Agrobacterium-Mediated Transformation Of Malus Robusta With Rirol Gene

In order to obtain some transgenic apple rootstocks of dwarfing, iron-deficiency tolerance, cold, drought and saline resistance, the trinal fusion gene (Rirol) was used as the purpose gene, transformation of Malus robusta by Agrobacterium-mediated, optimized the transformation system of Malus Robusta, established a high-efficient transformation system, obtained the transgenic plants with Rirol gene of Malus robusta. The experiment contents and the research results as follows:1. A high-efficient transformation system of Malus robusta was developed:Effect of the type of Agrobacterium strain, vir induce factor, time of adding kanmycin, plant whitening process, and the pH value of suspention liquid of Malus robusta transformation were studied. Established a transformation system of taking LBA4404 as infection strain, with MS₀+0.1 mmols/ L of KH₂PO₄+1.0 %cane sugars+0.5% glucose+ the 1 mmol/ L proline+75μmol/ L OH- AS (pH5.6) for the suspention liquid to activate the strain, leaves were dipped into agrobacterium suspension, then were coculture on regeneration culture medium (MS + BA4.0mg/L + NAA0.2mg/L) for two days, selection culture on selective medium (MS+BA4.0mg/L+NAA0.2mg/L+Km50mg/L) after seedling height was 2 centimeters. The result indicted that the appearing frequency of resistance seedling was 8.4%.2. Obtained the transgenic plants with Rirol gene of Malus robusta:84 putative transgenic Malus robusta lines with the Roril gene via selecting of kanamycin were obtained during optimizing process described above. Among them a total 65 positive lines via selecting of GUS. 24 of which were verified by PCR amplification, Among them, 19 of which were confirmed to carry the same fragment as positive plasmid. Random take 5 PCR positive lines, 4 of them were confirmed to carry the transgene by Southern blot analysis. According the result of PCR and Southern blot analysis, the Rirol gene had been integrated into the plant genome of transgene Malus robusta. All these 4 Southern positive lines could be detected the aim fragment by RT-PCR, the result indicated...