Genetic transformation of Verticordia grandis (Myrtaceae) using wild-type Agrobacterium rhizogenes and binary Agrobacterium vectors

Abstract

Verticordia grandis is an Australian native plant of the Myrtaceae family much prized for its display of bright red flowers. This paper describes a system for the transformation and regeneration of transgenic Verticordia grandis. The susceptibility of V. grandis to Agrobacterium rhizogenes was shown by simultaneous wounding and inoculation of the stems of shoot explants with 4 wild-type strains of A. rhizogenes. Shoots inoculated with 3 of the 4 strains developed abnormal tissues containing the opine (agropine or mannopine) characteristic of the bacterial strain used. A regeneration system for V. grandis was developed, using leaf discs excised from the petiole region of micropropagated shoots. These discs were used for transformation studies using two plasmid vectors in either the wild-type A. rhizogenes strains or a non-oncogenic A. tumifaciens strain, LBA4404. The plasmid vectors (pBI121 and pKiwi) contained chimeric kanamycin resistance genes, neomycin phosphotransferase II (NPTII) and the bacterial β-glucuronidase (GUS) uidA gene. Leaf discs were inoculated by wounding and selected for growth on kanamycin-containing medium. Regenerated shoots were transferred to root induction medium containing kanamycin and those plants which produced roots were regarded as potential transformants. These plants were assayed for GUS activity and transformation was confirmed by Southern DNA hybridisation and by PCR amplification of the GUS gene. These results represent the first report of transformation and subsequent regeneration of a plant from the economically important Myrtaceae. © 1995.