 |
Rural Industries Research & Development Corporation |
|| Home || Search || Contact || Publications Eshop || Privacy Statement ||
Rural Industries Research & Development Corporation
The Breeding Biology of Waxflowers (01/156 UWA 35A)by Guijun Yan
July 2001
RIRDC Publication No 01/156 RIRDC Project No UWA 35A
Chromosome numbers of 28 genotypes from 20 different original and secondary populations were examined. The chromosomes of C. uncinatum are small, about 1 µm when fully condensed and the detailed study requires the use of a 100x oil immersion lens.
Among the 28 genotypes, UA100 from Kings Park was tetraploid with 44 chromosomes (2n=44) while UA557 from Midlands Road was triploid with a count of 33 chromosomes (2n=33). The remaining 26 genotypes from 20 populations or cultivars were diploid with 22 chromosome (2n=22).
The polyploid plants had larger flowers (>25 mm), but plants with larger flowers were not necessarily polyploid. Pollen size was not correlated with ploidy level, but increased as the flower size increased.
No correlation between leaf length and ploidy level was observed. The number of apertures on the pollen surface however increased with ploidy level. Therefore, the number of pollen apertures (colpi) may be used as an indicator of ploidy level in C. uncinatum and possibly in related species and genera.
This hypothesis needs to be further investigated using larger samples.
Diploid plants produced highly viable pollen and the triploid plants produced sterile pollen only. The tetraploid plant produced pollen with reduced viability but probably would be adequate for use in breeding. These results are consistent with the previous research that the basic chromosome number in Chamelaucium is x=11 and that in C. uncinatum the common state is diploid. However, higher ploidy levels do occur and this has implications both for evolution of the genus and in practical breeding programs.
Chromosome doubling via colchicine treatment has been trialed on three genotypes of waxflowers under in vitro conditions. Colchicine was applied by two methods: by immersing shoots in a liquid medium containing one of four levels of colchicine: 0.0005%, 0.005%, 0.05% and 0.5% and by regeneration from stem segments on a tissue culture medium containing one of four levels of colchicine: 0.0002%, 0.001%, 0.005% and 0.025%.
Colchicine at high concentrations was found to be harmful to in vitro cultured waxflower plants. The treatment using 0.025% for regeneration or 0.5% for immersion decreased the survival rate of stem segments and growing shoot tips, and also reduced shoot proliferation. Colchicine treatment also caused the waxflower plantlets to grow wider, and led to forked and rosetted leaves and abnormal shoot tips.
The plantlets were assessed for chromosome doubling by the microscopy of root tips. The relationship between chromosome doubling and morphological and anatomical characteristics was also determined. Two tetraploid plantlets had been generated from eight ‘Esperance Pearl’ shoots immersed in the 0.05% colchicine solution, this represents a 25% success rate. The tetraploid plantlets were also found to have larger wider leaves with fewer larger stomata than the diploid, which can be used as indicators of ploidy level.
This research has demonstrated that ploidy of waxflowers can be doubled using colchicine. The immersion method using a 0.05% w/v colchicine solution gives the best result. Leaf width and stomata size and density can be used as indicators of increased ploidy level.
PCR amplification of three chloroplast DNA regions followed by restriction of the amplified products was used to identify restriction fragment length polymorphisms (RFLP) in 18 Chamelaucium uncinatum and C, megalopetalum genotypes. Five chlorotypes were observed among the two species studied. In C. uncinatum three site- and one length-mutations were observed, corresponding to three chlorotypes and in C. megalopetalum, two different chlorotypes were observed. Phylogenetic analysis based on the RFLP data produced a single most parsimonious Wagner tree, which is exactly the same as a Dollo tree in tree length (39 steps) and tree topology. The result suggests that intermediate types exist between the two species and a different chlorotype (MB05) was observed.
Based on the cpDNA polymorphisms between the female and the male parents, cpDNA inheritance was studied in 17 intraspecific hybrids and 33 interspecific hybrids. Maternal cpDNA inheritance was observed in all cases as in most of the angiosperms. This research result is being used to validate the exact parentage of natural hybrids and to confirm hybridity in our breeding program.
Investigations of pollen pistil interactions and seed set indicated that both pre- and postzygotic hybridisation barriers exist in Chamelaucium alliance. Intraspecific, interspecific and intergeneric crosses involving nine species from the genera Chamelaucium, Verticordia and Darwinia were conducted. Pollen pistil interactions and the formation of seed were studied in order to locate any hybridisation barriers that may exit. Whilst seed set was not observed from 16 crosses indicating no hybridisation barriers, the presence of barriers was observed in the remaining 55 crosses. The long styles of D. squarrose and D. spp (novo) were responsible for the incompatibilities when used as female parents, as the pollen tubes of the shorter styled species fail to transcend the longer styles. According to these results, methods to overcome the hybridisation barriers were suggested to facilitate the combination of desirable traits in new hybrids.
Pistil of C. uncinatum is unicarpellate and contains six, rarely eight ovules. The ovules, which arrange in two rows sharing the same funicle, are bitegmic, anatropous, and crassinucellate. A zigzag-shaped micropyle is formed by both the integuments. The archesporial cell originates subdermally in the ovule promordium. The embryo sac development conforms to Polygonum type. Mature embryo sac is constituted by an egg cell, two synergids with filform apparatus, two polar nuclei, one upper and one lower, and three antipodals, which are ephemeral. The walls of megasporocyte and megaspore tetrad do not deposit callose and the well-known central vacuole is absent in both 2- and 4-nucleate embryo sac. Starch grains are rich in integuments, nucellus as well as mature embryo sac. No storage protein and lipid droplet was viewed in the ovular tissue at the stages. In some cases, the embryo sac and ovule may degenerate. Some of the specific embryological features may indicate the taxonomic difference of Chamelaucium alliance from the other members of Myrtaceae.
Hybrid embryos of Chamelaucium alliance could be rescued as early as three days after pollination.
The highest rescuing rate was achieved at day 18 after pollination. Rescued young embryos could develop in MS medium and grew into hybrid plants. Close examination of embryo production of different cross combinations indicated that hybridisation barriers might exist for some cross combinations. Certain male and female plants were recognized to be highly productive when used as parent for interspeicific and intergeneric hybridisation.
|
