The
              Report

No. 109: Waxflower breeding for new and better nativeflowers


The full report
 This Short Report is a summary of a full report Breeding Biology of Waxflowers, RIRDC Publication Number 01/156, UWA-35A, by Dr Guijun Yan, Plant Sciences, University of Western Australia. Phone: 08 9380 1240; Fax: 08 9380 1108.  The full report is available in hardcopy for $10 from RIRDC (ph: 02 6272 4819 or from our eshop) or can be downloaded for free from RIRDC’s website: www.rirdc.gov.au/fullreports/wnp.html
Research into the breeding biology of waxflowers should significantly improve hybridisation of new and novel native flowers for the Australian cutflower industry.

At present Australia produces less than 10 per cent of the world’s $440 million annual sales of Australian native cut flowers. Most are supplied from Israel, the USA, Mexico and South Africa.

But with more than 25,000 different species of flowering native plants, Australia retains rich resources from which to grow and market, license or sell, new plant varieties around the world.

Research undertaken by Dr Guijun Yan of the University of Western Australia’s Plant Sciences Department in the faculty of Agriculture, investigated scientific aspects of breeding biology and reported the results and methodologies devised in waxflower breeding programs.

The Rural Industries Research and Development Corporation (RIRDC) initiated and funded the waxflower breeding research, of which the biological study is an integral part.

 The Study

In investigating breeding biology of waxflowers (Chamelaucium), there was special emphasis on: • chromosome numbers;

In cytogenetic studies of the waxflower, chromosome numbers and ploidy levels of 28 selected genotypes of Geraldton Wax (Chamelaucium uncinatum) were investigated.

These were collected from wild populations and cultivated plantations. Cuttings were rooted in a glasshouse under mist spray and their root tips examined for chromosome numbers and ploidy levels.

Clones were selected for examination to investigate any relationships between flower size, leaf length and plant ploidy levels.

Pollen also was studied using a bright field microscope to determine variations in size, shape and viability of the different clones at varying ploidy levels.

In another part of the project, in-vitro chromosome doubling of waxflower plantlets was involved. This sought to overcome the frequently occurring problem where wide crossing or hybridisation across ploidy levels produces sterile progeny.

Chromosome doubling to induce polyploiding (two or more single sets of unpaired chromosomes) is widely used in other plant breeding programs to restore fertility. Plants with doubled chromosome numbers can also produce enlarged flowers.

Developing a protocol to achieve this doubling was an objective of the project along with another major aim to induce polyploid plants to be used in the breeding program.

In the molecular work, the study sought to identify chloroplast DNA inheritance to assist parent selection in breeding programs and to use it as a tool to sort out the parentage of natural collections.

Efficient and successful wide crossing of plants requires an understanding of the genome (clone) interaction between the two plants involved and to provide an understanding of this, investigation of pollen/ style interaction is an important initial step.

The final part of the study involved embryology and early embryo rescue.

This approach seeks to overcome postzygotic hybridisation barriers in wide crosses. Mature waxflower seeds could not be germinated readily, so embryo rescue was adopted, ie, embryo germination under tissue culture conditions.

This included steps such as collecting developing embryos six weeks after pollination, germinating the young embryos in an MS medium (a mixture of salts of Murashige and Skoog), subculturing and rooting the seedlings in culture before eventually transferring them into soil in the glasshouse.

The experiment also aimed at understanding the embryo and endosperm development of waxflower hybrids.


The Results Among the 28 clones studied, one was tetraploid with 44 chromosomes, another triploid with a count of 33 chromosomes and the remainder was diploid with 22 chromosomes.

The work revealed that increasing ploidy levels could be identified by increased numbers of pollen apertures. Diploid plants produced highly valuable pollen while the triploid plant’s pollen was sterile. The tetraploid plant produced pollen with reduced viability but probably still adequate for use in breeding.

The chromosme doubling experiments demonstrated that ploidy of waxflowers could be doubled using the right concentration of the poisonous alkaloid, colchicine. Indicators of increased ploidy level after treatment with this alkaloid are leaf width and stomata size and density.

Results showed the chloroplast DNA inheritance came from the maternal plant in all cases.

Summary

This research has demonstrated that ploidy of waxflowers can be doubled using the alkaloid colchicine. Methods and strengths of the alkaloid that produce the best result have been identified. Indicators of increased ploidy level after treatment are leaf width and stomata size and density.

The maternal chloroplast DNA observation is being used to validate the exact parentage of natural hybrids and to confirm hybridity in the breeding program.

Investigation of pollen pistil interactions and seed set suggested methods to overcome the hybridisation barriers to enable the combination of desirable traits in new hybrids. The work identified the characteristics of pistil in Chamelaucium uncinatum and revealed some of the embryological features that may indicate the taxonomic difference between waxflower and other members of the myrtaceae family.

Finally the work demonstrated that hybrid embryos could be rescued as early as three days and as long as day 18 after pollination.

Rescued young embryos could develop in the MS medium and then grow into hybrid plants.

Close examination of embryo production in different cross combinations indicated there may be hybridisation barriers for some cross combinations.

And certain male and female plants were recognised to be highly productive when used as parents for interspecific and intergeneric hybridisation.


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