Transgenic patchouli plants produced by Agrobacterium-mediated transformation
Transgenic patchouli plants produced by Agrobacterium-mediated transformation
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DOI：
10.1007/s11240-005-1039-7
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发表时间：
2005-09
期刊：
影响因子：
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通讯作者：
Y. Sugimura;N. Kadotani;Y. Ueda;K. Shima;S. Kitajima;T. Furusawa;M. Ikegami
中科院分区：
文献类型：
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作者：
Y. Sugimura;N. Kadotani;Y. Ueda;K. Shima;S. Kitajima;T. Furusawa;M. Ikegami
以最重要的香料作物广藿香（Pogostemon cablinBenth.）为材料，建立了一套成功的农杆菌转化体系。为了避免积累在叶片组织中的广藿香油对农杆菌感染的抑制作用，将体外再生的完整植株用作外植体来源，这些植株的叶片组织中不含或含有微量的广藿香油。用两个A.含有不同嵌合质粒的根瘤菌菌株。叶片外植体感染A. EHA 101/pIG 121-Hm是一株携带β-葡萄糖醛酸酶（GUS）和潮霉素磷酸转移酶（HPT）基因的根瘤菌。共培养3天后，用50 mg l-1潮霉素B选择，选择具有不定芽的绿色愈伤组织，从中再生出具有根的推定转化体。组织化学检测表明，GUS基因在转化子的各个器官中都有表达，并检测到高活性的GUS基因。使用编码广藿香温和花叶病毒外壳蛋白前体基因（CP-P）和新霉素磷酸转移酶（NPTII）基因的另一个菌株LBA 4404/pBI 121-PaCP 1，在共培养7天并用100 mg l− 1卡那霉素选择后也获得了推定的转化体。利用转化子的总DNA，通过PCR反应检测CP-P的全长。通过比较两株菌株的感染情况，发现LBA 4404/pBI 121-PaCP 1感染成功的关键是延长共培养时间和增加筛选药物的剂量。
A successful transformation procedure usingAgrobacteriumwas established for the most important aromatic crop, patchouli (Pogostemon cablinBenth.). To avoid inhibition ofAgrobacteriuminfection by patchouli oil accumulated in leaf tissues, complete plants regeneratedin vitrowhich possessed no or trace amounts of patchouli oil in leaf tissues were used as an explant source. Conditions for transformation were examined using twoA. tumefaciensstrains containing a different chimeric plasmid. Leaf explants were infected withA. tumefaciensstrain EHA101/pIG121-Hm carryingβ-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes. Following co-cultivation for 3 days and selection by 50 mg l−1hygromycin B, greenish calli with adventitious shoots were selected, from which putative transformants with roots were regenerated. Histochemical assay showed that GUS expression is detected in every organs of transformants, which was confirmed by the detection of high activity of GUS. Using another strain LBA4404/pBI 121-PaCP1 encoding the coat protein precursor gene of patchouli mild mosaic virus (CP-P) and neomycin phosphotransferase (NPTII) gene, putative transformants were also obtained after co-cultivation for 7 days and selection by 100 mg l−1kanamycin. Using total DNAs from the transformants, the full length of CP-P was detected by PCR reaction. Comparing between two strains examined,it was noted that prolonged co-cultivation period and higher dose of a selection drug were indispensable for successful infection with LBA4404/pBI 121–PaCP1.