Introduction and expression of marker genes in sandalwood (Santalum album L.) following Agrobacterium-mediated transformation

Abstract

Sandalwood (Santalum album L.) is a tropical tree of importance for its scented heartwood and 'sandalwood oil'. This study for the first time, reports a system for the introduction and expression of foreign genes in sandalwood. Transgenic plants were regenerated from embryogenic cultures derived from transformed somatic embryos. A highly proliferative system of somatic embryo regeneration of this species established in our lab was combined with its susceptibility to Agrobacterium tumefaciens infection. The torpedo-cotyledonary stage embryos were inoculated by pricking and cells of disarmed Agrobacterium strains carrying β -glucuronidase uidA and neomycin phosphotransferase II (NPTII) genes on the binary vectors pKIWI105, pBI121, pIG121-Hm were directed at the wound sites. In total, 20% of inoculated embryos callused and developed second generation embryos. The transformation status of the regenerants was analyzed by growth in the presence of kanamycin, GUS and NPTII assays, PCR amplification and genomic DNA hybridization of the GUS gene. In order to avoid background GUS activity due to incomplete elimination of Agrobacterium by the antibiotic treatment, the GUS gene construct pIG121-Hm and pKIWI105 that show expression only in eukaryotic environment were chosen. Putative transgenic embryos identified after 2 months were allowed to develop into rooted plants. Plant regeneration was also attempted by direct germination or by repetitive embryogenesis of inoculated embryos. In an alternative method, seedling hypocotyls were inoculated and multiple shoot organogenesis obtained from the infected site on the seedlings. The methods presented here for the generation of transgenic sandalwood represent additions to the slow-growing list of transformation and regeneration protocols that have been reported for broad-leaved tropical trees.