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.

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.

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.

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.

Objective To explore the effect of γ-aminobutyric acid(GABA)on the growth and related physiological metabolism of plantlets of Tetrastigma hemsleyanum Diels et Gilg from Huaiyu Mountain under low temperature stress,which provides a basis for its application of anti-freezing and cold resistance in the wild-imitation cultivation of T.hemsleyanum Diels et Gilg from Huaiyu mountain.Methods Plantlets of T.hemsleyanum Diels et Gilg from Huaiyu Mountain,which were treated with GABA under low temperature stress,were selected as research objects.The growth indicators,physiological indexes,the relative expression levels of chalcone synthase and flavonol synthase genes were measured using plant tissue culture,physiological and biochemical methods and qRT-PCR,respectively.Results Compared with the control group without GABA,the proliferation coefficient,plant height,fresh weight,dry weight,and tissue water content of the two cultivated plantlets of T.hemsleyanum Diels et Gilg from Huaiyu Mountain("Huaiyu 1"and"Huaiyu 2")reach their maximum value when 0.5 g·L-1 GABA was added to the culture medium.Moreover,content of soluble protein(SP),soluble sugar(SS),proline(Pro),reduced glutathione(GSH),ascorbic acid(AsA),indole acetic acid(IAA),zeatin nucleoside(ZR),gibberellin(GA3),jasmonic acid(JA),the activity of superoxide dismutase(SOD),peroxidase(POD),ascorbic acid peroxidase(APX)and glutathione reductase(GR),and the relative expression level(RQ value)of chalcone synthase(CHS)gene were significantly increased.Moreover,relative conductivity,content of abscisic acid(ABA),hydrogen peroxide(H2O2),malondialdehyde(MDA),rate of O2·-production,and relative expression levels(RQ value)of flavonol synthase(FLS)gene were obviously decreased.The growth of plantlets of T.hemsleyanum Diels et Gilg from Huaiyu Mountain,which were treated with GABA under low temperature stress,was positively correlated with the contents of IAA,ZR,GA3,JA,SS,AsA,GSH,and the expression of CHS genes.Conclusion Appropriate concentration of GABA can regulate the contents of endogenous plant hormones and osmoregulation substances,antioxidant substances,and the expression of key genes in secondary metabolites,so as to promote the growth of plantlets of T.hemsleyanum Diels et Gilg from Huaiyu Mountain under low temperature stress.