早熟禾的组织培养和基因枪介导的基因转化体系的初步建立

Tissue culture and Genetic Transformation of Kentucky Bluegrass ( Poa pratensis) via Microprojectile Bombardment

分析了不同质量浓度的２，４－二硝基苯酚（简为２，４－Ｄ）对早熟禾愈伤组织诱导和愈伤组织分化成苗的影响，３ ｍ ｇ／Ｌ和０．１ ｍ ｇ／Ｌ ２，４－Ｄ分别是本试验所用早熟禾基因型（Ｐｏａ ｐｒａｔｅｎｓｉｓＬ ｃｖ． ｎｕｂｌｅ）诱导形成愈伤组织和愈伤组织分化成苗的最适质量浓度．利用基因枪将含ＧＵＳ基因的ｐＧＡ４７０和ｐＡｃｔ１－Ｄ的质粒导入愈伤组织，通过组织染色法检测到了Ｇｕｓ基因的瞬时表达．含ＮＰＴⅡ基因的ｐＧＡ４７０质粒和含ｂａｒ 基因的ｐＤＭ３０２质粒转化的愈伤组织，分别在含１００ ｍ ｇ／Ｌ卡那霉素和含３ ｍ ｇ／Ｌｐｈｏｓｐｈｉｎｏｔｈｒｉｃｉｎ（ＰＰＴ）的选择性培养基上培养，获得了抗卡那霉素和抗ＰＰＴ的愈伤组织；

Factors affecting callus induction from mature seeds and plantlet regeneration in Kentucky Bluegrass ( Poa pratensis L) were optimised using a modified medium containing MS salts and B5 organic elements and the MS medium respectively. A 2,4 D concentration of 3 mg·L -1 was optimal for callus induction, and 0.1 mg·L -1 for plantlet regeneration. Transient expression of the GUS gene was observed on calli transformed with pGA470 which contains GUS gene driven by the 35S promoter, and pAct1 D which contains GUS gene driven by the rice actin 1 promoter. Putative stable transformants were selected from calli transformed with pGA470, containing the NPT Ⅱ gene driven by the 35S promoter, and pDM302, containing the bar gene driven by the rice actin 1 promoter, and cultured on media containing 100 mg·L -1 kanamycin and 3 mg·L -1 phosphinothicin respectively. A stable expression of NPT Ⅱ gene was detected in the 12th sub cultural callus.