The Breeding Biology of Waxflowers

A report for the Rural Industries Research and Development Corporation by Guijun Yan

July 2001

RIRDC Publication No 01/… RIRDC Project No UWA 35A © 2001 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 0 642 (…RIRDC to assign) ISSN 1440-6845

Title of your publication Publication No. 01/ Project No. UWA-35A

The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details (Name) Dr Guijun Yan (Address) Plant Sciences Faculty of Agriculture The University of Western Australia Nedlands, WA 6009 Phone: (08) 9380 1240 Fax: (08) 9380 1108 Email: [email protected]

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au

Published in June 2001 Printed on environmentally friendly paper by Canprint

ii Foreword

The continued growth of Australian native plants as cutflowers depends largely on the breeding of new cultivars which are attractive, vigorous and productive, durable in transit and storage, resilient to pests and diseases, and with a greater seasonal spread of production. The understanding of the breeding biology is the key to success in these matters and can increase the breeding efficiency considerably.

With the support of RIRDC, a waxflower breeding program was initiated in Western Australia in 1995. This program aims at making wide hybridisations (interspecific and intergeneric) to produce new or novel cutflower cultivars for the Australia industry. The study of the breeding biology in Waxflowers is an integral part of that program.

This publication considers the breeding biology of waxflowers (Chamelaucium) with special emphases on chromosome numbers, ploidy levels, ploidy manipulation, chloroplast DNA inheritance, identification of natural and artificial hybrids, hybridisation barriers, embryology and early embryo rescue. It discusses how this information and methodologies can be used in waxflower breeding programs to increase the breeding efficiency.

This project was funded from RIRDC Core Funds, which are provided by the Federal Government.

This report, a new addition to RIRDC’s diverse range of over 700 research publications, forms part of our Wildflowers and Native Plants R&D program, which aims to identify and develop new fresh products.

Most of our publications are available for viewing, downloading or purchasing online through our website:

• downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/eshop

Peter Core Managing Director Rural Industries Research and Development Corporation

iii Acknowledgements

I wish to thank the following organisations and people for their contributions to this project and the final report (Alphabetical order):

Agriculture Western Australia King Park and Botanic Gardens Sunglow Flowers John Considine Anousk Cousin a Ben Croxford Simone Cunneen Lindsay Forrester Nina Foulkes-Taylor Nic George Digby Growns Lorreine Lawson Xuanli Ma Chris Newell Fucheng Shan Philip Watkins Mark Webb Shubiao Wu

iv Abbreviations

BAP 6-benzylaminopurine c-mitosis colchicine-mitosis °C degrees Celsius DNA deoxyribose nucleic acid F1 first generation hybrid F2 second generation hybrid FLP orcein formic lacto-propionic orcein g/L grams per litre IBA indole-3-butyric acid L-I layer first germlayer (outermost) L-II layer second germlayer L-III layer third germlayer (innermost) Mmolar mL millilitre mm millimetre mM millimolar µM micromolar MS mixture of salts of Murashige and Skoog (1962) n.a. not applicable NAA naphthalene acetic acid NaOH sodium hydroxide p p-value PCB para-dichlorobenzene pers. comm. personal communication std dev. standard deviation w/v weight by volume x basic ploidy number 2n somatic ploidy number 2x diploid 4x tetraploid

v Table of contents

Foreword...... iii

Acknowledgements ...... iv

Abbreviations ...... v

Executive Summary...... viii

1. Cytogenetic studies in Chamelaucium...... 1 1.1 Introduction ...... 1 1.2 Materials and methods...... 2 Chromosome examination...... 2 The flower size and leaf length ...... 2 The morphology and viability of pollens ...... 3 1.3 Results ...... 4 Chromosome number and ploidy level...... 4 The flower size and leaf length ...... 4 The morphology and viability of pollens ...... 4 1.4 Discussion...... 8 Chromosome number and intraspecific ploidy variation of Chamelaucium ...... 8 The origin of polyploids in Chamelaucium uncinatum ...... 8 Pollen morphology and viability of C. uncinatum ...... 8 Ploidy diversity and implication for breeding of C. uncinatum ...... 9 1.5 References ...... 9

2. Chromosome doubling of waxflower plantlets regenerated in vitro ...... 11 2.1 Introduction ...... 11 2.2 Materials and Methods ...... 11 Plant material...... 11 Regeneration procedures ...... 11 Immersion procedures ...... 12 Assessment of the plant material...... 12 Assessment of mitotic chromosomes in root tips ...... 12 Statistical analysis ...... 13 2.3 Results ...... 13 Survival rate after colchicine treatment...... 13 Shoot proliferation...... 14 Morphological assessment of plant material ...... 14 Chromosome assessment...... 15 Anatomical assessment of tetraploid plant material ...... 15 2.4 Discussion...... 16 Colchicine doubles chromosomes in somatic waxflower tissue...... 16 Colchicine concentration and mortality...... 16 Combining colchicine and in vitro culture ...... 17 Anatomical assessment - ploidy indicators ...... 17 2.5 References ...... 18

3. Molecular studies in waxflowers...... 21 3.1 Introduction ...... 21

vi 3.2 Materials and methods...... 22 Plant materials ...... 22 DNA extraction ...... 22 PCR protocol...... 22 Restriction ...... 22 Electrophoresis ...... 22 Data analysis...... 22 3.3 Results ...... 23 Variation of chloroplast DNA ...... 23 The trnF-trnVr spacer restriction...... 23 The trnC-trnD spacer restriction...... 23 Intraspecific chloroplast DNA inheritance in C. uncinatum ...... 24 Interspecific chloroplast DNA inheritance...... 24 3.4 Discussion...... 24 Chamelaucium chloroplast DNA variation ...... 24 Chloroplast DNA inheritance...... 25 3.5 References ...... 25

4. Receptivity timeline and pollen/pistil interactions...... 35 4.1 Introduction ...... 35 4.2 Materials and methods...... 35 4.3 Results ...... 36 4.4 Discussion...... 40 4.5 Reference...... 41

5. Studies On The Embryology And Early Embryo Rescue To Support Waxflower Breeding...... 42 5.1 Introduction ...... 42 5.2 Materials and methods...... 42 Embryology...... 42 Early embryo rescue...... 43 5.3 Results and discussion ...... 44 Floral Structure...... 44 Ovular Structure ...... 44 Megasporogenesis and Embryo Sac Development ...... 44 Embryo rescue in vitro ...... 45 5.4 Reference...... 46

vii Executive Summary

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

viii 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.

ix 1. Cytogenetic studies in Chamelaucium

1.1 Introduction

Geraldton Wax (Chamelaucium uncinatum Schauer) is one of the 31 species that comprise the genus Chamelaucium in the family Myrtaceae (Marchant and Keighery, 1995). The hardy evergreen shrub, which is rarely more than five meters high, occurs naturally through the coastal regions from Perth (32°S) to Kalbarri (27°S) in Western Australia. Chamelaucium uncinatum is the most well known member in the genus Chamelaucium and it forms the backbone of the Australian native flower industry (Considine and Growns 1997). Most of the present Geraldton Wax cultivars are direct selections from the wild. Recently, an extensive breeding program commenced as the result of a joint effort between the University of Western Australia and Agriculture Western Australia. The goal of this breeding program is to introduce bright flower colours and to extend the flowering time through narrow and wide crosses within C. uncinatum and with its related taxa. Preliminary results have shown that some cross combinations were more successful than others and a great morphological diversity, such as flower size, flowering time and plant vigour exists in the species (Considine and Webb 1998, Egerton-Warburton, et al. 1998, Growns, et al. 1999).

Knowledge of basic chromosome number and ploidy level is important in a breeding program. Cytological studies in the Myrtaceae have shown that all major divisions of the family and a great majority of the genera are based on n = 11. Polyploid variation is common in fleshy fruited tribe Myrtoideae, less common in dry-capsule-fruited tribe Leptospermeae and rare in dry-nut-fruited tribe Chamelauciinae (Smith-White 1942, 1948, 1950, 1954, Rye 1979). However, dysploidy, chromosome number reduction was found to be the main trend of cytoevolution in the genera Darwinia and Verticordia of Chamelauciinae (Rye 1979, Tyagi et al. 1991). Chromosome numbers and ploidy levels of 22 genotypes from 11 species of Chamelaucium have been reported and most were diploid with 2n = 22 except three tetraploid and two hexaploid plants in species C. ciliatum (Table 1.1). The only count from the meiosis of pollen mother cells showed that C. uncinatum is n=11.

Table 1.1 Summary of the published chromosome numbers in Chamelaucium Taxon Chromosome Ploidy Level Number of Reference No. 2n(n) genotype observed C. axillare F. Muell. (11) 2x 1 Smith-White, 1950 C. axillare F. Muell. 22 2x 1 Rye, 1979 C. brevifolium Benth. c. 22(11) 2x 2 Rye, 1979 C. chlorinum Marchant & (11) 2x 1 Rye, 1979 Keighery ms. C. ciliatum Desf. 22(11) 2x 4 Rye, 1979 C. ciliatum Desf. c. 44(22) 4x 3 Rye, 1979 C. ciliatum Desf. 66 6x 2 Rye, 1979 C. conostignum Marchant 22 2x 1 Rye, 1979 & Keighery ms. C. drummondii Meisn (11) 2x 1 Smith-White, 1954

1 C. forrestii F. Muell. 22 2x 1 Rye, 1979 C. halophilum Marchant & 22(11) 2x 2 Rye, 1979 Keighery ms. C. hamatum Marchant & 22 2x 1 Rye, 1979 Keighery ms. C. pauciflorum (Turcz.) (11) 2x 1 Rye, 1979 Benth. C. uncinatum Schauer (11) 2x 1 Smith-White, 1954

Intraspecific ploidy variation is widespread in many plants (Harlan and deWet, 1975; deWet, 1980; Veilleux, 1985; Bretagnolle and Thompson, 1995, Yan et al. 1994). It is important to carefully match parents based on their ploidy levels in a breeding program in order to produce viable hybrids (Yan et al. 1997). It is also well known that changes in morphology frequently accompany changes in ploidy (Singh, 1993) and the clones examined in this study were selected with this in mind. In this research, chromosome numbers and ploidy levels of 28 selected genotypes from natural selections were examined. Flower and leaf size, pollen morphology and viability, and the relationships between genotypes at different ploidy levels were also investigated.

1.2 Materials and methods

Plant materials were collected from wild populations (Considine and Webb 1998) or cultivated plantations. Cuttings were rooted in a glasshouse under mist. The genotypes collected from wild populations were allocated with a two letter code followed by a serial number. The two letter code refers to the species, here UA=Chamelaucium uncinatum and the number following the two letters refer to the serial number of the collections in chronological order. The genotype or cultivar name and the origin are listed in Table 1.2.

Chromosome examination

Root tips from the plants propagated by cutting were collected and held in saturated aqueous paradichlorobenzene for 4 hrs at room temperature. Samples were then fixed in Carnoy’s I fixative (95% ethanol : acetic acid = 3:1 v/v) for 24 hrs. After being hydrolysed in 1M HCL at 60° C for 8-10 mins, the samples were stained in Feulgen solution for 2 hrs, and then squashed in a drop of FLP orcein on microscope slide (Jackson, 1982). The slides were heated and then pressed firmly to flatten cells. All observations were made using a Zeiss Axioplan Microscope MC 80 and photographs were taken on Pan F film using a 35 mm camera. Only metaphase stages in which individual chromosomes were clearly distinguishable were used for making counts. At least 10 dividing cells were counted for each sample to determine the chromosome number and ploidy level.

The flower size and leaf length

To investigate the possible relationship between flower size, leaf length and plant ploidy, five plants within a genotype (clone) were selected randomly. The diameter of 6 flowers and the length of 6 mature leaves per plant were measured.

2 Table 1.2 Ploidy levels in Chamelaucium uncinatum

Genotype code Chromosom Ploidy Origin (WA) or cultivar. e number name (2n) UA100 44 4x Kings Park UA636 22 2x Kings Park UA557 33 3x Midlands Road UA557-16 22 2x Midlands Road UA559 22 2x North Perth UA514 22 2x Cervantes UA528 22 2x Yanchep UA549 22 2x Cataby UA552 22 2x Cataby UA578 22 2x Kalbarri UA583 22 2x Kalbarri UA606 22 2x Chapman River UA651 22 2x Winchester UA689 22 2x Dongara UA772 22 2x Jurien UA776 22 2x Jurien UA786 22 2x Watheroo UA824 22 2x Arrowsmith River UA827 22 2x Geraldton UA831 22 2x Yarder Gully UA882 22 2x Northampton UA883 22 2x Northampton UA887 22 2x Northampton Manning White 22 2x Cultivar Lady Stephanie 22 2x Cultivar Purple Pride 22 2x Cultivar Jurien Brook 22 2x Cultivar Semi Double 22 2x Cultivar

The morphology and viability of pollens

Pollen was examined using a bright field microscope to determine the variation in size, shape and viability of different genotypes at different ploidy levels. The pollen was stained with PICCH (Propionic acid-iron-carmine-chloral hydrate) to visualise the presence of cytoplasm in the pollen grains, as well as the size and shape of pollen grains (Yan, 1996) . Pollen size was measured with an eyepiece micrometre. Fifty grains from each genotype were measured to give information on the size of pollen and the size variation. Pollen germination was initiated by thinly spreading pollen on a one square centimetre strip of absorbent cellolose acetate film (Fowlers Vacola). These were then placed in a petri dish, on several layers of filter paper soaked in 20% sucrose solution. The petri dish was sealed and placed in a constant temperature room, at 20°C overnight to allow for germination. All tools and containers used in this experiment were sterilised in 100% ethanol before use. The plastic film with germinated pollen was put on a microscope slide, stained with a drop of 0.1M aniline blue in phosphate buffer (pH 7) and observed under a fluorescent microscope using BP-450-490 exciter filter. Percentage of pollen germination was determined by counting 5 fields under the microscope at 10x10 times

3 1.3 Results

Chromosome number and ploidy level

The chromosome number of 28 genotypes from 20 different original and secondary populations were determined in this study (Table 1.2). 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 (Fig. 1.1). Among the 28 genotypes, UA100 from Kings Park was tetraploid with 44 chromosomes (2n=44) (Fig. 1.1b) while UA557 from Midlands Road was triploid with a count of 33 chromosomes (2n=33) (Fig. 1.1a). The remaining 26 genotypes from 20 populations or cultivars were diploid with 22 chromosome (2n=22) (Fig. 1.1 c, d, Table 1.2).

