Establishment Of A Transgenic System For Pinellia Integrifolia

Pinellia integrifolia is a special wild perennial herb only found in China,bulbils aren't formed on the individual plants of P.integrifolia in the natural environment.In this paper,the tuber,petiole and leaf of P.integrifolia were used as materials to establish its regeneration system,the expression vector p1301-PteMFT was established by cloning PteMFT of Pinellia ternata,and the aseptic seedlings of P.integrifolia,obtained from the generation system,were used as explants to establish the transgenic system.The main results are as following:1.Establishment of efficient regeneration system for P.integrifoliaTuber,petiole and leaf were used as explants to induce callus formation.Results showed that tubers were the best explants for callus induction,followed by petiole and leaf in that order.The callus induction and proliferated was on MS+1.0mg·L^-1 6-BA+1.0 mg·L^-1 2,4-D;Furthermore,the greatest differentiation and proliferation of shoot was observed on MS+2.5mg L^-1 6-BA+0.3 mg·L^-11 IBA,the differentiation rates reached a maximum:92%,92.30%and 94.74%.The maximum shoot proliferation of multiple were 12.80,8.85 and 10.71,respectively.And the bud growth was better and stronger;This culture condition was the best for shoot differentiation and proliferation;The best rooting medium was MS+0.5 mg·L^-1 IBA+0.5 g·L^-1activated carbon,the rooting rate in this medium was 100%,the average number of main roots were 8-15 and their overall length was 10 cm.The culture period of the P.integrifolia was also shortened to 2.5 months.2.Construction of p1301-PteMFT expression vectorPteMFT gene of Pinellia ternata was cloned successfully;A new expression vector?p1301-PteMFT?containing the target gene?PteMFT?was constructed and transfered into Agrobacterium tumefaciens EHA105.The expression vector was proved by PCR detection.3.Establishment of genetic transformation system for P.integrifoliaIn this work,the effects of Kanamycin?Kan?and Hygromycin?Hyg?concentrations on callus induction rate from P.integrifolia tubers and petioles were studied.The results showed that the tuber and petiole of P.integrifolia were not Kan sensitive as the increase in Kan concentration did not decrease callus induction rates.However,the callus induction rate of petiole decreased to 85.71%,when the Kan concentration reached 500 mg·L^-1.On the other hand callus induction rates dropped to0%for both petioles and tubers at 40 mg·L^-1 of Hyg.At 20 mg·L^-1 of Hyg concentration,the callus induction rates of petiole and tuber decreased sharply to 7.5%and 6.67%,respectively.Therefore,20 mg·L^-1of Hyg concentration was selected as compressive strength for subsequent screening.The tubers and petioles from the aseptic seedlings of P.integrifolia,obtained from there generation system,were used as explants to establish the transgenic system.The effects of pre-culture time,OD₆₀₀value,co-culture time and infection time on the genetic transformation of tuber and petiole were studied.The results showed that the genetic transformation suitable conditions of tuber:tubers pre-cultured for 6 d,which incubated for 15 min by Agrobacterium tumefaciens which OD600 is about 0.8,and then were placed on the co-culture medium with 3 d co-culture periods in darkness.The genetic transformation suitable conditions of petioles:petioles pre-cultured for 2 d,which incubated for 15 min by Agrobacterium tumefaciens which OD600 is about 0.8,and then were placed on the co-culture medium with 3 d co-culture periods in darkness.In this study,we could not obtaine transgenic plants and only preliminary GUS staining was performed.