Shoot Regeneration From Leaves And VcANS Gene Transformation Of Lingonberry

The influence of 5 kinds of plant growth substance and 4 culture media on regenerating adventitious bud from plantlet leaves in vitro were studied to provide the basis for the establishment of high efficient regeneration system with 3 lingonberry(Vaccinium vitis-idaea L.)cultivars ‘Ida',‘Sanna'and ‘Bruch Rousi'.In this study the experimental conditions of genetic transformation of VcANS gene of lingonberry ‘Ida' were established by Agrobacterium-mediated method,aiming to provide the basis for the establishment of high efficient regeneration system and genetic transformation technology system.The results are as follows:1.The optimum culture medium was WPM medium,which was the best medium of WPM,MS,1 / 2MS and 1 / 4MS.The optimum culture medium was WPM medium.The optimal combination of growth regulators was: WPM + ZT 4.0 mg / L + IBA 0.1 mg / L.‘Ida' was up to 99.2% at TDZ 2.0 mg / L,and the highest regeneration rate was 28.3 / leaf under the treatment condition of ZT 5.0 mg / L.‘Sanna'was inducedn rate was 93.3% at TDZ 2.0 mg / L and the highest regeneration rate was 35.2/ leaf ‘Rossi' was induced at ZT 4.0 mg / L + NAA 0.5 mg / L The highest rate was 88.9%,and the regeneration frequency was 29.9 / leaf at ZT 5.0 mg / L.2.The growth status of red bean 'orange' was the best in all kinds of growth regulators.Therefore,the follow-up genetic transformation was carried out with 'Ada' sub-seedlings as recipient material.3.The plant expression vector pBASTA-VcANS was successfully constructed.4.The optimum concentration of cefotaxime sodium(Cef)was 250 mg/L,the herbicide glufosinate is the optimized for the transformant selection system,and the optimum concentration was 0.9 mg/L,the VcANS gene was transformed into lingonberry ‘Ida' by using the Agrobacterium-mediated methods,the genetic transformation systemas follows: Leaves from25~30d old stem were used as explants for culturing,using expanding leaves with the adaxial side touching the pre-culture medium,and maintained for 3d in darkness at 25? later,then taking out the pre-cultured leaves carefully were infectd with A tumefaciens diluted by liquid WPM for 10 min.After the infection take out the leaves and dry out the extra bacteria liquid then maintain for2 d in darknesson the co-culture medium at 25?;The infected leaves were rinsing with liquid medium(WPM + ZT 4.0 mg/L + IBA 0.1 mg/L + AS100 mg/L + 3% sugar).The the explants were transferred to selection medium(WPM + ZT 4.0 mg/L+ IBA 0.1 mg/L + 8 g/L agar powder + 3%sugar + 250 mg/L cef + glufosinate 0.9 mg/L)for 2d in the darkness at 25?.leaves weretransferred into the selection medium.After dark culture for 2 days,the leaves were cultured under light culture.After 30 days,the adventitious buds were transplanted into resistant medium(WPM+ ZT 4.0 mg/L+ IBA 0.1 mg/L + 8g / L agar powder + 3% sugar+250 mg/L Cef).5.There were 20 glufosinate-resistant shoots were obtained under the condition of the presence of selective glufosinate,using Agrobacterium tumefaciens-mediated genetic transformation method,1positive clones were carried out PCR.