The flower size and leaf length

Flower and leaf sizes from 6 different Geraldton Wax genotypes including diploid, triploid and tetraploid genotypes are shown in Table 1.3. Genotypes with high ploidy levels have larger flowers. The tetraploid (UA100) had an average flower diameter of 27.0 mm, the triploid (UA557), 24.7 mm, while the flower sizes of diploids ranged from 13.6 mm to 27.0 mm. Thus polyploids tend to have larger flowers but plants with large flowers are not necessarily polyploid. For instance, the average flower diameter of UA883 is 27.0 mm, but it is a diploid. Among the investigated genotypes, leaf length of diploids varied from 23.7 mm to 60 mm, while that of the polyploids fell into the same range (Table 1.3).

The morphology and viability of pollens

Pollen grains from genotypes at different ploidy levels differ in shape (Fig. 1.2). Diploids have pollen that is planar and is shaped as an equilateral triangle (97.5%-100%) with an aperture at each vertex (Fig. 1.2a). A few rare ellipsoidal pollens with two apertures were found but no pollen grains with more than three apertures were found in the diploid genotypes studied. The triploid UA557 produced mainly triaperturate pollen grains and a few ellipsoidal pollen grains, but 12 % of its pollen was in the shape of a square with 4 apertures, one at each vertex (Fig. 1.2b). The tetraploid, UA100, showed diversity of pollen shapes ranging from 2 to 5 polar apertures. Most of the pollen grains (75.9%) were square shaped with four apertures (Fig. 1.2c). These results indicate that the number of pollen apertures increase as the ploidy level of the plants increases.

Pollen diameters varied considerably among the different genotypes. Among the seven genotypes studied, the tetraploid UA100 produced the largest pollen grains (42.4µm) as well as the largest standard deviation (4.4 µm) (Table 1.4). The second largest pollen was produced by a diploid UA883 (41.8µm) with a standard deviation of 3.8 µm. However, the number of pollen apertures did not increase as their pollen size increased. The triploid UA557 generated the third largest pollen grains (34.2µm) and its standard deviation was 2.2 µm. The other diploids had smaller mean pollen grains size and a smaller standard deviation.

Cytoplasm content in the pollen was evaluated for seven different genotypes. All the diploids had a high percentage of pollen grains containing cytoplasm ranging from 83.3% to 97.3%. The tetraploid UA100 had lower proportion of pollen grains with cytoplasm (61.9%), while the triploid UA557 had only 47.3% of pollen grains with cytoplasm (Table 1.5).

Pollen viability was high in all diploid genotypes examined, with a pollen germination rate of about 80%-90% (Fig. 1.2d). The tetraploid UA100 had a 40%-50% germination rate (Fig. 1.2e) and that of the triploid UA557 was low (1%) in comparison to the diploids and the tetraploid (Fig. 1.2f). Those pollen tubes of UA557 which grew were much shorter than those of both diploids and the tetraploid.

4 Table 1.3. Flower & leaf size of C. uncinatum Genotypes Flower Standard Leaf Standard size deviation length deviation (mm) (mm) UA100 (4x) 27.0 0.79 39.1 4.0 UA557 (3x) 24.0 1.29 34.8 4.4 UA882 (2x) 27.0 0.82 58.0 4.1 UA883 (2x) 24.0 0.93 60.0 3.9 Manning White 20.0 0.83 32.6 3.3 (2x) Alba (2x) 20.0 0.96 45.1 4.9 Purple Pride (2x) 17.3 0.83 34.4 3.6 Jurien Brook (2x) 13.6 0.57 23.7 3.2

Table 1.4 Pollen diameter and variation of C. uncinatum

Genotype Sample Average Maximum Minimum Standard deviation (µm) (µm) (µm) UA100 50 42.4 55.0 35.0 4.4 UA557 50 34.2 45.0 22.5 2.2 UA883 50 41.8 50.0 32.5 3.8 Purple 50 37.5 40.0 32.5 2.3 Pride UA692 50 35.3 38.8 21.3 1.1 UA595 50 32.7 37.5 27.5 1.0 UA720 50 29.3 32.5 25.0 1.9

Table 1.5 The percentage of pollen grains with cytoplasm

Genotype Fields observed Total number Number of % of pollen with under microscope of pollens pollen with cytoplasm studied cytoplasm UA100 14 257 159 61.9 UA557 5 169 80 47.3 UA883 24 299 271 90.6 UA720 6 392 376 95.9 Purple 5 270 225 83.3 Pride UA692 5 150 146 97.3 UA595 4 100 84 84.0

5 Figure 1.1 Chromosome numbers of Chamelaucium uncinatum (Bar represents 5 µm) a. UA557, 2n=3x=33 b. UA100, 2n=4x=44 c. UA549, 2n=2x=22 d. UA887, 2n=2x=22

6 Figure 1.2. Pollen shape and germination in Chamelaucium uncinatum (Bars represent 10 µm) a. Triaperturate pollen of diploid UA720 b. Tetraaperturate pollen (right) of triploid UA557 c. Tetraaperturate and pentaaperturate pollen of tetraploid UA100 d. Pollen germination of diploid 'Purple Pride' e. Pollen germination of tetraploid UA100 f. Pollen germination of triploid UA557

7 1.4 Discussion

Chromosome number and intraspecific ploidy variation of Chamelaucium

The results of this study demonstrate that while x=11 is confirmed as the basic chromosome number, diversity in ploidy level may be the rule rather than the exception. All major divisions of the Myrtaceae and a great majority of its genera reportedly based on n=11 although a few dysploid series were found in a few genera (Smith-White, 1942 & 1948; Rye, 1979; Tyagi, et al., 1991). Among the related genera to Chamelaucium of the subtribe Chamelauciinae, the chromosome numbers varied widely in genera Darwinia and Verticordia because of dysploid chromosome number reduction. However, the basic chromosome number remained most constant with n=11 in the genera Chamelaucium and Pileanthus. We found no dysploid chromosome number changes among 48 genotypes (unpublished) selected from species within the genus Chamelaucium.

The report here of the occurrence of a tetraploid UA100 with 2n=44, a triploid UA557 with 2n=33 and all other diploids with 2n=22 is consistent with previous research indicating that the basic chromosome number in Chamelaucium is x=11. Although the number of the examined species in the genus Chamelaucium is still limited, diploid, tetraploid, hexaploid (Rye, 1979) and pentaploid (Shan, et al., unpublished) plants occur in the C. ciliatum. The occurence of this ploidy series in C. ciliatum and C. uncinatum strongly supports the conclusion that polyploidy is common and successful at both the intraspecific and interspecific level within the family Myrtaceae (Rye, 1979). Intraspecific ploidy variation has been proved to be common only when large number of plants are studied within a species (Yan et al. 1994). About 20 plants have been used to study the ploidy levels in C. ciliatum and about 30 plants have been used in C. uncinatum. In both cases, ploidy series has been found. For other species, polyploid may also exist but they were not detected mainly because only a few plants were sampled for the cytogenetic study.

The origin of polyploids in Chamelaucium uncinatum

In plants, polyploidy has two possible origins, asexual or somatic doubling, and sexual or the formation of unreduced gametes (2n gametes) during meiosis, with the later being common (Harlan & deWet, 1975; deWet, 1980; Iwanaga & Peloquin, 1982). The occurrence of odd polyploid levels are associated with interploidy hybridisation or apomixis (Rye, 1979) as evidenced by studies in Myrtoideae (Davis, 1966) and in the Callistemon rigidus-linearis-pinifolius complex (Smith-White, 1948). Therefore the tetraploid UA100 may be derived from either somatic chromosome doubling or through 2n gametes. Tetraploidy will occur if two unreduced gametes are mated. The triploid UA557 may have originated from the mating of a normal haploid and an unreduced 2n gamete or from a 2n gamete produced by a tetraploid parent. More work at cytological and molecular levels needs to be done to determine the role of unreduced gametes, apomixis and spontaneous somatic doubling as processes leading to ploidy series.

Pollen morphology and viability of C. uncinatum

This study has also shown that the number of apertures in a pollen grain can be used as an index of ploidy in Chamelaucium. The shape of pollen of Myrtaceae members is typically triaperturate (Erdtman 1986), and no variation has so far been recorded in the literature. Pollen shape, size, aperture position and the patterns on the surface are all important characteristics for the study of plant classification and evolution. From the results presented in this paper, it can be concluded that pollen also shows considerable diversity in shape, size and aperture position and number even within the one polyploid genotype, as in UA100 and UA557. Pollen size was not correlated with the ploidy level, but increased as the flower size increased. The number of apertures on the pollen increased as the ploidy level rose, but did not necessarily increase with pollen size. The variation of pollen morphology was apparently correlated with the meiotic process and the biological significance of the increase in aperture number and its relationship to meiotic chromosome arrangements remains to be investigated.

8 The lack of cytoplasm in many pollen grains of triploid UA557 is undoubtedly a result of unbalanced chromosome complements. Pollen from UA557 is sterile and the plant has not produced mature seed. The tetraploid UA100 also produces pollen with low viability suggesting that abnormalities also exist during the process of meiosis of this clone. The nature of the tetraploid as to whether it is an auto- or allo-tetraploid needs to be clarified using genome analysis technique (Yan, 1996).

Ploidy diversity and implication for breeding of C. uncinatum

The existence of ploidy differences in C. uncinatum imposes some restrictions in a breeding program as interploidy hybridisation is either difficult or results in odd ploidy sterile hybrids (Nishiyama & Innomata, 1996; Johnston et al., 1980). For normal breeding purposes, crosses should be made between plants at the same ploidy level though this does not guarantee fertility of the hybrid. The existence of tetraploid Geraldton Wax also opens a new avenue to breeding at tetraploid level as well as at diploid level and thus perhaps resolving the unbalanced pairing at meiosis which presumably occurs in both auto-tetraploids and hybrid diploids.

1.5 References

Bretagnolle, F., and Thompson, J. D. (1995). Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytologist 129: 1-22. Considine, J.A., and Growns, D. (1997). Waxflower. In: 'RIRDC Compendium of New Crops'. (Eds Hyde, K.) pp 512-520. (Rural Industries Research and Development Corporation: Canberra.) Considine, J.A., and Webb, M. (1998). Collection and Selection of Chamelaucium species for floriculture / Evaluation of Chamelaucium uncinatum for Floriculture. Technical Report. 5. Perth, Western Australia. The University of Western Australia. Davis, G. L. (1966). Systematic Embryology of the Angiosperms. John Wiley & Sons: New York. deWet, J. M. J. (1980). Origins of Polyploids, In: Lewis W. H. (ed) Polyploidy: Biological Relevance. New York: Plenum Press pp. 3-15. Egerton-Warburton, L. M., Ghisalberti, E. L., and Considine, J. A. (1998). Infraspecific variability in the volatile leaf oils of Chamelaucium uncinatum (Myrtaceae). Biochemical Systematics and Ecology 26: 873-888 Erdtman, G., (1986). Pollen Morphology and Plant Taxonomy-Angiosperms. E.J. Brill, Leiden, The Netherlands 11-18, 280-282. Growns D. J., Chris Newell, John Considine, and Guijun Yan. 1999. Waxflower selection, breeding and development - an overview. IV International Symposium on New Floricultural Crops. Chania - Crete, Greece 22 -27 May, 1999 Harlan J. R., and deWet JMJ (1975). On Ö Winge and a prayer: the origins of polyploidy. Botanical Review 41: 361-390. Iwanaga, M., and Peloquin, S. J. (1982). Origin and evolution of cultivated tetraploid potatoes via 2n gametes. Theoretical and Applied Genetics 61:161-169. Jackson, R. C. (1982). Polyploidy and diploidy: new perspectives on chromosome pairing and its evolutionary implications. American Journal of Botany 69: 1512-1523. Johnston, S. A., den Nijs, T. P. M., Peloquin S. J., and Hanneman R. E. Jr. (1980). The significance of genetic balance to endosperm development in interspecific crosses. Theoretical and Applied Genetics 57: 5-9. Marchant, N. G., and Keighery, G. J. (1995). Revision of the genus Chamelaucium (Myrtaceae). Nuytsia (in press) Nishiyama, I., and Inomata N. (1966). Embryological studies on cross incompatibility between 2x and 4x in Brassica. Japanese Journal of Genetics 41: 27-42. Rye, B. L. (1979). Chromosome number variation in the Myrtaceae and its taxonomic implications. Australian Journal of Botany 27:547-573. Singh, R. J. (1993) Plant Cytogenetics. CRC Press Inc. Smith-White, S. (1942). Cytological studies in the Myrtaceae I: Microsporogenesis in several genera of the tribe Leptospermoideae. Proceedings of the Linnean Society of N.S.W. 67:335-342.

