The development of regeneration and transformation systems for Eucalyptus spp.

Abstract

In South Africa, Eucalyptus breeding programmes are aimed at the selection of fastgrowing varieties, with appropriate wood characteristics and/or resistance to pests and diseases. However, the slow growth rate, long generation time and heterozygosity of trees make this a difficult task. Such problems may be overcome by the adoption of a biotechnological approach for plant propagation and modification. Towards this end, the aims of this investigation were to establish protocols for the micropropagation of Eucalyptus grandis and for the Agrobacterium-mediated transformation and subsequent plant regeneration of this important species. The usefulness of transformed cells and/or tissues is dependent upon the availability of methods for their regeneration into plants. Consequently, methods for plant regeneration via indirect organogenesis from leaf discs and cell suspension cultures were investigated. Organogenic calli were produced from leaf explants on MS medium with 16 mg.1-1 &l:em•calcltrate, 20 g.I-I sucrose, 1mg.I-I NAA and 0.05 mg.1--<1 BA. Shoots were induced on MS medium containing 1 mg.1-1 ZEA and 0.2 mg.1-1 IAA, and subsequently rooted on medium containing MS nutrients supplemented with 1 mg.1- 1 IAA. Cell suspension cultures were established but not regenerated via indirect organogenesis. Additionally, various media were investigated in order to obtain somatic embryos from cell suspension cultures. The MS media supplemented with 30 g.1-1 sucrose, 12 mg.1- 1 ABA and/or 40 g.1-1 PEG were found to be most suitable, resulting in the production of embryoids; germination results are not available at this stage. In order to establish methods for the transformation of both leaf discs and cell suspension cultures of Eucalyptus, a triparental mating was performed between Escherichia coli pnT119 (donor), A. tumefaciens LBA4404 (recipient), and E. coli HBI0l::pRK2013 (helper), resulting in the transconjugant LBA4404 (pnT119); insertion of the pJIT119 plasmid was demonstrated using agarose gel electrophoresis. The transconjugant CS8C1 (pMP90) (pJIT119) was also used. Protocols for the transformation of both leaf discs and cell suspension cultures were established, and resulted in the production of putatively transformed calli which were GUS positive and with stood selection on kanamycin (50 Ilg.mr1) and/or sulfadiazine (50 Ilg.mr1). Also, Southern blotting analysis indicated that the gene transfer process was successful. Due to difficulties in the regeneration of plants from transformed calli transgenic plants were not obtained. Future research strategies and applications of the developed protocols to Eucalyptus breeding programmes are discussed.