds from D.bulbifera shoot tips was 1/2 MS+NAA 0.5 mg/L;The best matrix of regeneration plantlets from D.bulbifera shoot tips was perlite-vermiculite (2∶1);RT-PCR Method was primary mean to detect potato virus Y (PVY) of regeneration plantlets from D.bulbifera shoot tips,and average rate of PVY-free was 86.5% by RT-PCR.Conclusion The virus-free culture system of D.bulbifera shoot tips is established for the first time,providing a technological basis for the rapid propagation and factory production of D.bulbifera virus-free plantlets.

Primary study on proliferation and rooting of stem with a bud in Dioscorea bulbifera / 中草药

Objective To study the effects of several factors on bud proliferation and rooting of Dioscorea bulbifera stem with a bud.Methods Single factor test and plant tissue culture methods were applied.Results The combination of 6-BA or KT and NAA helped the proliferation of stem with a bud in D.bulbifera;high concentration sucrose led to callus growth of stem with a bud in D.bulbifera which didn′t help the proliferation of stem with a bud in D.bulbifera;Liquid culture also helped the proliferation of stem with a bud in D.bulbifera;In a certain range,the increase of NAA concentration helped the rooting of stem with a bud in D.bulbifera.But it will inhibit the rooting with concentration in a higher level.Conclusion The best proliferation culture medium of D.bulbifera,stem with a bud is MS + KT 2 mg/L +NAA 0.5 mg/L or MS + 6-BA 2 mg/L + NAA 0.5 mg/L;For proliferation of D.bulbifera stem with a bud,the best sucrose concentration is 30 g/L sucrose,the best culture mode is liquid culture;The best rooting culture medium of D.bulbifera stem with a bud is MS + NAA 2 mg/L.

Cryopreservation technique of Dioscorea opposita germplasm by vitrification / 中草药

Objective To study the cryopreservation technique of Dioscorea opposita germplasm resources by vitrification. Methods Stems with buds of B# D. opposita germplasm were used as materials for cryopreservation by vitrification and the survival rates were examined by culture method in order to optimize a cryopresevation by vitrification technical system for D. opposita germplasm. Results A best procedure of cryopresevation by vitrification was as below: At first, the aseptic plantlets of stems with buds of B# D. opposita subcultured in vitro for 60 d were treated at 4 ℃ for 7 d. The stems with buds of D opposita about 1-1.5 cm in length were cut and precultured at 4 ℃ for 2 d in 5% sucrose and 3% mannose media, they were dehydrated with 60% vitrification solution (PVS_1: 22% glycerol+13% glycol+13% polyethyleneglycol+10% dimethyl sulfoxide) at 0 ℃ for 60 min and then dehydrated with 100% vitrification solution at 0 ℃ for 60 min. At last, the stems were immersed immediately into liquid nitrogen directly and conserved for 24 h. After rapidly thawing in a water bath at 37 ℃, the stems were washed four times with MS medium supplemented with 7% sucrose and 10 min each time, then transferred and re-cultured on the MS medium supplemented with KT 2 mg/L and NAA 0.02 mg/L. The survival rate was up to 75%. The regenerated plantlets of D. opposita showed no more difference than the normal plantlets in morphology. Conclusion This test sets up successfully a cryopresevation by vitrification system technique for D. opposita germplasm, which provides an effective way for long-lasting conservation in vitro of D. opposita germplasm resources.