He Hong, Xu Hong-hua
College of Chinese Materia Medica, Guangzhou University of Traditional Chinese Medical and Pharmaceutical Sciences, Guangzhou 510405, Guangdong, China.
Zhongguo Zhong Yao Za Zhi. 2002 Oct;27(10):733-5.
To establish an effective system for the Agrobacterium-mediated genetic transformation of M. officinalis, for laying a foundation for the improvement of breeds and introduction of foreign objective genes.
The explants used for culture were the nodular stem segments from M. officinalis. Agrobacterium tumefaciens strain was EHA101, containing vector plasmid pGA482GG. The GUS gene and NPT II gene were introduced into the plasmid.
MT basal medium with BA 1 mg.L-1 was effective to inducing the direct shoot formation, and the frequency of shoot formation was 97.8%. As BA concentrations increased, the ability of shoot formation decreased. The explants oriented with their apical ends protruding from the medium produced more shoots than when they were placed with their basal end upright or were placed horizontally. The optimal rooting medium for regenerating shoots was MT basal medium supplemented with 0.2 to 0.5 mg.L-1 NAA, and a root induction rate over 80.0% was observed. The selection pressure for kanamycin was 50 mg.L-1. Cefotaxime was used as antibiotics, and the concentration was 300 mg.L-1. After 1.5 months, 14.8% resistant shoots were emerged from the explants. Histochemical GUS assay showed that 22.2% of the resistant plants were GUS-positive.
Plant regeneration system and Agrobacterium-mediated genetic transformation have been established for M. officinalis in vitro.
建立一种有效的用于厚朴农杆菌介导遗传转化的体系，为品种改良和导入外源目的基因奠定基础。
用于培养的外植体为厚朴的结节状茎段。根癌农杆菌菌株为EHA101，含有载体质粒pGA482GG。将GUS基因和NPT II基因导入该质粒。
含1 mg.L-1 BA的MT基本培养基对直接诱导芽形成有效，芽形成频率为97.8%。随着BA浓度增加，芽形成能力下降。外植体顶端伸出培养基的方式比基部直立放置或水平放置产生更多的芽。再生芽的最佳生根培养基是添加0.2至0.5 mg.L-1 NAA的MT基本培养基，观察到根诱导率超过80.0%。卡那霉素的选择压力为50 mg.L-1。头孢噻肟用作抗生素，浓度为300 mg.L-1。1.5个月后，外植体产生了14.8%的抗性芽。组织化学GUS检测表明，22.2%的抗性植株为GUS阳性。
已在体外建立了厚朴的植株再生体系和农杆菌介导的遗传转化体系。