9 Smith-White, S. (1948). Cytological studies in the Myrtaceae II: Chromosome numbers in the Leptospermoideae and Myrtoideae. Proceedings of the Linnean Society of N.S.W. 73:16-36. Smith-White, S. (1950). Cytological studies in the Myrtaceae. III: Cytology and phylogeny in the Chamaelaucoideae. Proceedings of the Linnean Society of N.S.W. 75:99-121. Smith-White, S. (1954). Cytological studies in the Myrtaceae IV: The sub-tribe Euchamaelaucinae. Proceedings of the Linnean Society of N.S.W. 79:21-28. Tyagi, A. P., McComb, J., and Considine, J. (1991). Cytogenetic and pollination studies in the Genus Verticordia DC. Australian Journal of Botany 39:261-272. Veilleux, R. (1985). Diploid and polyploid gametes in crop plants: mechanisms of formation and utilisation in plant breeding. Plant Breeding Review 3: 253-288. Yan, Guijun (1996). Cytogenetic and molecular studies of Actinidia genomes. PhD thesis. The University of Auckland, New Zealand. Yan, Guijun, Ferguson, A. R., and McNeilage, M. A. (1994). Ploidy races in Actinidia chinensis. Euphytica 78: 175-183. Yan, Guijun, Ferguson, A. R., McNeilage, M. A., and Murray, B. G. (1997). Numerically unreduced (2n) gametes and sexual polyploidisation in Actinidia. Euphytica 96: 267-272.

10 2. Chromosome doubling of waxflower plantlets regenerated in vitro

2.1 Introduction

Waxflowers are indigenous to Western Australia and has become an important floriculture crop worldwide (Considine and Growns 1997). The worldwide value of the waxflower is likely to exceed $200 million in the future and is currently worth about $15 million to Australian exporters. Recently, an extensive breeding program commenced as the result of a joint effort between the University of Western Australia and Agriculture Western Australia. The goal of this breeding program is to introduce bright flower colours and to extend the flowering time through narrow and wide crosses within C. uncinatum and with its related taxa (Growns, et al. 1999). Unfortunately, like other plant species, wide crosses or hybridisation across ploidy levels frequently produces sterile progeny which can be propagated by apomixis or other vegetative means but prevent from further improvement (Liedl and Anderson, 1993). Chromosome doubling to induce polyploidy has been widely used in plant breeding programs to restore fertility in sterile genotypes and to overcome crossing barriers (Anderson et al. 1991; Griesbach and Bhat 1990; Lu and Bridgen 1997; Mozafari et al. 1997). Plants with doubled chromosome number can also produce enlarged flowers (Blakeslee and Avery, 1937).

Colchicine is the most commonly used agent to achieve chromosome doubling (Hassawi and Liang 1991; Geoffriau et al. 1997). Somatic tissues such as nodes and terminal meristems have been used successfully for in vitro chromosome doubling using colchicine as a pre-treatment or in the growth medium. The simplest method of applying colchicine is by immersing a young shoot in an aqueous solution (Blakeslee and Avery 1937; Eigsti and Dunstin, 1955). It is also believed that regeneration of plants from single cells from a stem segment in media containing colchicine may reduce the incidence of chimera.

One objective of this study is to develop a protocol to double the chromosome number of waxflowers using colchicine in combination with in vitro culture techniques. Both immersion and regeneration methods were used. The other major aim is to induce a few polyploid plants to be used in the present breeding program.

2.2 Materials and Methods

Plant material

Three genotypes of waxflowers were used in the experiments - an interspecific hybrid, ‘Esperance Pearl’ (EP), which was from a cross between C. uncinatum Schauer and C. megalopetalum F. Mueller ex Bentham and two open pollinated hybrids, 5001-3 and 5001-8, from ‘Manning White’ (C. uncinatum). These genotypes were established in tissue culture and then used for subsequent colchicine treatments.

To double the ploidy level of the three genotypes of waxflower the colchicine was applied to two types of plant material: (1) 5 mm stem sections containing at least one node were used in the regeneration experiment; and, (2) 15 mm shoots, including its apical meristem, were used in the immersion experiment.

Regeneration procedures

11 The 5-mm stem segments were plated ten per petri dish on MS medium of pH 5. 8, with 1 µM 6- benzylaminopurine (BAP), 30 g/L sucrose and gelled with 2. 5 g/L Phytagel. The medium also contained one of five levels of colchicine: 0, 0.0002% (w/v), 0.001% (w/v), 0.005% (w/v) or 0.025% (w/v). The treatments were cultured for 6 weeks in the dark at 25°C before subculture.

Immersion procedures

Two MS media were used for this experiment: (1) immersion media; and, (2) shoot growth media. The 15 mm long shoot tips were immersed in MS solutions of pH 5.8 containing 1 µM BAP, 30 g/L sucrose and one of five levels of colchicine: 0, 0.0005% (w/v), 0.005% (w/v), 0.05% (w/v) or 0.5% (w/v). These were shaken in an Innova 4080 incubator shaker at 25°C and 50 rotations per minute for 5 hours. Each shoot was transferred to a 30 mL polycarbonate culture tube containing 10 mL of MS medium with 0.3 µM BAP, 20g/L sucrose. The medium was adjusted to pH 7. 0 with NaOH and gelled with 8 g/L course agar powder (APS Ajax Finechem). The tubes were kept in a temperature controlled room to be maintained at 25°C ± 1°C. The photoperiod was 16 hours under fluorescent light providing a fluence rate of 30 µmol/m-2/s-1 and then 8 hours in dark.

Assessment of the plant material

The plant material was initially graded according to shoot health. For the immersion experiment, leaf colour (green or yellow) was assessed 4 days after the treatment. The assessments of shoot health were also made on day 20 and day 40 of the plants from the immersion experiment and at day 67 of the shoots from the regeneration experiment. Five levels were used to grade plant health from excellent to dead, these being: excellent, good, poor, very poor or dead. Also any deformed growth was recorded. Deformed growth includes thickened leaves exhibiting varying degrees of thickening, puckering and malformation. Hyperhydric shoots were also recorded. The number of shoots per stem segment, per regeneration treatment (RT) and per immersion treatment (IT) was recorded on day 40, day 91 (IT only) and day 171. The plantlets were assessed as alive or dead and normal, abnormal or hyperhydric. Also any inordinately large plantlets were sought out and recorded.

The plantlets from which roots had been harvested were used for measurements of leaf length, width, stomata guard cell length and stomata density. A metric ruler was used to measure the leaf length (mm). A light microscope fitted with micrometre was used to measure the leaf thickness (mm), stomata density and guard cell length.

Assessment of mitotic chromosomes in root tips

Chromosome numbers of plantlets were studied based on a published procedure by Yan (1996). Fast growing roots were collected and pretreated with saturated aqueous para-dichlorobenzene (PDB) solution. After 18 hours at 4°C the roots were fixed with Carnoy’s I solution (95% ethanol/acetic acid 3:1) for 24 hours at room temperature. The root tips were hydrolysed with 1M hydrochloric acid at 60°C for 10 minutes. The roots were stained with Feulgen solution for 2 hours. The roots were transferred to a microscope slide and the darkly stained tips, about 0.2 mm, were trimmed. A drop of FLP orcein was added to the root tip and a blunt ended metal was used to gently break-up the root tip into cell suspensions (Jackson, 1973). A cover slip was applied to squash the root tip on the microscope slide and the slides were examined for chromosomes under a light microscope.

12 Statistical analysis

The results were analysed using ANOVA and regression packages in Minitab. A 95% confidence level was used (p < 0.05). Fisher’s individual comparisons were determined and microbial infection were where the same are denoted a, b, c or d in the tables. The plant materials discarded due to not included in the analysis.

2.3 Results

Survival rate after colchicine treatment

A high concentration of colchicine reduced the survival rate of the plant material from both the regeneration and immersion experiments. All three materials reacted similarly to the concentration of colchicine.

All the stem segments plated on the media containing 0.025% colchicine died (Table 2.1). The survival rates on media containing colchicine concentration of 0.0002% and 0.001% media were not different than that of the control. For all genotypes, the survival percentages were lower on the 0.005% media than that on the 0.001% media.

Table 2.1 Survival rates of stem segments on the regeneration medium containing different concentrations of colchicine as assessed at day 40 after treatment

Concentration EP (Stdev.) 5001-3 5001-8 Average of all (%) (Stdev.) (Stdev.) 0.0000 55. 0 (7. 1) a 50.0 (42. 4) ab 90.0 (10.0) a 68. 6 (27. 3) a 0.0002 50.0 (14. 1) a 65. 0 (21. 2) a 80.0 (26. 5) a 67. 1 (22. 9) a 0.001 80.0 (14. 1) a 80.0 (17. 3) a 86. 7 (15. 3) a 82. 5 (13. 9) a 0.005 60.0 (00.0) a 50.0 (14. 1) ab 20.0 (17. 3) b 40.0 (22. 4) b 0.025 00.0 b 00.0 b 00.0 b 00.0 c

The survival of shoot tips was adversely affected by immersion in the 0.5% colchicine solution only, with the survival of shoot tips falling to an average of 70.3% of all the shoots tips (Table 2.2). Immersion in solutions containing colchicine concentrations ranging from 0 - 0.05% did not affect the survival of the shoot tips.

Table 2.2. Survival rates of explants on the multiplication medium after immersion in different concentrations of colchicine as assessed at day 40 after treatment

Concentration EP (Stdev.) 5001-3 5001-8 Average of all % (Stdev.) (Stdev.) 0.0000 100.0 (00.0) a 87. 5 (17. 7) a 100.0 (00.0) a 95. 8 (10.2) a 0.0005 83. 4 (23. 5) ab 100.0 (00.0) a 100.0 (00.0) a 94. 5 (13. 6) a 0.005 83. 4 (23. 5) ab 100.0 (00.0) a 100.0 (00.0) a 94. 5 (13. 6) a 0.05 100.0 (00.0) a 100.0 (00.0) a 100.0 (00.0) a 100.0 (00.0) a 0.5 58. 35 (11. 8) b 75. 0 (35. 4) b 77. 5 (3. 5) b 70.3 (19. 2) b

13 Shoot proliferation

Colchicine treatment of EP on regeneration medium significantly reduced the mean number of shoots per living stem segment. The number of shoots produced by the genotypes 5001-3 and 5001-8 per living stem segment was the same on all the media. Both 5001-3 and 5001-8 produced callus alongside shoots, whereas EP produced shoots only.

The number of shoots produced by a shoot immersed in a colchicine solution tended to be less for those immersed in 0.05% and 0.5% colchicine solutions than that produced by the control. The mean number of shoots produced by a shoot immersed in a 0.5% colchicine solution was approximately half that of the control for all genotypes. The number of shoots produced by the shoot tips immersed in the 0.0005% and 0.005% colchicine solutions were not significantly different than that of the control.

Morphological assessment of plant material

All of the shoots produced by the regeneration technique were hyperhydric at day 40 and at day 67. As at day 171 many of the plantlets of 5001-3 showed the same symptoms of hyperhydricity. Plants with thick, forked, rosetted and forked leaves were recorded in Table 2.3.

Table 2.3. Morphological abnormalities of plantlets at day 171 in regenerated experiment

Genotypes and colchicine Number of plantlets with: treatment Leaf abnormality Rosetted leaves Percentage Concentration No. of Increased Forked Three in Four in of plantlets Width a whorl a whorl Abnormality EP 0.0000 12 0 0.0002 10 0 0.001 8 1 1 12. 5 0.005 1 0 5001-3 0.0000 22 2 4 1 31. 8 0.0002 16 2 10 1 81. 3 0.001 39 4 1 17 3 64. 1 0.005 1 0 5001-8 0.0000 47 0 0.0002 29 0 0.001 30 4 5 1 33. 3 0.005 4 1 25. 0

By day 4, all of the shoot tips immersed in 0.05% or 0.5% colchicine had turned yellow in colour, the leaves eventually died. Deformation of leaves was observed in the plants that were immersed in the high colchicine solutions of 0.05% and 0.5%. Most of the deformation of the initial new growth apparent at day 20 was transient, as growth returned back to normal by day 40. The deformed new growth consisted of thickened leaves exhibiting varying degrees of thickening, puckering and malformation (Table 2.4).

