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Intra-specific and inter-specific hybridisation between Santalum Spicatum and S. Album

Rugkhla, Acharee (1997) Intra-specific and inter-specific hybridisation between Santalum Spicatum and S. Album. PhD thesis, Murdoch University.

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Several approaches have been applied to develop procedures for genetic improvement of Western Australian sandalwood (Santa/um spicatum) and Indian sandalwood (S. album): conventional crossing, protoplast fusion and Agrobacterium-mediated gene transfer.

In the conventional approach, the possibility of producing inter-specific and intra-specific hybrids of these species was investigated. The reproductive biology of the species ie. flower morphology, stigma and ovular receptivity and sexual compatibility between genotypes and species, was studied. The results showed that both sandalwood species are obligate out-crossing species with pre- and postfertilisation barriers preventing self and inter-specific pollination. For both intra- and inter-specific crosses, initial fruit set was low with 70-100% fruit abscission. Reasons for failure of fruit set included lack of fertilisation, lack of an embryo sac in some S. album flowers, delayed endosperm development and necrosis of placenta tissue. An ovary culture technique was developed and which could be used to rescue embryos of intra-specific crosses 4-7 months after pollination, but not putative inter-specific hybrid fruit, which abscised at 1-3 months old. Putrescine spray increased fruit set and delayed fruit abscission for both intra-specific and inter-specific crosses. However, only a few putative hybrid fruits were retained until 4 months after pollination; they were not harvested for ovary culture but allowed to develop to maturity. These mature putative hybrid seeds did not germinate but their endosperms were confirmed to be hybrid by random amplified polymorphic DNA (RAPD) analysis. RAPD results indicated the wide genetic distance between S. spicatum and S. album.

A tissue culture approach was conducted in parallel with conventional crossing. Direct somatic embryogenesis of S. album and S. spicatum was obtained by including thidiazuron in the culture medium. The published protocol of S. album regeneration through protoplasts could not be reproduced due to necrosis of the microcalli. An improved method of protoplast culture was developed by including thidiazuron and 2,4-D in the protoplast culture medium to induce direct somatic embryo formation in S. album. Reproducible protoplast fusion for S. album and S. 5picatum was accomplished and a high proportion of binary heterokaryons obtained. The heterokaryons grew to non-morphogenic microcalli while homokaryons of S. album developed to somatic embryos.

The efficient in vitro regeneration of S. album via direct somatic embryos was combined with an Agrobacterium-mediated gene transfer procedure. Putative transgenic somatic embryos and plantlets were obtained and confirmed to be transgenic by the gus histochemical assay.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological and Environmental Sciences
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): McComb, Jen and Jones, Michael
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