杉木愈伤组织的高效诱导和瞬时转化体系的建立

Establishment of High-efficiency Callus Induction and Transient Transformation System of Chinese Fir

本研究参考已有的杉木(Cunninghamia lanceolata)体细胞胚胎发生系统,建立了以杉木针叶为外植体的愈伤组织高效诱导体系和原生质体瞬时转化体系,并初步探讨了细胞壁再生和以茎段为受体材料的稳定遗传转化技术。以杉木针叶为外植体,在添加有1.0 mg/L 2,4-二氯苯氧乙酸(2,4-D)、2.0 mg/L 6-苄氨基嘌呤(6-BA)、1.0 mg/L 6-糠基氨基嘌呤(KT)以及0.5 mg/L萘乙酸(NAA)的MS培养基诱导愈伤组织,愈伤组织形成率可以达到87.5%;对分离得到的杉木次生木质部原生质体采用40%PEG-4000介导法进行原生质体瞬时转化,外源基因的转化率达到30%,并实现了原生质体多次分裂形成细胞团;以上结果表明,以杉木针叶作为外植体可高效诱导形成愈伤组织,且在增殖培养基上生长状态良好。本研究建立了杉木原生质体瞬时转化体系并实现了原生质体的细胞壁再生和分裂。

In this study,we integrated multiple somatic embryogenesis systems of Chinese fir(Cunninghamia lanceolata),to establish a high-efficiency callus induction system with needle leaves as explants.We also developed a transient transformation system of protoplasts,and preliminarily explored cell wall regeneration and cell division.Finally,A simple protocol was explored for Agrobacterium tumefaciens-mediated stable genetic transformation of Chinese fir using shoot sections as explants.We established 87.5%high efficiency rate of callus induction using needle leaves as explants with MS medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D),2.0 mg/L 6-Benzylaminopurine(6-BA),1.0 mg/L 6-Furfurylaminopurine(KT),and 0.5 mg/L naphthalene acetic acid(NAA).Browning phenomenon of callus in tissue culture can be reduced by addition of 2,4-D during callus subculture.In total,30%of transformation efficiency for exogenous gene was achieved using PEG4000-mediated transient transformation system and protoplasts could undergo multiple divisions to form cell clusters.Needle leaves can be efficiently induced into callus and grow well on the proliferation medium. An efficient transient transformation system for protoplasts has been established. Cell wallregeneration and division have been achieved for protoplasts.