Study On Tissue Culture System Of Polygonatum Cyrtonema

Polygonatum cyrtonema is a perennial herb of the genus Polygonatum of the Liliaceae family.It is characteristic of thickening rhizome and is one of the three basic plants in the 2015 edition of the Chinese Pharmacopoeia.In the background of great health,the P.cyrtonema industry has entered a new era of rapid development,but traditional seed or tuber reproduction methods encounter the problems such as waste of resources and species degradation.The tissue culture technology of P.cyrtonema has been reported using tuber as explant,but suffering from high pollution,low reproductive coefficient,long production cycle,etc.It is difficult to meet the industrial demand for high quality seedlings.In this study,on the one hand,tissue culture system was optimized using P.cyrtonema tubers as explants.On the other hand,a new way of rapid propagation was established using young leaves as explants,and the cytological change of callus induction and differentiation was investigated,which provided technical support and genetic transformation basis for the production of high-quality P.cyrtonema seedlings.The main findings are as follows:(1)Explant sterilization optimization test showed that the tuber contamination rate without pretreatment with carbendazim was 100%.The combinations with pretreatment by 1‰ carbendazim for1 hour,sequentially followed by 0.15% mercuric chloride(10min)and 2.5% sodium hypochlorite(5min)led to significantly decreased pollution rate,only 29.33%;(2)Screening experiments showed the optimum medium for tuber adventitious bud differentiation was MS+6-BA 1.0 mg·L-1+NAA 1.0 mg·L-1+TDZ 1.0 mg·L-1,the differentiation rate was 88.67%,the average number of buds was 3.58,and the buds grew well;(3)Tissue culture with leaves of P.cyrtonema plantlets as explants showed that the callus induction rate reached 80% on MS+6-BA 1.5 mg/L-1+2,4-D 0.2 mg/L-1 medium at 30-day-old of leaf base,and the optimum differentiation medium for adventitious buds of leaves was MS+6-BA 4.0 mg/L-1+2,4-D0.2 mg/L-1 with 90% differentiation rate and the average number of differentiated buds was 5.16;.The optimum medium for adventitious bud proliferation was MS+6-BA 2.0 mg.L-1+NAA 0.1-0.2 mg.L-1;the rooting rate of trifoliate seedlings in 1/2MS+IBA 2.0 mg.L-1 medium was the highest(94%),the average number of roots was 8.3,and the average root length was 4.07 cm;the rooting seedlings transplanted in peat-vermiculite 1:1 medium grew well and the survival rate was over 85%;(4)The cytological observation of the in vitro tissue culture process of the tube seedling leaves showed that the leaf base usually produce callus other than leaf tip and mid-leaf region.The cells at the leaf base have the characteristics of strong meristematic ability.The adventitious buds have two origins:exogenous and endogenous origin,which are formed by callus surface cells and callus inner meristem,respectively,and the exogenous origin occurs earlier than endogenous origin.