樱桃矮化砧木‘吉塞拉6号’基因转化体系的建立

Development of Transformation System in Cherry Dwarf Rootstock 'Gisela 6'

采用‘吉塞拉6号’甜樱桃矮化砧木(Prunus cerasus×P.canescens)离体叶片外植体,在再生培养基附加生长素的条件下通过根癌农杆菌(Agrobacterium tumefaciens)EHA105(p35SGUS intron)介导研究了β-葡萄糖醛酸酶基因(GUS)的瞬时表达、稳定表达和转基因植株再生,证明了培养基中生长素(IBA或NAA)的存在可促进基因转化,转化效率比对照提高2倍以上。将500个叶片外植体与EHA105(p35SGUS intron)株系在含有生长素的培养基中共培养,获得了11个转基因株系。采用PCR分析和South-ern Blotting核酸杂交,确定GUS基因已整合到矮化砧木‘吉塞拉6号’植株的染色体上。组织化学染色确定了GUS基因在植株体内的表达。

Transient and stable β-glucuronidase （GUS） expressions and transgenic plant regeneration from the leaf segment explants of the triploid sweet cherry dwarf rootstock Gisela 6 （Prunus ceransus x P. canescens） on the medium with or without auxins were examined using Agrobacterium tumefaciens supervirulent strains EHA105 （p35SGUS intron） during transformation. Explants with auxins （IBA or NAA） yielded more than twice as many GUS-expressing zones and calli compared with ones on the medium without auxin. High efficiency transformation system was established. On regeneration medium with 0. 5 mg · L^-1 IBA, eleven transgenic plants with GUS gene were obtained from 500 explants after cocuhivation with EHA105 strain. The integration of the GUS gene into cherry genome was confirmed by PCR and Southern Blotting analysis. The gene expression was confirmed by GUS-histochemistry staining.