Apomixis in Taraxacum an embryological and genetic study promotor: Professor dr. R.F.Hoekstra , hoogleraar in de genetica, met bijzondere aandacht voor de populatie- en kwantitatieve genetica co-promotoren: Dr. P.J.va n Dijk, senior onderzoeker bijhe t Nederlands Instituut voor Oecologisch Onderzoek, Centrum voor Terrestrische Oecologie (NIOO- CTO) te Heteren, en Dr. J.H.d e Jong, universitair hoofddocent bijhe t Departement Plantenwetenschappen, Wageningen Universiteit promotiecommissie: Prof. Dr. S.C. de Vries Wageningen Universiteit Prof. Dr.J.L .va n Went Wageningen Universiteit Prof. Dr.J.M.M . van Damme NIOO-CTO Heteren Peter van Baarlen Apomixis in Taraxacum an embryological and genetic study Apomixie in Taraxacum een embryologische en genetische studie Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. Dr. Ir. L. Speelman, in het openbaar te verdedigen op dinsdag 11 September 2001 des namiddags te vier uur in de Aula Baarlen, Peter van Apomixis in Taraxacum / Peter van Baarlen Thesis Wageningen University. - With references - With summary in Dutch Subject headings: apomixis/diplospory/embryology/polyploidy Typeset in ll-14pt Book Antiqua ISBN 98-5808-473-6. UNO' ^o\3c?2 STELLINGEN 1. - Het verstoren van de paring van homologe chromosomen tijdens de eerste meiotische profase, parthenogenetische eicel ontwikkeling en autonome endosperm ontwikkeling in apomictische paardebloemen is te verklaren door aan te nemen dat bepaalde chromosoom-specifieke eiwitten verschillen van hun "sexuele" analogen. dit proefschrift 2. - Het grote evolutionaire succes van paardebloemen kan verklaard worden door hun vermenging van de voordelen van sexuele en asexuele reproductie. dit proefschrift 3. - De hypothese van Vinkenoog en collega's: autonome endosperm ontwikkeling in autonome, polyploi'de apomicten is mogelijk bewerkstelligd via hypomethylatie van het genoom, is zeer lastig te testen daar genoominteracties, imprinting en aanwezigheid van "modifiers", alle drie karakteristiek voor apomicten, gepaard kunnen gaan met methylatie en ook endosperm ontwikkeling kunnen bei'nvloeden. Vinkenoog et al., 2000. Hypomethylation promotes autonomous endosperm development and rescues postfertilization lethality infie mutants. Plant Cell 12, 2271-2282. 4. - De verregaande specialisatie en complexiteit van de stand van het huidige onderzoek maakt het wenselijk dat wetenschappers zelf het initiatief nemen hun bevindingen en inzichten te communiceren naar het algemene publiek, bijvoorbeeld via Internet en columns in tijdschriften en kranten. 5. - Wageningen Universiteit zou zich tot doel moeten stellen met haar voorlichting en image niet louter middelbare scholieren aan te spreken teneinde de instroom van propaedeuse studenten te vergroten, maar om een duidelijk gezicht te hebben naar de gehele samenleving. 6. - De invloed van TV en overige visuele media waaronder het Internet kunnen ertoe leiden dat jongere wetenschappers minder gebruik maken van de oudere, voornamelijk tekst-georienteerde vakliteratuur. 7. - De mate van inbedding van evolutionair-biologische theorieen in vakgebieden als genoomonderzoek en celbiologie is een maat voor de evolutie van het respectievelijke vakgebied. 8. - Wetenschap kan, net zoals de muziek, beschouwd worden als een uitdrukkings- vorm waarbij het weglaten van fragmenten gebruikt wordt om andere fragmenten meer nadruk te geven. Stellingen behorende bij het proefschrift "Apomixis in Taraxacum - an embryological andgenetic study" van Petervan Baarlen. -11 September 2001, Wageningen jij hebt de dingen niet nodig omte kunnen zien de dingen hebbenjou nodig om gezien te kunnen worden Anonymous, Openbare Bibliotheek Rotterdam Content 1. General introduction 2. Ribosomal DNA spacer variation in apomictic Taraxacum officinale 13 3. Meiotic recombination in sexual diploid and apomictic triploid dandelions (Taraxacum officinale L.) 29 4. Comparative cyto-embryological investigations of sexual and apomictic dandelions (Taraxacum) and their apomictic hybrids 47 5. The occurrence of reciprocal apomixis-recombinants indicates that autonomous apomixis in the common dandelion (Taraxacum officinale) is regulated by multiple dominant genes 63 6. The influence of sexual-apomictic genome interactions on apomictic gametophyte development 79 7. General discussion 95 8. References 107 9. Summary 117 10. Samenvatting 121 11. Nawoord 125 12. Curriculum vitae 127 General Introduction Peter van Baarlen Summary In this chapter I will present an overview of the main differences between sexual and asexual seed production in the genus Taraxacum (dandelions). Several cytogenetic and plant developmental biology terms and concepts that will be used throughout this thesis will be introduced. The Taraxacum