14 Table 2.4. Morphological abnormalities of plants at day 171 in immersion experiment Genotypes and colchicin Number of plantlets with: treatment Leaf abnormality Rosetted leaves Concentration No. of Increased Forked Three in Four in Percenta plantlets Width whorl Whorl ge EP 0.0000 16 0 0.0005 10 0 0.005 3 1 33. 3 0.05 27 3 11. 0 0.5 2 0 5001-3 0.0000 59 0 0.0005 53 1 1 3. 8 0.005 60 5 8. 3 0.05 49 3 1 2 12. 2 0.5 28 3 3 21. 4 5001-8 0.0000 56 0 0.0005 57 3 5. 3 0.005 38 3 7. 9 0.05 49 4 8. 2 0.5 26 2 7. 7

Chromosome assessment

Root squashes of 39 abnormal plantlets revealed three tetraploid plantlets and all the rest are diploid. Two tetraploids came from a different shoot among the eight EP shoots immersed in a 0.05% colchicine solution, this represents a 25% success rate with the immersion of shoots in the 0.05% colchicine concentration. One tetraploid plantlet was also regenerated from MS media containing 0.01% colchicine. No tetraploid plantlet was found from the other two genotypes treated.

The induced tetraploid plants clearly showed 44 chromosomes in their root tips (Figure 2.1a) as compared to the undoubled diploid plants (Figure 2.1b).

Anatomical assessment of tetraploid plant material

Compared to diploid plantlets the tetraploid plantlets have leaves that are longer (Figure 2.1e) and thicker with fewer larger stomata (Figure 2.1c, d) (Table 2.5).

Table 2.5. Anatomical differences between tetraploid and diploid plantlets of Esperence Pearl Genotype EP Leaf Stomata Length (mm) Width (mm) Length Density Ploidy Mean S. D. Mean S. D. Mean S. D. Mean S. D. Diploid (2x) 12. 5 2. 6 0.54 0.07 16. 4 1. 3 16. 8 3. 7 Tetraploid (4x) 14. 9 1. 9 0.66 0.06 18. 9 1. 0 7. 8 3. 0 Difference: 20% increase 22% increas 15% increase 54% decrease

15 The tetraploid plants tended to be smaller than diploid plants and the central leader was not as dominant as the diploid (Figure 2.1f).

2.4 Discussion

Colchicine doubles chromosomes in somatic waxflower tissue

In this study, colchicine promotes chromosome doubling of in vitro waxflower plant material. Root tip squashes revealed that chromosome doubling had occurred in three waxflower shoots of Esperance Pearl (EP). This represents a success rate of at least 25% (2/8) with the immersion of shoots in the 0.05% colchicine concentration and 9% (1/11) success with the regenerated shoots in the 0.001% colchicine concentration. As only the morphologically abnormal plants were observed under microscope and not all the plant material from all treatments has been assessed, the actual pecentage of polyploidisation may be lower.

The ploidy level of the roots may not signify that the plantlet is entirely tetraploid, the plantlets may be chimerical. As a low frequency of chimeras have been found in shoots from colchicine experiments (Cardi et al. 1993; Cohen and Yao, 1996; Bouvier et al. 1994) it is not certain the waxflower plantlets are entirely tetraploid. However, chimerical plants have been detected by root tip analysis of colchicine treated plants (Cohen and Yao 1996). Therefore, as the roots were comprised of tetraploid cells only it is probable that the L-III germlayer is not chimerical or mixoploid. However, plant apices have three independent germlayers from the outside in they are: the first layer (L-I), which forms the epidermis; the second layer (L-II), which forms the leaf and reproductive tissue; and, the third layer (L-III), which forms the central core (Satina and Blakeslee, 1941). Still the experiment is a success as the root material can be useful in a breeding program, as plant cells are totipotent any part of the plant in which doubling has occurred can be induced to grow out into a 4x branch and bear seeds or the branch may be propagated vegetatively (Blakeslee and Avery 1937).

Plants with doubled chromosome number have occasionally appeared spontaneously in experimental culture and rarely appear in nature (Blakeslee and Avery 1937). The tetraploid root tips, which came from plantlets growing on an MS media, are unlikely to be due to experimental culture the IBA or BAP as no polyploid was found amongst the controls. Also a 25% rate of occurrence is not what we would consider to be a rare spontaneous mutation. Chromosome number instability can occur in plant cells cultured as callus or cell suspensions, which can result in high incidences of aneuploidy and polyploidy (Lavia et al. 1994). This is not likely to be the case either as no callus was produced by the Esperance Pearl (EP) material anyway and the other two genotypes produced callus but no polyploid was found.

Colchicine concentration and mortality

The high concentrations of colchicine were harmful to in vitro cultured waxflower plants by an increased death rate and a decrease in shoot proliferation. This supports that colchicine can cause death due to its toxic effect (Navarro-Alvarez et al, 1994) and that it can also severely decrease regeneration ability (Challak and Legave, 1996).

16 The time of exposure to the colchicine would determine whether it is a lethal dose, as stem segments exposed to 0.025% for 6 weeks died but none of the shoot tips exposed at 0.05% for 5 hours died. Yet the shoot tips immersed in the 0.05% received a phytotoxic dose of colchicine that cause the leaves to yellow and eventually to die and shoot proliferation was generally reduced to about half that of the control. This shows that the plant material can regenerate after a toxic dose of colchicine.

Combining colchicine and in vitro culture

The in vitro nature of the experiment may have had a beneficial impact on the chromosome doubling. This would be due to the non-woody nature of the material used and the rapid growth and cell division brought on by the addition of the cytokinin 6-benzylamine (BAP) to the media. It can be difficult to obtain totally polyploid plants by using colchicine on woody plant material (Dermen, 1945) and as the in vitro waxflower material was not woody this should have been avoided. Cytokinins, such as BAP, stimulate cell division, induce shoot formation and axillary shoot proliferation (Torres 1989) and the success of colchicine treatment is reliant upon rapid cell division of the treated meristem of a relatively long period, preferably during a growth flush (Ackerman and Dermen 1972). Therefore, the addition of BAP to the shoot growth media should have added the experiment by promoting proliferation of shoots, thus providing new non- woody growth. Also rapid propagation of axillary shoots of any chimerical plants tends to produce two populations of shoots, that are solid diploid and tetraploid (Cohen and Yao 1996).

Anatomical assessment - ploidy indicators

The leaf being generated from the L-II germlayer of the plant apices like the reproductive tissues (Satina and Blakeslee 1941) could probably indicate that the reproductive tissue is tetraploid. It was found that the tetraploid plantlets had leaves that were 20% longer and 22% wider than the diploid plantlets. This is visual difference that is easy to determine by sight alone when examining the plants alongside one another in tissue culture. Isolating the plants by this means would substantially reduce the time performing cytological examination on diploid material when tetraploid material is sought. In this case, the cytological examination would be performed to confirm tetraploidy rather than find it. Also as the leaf size is correlated with the tetraploidy of the roots, it seems that both the L-II and L-III germlayers are affected, therefore the plants should reproductively function as tetraploids.

The epidermis is generated from the L-I germlayer of the plant apices (Satina and Blakeslee 1941). It was found that the leaves of the tetraploid plantlets had 54% fewer stomata that were 15% shorter in length than that of the diploid plantlets. These differences can only be observed by microscopy and in doing so it is much easier to observe half as many stomata than it is to measure the stomata length. These anatomical difference are not as easy to spot as the increase in leaf length and thickness and therefore are not recommended as the first step in singling out plants for cytological examination. The stomata are still worth examining as the leaf epidermis would be generated from the L-I germlayer and could be correlated to the ploidy of the L-I germlayer. Should this be the case then such examination is a step towards establishing that the plant is not sectorial chimera. However, we expect that stomata density per field of vision is also related to leaf thickness, in that it decreases as the leaf thickness increases, and therefore may reflect the ploidy of the L-II germlayer.

Incidences of leaves exhibiting varying degrees of thickening, puckering, malformation and mixed stomatal sizes developing at nodes of treated plants have been reported after treatment with colchicine (Hull and Britton 1956). The shoot tips treated by immersion in 0.05%

17 colchicine generally exhibited a deformation of the new growth similar to that described by Hull and Britton (1956), but it was transient. Chimerical plants are often at first malformed in growth, with characteristically thick and roughened leaves, due to different growth-rates of the 2x and 4x tissue (Blakeslee and Avery 1937). However, no chimerical plantlets were found in this research. The new growth may have represented chimerical axillary shoots, which grew to produce two populations of shoots that are solid diploid and tetraploid, in a similar manner to that described by Cohen and Yao (1996).

A tetraploid plant typically has a more rugged appearance, looks sturdier, has certain giant-like features, the rate of growth is slower and it is shorter than its diploid parent (Eigsti and Dunstin, 1955). Of the tetraploid plants found this was true of one but not the other, which had larger features but grew as well as the diploid plantlets. Also the assessment of the shoots produced by the regeneration method was made difficult by the shoots being hyperhydric. The symptoms of which, are shorter internodes which make leaves appear rosetted (i. e. three or four leaves in a whorl), leaves that are thickened, elongated, curled and/or wrinkled, brittle, translucent and of abnormal colour (Debergh et al. 1992). It seems hyperhydricity may prevent the spotting of any morphological changes due to polyploidy and therefore it is advisable to avoid using a media that promotes a high incidence of the condition.

2.5 References

Ackerman WL & Dermen H (1972) A fertile colchiploid from a sterile interspecific Camellia hybrid. The Journal of Heredity. 63: 55-59 Anderson JA, Mousset-Declas C, Williams EG & Taylor NL (1991) An in vitro chromosome doubling method for clovers (Trifolium spp. ). Genome. 34: 1-5 Blakeslee AF & Avery AG (1937) Methods of inducing doubling of chromosomes in plants. The Journal of Heredity. 28: 393-411

Bouvier L, Fillon FR & Lespinasse Y (1994) Oryzalin as an efficient agent for chromosome doubling of haploid apple shoots in vitro. Plant Breeding. 113: 343-346

Cardi T, Iannamico V, D’Ambrosio F, Filoppone E & Lurquin PE (1993) In vitro regeneration and cytological characterization of shoot from leaf explants of three accessions of Solanum commersonii. Plant Cell, Tissue and Organ Culture. 34: 107-114

Challak L & Legave JM (1996) Oryzalin combined with adventitious regeneration for an efficient chromosome doubling of trihaploid kiwifruit. Plant Cell Reports. 6: 97-100

Cohen D & Yao JL (1996) In vitro chromosome doubling of nine Zantedeschia cultivars. Plant Cell, Tissue and Organ Culture. 47: 43-49. Considine, J.A., and Growns, D. (1997). Waxflower. In: 'RIRDC Compendium of New Crops'. (Eds Hyde, K.) pp 512-520. (Rural Industries Research and Development Corporation: Canberra.) Debergh P, Aitken-Christie J, Cohen D, Grout B, von Arnold S, Zimmerman R & Ziv M (1992) Reconsideration of the term ‘vitrification’ as used in micropropagation. Plant Cell, Tissue and Organ Culture. 30: 135-140

Dermen H (1945) The mechanism of colchicine-induced cytohistological changes in cranberry. American Journal of Botany. 32: 387-391

Eigsti OJ & Dustin P (1955) Colchicine: in agriculture, medicine, biology and chemistry. (Iowa State College Press: Ames, Iowa, USA. )

18 Geoffriau E, Kahane R, Bellamy C & Rancillac M (1997) Ploidy stability and in vitro chromosome doubling in gynogenic clones of onion (Allium cepa L. ). Plant Science. 122: 201-208 Griesbach RJ & Bhat RN (1990) Colchicine-induced polyploidy in Eustoma grandiflorum. HortScience. 25: 1284-1286. Growns D. J., Chris Newell, John Considine, and Guijun Yan. 1999. Waxflower selection, breeding and development - an overview. IV International Symposium on New Floricultural Crops. Chania - Crete, Greece 22 -27 May, 1999 Hassawi DS & Liang GH (1991) Antimitotic agents: effects on double haploid production in wheat. Crop Science. 31: 723-726 Hull JW & Britton DM (1956) Early detection of induced internal polyploidy in Rubus. Proceedings American Society for Horticultural Science. 55: 171-177 Jackson, R. C. (1973). Chromosomal evolution in Haplopappus gracilis: a centric transposition race. Evolution 27, 243-256. Lavia G, Fernandez A & Marquez G (1994) Chromosome doubling in Turnera ulmifolia (Turneraceae) induced by regeneration of plants from in vitro cultured leaf explants. Plant Systematics and Evolution. 192: 41-48 Liedl BE & Anderson NO (1993) Reproductive barriers: identification, uses, and circumvention. IX. Polyploidy. Plant Breeding Reviews. 11: 98-105

Lu C & Bridgen MP (1997) Chromosome doubling and fertility study of Alstroemeria aurea x A. caryoplyllaea. Euphytica. 94: 75-81 Mozafari J, Wolyn DJ & Ali-Khan ST (1997) Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy. Plant Cell Reports. 16: 329-333

Navarro-Alvarez W, Baenziger PS, Eskridge KM, Hugo M & Gustafson VD (1994) Addition of colchicine to wheat anther culture media to increase doubled haploid plant production. Plant Breeding. 112: 192-198 Satina S & Blakeslee AF (1941) Periclinal chimeras in Datura stramonium in relation to development of leaf and flower. American Journal of Botany. 28: 862-871. Torres KC (1989) Tissue culture techniques for horticultural crops. (Van Nostrand Reinhold: New York, USA Yan G (1996) Cytogenetic and molecular studies of Actinidia genomes. Thesis (Doctor of Philosophy). Plant Sciences, The School of Biological Sciences, The University of Auckland, New Zealand

19 a b

c d

e f

Figure 2.1. a. Root-tip squash of mitotic chromosomes of induced tetraploid 'Esperence Pearl', 2n=4x=44. b. Root-tip squash of mitotic chromosomes of 'Esperence Pearl', 2n=2x=22. c. Stomata of induced tetraploid 'Esperence Pearl'. d. Stomata of diploid 'Esperence Pearl'. e. Comparison of leaf sizes of diploid 'Esperence Pearl' (left 4 leaves) and induced tetraploid 'Esperence Pearl' (right 4 leaves). f. Plants of diploid 'Esperence Pearl' (left) and induced tetraploid 'Esperence Pearl' (right).

