Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds

• Published in: Plant cell reports 2008-09, Vol.27 (9), p.1509-1519
• Main Authors: Sailaja, M, Tarakeswari, M, Sujatha, M
• Format: Article
• Language: English
• Subjects: Biolistics - methods; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Cell Biology; DNA, Plant - genetics; Embryos; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Genetic engineering; Genetic technics; Genetic Transformation and Hybridization; Genotype; Glucuronidase - genetics; Helium; Life Sciences; Methods. Procedures. Technologies; Phosphotransferases (Alcohol Group Acceptor) - genetics; Plant Biochemistry; Plant Sciences; Plant tissues; Plants, Genetically Modified - embryology; Plants, Genetically Modified - genetics; Plasmids; Pressure; Ricinus communis - embryology; Ricinus communis - genetics; Seeds; Seeds - embryology; Seeds - genetics; Statistical analysis; Transformation, Genetic; Transgenes; Transgenic animals and transgenic plants; Transgenic plants
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-008-0580-3

The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ thidiazuron (TDZ) and subjected to bombardment after 5-7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding β-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 μm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l⁻¹ BA and hygromycin (20, 40 and 60 mg l⁻¹) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T₀ and T₁ plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.