Development of in Vitro Techniques As Supportive Tools for Breeding and Mass Clonal Propagation Of

Development of in vitro techniques as supportive tools for breeding and mass clonal propagation of Leucocoryne spp. (Amaryllidaceae) Alejandro Félix Altamira Bravo 2016 1 Pontificia Universidad Católica de Chile Facultad de Agronomía e Ingeniería Forestal Development of in vitro techniques as supportive tools for breeding and mass clonal propagation of Leucocoryne spp. (Amaryllidaceae) Alejandro Félix Altamira Bravo Thesis to obtain the degree of Doctor Ciencias de la Agricultura Santiago, Chile, December 2016 2 Thesis presented as part of the requirements for the degree of Doctor en Ciencias de la Agricultura, approved by the Thesis Committee _____________________ Gloria Montenegro , Advisor __________________ Dr. Patricio Arce Johnson ___________________ Dr. Levi Mansur Santiago, December 2016 3 In memory of my grandmother Adela, with whom I spent many days learning to appreciate and propagate plants during my childhood. 4 This work was supported by CONICYT doctoral g rant number 21110937 and FIA Project PYT - 2012 - 0079 . 5 ACKOWLEDGEMENTS I would like to thank all those who were members of my committee, Professors Gloria Montenegro, Patric i o Arce, Levi Mansur and Eduardo Olate for all their valuable contributions in the development of this thesis. I would also like to thank Profe ssor Marlene Gebauer for her infinit e support a nd for allowing me to finish this thesis in her l aboratory. I also thank the ex - members of the “Laboratorio de cultivo in vitro y Ornamentales” with whom I shared during the development of this thesis , especially Nicole Arenas and Gonzalo Gutierrez for their friendship and support . Th anks to my parents Sergio and Mó nica and my sisters Camila and Josefa for their love and unconditional support . And especially I want to thank my partner Mario for all th ese years of friendship, company, support and challenges that we have overcome together . 6 CONTENTS CHAPTER 1. General Introduction ................................ ................................ ........... 8 CHAPTER 2. An efficient clo nal micropropagation protocol for Leucocoryne, a geophyte genus endemic of Chile ................................ .................... 26 CHAPTER 3. An efficient in vitro production system for Leucocoryne sp p . plant s initiated from seeds ................................ ................................ .......... 68 CHAPTER 4. Conclusion s ................................ ................................ ...................... 92 7 CHAPTER 1 GENERAL INTRODUCTION 8 1. Description of Leucocoryne genus. Leucocoryne genus belongs to the Amaryllidaceae family and is endemic to Chile. There are 17 described taxons in the genus (15 species and 2 subspecies), however its taxonomic classification is still confusing due to its high morphological variability and the occurrence of natural hybrids . Consequently some authors mention up to 45 species as part of the genus (Muñoz and Moreira 2000; Riedemannn and Aldunate 2001; Mansur 2002; Zoellner 2002; Mansur and Cisternas 2005; Zuloaga et al. 2009; Ola te and Schiappacasse 2013) . The n atural life cycle of Leucocoryne can vary depending on the species but, in general, the plant takes three years from seed to floral bulb size (Riedemannn and Aldunate 2001; Mansur 2002) . In general, this genus present s high adaptation to dry climates and is widely distributed along Chile, from lat 20°S in deserted zones until lat 41°S in more rainy temperate zones, with populations ranging from mild coastal habitats to more cold er mountainous areas where the soil is cover with snow for several months, at altitudes of 2.800 m.a.s.l . in the Andes mountains. However, most of the Leucocoryne populations grow in coastal areas in Central Chile (lat 30° - 35°S), in dry climates that show a marked rainy season from May to August and a 70 mm average annual rainfall (Zoellner 1972; Kim et al. 1998; Mansur et al. 2004; Olate and Schiappacasse 2013) . Plants of this genus are considered geophyte s due the presence of an underground storage structure, which in the case of Leucocoryne corresponds to a 9 tunicated bulb. These bulbs ha ve a spherical or oval shape, reaching 1.5 to 2.5 cm in diameter, covered by brown dry membranes and having up to twenty fleshy scales on the inside. B ulbs have a basal plate from which adventitious roots develop (Hartmann et al. 1997; Zoellner 2002) . Leaves have a flaccid, glabrous and semi - fleshy appearance. From the b ulbs a single floral scape grow whose height can vary between 30 and 80 cm. Its apical inflorescence it is an umbel that can have from 3 - 4 to 8 - 12 flowers depending on the species, althoug h 5 - 15 flowers have also been described. Flowers are perfect, with the stigma and stamens attached to the floral tube, with 2.5 - 6 cm in diameter, six tepals 1.4 - 2 cm in length exhibiting colors from white, blue - sky, purple, violet and others (Zoellner 1972; Mansur 2002; Schiappacasse et al. 2002) . The a ndroecium is composed