MECHANISMS OF VEGETATIVE PROPAGATION IN BULBS a molecular approach Natalia M. Moreno-Pachón Thesis committee Promotor Prof. Dr R.G.H. Immink Special professor Physiology of Flower Bulbs Wageningen University & Research Co-promotor Dr H.W.M. Hilhorst Associate Professor, Laboratory of Plant Physiology Wageningen University & Research Other members Prof. Dr B.J.G. Scheres, Wageningen University & Research Prof. Dr R. Offringa, University of Leiden Dr C. Philipsen, Dummen Orange, De Lier Dr P.F.P. Arens, Wageningen University & Research This research was conducted under the auspices of the Graduate School Experimental Plant Sciences MECHANISMS OF VEGETATIVE PROPAGATION IN BULBS a molecular approach Natalia M. Moreno-Pachón Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Thursday 12 October 2017 at 11 a.m. in the Aula. Natalia M. Moreno-Pachon MECHANISMS OF VEGETATIVE PROPAGATION IN BULBS a molecular approach 178 pages PhD thesis, Wageningen University, Wageningen, the Netherlands (2017) With references, with summary in English ISBN: 978-94-6343-701-1 DOI: https://doi.org/10.18174/423177 TABLE OF CONTENTS CHAPTER 1 General introduction.......................................................................................................... 7 CHAPTER 2 Transfer of knowledge about flowering and vegetative propagation from model species to bulbous plants ................................................................................................................. 19 CHAPTER 3 Elucidating and mining the Tulipa and Lilium transcriptomes .............................................. 37 CHAPTER 4 Role of Tulipa gesneriana TEOSINTE BRANCHED 1 (TgTB1) in the control of axillary bud outgrowth in bulbs ........................................................................................................................... 59 CHAPTER 5 Molecular analysis of de novo shoot meristem regeneration in lily bulb scales ..................... 77 CHAPTER 6 Histological origin of regenerated bulblets in lily bulb scales ............................................. 105 CHAPTER 7 General discussion ......................................................................................................... 123 REFERENCES ............................................................................................................... 133 SUMMARY ................................................................................................................... 163 ACKNOWLEDGEMENTS ............................................................................................. 167 CURRICULUM VITAE .................................................................................................. 171 LIST OF PUBLICATIONS .............................................................................................. 173 EDUCATION STATEMENT ........................................................................................... 175 CHAPTER 1 General introduction Chapter 1 Description of used terms Bud: shoot meristem covered by few leaves. A meristem covered by scales in the case of bulbs. For bulbs, bulblet and bulbil are synonyms of bud. Bulb: main storage organ of bulbous plants. Bulblet: vegetative adventitious meristem arising from underground organs and covered by a few scales. Bulbil: vegetative axillary meristem covered by a few scales and located in the axil between a leaf and the stem. Daughter bulb: New bulb produced in the axil of a scale of the mother bulb through vegetative propagation. Plant reproduction Reproduction, the biological process of creating a new individual, is essential for the survival of the species. While most animals rely on sexual reproduction for this purpose, plants have the capacity to reproduce sexually through seeds or asexually through vegetative shoot meristems (Hartmann 2014). This trait might have been developed in response to sessility, to overcome the mortality by harsh environmental conditions. While seeds can disperse and germinate when the right environmental conditions are met, their mortality is generally higher than plants formed by asexual reproduction, because the mother plant is no longer providing nutrients and mechanical protection (Ikegami et al. 2012). Moreover, plants originating from vegetative meristems are identical to the mother plant, and as such represent clones. Cloning is a desired trait in agriculture and horticulture, allowing to fix and maintain unique desirable genetic compositions. Vegetative shoot meristems can be categorized according to their developmental process and location on the plant as shoot apical meristems (SAM), axillary meristems and adventitious meristems (Figure 1A). The SAM develops during embryogenesis and is located at the tip of the plant (Long and Barton 2000). Axillary meristems are located in the leaf axils and develop during post-embryonic shoot development into axillary buds that either continue growing into lateral shoots or fall into a period of arrest (Schmitz and Theres 2005), commonly referred to as dormancy. Adventitious meristems arise from differentiated tissues such as leaves, stems or roots (Kerstetter and Hake 1997). Those meristems may originate from a pre-existing group of cells descended from embryonic cells that did not lose their meristematic identity during the development of the plant (Hartmann 2014). This is e.g. the case in Kalanchoe species, commonly known as mother of thousands and producing plantlets from the notches around the leaf margin (Garcês et al. 2007). Adventitious meristems can also originate de novo from 8 General Introduction differentiated tissues that undergo dedifferentiation to create a new population of meristematic cells from where new plant organs will form (Hartmann 2014). Figure 1. Description of a non-bulbous and a bulbous plant. A. Vegetative meristems in a non- bulbous plant classified based on their developmental process and position in the plant. SAM: shoot apical meristem. AxM: Axillary meristem. AdM: Adventitious meristem. B. Anatomy of tulip and lily bulbs. Bulb are compressed plants where the scales are homolog to the leaves and the basal plate to the stem. Reproduction in bulbous plant species Both axillary and adventitious meristems constitute an important source of vegetative propagation in species where sexual reproduction takes considerably much longer or is not very successful (reviewed in (Leeggangers et al. 2013)). The first case occurs in flower bulbs. Flower bulbs, also known as ornamental geophytes, are herbaceous perennial plants with a bulb as storage organ. Other geophytes have storage organs such as, corms, tubers, rhizomes or enlarged hypocotyls that assure the survival of the plant during winter or dry periods (Hartmann 2014; Kamenetsky 2012; Okubo and Sochacki 2012). Among the diverse geophytes, bulbous plants are of special interest for the Netherlands because of the economic importance of two of its members: lily and tulip (Benschop et al. 2010). In both species the bulb contains specialized scales that serve as the primary storage tissue. Such scales are modified leaves attached to a modified short stem called the basal plate, which at the same time bears roots and at least one axillary bud (De Hertogh et al. 1983; De Hertogh and Le Nard 1993b) (Figure 1B). 9 Chapter 1 What is known about vegetative propagation in tulip and lily? Tulip and lily can sexually propagate through seeds but once they sprout, their juvenile and adult vegetative stage can last three to seven years, depending on the genotype and growth conditions, until they finally transition to the reproductive stage (Minas 2007; Shahin et al. 2012). On the contrary, once reached the adult vegetative stage, through vegetative propagation the new plantlets, also referred as bulblets or daughter bulbs, can reach the reproductive state in one growth cycle. On which basis a bulb decides to invest most of its resources in either sexual reproduction via flowers and seeds or vegetative propagation via daughter bulbs or bulblets is not known, but the different reproduction strategies provide these bulbous species a way to adapt to harsh conditions and to optimize survival. While tulip naturally propagates asexually by the outgrowth of axillary meristems of the mother bulb into daughter bulbs, lily is vegetative propagating mainly by regenerating de novo meristems from the underground stem or roots (Figure 2A-B). One growth cycle of an adult bulb takes a year in both species; however, they differ in the time of flowering. In cultivation, tulip bulbs are planted in late autumn, the shoot apical meristem sprouts by the end of the cold period (winter), the floral bud opens in spring, and the resulting daughter bulbs are lifted from the ground in early summer (De Hertogh et al. 1983) (Figure 2A). On the other hand, lily bulbs are planted in spring, flowering takes place in summer and the daughter bulbs are lifted in early winter (De Hertogh and Le Nard 1993b) (Figure 2B). Figure 2. Native vegetative propagation of tulip and lily bulbs. A. Growth cycle of tulip bulbs in relation to their vegetative propagation through axillary bud outgrowth. B. Growth cycle of lily bulbs in relation to their vegetative propagation through adventitious regeneration. Native vegetative propagation by axillary bud outgrowth in tulip bulbs A mature