Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The exomorphology and size of Leucadendron pollen was examined using a scanning electron and light microscope respectively. Pollen was found to have a consistent triangular shape with three apertures. Pollen grain size however, show difference between species, sections and subsections on Leucadendron. Pollen of all species examined had a woven appearance like the intertwined threads of a fabric with orbicules present on the surface of L. chamelaea, L. elimense subsp. elimense and L. galpinii. Pollen viability was successfully assessed using a solidified agar medium containing 2g agar and 109 sucrose. Pollen germination for all species was found to be above 55% viability. A diallellayout of crosses has demonstrated conclusively that fecundity differs when crossing between species of the genus Leucadendron. Artificial hand pollination was applied successfully on Leucadendron and showed repeatedly that seed set following intraspecific crosses between the male and female inflorescence of the same species gave the same high rate of seed set as found in nature. However, seed numbers declined sharply when crossing between species of the section 'Leucadendron'. Seed set following crosses between species of different sections or sub-sections was the lowest and in most cross combinations there was no seed harvested or no seed germination. The diallellayout was useful in identifying incompatible species and for locating possible incompatibility barriers to interspecific seed development. The morphology of the stigma was examined with a scanning electron microscope. Stigma appearance of all species had a consistent round to oval shape, except for L. rubrum, which had an elongated shape. Stigma surfaces of all species were densely covered with a large number of unicellular papilar cells on the swollen base. The aniline blue staining technique, together with the fluorescent microscope technique was used to follow the growth of the pollen tube following compatible and incompatible cross combinations. Pollen on the stigmas of compatible and incompatible species examined showed signs of germination. Pollen tubes grew between the papilla cells in all directions and only the most vigorous ones reached the upper part of the style. From the upper region of the style, yellow green tubes grew cohesively in the middle of the style towards the ovule. In compatible combinations a not more than 4 tubes reached the ovule region, but was difficult to observe when they entered the micropyle for fertilization. In incompatible species a large number of abnormalities occurred beyond the upper region of the style. / AFRIKAANSE OPSOMMING: Die morfologie en grootte van Leucadendron stuifmeel is deur middel van 'n skandeerelektron - en ligmikroskoop bestudeer. Baie klein verskille in stuifmeel morfologie het voorgekom. Diverse verskille in stuifmeelgrootte het wel voorgekom tussen spesies, groepe en subgroepe van Leucadendron. Stuifmeelvorm was deurgaans driehoekig en die oppervlakte van die stuifmeelkorrel het die voorkoms van geweefde vesels gehad. Klein, bolvormige struktuurtjies was teen verskillende digthede oor die stuifmeeloppervlak van L. chamelaea, L. elimense subsp. elimense en L. galpinii versprei. Stuifmeelkiemkragtigheid is bepaal deur dit op soliede agar medium te ontkiem en was deurgaans bo 55% kiemkragtig. Onderlings dialleliese kruisings van Leucadendron spesies het variasie in saad set getoon. Handbestuiwing is suksesvol uitgevoer en saadset in intraspesie kruisings hoog en soortgelyk aan natuurlike bestuiwing. Saadset en saad ontwikkeling het drasties verswak toe verder vewante spesies as ouers gebruik. As gevolg van hulondeurdringbare saadhuid is neutagtige sade gewoonlik moeiliker ontkiembaar. Die diallel uitleg was ook nuttig om verenigbare en onverenigbare kruisingskombinasies te identifiseer en om onverenigbaarheidskanse op te spoor. 'n Skandeerelektronmiskoop is gebruik om die morfologie van die stigma te bestudeer. Stigmas was deurgaans rond tot ovaalvormig, behalwe die van L. rubrum wat 'n verlengde voorkoms gehad het. Die stigma bestaan uit 'n groot aantal eensellige papilla, wat dig teen mekaar gepak is op 'n geswolle basis. Aniline-blou fluoresserende kleurstof en 'n fluoressensie mikroskoop is gebruik om die pad van die stuifmeelbuis in verenigbare en onverenigbare kruisingskombinasies in Leucadendron te volg. Stuifmeelontkieming het in alle kruisingskombinasies geskied. Stuifmeelbuise het in alle rigtings tussen die papilla gegroei en slegs die mees kiemkragtige stuifmeelbuise het die boonste deel van die styl bereik. In die styl het die buise dig teen mekaar gegroei en was dit moeilik telbaar. 'n Maksimum van vier buise het die vrugbeginsel bereik, maar dit was moeilik om verder te volg nadat hulle die poortjie bereik het. In onverenigbare kruisingskombinasies het stuifmeelbuise abnormale groeipatrone in die boonste gedeelte van die styl getoon.

