Saini R, Jaiwal P K
Department of Biosciences, M.D. University, 124001, Rohtak, India.
Plant Cell Rep. 2003 Jun;21(9):851-9. doi: 10.1007/s00299-003-0574-0. Epub 2003 Mar 22.
Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.
绿豆是尚未被转化的大粒谷物豆类之一。我们在此首次报道了用携带二元载体pCAMBIA2301的根癌农杆菌接种子叶节外植体后，获得形态正常且可育的转基因植株，该载体含有新霉素磷酸转移酶（nptII）基因和一个被内含子打断的β-葡萄糖醛酸酶（GUS）基因（uidA）。在含有卡那霉素的培养基上筛选并生根的转化绿芽，通过聚合酶链反应（PCR）分析检测nptII和uidA基因呈阳性。这些芽在土壤中定植并生长至成熟以收集种子。发现接种根癌农杆菌之前对外植体进行机械损伤、由于从外植体上移除子叶导致再生时间延迟以及在生根阶段进行第二轮筛选对转化至关重要。对T(0)代植株的分析表明uidA已表达并整合到植物基因组中。通过组织化学分析检测了叶片、根、花、花药和花粉粒中的GUS活性。对T(1)代后代的PCR分析揭示了孟德尔转基因遗传模式。转化频率为1%，产生转基因植株需要6至8周。
