GENETIC-TRANSFORMATION OF VERTICORDIA-GRANDIS (MYRTACEAE) USING WILD-TYPE AGROBACTERIUM-RHIZOGENES AND BINARY AGROBACTERIUM VECTORS

Verticordia grandis is an Australian native plant of the Myrtaceae fam ily much prized for its display of bright red flowers. This paper desc ribes a system for the transformation and regeneration of transgenic V erticordia grandis. The susceptibility of V. grandis to Agrobacterium rhizogenes was shown by simultaneous wounding and inoculation of the s tems of shoot explants with 4 wild-type strains of A. rhizogenes. Shoo ts inoculated with 3 of the 4 strains developed abnormal tissues conta ining the opine (agropine or mannopine) characteristic of the bacteria l strain used. A regeneration system for V. grandis was developed, usi ng leaf discs excised from the petiole region of micropropagated shoot s. These discs were used for transformation studies using two plasmid vectors in either the wild-type A. rhizogenes strains or a non-oncogen ic A. tumifaciens strain, LBA4404, The plasmid vectors (pB1121 and pKi wi) contained chimeric kanamycin resistance genes, neomycin phosphotra nsferase II (NPTII) and the bacterial P-glucuronidase (GUS) uidA gene, Leaf discs were inoculated by wounding and selected for growth on kan amycin-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 assa yed 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 regenerati on of a plant from the economically important Myrtaceae.