‘克新13号’马铃薯植株再生和转化技术

Plant Regeneration and Transformation of the Potato Cultivar'Kexin No.13'

为研究马铃薯植株再生和转化技术,以‘克新13号’的茎段、叶片和微型薯为外植体,接种到不同激素配比的培养集中进行愈伤组织诱导和分化,再采用农杆菌转化法,初步建立了马铃薯遗传转化再生体系。结果表明:茎段是‘克新13号’马铃薯比较理想的外植体材料,最佳的愈伤组织诱导培养基为4.4 g/L MS+30 g/L蔗糖+8 g/L琼脂+2 mg/L 6-BA+0.5 mg/L 2,4-D+0.4 mg/L KT,愈伤组织诱导率在95%以上;最佳的分化培养基为4.4 g/L MS+30 g/L蔗糖+8 g/L琼脂+2 mg/L 6-BA+3 mg/L KT+0.5 mg/L GA_(3)+0.1 g/L肌醇,出苗率为56.25%;外植体预培养2 d后,用OD_(600)=0.5的农杆菌菌悬液侵染7 min,再共培养3 d,以50 mg/L Kan和200 mg/L Tim为抗生素进行筛选,能成功获得含目的基因Cas9p的转化植株。

This study aims to explore the potato plant regeneration and transformation technology.The stem segment,leaf,and minituber of'Kexin No.13'were used as explants and inoculated into the media with different plant hormone ratios for callus induction and differentiation.Furthermore,the Agrobacterium tumefaciens-mediated transformation method was employed to establish a genetic transformation and regeneration system for potatoes.The results showed that the stem segment was the ideal explant of'Kexin No.13'.The optimal medium for callus induction was 4.4 g/L MS+30 g/L sucrose+8 g/L agar+2 mg/L 6-BA+0.5 mg/L 2,4-D+0.4 mg/L KT,in which the callus induction rate reached above 95%.The optimal differentiation medium was 4.4 g/L MS+30 g/L sucrose+8 g/L agar+2 mg/L 6-BA+3 mg/L KT+0.5 mg/L GA_(3)+0.1 g/L inositol,in which the emergence rate was 56.25%.The transformation experiment was carried out in a procedure of explant pre-culture for 2 d,soaking in the A.tumefaciens suspension(OD_(600)=0.5)for 7 min,and co-culture for 3 d.Subsequently,50 mg/L kanamycin and 200 mg/L timentin were used for screening of the seedlings carrying the target gene Cas9p.