[Truncated abstract] Grevillea and Leucadendron belong to Proteaceae and both have economic importance to the floriculture industry. Grevillea is a highly diverse genus endemic to Australia and very attractive for landscaping. Leucadendron is a South African Proteaceae but is cultivated in Australia and is well known as a cut flower. This thesis focuses on the application of DNA-based molecular markers to these genera. Several groupings within Grevillea were suggested by previous researchers based on morphological characteristics. In this thesis the monophyly of the groupings among 12 Grevillea species from New South Wales was tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses. To test the robustness of the data, UPGMA using Jaccard similarity, Neighbor Joining using total character difference and Wagner parsimony analyses were undertaken. The relationship trees generated supported monophyly of the groupings. Chloroplast DNA (cpDNA) was used to develop phylogenetic relationships among Leucadendron species. Inheritance and variation of cpDNA were evaluated using PCR-RFLP. The study demonstrated that cpDNA was inherited maternally and a phylogenetic tree of Leucadendron species using parsimony analysis was constructed. ... A fingerprinting study conducted using ISSR, produced a dendrogram showing the relationships among 30 cultivars. From the results, i a fingerprinting key was developed. Three examples of synonymous cultivar pairs were identified. In Leucadendron the male and female flowers develop on separate plants, and sex identification is only possible at time of flowering. ISSR, suppression subtractive hybridisation (SSH), and SSH combined with mirror orientation selection (MOS) were used in attempts of identifying sex-dependent DNA fragments at earlier stages of plant development. Neither of these techniques was able to identify sex-specific markers in Leucadendron. Nevertheless, the results did indicate that cpDNA copy number may differentiate male and female plants. Also, it was demonstrated that the genomes of male and female plants are quite homologous, which increases the difficulty in identifying sex-specific sequences. This thesis highlights the potential of DNA-based markers to determine species relationships in Grevillea and Leucadendron, as well as to identify Leucadendron cultivars. The information produced during the research for this thesis provides a basis for Grevillea and Leucadendron variety development and may be used to assist the design of interspecific crosses, to identify cultivars and the parents of hybrids. In addition, the results offer insights into the likelihood, problems and strategies of finding sex-specific markers for genes controlling sex in Leucadendron. ii

Flowers from members of the genus Leucadendron have colourful bracts and long vase life that make them highly desirable cut-flowers. Breeding programs based on interspecific hybridization would encounter difficulty if pre- or post-fertilization barriers exist in the distant crosses. Embryo rescue is one of the commonly used approaches to overcome post-fertilization barriers in wide hybridization. In this study, intersectional and intersubsectional hybridization of Leucadendron was attempted. Observation of pollen-pistil interactions revealed that post-zygotic rejection was the main reason for the incompatibility of the crosses, therefore embryo rescue was adopted and a protocol was developed to raise the hybrids. To better understand the genome structure in the genus, karyotypes of selected species were analyzed. Chromosome examination indicated that all (27) Leucadendron species examined were diploid and had a chromosome number of 2n = 26. The chromosomes were small in size and had predominantly median to submedian centromeres. The karyotypes of the species were rather symmetrical and seemed to be primitive according to Stebbins' karyotype classification. DNA based PCR-RFLP and RAMP markers were developed to identify Leucadendron hybrids at an early age. RAMP analysis showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP. The occurrence of PCR recombination also proved to be a troublesome issue when using the PCR-RFLP method, whereas the clarity of the interpretion of the RAMP method was not influenced by PCR recombination. Interspecific hybridization in a breeding program can provide valuable information on grouping of the species for systematic purposes. Regression analysis between cross success rate and cpDNA character difference revealed that there was a highly significant correlation between them. Patterns of success for intersectional hybridizations in Leucadendron were generally consistent with current taxonomic hypotheses regarding the sectional division of the genus. Success was generally lower for intersectional crosses than for intrasectional crosses.