摘要
利用野生型发根农杆菌15834菌株感染小冠花15日龄无菌苗子叶和下胚轴切段,建立了高效的发状根培养及其体细胞胚胎发生再生体系。发状根可直接从受伤的外植体表面产生,也能在外植体诱导的愈伤组织上发生,在无外源激素的MS固体和液体培养基上,转化根能自主生长,表现出典型的发根特征。用适宜浓度的乙酰丁香酮处理对数生长期的农杆菌菌液2h,感染预培养2d的子叶获得了最高的转化频率(87.4%)。在附加0.2mgL2,4_D,0.5mgLNAA和0.5mgLKT的MS培养基上,发状根能100%形成胚性愈伤组织,并于含0.5mgLKT,0.2mgLIBA和300mgL脯氨酸的MS培养基上顺序经过体细胞胚胎发育的各个典型时期,转换成完整植株。再生植株除具有发达的侧根外,其它形态特征与未转化植株未见明显的差异,但在获得的5个转化克隆中,其中1个的发状根及其再生植株叶片中有毒物质3_硝基丙酸的含量显著下降,分别为未转化对照的57.68%和58.17%。冠瘿碱纸电泳检测和rolB基因PCR扩增检测均证明农杆菌Ri质粒上的T_DNA已经整合到小冠花转化细胞的基因组中。
An efficient system of genetic transformation and plant regeneration via somatic embryogenesis was established in crownvetch （ CoroniUa varia L. ） by infecting the segments of cotyledons and hypocotyls of 15d-old seedlings with Agrobacterium rhizogenes strain 15834. Hairy roots were produced directly from the wounded surface of the explants or via calluses on hormonefree Murashige and Skoog （MS） medium after infection by A. rhizogenes. Transformed roots grew rapidly either on solid or liquid MS medium, and exhibited typical hairy root phenotypes. The highest transformation frequency （87.4%）was achieved by pre- culturing cotyledons for 2d and pre-treating the A. rhizogenes with suitable concentration of acetosyringone at logarithmic phase （OD6oo = 0.8）. The embryogenic calluses with 100% induction frequency were induced from hairy roots on MS medium containing 0.2mg/L 2,4-D, 0.Smg/L NAA and 0.Smg/L KT. Globular-, heart-, torpedo-, and cotyledon shaped somatic embryos were produced orderly and developed into plantlets when transferred the embryogenic calluses on MS medium supplemented with 0.Smg/L KT, 0.2mg/L IBA and 300mg/L proline. The transformed plants did not show differences in morphology except abundant lateral root branches compared to the non-transformed plants. However, the contents of 3-nitropropanic acid in hairy roots and leaves of one of 5 transformed clones were 57.68% and 58.17% in roots and leaves of untransformed plants, respectively. Opine paper electrophoresis revealed the integration and expression of TR-DNA. PCR analysis confirmed that the TL-DNA including 654 bp rol B sequence was inserted into the genome of transformed hairy roots and their regenerated plants.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第1期107-113,共7页
Chinese Journal of Biotechnology
基金
陕西省自然科学基金项目(No.2003C108)
省重点实验室重点项目(No.05JS48)~~
关键词
小冠花
发根农杆菌
发状根
体细胞胚胎发生
植株再生
Crownvetch, Agrobacterium rhizogenes, hairy root, somatic embryogenesis, plant regeneration