of three or six fertile stamens and three fleshy staminodes (infertile stamens) 6 mm in length, with color varying between white, yellow, greenish or two - colored. The g ynoecium has a cylindrical superior ovary, with a short style and a capitate stigma. Fruit corresponds to a tricarpellary dehiscent capsule, and ea ch carpel has several seeds. In general, 15 - 40 seeds per fruit are produced, which are small with sizes of 0.1 - 0.2 cm in diameter (Muñoz and Moreira 2000; Mansur 2002; Schiappacasse et al. 2002; Zoellner 2002; Verdugo 2013) . Besides the diversity in shape and colors, it has been genetically verified the existence of diversity in terms of chromosomal number, which can vary from 2n = 10 in Leucocoryne purpurea to 2n = 18 in Leucocoryne coq uimbensis , with hybrids showing 2n = 14, 2n = 20 y 2n = 22 (Mansur 2002; Araneda et al. 2004; Salas and 10 Mansur 2004) . Also, it has been reported six Leucocoryne spec ies as self - incompatible, which would explain the high genetic variability (Mansur 2002; Mansur et al. 2004) . 2. Advances in Leucocoryne domestication and commercial use. The Leucocoryne genus is known in Chile by the common name "Huilli" and internationally as "Glory - of - the - S un". Because the high phenotypic variability of flowers ( color s , shapes and aroma ) and also because its long vase life, Leucocoryne has become a great alternative as an ornamental plant, either as cut flower, potted or garden plant (Bridgen 2000; Mansur 2002; Olate and Schiappacasse 2013; De la Cuadra et al. 2016) . The o rnamental value of the different species has led to several studies and domestica tion efforts and even incipient commercial ization of them in countries like Japan, Netherlands, Israel, New Zealand and USA (Bridgen 2000; Lancaster et al. 2000; Walton et al. 2008; Olate and Schiappacasse 2013; De la Cuadra et al. 2016) . This interest is similar to the one occ urred with Alstroemeria genus, which has been used by companies in other countries , for selecting, breeding and commercialization with high economic succe ss, using Chilean germplasm . The se are example s of importance of identifying, selecting, propagating , breeding and registration of plant material belonging to Chilean native genetic resources as Leucocoryne , which could also lead to economic benefits and improve the conservation of endangered population s (Jorquera et al. 2007) . 11 In orde r to tak e advantage of the commercial potential and also to conserve Leucocoryne , which has already been affected by antrophic intervention of their environment , conservation programs have been developed in Chile. S ustainable germplasm banks, genetic and a gronomic studies for future uses in landscaping, and also breeding programs for rel easing new cut - flower cultivars have been developed by researchers at the Pontificia Universidad Católica de Valparaíso . To date, three cut - flower cultivars ( 'Elena', 'Gabri ela' and 'Paulina' ) have been patented (Verdugo and Teixeira 2006) . Nevertheless, an efficient mass propagation system is still a pending challenge for successfully commercialization of these new cultivars (Bridgen 2000; Mansur 2002; Araneda et al. 2004; Olate and Schiappacasse 2013) . 3. Propagation techniques 3.1 Seed propagation Seed propagation of Leucocoryne naturally occurs when seeds are released from its dehiscent capsule sometime during the spring . Seeds stay on the soil surface during all summer and part of the autumn until the next rain y season (autumn and winter) , with temperatures ranging 10 to 15 °C (Mansur 2002; De la Cuadra et al. 2016) . Once germination occurs, plants develop a single leaf and after 45 days a small bulb starts to grow. This bulb can weight 0.06 g in average after 100 days since germination , and then it enters in dormancy until the next season. During the next autumn these small bulbs could produce shoot s even in absence of water, 12 using for that their reserves getting then ready for the winter rains. During t his second growing season two leaves will appear and the plant will stay active for 100 - 120 days producing more bulb weight increas e until it become s dormant again until next rainy season. Only during the third or fourth growing season the bulbs will rea ch 0.3 g in weight and the flowering process will happen and new seeds will be produced (Riedemannn and Aldunate 2001; Mansur 2002; De la Cuadra and Mansur 2004) . Germination studies have been conducted on L. coquimbensis, L. ixioides and L. purpurea , which have determined that water imbibition for one day and subsequent cold stratification at 7°C for 7 weeks results in high er germination rates ( over 90% ) compared to non - stratified se eds (Salazar 2001; Schiappacasse et al. 2002) . Similar studies have also achieved high germination rates by imbibition of Leucocoryne seeds for 96 h and s ubsequ ent culture at 10 - 15°C, without a stratification period , higher than germination observed at 20°C and no germination at 25°C (J ara et al. 2006; De la Cuadra et al.

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