type of apomixis will be described and I will highlight its main deviations from sexual reproduction in this species. A synopsis will be given of earlier investigations that have partially led to the research questions that form the basis of this thesis. Sexual plant reproduction involves double fertilisation The life cycle of diploid sexually reproducing plants alternates between a diploid vegetative or sporophytic phase and a haploid generative or gametophytic phase. Within the female reproductive centre, the megagametophyte, the nuclei from two differentiated cells, the haploid egg cell and the diploid central cell, need to be fertilised by haploid pollen sperm nuclei before reproduction can initiate. Thus, sexual plants can only reproduce after fertilisation of two distinct cell types of different ploidy level. The female gametes (egg cells) and male gametes (sperm cells) become haploid after two meiotic divisions, one reductional division, halving the chromosome number in the daughter cells and one equational division halving the chromosomes into two chromatids. Fertilisation takes place in a specialised 7-cell structure of maternal origin, the embryo sac. Egg and central cell are both located in one single embryo sac (fig. 1.1). Immediately after embryo sac formation, two haploid nuclei appear in the central cell. Prior to fertilisation, these two nuclei fuse into a diploid nucleus in several angiosperm genera (Johri, 1963; Willemse & van Went, 1984). The fertilised egg cell starts dividing mitotically to produce a diploid embryo. In the fertilised central cell, the nucleus starts dividing to give triploid endosperm nuclei. Cellularisation takes place later, resulting into the endosperm, a specialised tissue that nourishes the embryo during seed formation. Sexual plants depend on fertilisation of egg and central cell nuclei in order to set seed since fertilisation restores the somatic diploid ploidy level and triggers endosperm and seed development. Embryo sac formation and fertilisation occur within the ovules. Megagametophyte and gamete formation starts with specialised cells within the ovules, the archespores (fig. 1.1b). Archespores transform into megaspore mother cells, the meiocytes that produce four megaspores after a standard type of female meiosis or megasporogenesis (recently reviewed in Porceddu et al., 1999; Schwarzacher, 1999). Megaspores first undergo three successive mitotic divisions without wall formation, a stage termed megagametogenesis. After this initial 8-nucleate (coenocytic) stage, cellularisation takes place and the 8-nucleate megaspores transform into embryo sacs. The type of embryo sac that applies to all plants in this study is the common Polygonum type of embryo sac (described in Johri, 1963; Willemse &va n Went, 1984;fig . 1.1a & 1.2). '/in f 11'• + • • • ^ * • • •'•' v • •• • •\ \ •. •'•7 Fig 1.1a. the Taraxacum embryo sac. The upper chalazal and bottom micropylar region are indicated, together with the contours of the central and egg cell. To the left of the egg cell, one of the two synergids is visible (not traced). Fig. 1.1b. the archespore (traced in white) that transforms into the megaspore mother cell. Asterisks * indicates the position of nuclei, with prominent nucleoli. Male gamete formation takes place in the anthers. Microspore mother cells, the male meiocytes, produce four microspores after male meiosis or microsporogenesis. Microspores eventually develop into pollen grains which are surrounded by characteristic thick cell walls. Within each pollen grain, mitotic divisions give rise to one vegetative cell and two generative sperm cells, each containing one vegetative nucleus or one generative sperm nucleus. One sperm nucleus will fertilise the egg cell nucleus, the other one the central cell nucleus. This scheme of gametophyte formation followed by fertilisation of reduced haploid egg and diploid central cell nuclei by haploid pollen nuclei does not only apply to sexual diploid Taraxacum but to most angiosperms. Apomixis is asexual reproduction through seed Although most angiosperm plants form seeds exclusively sexually, approx. 0.1% o f all angiosperms produce seeds without prior fertilisation of the egg cell and sometimes without fertilisation of the central cell (Mogie, 1992). This type of asexual plant reproduction through seed is termed gametophytic apomixis (Nogler, 1984) to distinguish it from vegetative asexual reproduction via production of shoots or runners. In this thesis, the conventional term apomixis will be used instead of gametophytic apomixis. Some important definitions that apply to different types of apomicts and that will be used throughout this thesis can be found in table 1 on page 5. Although there are several