20 3. Molecular studies in waxflowers 3.1 Introduction

Chamelaucium is one of the 22 genera within the Chamelaucium alliance, family Myrtaceae (Johnson & Briggs, 1984). It contains 31 species and six of them have been either bush- picked or cultivated as cut flowers (Marchant and Keighery 1995). Chamelaucium spp. is generally known as waxflowers and the most well known species are C. uncinatum, the Geraldton Wax and C. megalopetalum, the Large Waxflower. Chamelaucium is endemic to º º Western Australia and its natural range is from Kalbarri (27 40’S. 114 26’E) to Albany º º (35 02’S. 117 53’E) and along the south coast to Esperance in the southwest botanical province (Manning et al., 1996).

Genetic variation and evolution in a plant population can be caused by mutation, sexual recombination, migration, genetic drift and genetic selection (Henry, 1997). The genetic evolution of chloroplast genomes mirrors that of nuclear genomes. However, it evolves much slower. Both gene content and the order of chloroplast genes in the chloroplast genome are highly conserved (Palmer et al., 1988). Therefore, change in chloroplast DNA (cpDNA) sequence is an appropriate measure to resolve plant phylogenetic relationships. Chloroplast DNA variation has been widely used in systematic and evolutionary studies (Palmer, 1985; Soltis et al, 1992; Clegg & Zurawki, 1992; Downie & Palmer, 1992).

Most plants studied in angiosperms exhibit maternal plastid inheritance and plants in the gymnosperms exhibit paternal cpDNA inheritance (Birky, 1994). Even though both have some exceptions, but in all land plants examined so far, cpDNA is inherited clonally, through only one parent to offspring. In those plants where both parents contribute chloroplast to their offspring, the chloroplasts and their genomes do not seem to recombine, but simply sort out somatically (Mogensen, 1996). Chloroplast DNA inheritance has been studied in many plants such as Coffea (Lashermes, Cros et al. 1996), kiwifruit (Cipriani, Testolin et al. 1995), oak (Dumolin, Demesure et al. 1995), etc. Understand the mode of cpDNA inheritance will help us to find out the mode of gene recombination and provide evidence for evolutionary studies. Practically, It will be helpful in parent selection in breeding programs and as a tool in sorting out the parentage of natural collections.

The concept of the Chamelaucium alliance were introduced by Bentham (1867), and recently confirmed on the basis of morphological and anatomical characteristics (Johnson and Briggs, 1984). Although chromosome number and essential oil information has being incorporated into the system (Egerton-warburton et al., 1998a; Egerton-warburton et al., 1998b; Rye, 1979; Smith-white, 1950), DNA phylogeny has not been attempted previonsly. Information in these aspects together with the manner of inheritance of the cpDNA will provide evidence for evolutionary studies and facilitate breeding programs. In this study, cpDNA variation and evidence collected on the mode of cpDNA inheritance in Chamelaucium spp will be reported.

21 3.2 Materials and methods

Plant materials

Plant materials were collected at the Medina Research Station of Agriculture Western Australia and the Shenton Park Field Station of the University of Western Australia, both near Perth, Western Australia. Eighteen genotypes from two species, C. uninatum and C. magelopetalum and 17 intraspecific hybrids between different genotypes of C. uncinatum and 33 interspecific hybrids between C. uncinatum and C. megalopetalum were used in the study. The C. uncinatum genotypes used include cultivars ‘CWA Pink’, ‘Purple Pride’ and ‘5001’ and natural collections UA100, UA557, UA561, UA595, UA692, UA720, UA772, UA773, UA786 and UA827. The C. megalopetalum genotypes used were MB01.5, MB03.1, MB05, MB05.5 and MB99. Details of the hybrids used in the cpDNA inheritance experiment are presented in Table 3.1. Most of the parents were grown side by side with their hybrids in the research stations and some parents were collected from another block or from plants in pots in the shadehouse. Young leaves were collected from each plant and were ° maintained at -80 C until DNA extraction.

DNA extraction

Total DNA was extracted from fresh or frozen young leaves with CTAB buffer, separated with chloroform/isoamyl alcohol, centrifuged at 4000x g for 10 min, precipitated in isopropanol and washed with ethanol, according to the procedure described by Rogers & Bendich (1994).

PCR protocol

Three pairs of primers were synthesised (GibcoBRL, Table 3.2). Polymerase chain reaction were carried out in 50 µl volume, containing 40 ng of genomic DNA, 0.4 µM of a primer pair (0.2 µM each) , 200 µM dNTP (50 µM each), 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 mM MgCl and 1 unit of Taq polymerase (GibcoBRL). PCR was performed using a o Hybaid OmniGene Thermal Cycler with the following profile: one cycle of 2 min at 94 C, o o o 30 cycles of 30 s at 94 C, 1 min at 47.5 C to 57.5 C (depending on the primer pairs used), o 2-4 min (depending on the length of the fragment to be amplified) at 72 C, and one cycle of o 3 min at 72 C. A hot lid was used during the reaction.

Restriction

Aliquots of amplified samples were used in restriction endonuclease digests to identify diagnostic restriction site and length differences. The enzyme HaeIII, RsaI, and EcoRV (GibcoBRI) were used in trnF-trnVr and trnC-trnD amplified fragments.

Electrophoresis

The PCR product and the digests were electrophoresed in 1.6% agarose(GibcoBRL) gels and stained with ethidium bromide. The gels were then photographed on an UV box using a Kodak DC120 zoom digital camera and analysed using Kodak digital Science TM 1D image analysis software.

Data analysis

22 Phylogenetic analyses of the cpDNA data were performed by PAUP (Phylogenetic Analysis Using Parsimony, Beta version 4.0b2, Swofford, 1998). The restriction bands of different genotypes were scored as 0, 1 data set and were presented to PAUP as a Nexus file. Both Wagner (Farris 1970) and Dollo (Farris 1977) parsimonies were used to estimate phylogenetic relationships. Wagner parsimony implies that binary characters are freely reversible, so that site losses and gains are equally likely but Dollo parsimony permits each character state to originate only once. The most parsimonious trees were determined by implementing the Heuristic option of this program which performs an evaluation of tree topology to find the shortest trees. The tree was rooted by using an outgroup of two genotypes C. erythrochlora, a genotype of C. floriferum sp. diffusum and a genotype of C. forrestii.

3.3 Results

Variation of chloroplast DNA

Chloroplast DNA variation has been studied in 13 genotypes of C. uncinatum and 5 genotypes of C. megalopetalum.

The trnF-trnVr spacer restriction

A 3030bp fragment was amplified from trnF-trnVr spacer region in all genotypes within the two species except MB05, a genotype of C. megalopetalum from which a 3100bp fragment was obtained. Restriction of these fragments with HaeIII, RsaI and EcoRV identified five different banding patterns among the 18 genotypes used. With EcoRV, two fragments, 2225 and 805bp long were observed in all C. uncinatum genotypes and four of the C. megalopetalum genotypes. Two fragments of 2225 and 875bp were found in MB05, the reminding C. megalopetalum genotype.

HaeIII restriction of the same fragments identified two banding patterns in C. uncinatum and two banding patterns in C. megalopetalum. Four fragments, 1150bp, 870bp, 600bp and 410bp, were observed in all genotypes studied in C. uncinatum, except UA772 and UA773, in which the 600 fragment was replaced by a 360bp and a 240bp fragment. In C. megalopetalum, five bands sized 1150bp, 630bp, 520bp, 410bp and 320bp were observed, except MB05 which showed four fragments as 1150, 870, 670 and 410bp (Fig. 3.1).

The RsaI restriction revealed three banding patterns. Four fragments, 710, 445, 345 and 140bp were produced in all the genotypes tested, with the exception of UA561 and MB05. For UA561, where the 140bp fragment was replaced by a 210bp fragment and for MB05, four different fragments, 1100, 690, 425 and 140bp were produced.

The trnC-trnD spacer restriction

In the trnC-trnD region, a 3450bp fragment was amplified in all genotypes within the two species without variation. However, an additional band of 290bp was generated in genotype MB05. HaeIII digested the 3450bp fragment into two bands, 2780bp and 670bp in all genotypes of C. uncinatum, except UA561. No restriction site was found in the 3450bp fragment for UA561 nor in any genotype of C. megalopetalum.

23 The same 3450bp trnC-trnD fragment, restricted with RsaI, produced six fragments, 1030, 640, 440, 410, 160 and 80bp in all the genotypes of C. uncinatum. In four out of five C. megalopetalum genotypes, the first four bands were the same as in C. uncinatum but the bands 160 and 80bp were replaced by 180 and 20bp. Once again, MB05 produced a different banding pattern from the other genotypes. Six bands were produced from MB05(1010, 670, 430, 290, 180 and 20bp).

The fragments amplified from trnS-trnfM spacer region were 1700bp long in all genotypes with the exception of UA561, a genotype of C. uncinatum, in which a 1250 bp fragment was obtained. This direct length polymorphism was used in the analysis of intraspecific cpDNA inheritance but was not further analysed with restriction enzymes.

The amplification and restriction fragments length polymorphism is summarized in Table 3.3. The results indicate that at least five cpDNA haplotypes exist among the two species used – three haplotypes in C. uncinatum and two in C. megalopetalum.

Intraspecific chloroplast DNA inheritance in C. uncinatum

Chloroplast DNA inheritance of twelve intraspecific hybrids between UA773 and UA561 and 5 reciprocal hybrids of the same cross were analysed using fragment length polymorphism generated in trnS-trnfM region. All amplified fragments of the offspring of UA773 x UA561 were 1700bp long, the same as UA773, and the fragments from the reciprocal hybrids were 1250bp long, the same as UA561. Therefore, all hybrids showed the same banding patterns as the maternal parents.

Interspecific chloroplast DNA inheritance

Thirty-three progenies obtained from six interspecific cross combinations were tested using fragment amplified from trnF-trnVr region, restricted by HaeIII. All progenies from these crosses presented chloroplast DNA typical the maternal parent: Purple Pride x MB99/MBO5.5 (Fig. 3.2); CWA Pink x MBO1.5/MBO3.1 (Fig. 3.3); MBO3.1 X 5001 (Fig. 3.3) and MBO5.5 x UA827 (Fig. 3.3).

3.4 Discussion

Chamelaucium chloroplast DNA variation

Three pairs of primers were used to amplify cpDNA in C. uncinatum and C. megalopetalum. Considerable variation was found among 18 genotypes within the two species studied. The size of trnC-trnD fragment (3450bp) amplified was the same in all genotypes tested. No length mutation was observed. However, the size of the trnS-trnfM fragment in UA561 was 1254bp, but in other tested genotypes was approximately 1700bp, the same as in Quercus robur (Demesure et al., 1995). Length variation was also observed in trnF-trnVr region where a 3100bpfragment was amplified for MB05 and a 3030bp fragment was observed among all other genotypes tested . These length variation indicates that some insertion/deletion may have occurred in the respective regions.

The PCR-RFLP method has demonstrated as a useful tool for the amplification of both nuclear and chloroplast DNA and the study of DNA polymorphism. The effectiveness of this method in amplifying DNA was demonstrated also in other genera, such as Quercus, Ilex, Arundaria, Polystichum (Dumolinlapegue et al., 1997) and other species in Chamelaucium as C. axillare, C. ciliatum, C.floriferum ssp diffusum, C. erythrodhlorum, C.

24 forrestii, and C. roycei (Ma et al., unpublished). The amplified fragments and the enzymes used in the restriction showed a different capacity to identify the species and genotypes. The trnF-trnVr fragment was more informative than the trnC-trnD fragment. The fragment lengths of the different profiles for RsaI in both trnF-trnVr and trnC-trnD spaces do not add up to full length of the PCR amplified fragments. We attribute this to fragments comigrating or additional small fragment that are not visible on the gels.

