根癌农杆菌介导大果西番莲基因转化

Establishment of Transgenic and Regeneration System of Giant Granadilla (Passiflora quadrangularis L.)

以大果西番莲种子无菌苗的子叶和下胚轴为外植体,在添加不同质量浓度BA和IAA的MS系列培养基上进行不定芽的诱导,确定MS+BA2.0mg·L^(-1)+IAA0.1 mg·L^(-1)为最适的不定芽诱导培养基,下胚轴、子叶外植体诱导芽分化率分别为88.33％,90.00％。将诱导出的不定芽接种在添加不同质量浓度IBA和NAA的MS系列培养基上,进行不定根的诱导,确定MS+NAA0.5mg·L^(-1)+IBA0.2mg·L^(-1)为最适的不定根诱导培养基,生根率达92.86％。用三亲交配法将质粒pBICr先转移到E.coli DH_5α,然后转移到根癌农杆菌。利用含有NPT-Ⅱ基因和CaMV35S启动子控制下的黄瓜花叶病毒外壳蛋白(CMV-CP)反义基因的表达载体,通过根癌农杆菌介导法将其导入西番莲受体细胞,在质量浓度为50mg·L^(-1)的Kan^+选择压力下培养,获得大果西番莲CP转基因再生植株。

The establishment of regeneration system of Passiflora quadrangularis from cotyledon and hypocotyl explants and the transformation of this plant with reverse CMV-CP gene are described in this paper. MS based medium containing 2 mg·L-1 BA and 0.1 mg·L-1 IAA was confirmed the best medium for inducing shoots, and the medium of MS + 0.5 mg·L-1 NAA + 0.2 mg·L-1 IBA for rooting. The regeneration frequency reached 88.33 %-90.00 % using this system. pBI121 based expressive vector pBICr containing NPT-II gene cassette as well as anti-CMV CP sequence controlled by CaMV 35S promoter and NOS terminator, was transformed into P. quadrangularis mediated by Agro-bacterium tumefaciens. Putative transgenic plantlets were screened on kanamycin medium and confirmed by dot blot hybridization.