This thesis examined aspects of the reproductive and molecular biology of Eucalyptus marginata (jarrah). The aims were to develop protocols for controlled pollination, that could be used in clonal orchard trees to breed jarrah seedlings that have a known genetic resistance to Phytophthora cinnamomi (dieback), for use in rehabilitation after mining and logging. An intimate knowledge of the breeding biology of jarrah was necessary to achieve this aim. The project also aimed to increase knowledge of the genetic diversity and structure of jarrah, in order to make informed decisions regarding the collection of material to be used for clonal propagation. Previous research has had little success in producing viable seed from any controlled pollinations, but clonal material resistant to P. cinnamomi has been produced using tissue culture. The question posed in this thesis was 'Can we improve breeding and propagation techniques of jarrah?' 

Techniques were developed for testing of in vitro pollen viability and pollen storage, pollination and fertilisation success after controlled pollinations, including determination of stigma receptivity and development of bud isolation techniques using alfoil. The variation in female fertility between genotypes was examined. The use of paclobutrazol was explored as a method of increasing the level of viable seed production in clonal orchard trees. The use of fertiliser as well as the growth retardant was also explored to see if it increased the level of seed production even more. Genetic diversity, genetic differentiation and phylogeny within Eucalyptus marginata were examined using nuclear and chloroplast DNA analysis with Restricted Fragment Length Polymorphisms. 

While it was first thought that the fertilisation rate was quite low, it was confirmed that the fertilisation rate is similar to other eucalypt species. The zygote abortion rate was quite high in one clone, but one wild tree had a similar seed production rate to other eucalypt species. The zygote and endosperm appeared to be different in the clone and the wild tree observed. The level of seed production was examined in clones and wild trees and it was found that the level was often quite low, particularly in the clones (0 - 13% in clones, 0 - 18% in wild trees) in comparison with other Eucalyptus species, and varied between genotypes. The use of a growth retardant such as paclobutrazol may increase the production of viable seed, if it is applied during autumn. The results were inconclusive for the fertiliser/paclobutrazol experiment, since the paclobutrazol was applied during spring which was the worst time of year for increasing seed production. There were differences between genotypes in reaction to both the paclobutrazol and the fertiliser/paclobutrazol. Genetic diversity was moderate in comparison with other Eucalyptus species, and there was a low level of genetic differentiation between populations in the nuclear genome. No differentiation was observed between the morphologically recognised subspecies in the nuclear genome, but differentiation between the populations on the Swan Coastal Plain and populations on the Darling Plateau was seen in the chloroplast genome, indicating that there was historical separation of these two areas.

The conclusions arising from this work are that while controlled pollinations are possible in Eucalyptus marginata the clones that were used in these experiments have often behaved differently to the wild trees in the time of anthesis and levels of viable seed production, and in one clone (5J119) the zygote and endosperm nuclei appeared to be very different to the zygote and endosperm nuclei of a wild tree. Further investigation is necessary to see if these differences are related to the low level of seed production observed in the clonal populations. Paclobutrazol may be worth exploring further as a means of increasing seed production. Material to be used for rehabilitation and seed orchards can be collected from a wide area in the main distribution of the species, although trees on the Swan Coastal Plain are distinct from the trees in the main forest area in the chloroplast genome.

Reproductive and molecular biology of Eucalyptus marginata Donn ex Smith

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Reproductive and Molecular Biology of

/pdf/eucalypt-domestication-and-breeding-2nu4zylb7r.pdf

Eucalypt domestication and breeding

Techniques were evaluated to develop an effective controlled-pollination technique for jarrah (Eucalyptus marginata). Pollination success was scored by counting pollen tubes in squashed styles. The most effective method to isolate the flowers after emasculation was to wrap each in a twist of aluminium foil. Pollen application was best when several freshly-picked, newly-opened flowers were wiped across each receptive stigma. Stigmas were receptive from 3-6 days after anthesis and most pollen tubes were observed pollination in the middle of the flowering season. Very few mature fruits resulted from any controlled-pollution technique. Bud, flower and fruit-loss rates under natural conditions were recorded (in five clones and five wild trees) and 0-13% of buds remained on the trees to develop into fruits. The percentage of pollen germination from different trees was very variable. Pollen was most fertile in the middle of the flowering season, environmental conditions had little effect. Fresh pollen in anthers was more fertile than pollen separated using a fine sieve.

Pollination techniques, fruit loss and pollen viability in Eucalyptus marginata (Jarrah)

The eucalypt species Eucalyptus marginata which is harvested for high quality timber comprises three subspecies recognized by morphological characters; E. marginata ssp. marginata, ssp. thalassica, and ssp. elegantella. Genetic diversity and phylogenetic relationships between the subspecies were examined using anonymous nuclear RFLP loci, with Eucalyptus staerei included as an outgroup in the phylogenetic analysis. The level of diversity within the nuclear genome was lower than that found in comparative studies with other eucalypts (A = 2. 7, H4 = 0.345). Most of the variation occurred with ill the populations (96. 9%, H4 = 0.334) The two populations sampled for each of ssp. thalassica and ssp. elegantella clustered together in the UPGMA analysis, however there was little differentiation between the three subspecies overall (D = 0.029). Eucalyptus marginata was clearly distinct from its closest relative E. staerei (0 = 0.16). There is little genetic support for the separation of the subspecies.

Genetic diversity and phylogenetic relationships within Eucalyptus marginata (Myrtaceae)

The success of controlled pollination techniques was investigated for clones and wild trees of Eucalyptus marginata. The number of pollen tubes in the style was used as a measure of pollination success. The stigma was receptive from 3-11 days after anthesis, but it appeared that stigmatic receptivity may be highest from 4-7 days after anthesis. Flowers were isolated using aluminium foil squares, and fresh pollen was found to be more effective for pollination success than processed pollen. Results were inconclusive comparing self- to cross-pollination success, and no difference was found comparing pollination success in clones and wild trees. However clones generally tended to flower earlier than the wild trees, whatever their origins, and very few mature fruits resulted from any controlled pollinations in jarrah clones. Clones also produced a lower proportion of mature capsules from the flowers produced (Wheeler 2004).

/pdf/low-success-of-controlled-pollination-in-eucalytpus-21osziv081.pdf

Low success of controlled pollination in Eucalytpus marginata (jarrah)

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Pollination techniques, fruit loss and pollen viability in Eucalyptus marginata (Jarrah)

Genetic diversity and phylogenetic relationships within Eucalyptus marginata (Myrtaceae)

Low success of controlled pollination in Eucalytpus marginata (jarrah)