Different combinations of fragments and enzymes grouped the genotypes in a slightly different way. With HaeIII, both fragments generated two main groups, C. uncinatum and C. megalopetalum, but with the exception of UA561 and UA772, UA773 grouped with C. uncinatum and MB05 in C. megalopetalum. With RsaI, all genotypes in both species were grouped together, except UA561 and MB05 in the trnF-trnVr fragment. However, two main groups were generated based on the patterns in trnC-trnD fragment, with the exception of UA561 and MB05. Using combined data, a phylogenetic tree was constructed by PAUP (Fig. 3.4). This confirmed the present phylogenetic relationship within the two species studied. On the other hand, it indicated UA561, UA772, UA773, and especially MB05 are very possibly with different maternal background than other genotypes in same species. This result has demonstrated the usefulness of the restriction of PCR amplified products to confer data for phylogenetic studies.

Chloroplast DNA inheritance

In the 50 progenies from controlled inter- and intraspecific crosses, we only observed the chloroplast haplotypes of the female parents (Table 3.4). This is consistent with chloroplast DNA inheritance being predominantly maternal. In intraspecific study, a pair of reciprocal crosses was studied, and they both showed maternal inheritance. In the interspecific crosses, no strict reciprocal crosses were available. However, no matter C. uncinatum or C. megalopetalum was used as maternal parents, cpDNA was unequivocally inherited maternally. This suggests a strict maternal cpDNA inheritance in Chamelaucium or at least in these two species.

From the above evidence, we have concluded that chloroplast genome are transmitted via egg cells in Chamelaucium, the same as most angiosperms (Dumolin et al., 1995; Lashermes et al., 1996; Vivek et al., 1999 ). Moreover, phylogenetic studies based on chloroplast DNA should be able to trace the maternal evolutionary lineage in Chamelaucium. The results from this research can be used to trace the parentage of natural hybrids and can help in parent selection in our waxflower breeding program.

3.5 References

Bentham, G. (1867). “Flora Australiensis: a Description of the Plants of the Australian Territory,” Lovell Reeve & Co., London.

Cruzan, M. B., Arnold, M. L., Carney, S. E., and Wollenberg, K. R. (1993). cpDNA inheritance in interspecific crosses and evolutionary inference in lorisiana irises. American Journal of Botany 80, 344-350.

Demesure, B., Sodzi, N., and Petit, R. J. (1995). A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology 4, 129-131.

25 Dumolin, S., Demesure, B., and Petit, R. J. (1995). Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method. Theoretical and applied genetics 91, 1253-1256.

Dumolinlapegue, S., Pemonge, M. H., and Petit, R. J. (1997). An Enlarged Set of Consensus Primers For the Study of Organelle Dna in Plants. Molecular Ecology 6, 393-397.

Egertonwarburton, L. M., Ghisalberti, E. L., and Burton, N. C. (1998a). Intergeneric Hybridism Between Chamelaucium and Verticordia (Myrtaceae) Based On Analysis of Essential Oils and Morphology. Australian Journal of Botany 46, 201-208.

Egertonwarburton, L. M., Ghisalberti, E. L., and Considine, J. A. (1998b). Infraspecific Variability in the Volatile Leaf Oils of Chamelauciuun Uncinatum (Myrtaceae). Biochemical Systematics & Ecology 26, 873-888.

Henry, R. J. (1997). “Practical Applications of Plant Molecular Biology,” Chapman & Hall, London.

Johnson, L. A. S., and Briggs, B. G. (1984). Myrtales and Myrtaceae - a Phylogenetic Analysis. Annals of the Missouri Botanical Garden 71, 700-756.

Lashermes, P., Cros, J., Combes, M. C., Trouslot, P., Anthony, F., Hamon, S., and Charrier, A. (1996). Inheritance and Restriction Fragment Length Polymorphism of Chloroplast Dna in the Genus Coffea L. Theoretical & Applied Genetics 93, 626-632.

Manning, L. E., Considine, J. A., and Growns, D. J. (1996). Chamelaucium uncinatum (Waxflowers), Family Myrtaceae. In “Native Australian Plants” (K. A. Johnson and M. Burchett, eds.), pp. 124-151. UNSW Press, Sydney.

Mogensen, H. L. (1996). The Hows and Whys of Cytoplasmic Inheritance in Seed Plants. American Journal of Botany 83, 383-404.

Palmer, J. D., Jansen, R. K., Michaels, H. J., Chase, M. W., and Manhart, J. R. (1988). Chloroplast DNA variation and plant phylogeny. Annals of the Missouri Botanical Garden 75, 1180-1206.

Rogers, S. O., and Bendich, A. J. (1994). Extraction of total cellular DNA from plants, algae and fungi. In “Plant Molecular Biology Manual” (S. B. Gelvin, ed.), Vol. D1, pp. 1-8. Kluwer Academic Publishers, Belgium.

Rye, B. L. (1979). Chromosome Number Variation in the Myrtaceae and its Taxonomic Implications. Australian Journal of Botany 27, 547-573.

Smith-white, S. (1950). Cytological Studies in the Myrtaceae. Proceedings of the Linean Society of N.S.W. 75, 99-121.

Vivek, B. S., Ngo, Q. A., and Simon, P. W. (1999). Evidence for maternal inheritance of the chloroplast genome in cultivated carrot (Daucus carota L-ssp sativus). Theoretical & Applied Genetics. 98, 669-672.

26 1 2 3 4 5 6 7 8 9

2072 bp 1500 bp

600 bp

100 bp

Figure 3.1 HaeIII restriction of amplified trnF-trnVr fragment. Lanes (from left to right): 1. λ DNA/EcoRI+ HindIII molecular marker, 2. UA100, 3. Purple Pride, 4. UA692. 5. UA772, 6. UA773, 7. MB01.5, 8. MB05 and 9. 100bp molecular marker.

27 1 2 3 4 5 6 7 8 9 10 11 12 13 14

2072 bp 1500 bp

600 bp

100 bp

Figure 3.2 Maternal inheritance of the chloroplast genome in progeny from the cross between ‘Purple Pride’ x MB05.5. The fragment trnF-trnVr was digested with HaeIII. All progeny produced the same banding pattern as the female parent Purple Pride. The male parent MB05.5 produced a different banding pattern. Lanes (from left to right): 1 and 14. 100bp molecular marker, 2. Purple Pride (the female parent), 3—12. Hybrids, 13. MB05.5 (the male parent).

28 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19

2072 bp 1500 bp

600 bp

100 bp

Figure 3.3 Maternal inheritance of the chloroplast genome in progeny from the crosses of ‘CWA Pink’ x MB03.1 and MB05.5 x UA827. The trnF-trnVr fragment was digested by HaeIII. The progeny of cross between ‘CWA Pink’ x MB03.1 had the same banding pattern as ‘CWA Pink’ and the progeny of MB05.5 x UA827 had the same pattern as MB05.5, the female parent. Lanes (from left to right): 1, 13 &19. 100bp molecular markers, 2. ‘CWA Pink’, 3—11. Progeny of ‘CWA Pink’ x MB03.1, 12. MB03.1, 14. MB05.5 , 15—17. Progeny of MB05.5 x UA827, 18. UA827.

29 0 CWA

0 100

0 PP

0 557

0 827 1 0 595

0 692

0 720 3 0 786

0 5001 1 0 772 3 0 773 3 2 561

15 MB05

0 MB99

0 MB01.5 1 0 MB03.1 3 0 MB05.5

0 diffusum 2 0 forrestii

1 ery2x 3 1 ery3x

Figure 3.4 Maternal phylogeny of C. uncinatumm and C. megalopetalum as estimated by RFLPs of amplified chloroplast genome. The most parsimonious tree was constructed by the Wagner parsimony criterion. Numbers above the branch indicate the minimum number of mutational steps for that branch.

30 Table 3.1 Hybrids used in the study of chloroplast inheritance

Type of cross Cross combination No. of hybrids

Intra-specific UA561 x UA773 12

UA773 x UA561 5

Inter-specific Purple Pride x MB99 4

Purple Pride x MB05.5 10

CWA Pink x MB01.5 2

CWA Pink x MB03.1 7

MB03.1 x UA5001 7

MB05.5 x UA827 3

Total 50

31 Table 3.2 Chloroplast DNA primers

Primer Primer sequence (5’-3’) Amplify Anealing Refere Name distance temperature nce (bp) (0C) TrnSr GAG AGA GAG GGA TTC GAA CC 1700 62 C. TrnfM CAT AAC CTT GAG GTC ACG GG Demes ure et al (1995) TrnF CTC GTG TCA CCA GTT CAA AT 3492 57.5 S. TrnVr CCG AGA AGG TCT ACG GTT CG Dumol in- Laregu e et al (1997) TrnC CCA GTT CAA ATC TGG GTG TC 3000 58 C. TrnD GGG ATT GTA GTT CAA TTG GT Demes ure et al (1995)

32 Table 3.3 Summary of restriction sites found in cpDNA PCR fragments of Chamelaucium species in this study.

C. uncinatum C. megalopetalum Band C C P C C C C C C C C C C M M M M M (bp) W A P A A A A A A A A A A B B B B B A 1 5 8 5 6 7 7 5 7 7 5 9 0 0 0 0 0 5 2 9 9 2 8 0 7 7 6 9 1 3 5 5 0 7 7 5 2 0 6 0 2 3 1 . . . 1 5 1 5 T H 1150 r a 870 n e 670 F 630 - I 600 t I 520 r I 410 n 360 V 320 r 240 R 1100 s 710 a 690 445 I 425 345 210 140 T H 3450 r a 2780 n e 670 C I 290 - I t I r R 1030 n s 1010 D a 670 640 I 440 430 410 290 180 160 80 20 CWA—CWA Pink PP—Purple pride

33 Table 3.4 Inheritance of chloroplast DNA in Chamelaucium.

Crosses Number of CpDNA inheritance hybrids tested PMB Intra UA561 x UA773 12 0 12 0 specific UA773 x UA561 5 0 5 0

C. uncinatum (Purple Pride) x 40 4 0 C. megalopetalum (MB99)

Inter specific C. uncinatum (Purple Pride) x 10 0 10 0 C. megalopetalum (MB05.5)

C. uncinatum (CWA Pink) x 20 2 0 C. megalopetalum (MB01.5)

C. uncinatum (CWA Pink) x 70 7 0 C. megalopetalum (MB03.1)

C. megalopetalum (MB03.1) x 70 7 0 C. uncinatum (Manning White)

C. megalopetalum (MB05.5) x 30 3 0 C. uncinatum (UA827)

Total 50 0 50 0

P—paternal inheritance. M—maternal inheritance. B—biparental inheritance.

34 4. Receptivity timeline and pollen/pistil interactions

4.1 Introduction

Despite the difficulties involved, wide hybridization has been utilized for about three centuries in the deliberate improvement of plants. The first authentic record of an artificial cross between different species was that of one between the ornamentals Carnation and Sweet William by the Englishman Thomas Fairchild in 1711.

Wide hybridization has been utilized most extensively in the breeding of more spectacular ornamental plants, and many well-known varieties of rose, tulip, iris, dahlia, chrysanthemum and gladiolus can be traced back to a deliberate wide hybrid.

In Australia, both natural and deliberate wide hybridization have been exploited successfully in the development of the wide range of Grevillias which are now available to home gardeners. Often, a spontaneous hybrid with unique characteristics has been found in a natural habitat and then multiplied by asexual reproduction for commercial release.

Wide hybridization has also been utilized widely in the breeding of new fruit cultivars and totally new types of fruits. The genus Prunus, for example, comprises a wide range of species cultivated for their fruits: - P domestica (European plum), P armeniaca (apricot), P. persica (peach), P amygdalus (almond), and P. avicum (Cherry). One of the most famous plum varieties Santa Rosa was produced from crosses among P salicina, P simonii and P americana. A cross between plum and apricot resulted in the development of a new fruit type, 'plumcot". New types of citrus fruits such as the

“tangelo” (tangerine x grapefruit) and the "orangelo" (orange x grapefruit) have been obtained through wide hybridization.

Plants within the sub-family Chamelauciae (Myrtaceae) are an important element of the Western Australian flora and are particularly evident in the seasonal floral displays in the Kwongan heaths. This group is poorly studied, but as many of the flora occur in disjunct mosaic populations many of which appear to have evolved in substantial isolation during recent geological history. There are 31 species in the genus Chamelaucium and more than100 species in other related genera, which we can use to make wide hybridizations. Efficient and successful wide cross or genetic introgression requires an understanding of the genome interaction between the two plants involved. Investigation of pollen/style interaction is an important initial step to the understanding of genome interactions between the genotypes involved.

4.2 Materials and methods

Flowering times were gathered from observations of plants in the wild and domestic cultivars.

Pollen viability was assessed by growing pollen in vitro in a 20 % sucrose solution for 24 hours at 25oC. The pollen was then stained with aniline blue and observed under a microscope via fluorescence optics so that the number of grains that had germinated could be counted.

