Khan Raham Sher, Chin Dong Poh, Nakamura Ikuo, Mii Masahiro
Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba 271-8510, Japan.
Plant Cell Rep. 2006 Sep;25(9):914-9. doi: 10.1007/s00299-006-0125-6. Epub 2006 Apr 8.
Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. beta-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt (-), GUS(+) and excision(+) as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.
携带ipt型MAT载体pNPI132的根癌农杆菌菌株EHA105被用于培育形态正常的转基因蓝花烟草品种勃朗峰，采用ipt基因作为选择标记基因。β-葡萄糖醛酸酶（GUS）基因用作目标模型基因。MAT载体系统是一种阳性选择系统，它赋予转基因细胞再生优势，而不杀死非转基因细胞。将感染的外植体在不含激素和抗生素的MS培养基上培养，共培养约3个月后，65%的再生芽出现ipt多芽表型——形态异常的芽。共培养7个月后，从12个转基因ipt芽中产生了20个形态正常的芽。正常的芽在不含激素的MS培养基上生根良好。通过PCR和Southern杂交分析确定，90%的正常芽为ipt（-）、GUS（+）和切除（+）。这些结果表明，ipt型MAT载体系统可成功用于蓝花烟草，无需使用植物激素和选择性化学试剂即可生产无标记转基因植物。
