Agrobacterium Mediated Transfer of Bacillus thuringiensis Strain 6e Cry Gene to Ixora odorata var. Vulcan: A Comparison of Tissues

Ixora species are popular among landscape architects and gardeners as hedge plants and potted ornamentals. However, infestations by pests such as Ixora leaf webber, flower webber, green horned caterpillar and looper caterpillar have become a problem to the commercial exporters. Most chemical pest control methods adopted have hazardous effects on the environment and other beneficial insects and therefore, are unpopular among the buyers. As an alternative, Ixora odorata var. Vulcan was transformed with the cry gene of Bacillus thuringiensis (Bt) strain 6e by Agrobacterium mediated gene transfer. Leaf discs, four month old calli and in vitro grown I. odorata shoot tips (2 cm) were co-cultivated with LBA4404, harboring the Bt 6e cry cloned binary vector pLG121Hm. Co-cultivated shoot tips were transferred to the shoot multiplication medium (1⁄2 MS with 2 mg/L Benzyl Aminopurine (BAP) and 500 mg/L cefotaxime) and incubated at 2500 lux with a 16 hrs photoperiod. After four weeks, the axillary shoots were screened on 1⁄2 MS supplemented with 200 mg/L hygromycin. Hygromycin positive plants were used for the Polymerase Chain Reaction (PCR) analysis and Southern blot analysis. PCR analysis of the putative transformants, carried out with Gus specific primers, produced the expected 1.68 kb fragment in all the hygromycin positive transformants. Southern blot analysis of these with the Dig labeled Bt 6e cry probe produced positive results. The positive results obtained from the PCR analysis and the Southern blot confirmed genomic integration of the Bt 6e cry gene. The transformation efficiency of shoot tips was 10% whereas it was 25% and 20% respectively for callus and leaf disks transformation.