35 Artificual pollination was performed on plants at the Univeristy of Western Australia Research Station in Shenton Park between C. uncinatum genotypes 642, chosen as the female parent, and 720, used as the male parent. Freshly opened flowers from a bagged branch were used to collected pollen. The viability of the pollen was checked. Flowers from 642 were measculated prior to anthesis and covered with perforated plastic bags. To eliminated pollen vectors. 10 flowers were pollinated 0, 3, 6, 9, 10, 11, 12, 13, 15, 18, and 21 days after anthesis. Anthesis was taken as being when the style was fully elongated. 5 flowers were also set aside to be tested for contamination. 48 hours after pollination flowers were collected and placed in Canoys I solution (acetic acid : ethanol 1:3) for 24 hours and kept at – 20OC until assessment. Before being assessed they were rinsed in de-ionized water and placed in 8 M NaOH for 3 hours and then rinsed again with de-ionized water and stained with aniline blue and observed with fluorescence. The flowers were rated via a system where: 1 = pollen germination, 2 = penetration of stigma, 3 = penetration of style, 4 = 0 to 10 pollen tubes in the ovary, 5 = 10 or more pollen tubes in the ovary and 6 = seed set.

4.3 Results

Flowering times for genotypes and species of Verticordia, Chamelaucium and Pileanthus are given in table 4.1. The flowering times and durations are variable between genotypes and species.

Table 4.1 The known flowering times (F) and possible flowering times (PF) for species of Verticordia, Chamelaucium and Pileanthus..

Month

Species Variety J F M A M J J A S O N D

C. erythrochlorum PF F F F FPF

C. megalopetalum MB01.3 PF F

C. megalopetalum MB03 PF F

C. megalopetalum MB Late PF F

C. megalopetalum MB09 PF F

C. uncinatum 583 FPF

C. uncinatum 827 FPF

C. uncinatum 883 F F

C. uncinatum 765 PF F

C. uncinatum 781 PF F

C. uncinatum 692 FPF

36 C. uncinatum 528 FPF

C. uncinatum UWA PP FPF

C. uncinatum 831 FPF

C. uncinatum Hutt River F PF (KP)

C. uncinatum 514 FPF

C. uncinatum 785 PF F

C. uncinatum 772 PF F

C. uncinatum 5001 F F

P. auranticus PF FPF

P. filifolia FPFF

V. aurea F F

V. erythrochlorum PF F F F FPF

V. etheliana F F F F F

V. grandis F F F F F F F F F F F F

V. helichrysantha F F

V. leppidophylla F F F F

V. mitchelliana F F F F F

V. multiflora F F

V. serrata F F

The pollen viability for nine individuals from Chamelaucium and Verticordia are given in table 4.2. Pollen viability is variable between and within species.

37 Table 4.2 Pollen viability of genotypes within the Chamelaucium Alliance.

Genotype/Species Pollen Viability (% Germination)

C. uncinatum (Genotypes 514, 772, 773) >70%

C. uncinatum (Genotype 557) <20%

C. floriferum >70%

V. helmsii >70%

V. multiflora 20-50%

V. plumosa >70%

D. squarrosa 50-70%

D. Spp (novo) 50-70%

The receptivity of the stigma of C. uncinatum over time is presented in figure 4.1. The stigma is at its most receptive from around 6 to 18 days after anthesis.

6 5 4 3 2 1 Stimatic Receptivity 0 036910111213151821 Days After Anthesis

Figure 4.1 Pollen tube growth at different periods after anthesis from a cross between C. uncinatum 642 and C. uncinatum 720 where: 1 = pollen germination, 2 = penetration of stigma, 3 = penetration of style, 4 = 0 to 10 pollen tubes in the ovary, 5 = 10 or more pollen tubes in the ovary.

38 The seed set of C. uncinatum from a cross between genotypes is presented in figure 4.2. The maximum seed set is achieved when pollination is carried out 3 to 6 days after anthesis.

100 80 60 40

% Seed Set 20 0 0 3 6 9 10 11 12 13 15 18 21 Days After Anthesis

Figure 4.2 Seed set from flowers pollinated at different periods after anthesis in the cross C. uncinatum 642 and C. uncinatum 720.

The pollen tube growth for reciprocal intraspecific, interspecific and intergeneric crossess within the Chamelaucium alliance are given in table 4.3. Pollen tube growth varies depending on the male and female parents in a cross. The greatest pollen tube growth is achieved when C. uncinatum is the female parent. Although the stigma appears to be receptive for a long period of time, and is observed to be optimised between 6 to 18 days, seed set is optimised when the flowers are pollinated between 3 to 6 days after anthesis.

Table 4.3 Pollen tube score for intraspecific, interspecific and intergeneric crosses within the Chamelaucium alliance. Where 0 = No pollen germination, 1 = pollen germination, 2 = pollen tube penetration into stigma, 3 = pollen tube penetration into the style, 4 = pollen tube presence at ovary end of style, 5 = pollen tube presence in the ovary and 6 = seed set.

Male C. C. C uncinatum C. V. helmsii C. multiflora V. plumosa D. squarrosa D. sp. (Mt. uncinatum uncinatum 773 floriferum Success) Female 514 772 C. 566604443 uncinatum 514 C. 666606644 uncinatum 772 C uncinatum 666666566 773 C. 015501400 floriferum

C. helmsii 000000130

V. plumosa 424044444

39 D. squarrosa 540200555

D. sp. (Mt. 100000000 Success)

4.4 Discussion

There is a large variation in success of embryo production from reciprocal crosses between individuals from Chamelaucium, Verticordia and Pilianthus. As crosses become wider the success of the crosses decrease. For example intraspecific crosses tend to be more successful than interspecific crosses. The success of crosses within C. uncinatum does vary as well however, and there have been successful interspecific and intergeneric crosses. The variation in the success of crosses is not related to the geographical distance between wild populations of the plant. It is instead probably the result of other factors, such as ploidy and plant morphology. Few conclusions can be made about why no embryos were produced from some crosses. We can only conclude that there are hybridization barriers.

Of the 71 crosses conducted involving 9 species within the Chamelaucium alliance, epifluorescence microscopy indicated that 28 combinations showed immediate pollen-pistil incompatibility with pollen failing to germinate, or pollen tubes not penetrating the stigmatic surface. Such prefertilisation incompatibilities may be overcome by the use of mentor pollen (Pryzywara et al. 1989) or the addition of growth hormones to aid the germination of pollen and the growth of pollen tubes (Shrivastava and Chawla, 1993).

Three crosses, including D. Squarrosa x C. floriferum, displayed pollen tube arrest in the stigmatic or style tissue. The hybridisation barriers in some crosses may have been caused by differences in style length where the pollen tubes of the species with shorter pistils were unable to transcend the styles of species with longer pistils. The D. squarrose and D. spp. (novo) styles were much longer than the other genotypes and were responsible for the incompatibility. This may explain why the reciprocal crosses involving the two species were different.

Epifluorescence microscopy indicated that some crosses were apparently viable with pollen tubes reaching the ovary end of the styles. The lack of seed set from such crosses indicates that post- zygotic barriers may have been present preventing hybrid zygote formation. In vitro pollination (Zenkteler et al. 1987) or earlier embryo rescue are effective techniques for overcoming post-zygotic barriers.

Although breeding barriers have been detected in the Chamelaucium alliance in interspecific and intergeneric crosses, there remains great potential for the exploitation of breeding on all levels for the creation of new cultivars once work have been invested into overcoming these barriers.

The normal hybridization involves the processes of pollen landing on the stigma of a flower, pollen germinate and penetrate the stigma and grow down the style, pollen tube arrives at embryo sac and releases the sperm cells, sperm cell fertilizes the egg cell and form a new life – a hybrid. However, for wide hybridization, pollen may not be able to germinate on the stigma, pollen tube may grow too slowly down the style or may stop before it reaches the embryo sac. Therefore fertilization cannot occur and seed will not form.

40 In the breeding of waxflowers, the following cases were observed:

1. Pollen does not germinate on the stigma (Figure a). 2. Pollen germinates but no penetration (Figure b). 3. Pollen penetrates the stigma but grow too slowly along the style (Figure c, d). 4. Pollen grows normally, penetrate the style and grow to the other end of the style (Figure e, f).

Based on the above observations, specific techniques can be used to help the pollen tube to grow and do its work properly. These techniques include:

1. Style truncation: which is to cut the top part of the style including the stigma and put pollen closer to the embryo sac. This will help the slow growing pollen tubes to get to the embryo sac faster. Some time pollen does not like to germinate on the cut and a style grafting can be used. This technique involves cutting the middle part of the style off and graft the stigma back onto the cut and sow pollen on the stigma. 2. 3. Mentor pollen: which is killed pollen from the female parent used in the hybridization and mix the mentor pollen with the pollen from the male parent. This technique is hoping that some substances in the mentor pollen may help the pollen from the male parent to germinate and grow faster. 4. 5. Growing substances: Some growing substances can be used to mix with pollen from the male parent to help the pollen to germinate and grow. The substances often used are gibberellic acid

(GA3), Auxin (IAA, IBA, etc.) and cytokinin (BAP or Zeatin).

4.5 Reference

Pryzywara, L., D.W.R. White, P.M. Sanders, and D. Maher.. 1989. Interspecific hybrodisation of Trifolium repens with Trifolium hybridum using in ovulo embryo and embryo culture. Annals of Botany 64: 613-624.

Shrivastava, S. and H.S. Chawla. 1993. Effects of seasons and hormones on pre-and post- zygotic barriers on crossability and in vitro development between Vigna unguiculata and Vigna mungo crosses. Biologia Plantarum 35: 505-512.

Zenkteler, M., G. Maheswaran, and E.G. Williams. 1987. In vitro placental pollination in Brassica campestris and B. napus. Journal of Plant Physiology 128: 245-250.

41 5. Studies On The Embryology And Early Embryo Rescue To Support Waxflower Breeding

5.1 Introduction

Australia is blessed with the diversity of its unique flora. Over 25,000 different species of flowering plants have been recorded. Western Australia is especially rich in plant resources, with about 12,000 species found within its boundary, more than 78% are endemic. Increasing attention is being given to this treasure to conserve it and to realize its economic potential. The wildflower industry has been one of the fastest growing rural industries in Australia. Between 1981 and 1993 exports of wildflowers and native plants have grown from $2.9 million to around $23.1 million (RIRDC, 1994). In 1996/97 it reached $30 million (RIRDC, 1997). Western Australia accounts for about half of this, most of which is contributed by waxflower (Chamelaucium spp.).

Waxflower is one of the key commercial wildflowers in the world market. In the ladder of major Australian flowers exported in the world in 1995-96, in terms of volume and value, waxflower (Chamelaucium spp.) was the No. 1 (FECA, 1998). The major producer of waxflowers in the world is Israel, and the production is increasing in USA, Mexico and South Africa (Considine et al. 1998).

With the richest plant resources in our hands, combined with a strong industrial interest, Australia should be in a leading position in wildflower breeding and production in the world. However, Australia currently shares only a small part of the world market. In 1997, less than 10% of the $440 million annual sales of Australian native cut flowers were sourced from Australia (Considine et al. 1998). However, the cut flower, potted and amenity plant industries are ‘fashion’ industries and the economic life of a new cultivar may be as short as five years. This gives Australian industry an opportunity to recapture the market internationally, not only through production of new cut flowers, but also through the licensing and sale of new plant varieties to the world. Development and production of new plant variation to meet the specific and changing market are critical for the survival and profitability of Australian wildflower industry.

An extensive waxflower breeding program was set up in 1995 aiming at producing new varieties through intraspecific, interspecific and intergeneric sexual hybridisation. However, as postzygotic hybridisation barriers were assumed in wide crosses and mature waxflower seeds could not be germinated readily, an embryo rescue (embryo germination under tissue culture conditions) approach was adopted. The approach included a few steps such as collecting developing embryos six weeks after pollination, germinating the young embryo in MS medium, subculturing and rooting the seedlings in culture and transferring them into soil in the glasshouse. Because the embryos were rescued six weeks old, we were not sure whether embryo abortion had already happened before that time. If we could rescue the embryos at an earlier stage, more seedlings might be able to be produced. Armed with those questions, we have conducted this experiment. The experiment is also aiming at understanding the embryo and endosperm development of waxflower hybrids.

5.2 Materials and methods

Embryology

A commercial released variety of Chamelaucium uncinatum, Alba, was used in this study. The plants were grown on the campus of The University of Western Australia, Nedlands, Perth, Australia. The developing flower buds sized about 1mm to anthesis were collected on 17 August and 29 September, 1998. The collected buds at different stages were fixed in 2.5% glutaraldehyde in 0.05M phosphate buffer (pH 7.0) for 4 h, dehydrated in solvents, and then infiltrated and embedded in glycol methacrylate

42 (GMA) (O’Brien and McCully 1981). Sections at 2µm were cut on a Sorvall microtome with glass knife. 0.05% toluidine blue O in benzoate buffer (pH 4.4) was used to stain nucleus and cell wall, saturated Sudan black B in 70% ethanol for 4 h to detect lipid, 10% Amido black 10 B in 7% acetic acid to discriminate protein, periodic acid and Schiff Reaction (PAS) to identify polysaccharides and starch grains and 0.05% water soluble aniline blue in M/15 Na2HPO4 solution to view callose under epiflorescence microscope. Thin sections were also cut after Spurr resin embedding and stained with toluidine blue O in benzoate buffer (pH 9.0).

