Zhou J, Wei Z, Xu Z, Liu S, Luo P
National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, Shangha, China.
Chin J Biotechnol. 1996;12(1):39-45.
Excised cotyledons of Orychophragmus violaceus were used as explants for tissue culture. They were cultured on the MS medium supplemented with BA (3 mg/L) and NAA (0.2 mg/L). When the regenerated buds were 2 cm long, they were excised and transferred onto 1/2 MS medium with IBA (0.03 mg/L), then the whole plants were regenerated. The frequency of plant regeneration was 100%. Subsequently, the genetic transformation of O. violaceus was studied. After 2-3 days of cocultivation with Agrobacterium tumefaciens strain A208se (pTiT37, pROA93), the cotyledons were transferred onto the selection medium containing 25 mg/L Km and 250 mg/L Ap. After shoots emerged, they were excised and transferred onto the rooting medium containing 25 mg/L Km and 100 mg/L Cef. The roots were formed within 4-5 weeks. The whole plants were transplanted into pots and grew well. The frequency of plant regeneration was about 51%. The regenerated plants showed high enzymatic activities of beta-glucuronidase and neomycine phosphotransferase II. Southern blot analysis confirmed that NPTII gene had been stably integrated into the chromosomal genome of O. violaceus. The transformation frequency was 5.6%. The first transgenic plant of O. violaceus is being reported.
诸葛菜的离体子叶被用作组织培养的外植体。将它们接种在添加了BA（3毫克/升）和NAA（0.2毫克/升）的MS培养基上。当再生芽长到2厘米长时，将其切下并转移到含有IBA（0.03毫克/升）的1/2 MS培养基上，然后再生出完整植株。植株再生频率为100%。随后，对诸葛菜进行了遗传转化研究。与根癌农杆菌菌株A208se（pTiT37，pROA93）共培养2-3天后，将子叶转移到含有25毫克/升卡那霉素和250毫克/升氨苄青霉素的选择培养基上。芽出现后，将其切下并转移到含有25毫克/升卡那霉素和100毫克/升头孢霉素的生根培养基上。4-5周内形成根。完整植株移栽到花盆中，生长良好。植株再生频率约为51%。再生植株显示出较高的β-葡萄糖醛酸酶和新霉素磷酸转移酶II的酶活性。Southern杂交分析证实NPTII基因已稳定整合到诸葛菜的染色体基因组中。转化频率为5.6%。首次报道了诸葛菜的转基因植株。