Early embryo rescue

The experiment was conducted in 1998/1999 flowering season. Seven male parents and seven female parents were used. The cross combinations conducted are listed in Table 1. Table 1 Cross combinations conducted for the early embryo rescue Female Male UA559/771-4 UA831 UA559/771-4 CM9 UA559/771-4 PU UA559/771-5 C. floriferum UA559/771-5 MB01.3 UA773X561-9 UA831 773X561-9 MB01.3 559 UA831 559 CM9 571 MB01.3 571 V. etheliana 571 PU 768 C. floriferum 768 MB01.3 781 C. erythrochlora

All the plants used as females were grown at Medina Field Station and most of the male plants were grown in the Medina Field Station with some kept in the shade house of AgWest, South Perth.

Pollen was collected from flowers and was dried and stored in –20°C freezer before used. Pollen viability was tested just before hybridisation to make sure the pollen was viable. Flowers from the plants used female were hand emasculated, bagged and pollinated when the styles mature (normally 5-7 days after emasculation).

The pollinated fruits were collected 3, 6, 9, 12, 15, 18, 21, 24 and 27 days after pollination. Twenty fruits were collected each time, half of which are fixed in fixative and the other half were used for embryo culture.

The fruits used for embryo culture were surface sterilized and were dissected under stereo microscope to find the swollen ovules. The number of swollen ovules was recorded for statistical analysis. The total number of embryos or seedlings survived in culture were recorded two months after establishment for statistical analysis. Cricket Graph was used for the analysis to find the general trends.

The fixed materials were dissected under compound microscope to examine endosperm abortions.

43 5.3 Results and discussion

Floral Structure

Chamelaucium uncinatum flowers consist of five petals, five reduced sepals alternating with the petals and single whorl of 10 stamens alternating with 10 staminoides. In buds, the anthers locate alternately in two whorls; one lower and another higher. Generally, the development of the lower anthers is slightly slower than that of the higher ones. The anther connective tips extend at the end close to filament to form anther glands, which are on the upper side of the anther. The glands of the higher whorl of anther locate just around below the stigma prior to anthesis. When the flower is opening, the anthers with glands extend upwards and the glands rub on the stigma possibly to deposit gland substance on stigma surface and also into the anther locule to make pollen oily.

Ovular Structure

The pistil is unicarpellate and contains six, rarely eight ovules. The ovules, which arrange in two rows sharing the same funicle are bitegmic, anatropous, and crassinucellate.

The ovules initiate from the side-bottom of the ovary locule while the anther is at the microsporogenous cell stage. A parietal cell, one of the derivate cells of the archesporium (Figure 5.1a), divides several times to form a multi-cellular nucellus (Figure 5.1b). The inner integument originates dermally from near the base of the ovular primordium (Figure 5.1a), whereas the outer integument forms lately from the subdermal cells when the megasporogenous cell are formed. Both the integuments develop rapidly and enclose the nucellus when the megaspore is about to commence meiosis. As a result, a zigzaged micropyle is constituted by both inner and outer integuments.

At the early stage of differentiation, starch grains deposited in the funicular tissue and the integuments . Since then, the starch is rich in the ovule, in particular nucellar cells (Figures 5.1g-j). Protein granules and lipid droplets were not observed during the ovule development.

Megasporogenesis and Embryo Sac Development

The archesporium (Figure 5.1a) which originates from the subdermal cell of the ovule primordium divides periclinally to form a parietal cell and a sporogenous cell (Figure 5.1b). The latter develops into a megasporocyte (Figure 5.1c). Twin megasporocytes (Figure 5.1d) can be viewed in some of the ovules. The megasporocyte divides meiotically to form a linear tetrad in which three megaspores at micropylar end degenerate, and the remaining one at the chalazal end functions (Figure 5.1e) and develops into uninucleate embryo sac. At megasporocyte and tetrad stages, the walls of either the megasporocyte or the tetrad have no fluorescence with aniline blue staining, indicating the absence of callose on the wall. However, slight fluorescence is visible on the cell wall of nucellus near the tetrad (Figure 5.1f).

The uninucleate embryo sac divides mitotically for three times to form an eight nucleate one through, 2- and 4-nucleate stages (Figures 5.1g-i). A mature embryo sac is formed by the differentiation of three micropylar nuclei to egg apparatus (Figures 5.1j and k), three chalazal ones to antipodals, and middle two to polar nuclei. Therefore, the embryo sac formation attributes to Polygonum type. The nuclei of egg cell and synergids were stained very weakly with toluidine blue and only slightly with Amido black (Figure 5.1j), but clearly observed using Normaski Differential Interference Contrast microscope (Figure 5.1k). The filament apparatus was strongly stained by PAS reaction on the micropylar end of the synergid wall. During the development, the widely reported large central vacuole does not exist in both 2- and 4-nucleate embryo sac (Figures 5.1g-i). During the development, some of the ovules and embryo sacs may degenerate and some abnormality are formed such as narrow ovule and hence embryo sac, and poorly developed embryo sac elements (Figure 5.1l). This may be one of the reasons for low seed set rate in Chamelaucium uncinatum.

44 The embryo sac contains starch grains at maturity, mostly in the central cell and a few in egg cells but storage protein and lipid were not seen.

Embryo rescue in vitro

The total number of embryos rescued and plants produced from 300 fruits of different crosses were plotted against days after pollination and the result is shown on Figure 5.2. The number of embryos rescued increased as time passed by. At day three, only 45 embryos were rescued from 300 fruits. But at day 24, more than 150 embryos were rescued. There might be two reasons for this result. Firstly, the embryos at early stage of their development were so small that they are very difficult to handle and secondly smaller embryos might not be able to survive very well. On the whole, the number of embryos rescued reached a plateau after 12 days of pollination. However, the number of plants produced from 300 fruits peaked at day 18 after pollination. About 80 plants were produced by 300 fruits at day 18 after pollination and this gives about 27 hybrids from every 100 flowers pollinated. From this data, we conclude that about 18 days after pollination is the optimum time for the rescue of waxflower hybrids.

When we break the data into different cross combinations as intraspecific, interspecific and intergeneric crosses to examine the number of embryos rescued, we could not see any other trend except the number of embryos rescued increased as time passed by (Figure 5.3). However, when we plot the data of number of plants produced against the time, we could see clearly a peak at day 18 for interspecific crosses and a peak at day 18 for intergeneric crosses but the trend for intraspecific crosses was not very clear (Figure 5.4). As the breeding program is mainly aiming at interspecific and intergeneric crosses, this result again suggested that about 18 days after pollination is the optimum time for the rescue of waxflower hybrids.

Different cross combinations produced different number of embryos or seeds (Figure 5.5). There is no significant difference between the average number of embryos of intraspecific, interspecific and intergeneric crosses, however, certain cross combinations produced high number of embryos and some cross combinations produced only small number of embryos. Among the three intraspecific crosses attempted, there was not much variation between different combinations in the number of embryos produced. Considerable variation was observed among the interspecific crosses. One particular cross, (559/771-5)/MB01.3, produced more than 120 embryos out of 180 fruits but the cross between UA781 and C. erythrochlora only produced less than 10 embryos from 180 fruits collected. One intergeneric cross, UA571/V. etheliana, also produced very small number of embryos (Figure 5.5). From this result, we can conclude that hybridisation barriers may exist among some of the cross combinations and further research is needed to elucidate the situation.

Genotypes behaved differently when used as male parents. V. plumosa, UA831, MB01.3, MB09 and C. floriferum were proved to be good male parents and C. erythrochlora and V. etheliana to be unsuccessful male parents (Figure 5.6). There are two major reasons for the male parents to behave badly during cross. One reason is that the plants may not be able to produce viable pollens. For instance this is the case for C. erythrochlora. Some of the C. erythrochlora plants used in this experiment were triploid and they only produce sterile pollens. The other reason is clearly because of incompatibility. Plants also behave differently when used as female parents (Figure 5.7). The siblings, 559/771-4 and 559/771-5, were proved to be good female parents but UA781 behaved badly when used as a female parent.

It was technically demanding to conduct early embryo rescue as the embryos were very small at early stage. We mainly attempted rescuing the whole ovules at early stages of embryo development, day 3 to day 9. The swollen ovules, indicating embryo growth inside, were not apparent at early stage (Figure 5.8a). Therefore all the ovules were dissected out and planted in the MS medium.

45 About two weeks after planting on the media, the swollen ovules became clear (Figure 5.8b) and the small embryo could be dissected out of the swollen ovules (Figure 5.8c). From day 12 after pollination onwards, the swollen ovules became more and more apparent and the embryos dissected out of the swollen ovules were bigger (Figure 5.8d). The embryos rescued on the MS medium could germination (Figure 5.8e) and grew into a plant with a root and a green shoot (Figure 5.8f).

5.4 Reference

Considine, J. A. and Growns, D. (1998). Geraldton wax and relatives. In: 'The New Rural Industries - A Handbook for Farmers and Investors'. (Eds K. W. Hyde.) FECA. (1998). A snapshot of Australian floriculture & exports. (The Australian Government Publishing Service) O'Brien, T. P., and M. E. McCully, 1981 The study of plant structure : principles and selected methods, Melbourne : Termarcarphi. RIRDC. (1994). The Australian wildflower industry - a review. (The Australian Government Publishing Service) RIRDC. (1998). The Australian wildflower industry review, (The Australian Government Publishing Service)

46 47 Figure 5.1 Embryogenesis of Chamelaucium uncinatum ‘Alba’ Fig. a-e, and l are bright field photographs of GMA sections stained by toluidine blue; Fig. h is stained by toluidine blue and PAS; Fig. g, i and j are stained by Amido black and PAS. Fig. f is fluorescence photographs of GMA sections stained by aniline blue. Fig. k is photograph of Normaski Differential Interference Contrast of GMA section. a. Ovule with subdermal archesporium and inner integument just initiated. X540. b. Ovule with a sporogenous cell and integuments. X740. c. Megasporocyte before meiosis. X740. d. Twin megasporocytes. X740. e. Megaspore tetrad, showing three degenerating micropyle and one functional chalazal megaspores. X1100. f. megaspore tetrad has no callose wall around megaspores, but slight fluorescence is present on a chalazal nucellar cell. X 217. g. Two-nucleate embryo sac (ES) and starch grains in nucellar cells. X900. h. Four-nucleate ES, showing micropylar 2 nuclei only slightly stained by toluidine blue and no central vacuole in the ES. X1100. i. Same section as h but the nuclei are strongly stained by Amido black. X1100. j. Mature ES, showing an egg cell, a synergid and starch grains in nucellar cells. X370. k. Same section as j under Normaski differential interference contrast microscope. X217. l. Degenerating ES, showing narrow ES and abnormal composition inside. X217.

200 200

150 150

Plant produced 100 100 Embryo rescued

50 50 Embryo rescued or plants produced 0 0 3 6 9 12 15 18 21 24 27

Days after pollination

Figure 5.2 Embryo rescued and plants produced from 300 fruits of different cross

48 20 20

15 15

Intraspecific crosses (3)

10 10 Interspecific crosses (9)

Intergeneric crosses (3)

5 5 Number of embryos rescued

0 0 0 5 10 15 20 25 30

Days after pollination

Figure 5.3 Number of embryos rescued from different cross combinations of 20 fruits

10 10

7.5 7.5

Intraspcific crosses (3)

5 5 Interspecific crosses (9)

Intergeneric crosses (3)

2.5 2.5 Numbers of embryos rescued

0 0 0 5 10 15 20 25 30

Days after pollination

Figure 5.4 Number of plants produced from different cross combinations of 20 fruits

49 150

100

50 Number of embryos/seeds rescued

0 571/VP 559/831 559/CM9 Intraspecific Intergeneric 768/MB01.3 571/MB01.3 Interspecific 781/CERYTH 571/V. ETHYL 768/C.FLORIF (559/771-4)/VP (559/771-4)/831 (773X561-9)/831 (559/771-4)/CM9 (559/771-5)/cflori (559/771-5)/MB01.3 (773X561-9)/MB01.3

Cross combinations

Figure 5.5 Embryos/seeds rescued from 180 fruits of different cross combinations

50 100

75

50

25

0 P Number of embryos from fruits 180 Number embryos of V 831 . ethyl C.flori V C. eryth MB01.3 CM9 (MB09)

Male

Figure 5.6Embryo production of different male parents

125

100

75

50

25 Number of embryos in 180 fruits 0 559 571 781 768 559/771-4 559/771-5 773/561-9 Female parents

Figure 5.7 Embryo production of different female parents (Fecundity)

51 Figure 5.8 Early embryo rescue of hybrids from Chamelaucium alliance a. Ovules rescued nine days after pollination of 559/771-5 by C. floriferum, showing that swollen ovules could not be seen at early stage. Eight ovules were dissected out from this fruit instead of the normal six. b. Ovules rescued nine days after pollination of 559/771-5 by UA831. After about two months culture, the swollen ovules could be seen clearly. c. Ovules rescued 12 days after pollination of UA768 by C. floriferum. After about two months culture, a young embryo was diccested out from a swollen ovule. d. Ovules rescued 24 days after pollination of UA768 by C. floriferum, showing that the embryo dissected from a swollen ovule was much bigger than the embryo resuced earlier. e. Ovules rescued 15 days after pollination of UA559 by UA831, showing that the embryo dissected from a swollen ovule started to grow. f. Ovules rescued 15 days after pollination of UA559 by 831, showing that the embryo dissected from a swollen ovule developed a root and a shoot.

a b

c d

e f

52 1