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

Alejandro Félix Altamira Bravo

2016

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

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

In memory of my grandmother Adela, with whom I spent many days learning to appreciate and propagate plants during my childhood.

This work was supported by CONICYT doctoral g rant number 21110937

and FIA Project PYT - 2012 - 0079 .

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 .

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

CHAPTER 1

GENERAL INTRODUCTION

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

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) .

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. 2016) .

Due to the high genetic variability o f Leucocoryne and self - incompatibility in some of its species, seed propagation techniques ha ve great disadvantages to maintain interesting traits and massive propagati on of new registered cultivars. Despite this, seed propagation is still used as a valuable technique in breeding programs to obtain new plant segregants (Mansur 2002; Mansur et al. 2004; De la Cuadra et al.

2016) .

3.2 Vegetative propagation

Vegetative propagation consists in obtaining new plants from other plant organs like st ems, leaves, roots, bulbs, corms and rhizomes. The main advantage related to this techniques is that new developed plants are genetically identical to the propagated plant (Schiappacasse et al. 2002; Kumar et al. 2010) . In Leucocoryne , as in other geophyte genus, natural vegetative propagation occurs in the second or third year of growth , producing 1 - 2 lateral bulblets every one or two years depending on the genotype . I n some species and in particular environmental co nditions the plants can produce even more deep bulb s called "droppers"

(Riedemannn and Aldunate 2001; Mansur 2002; Schiappacasse et al. 2002) .

In some tunicate bulb species artificial propagation techniques allow to increase natural multiplication rates . These techniques are known as bulb - cutting methods and they consist essentially in to generate a mechanical damage to the basal plate or to the entire bulb. "Scooping" is one of these technique s that involve the complete elimination of the basal plate, the apical meristem and the base of the scales (Fig. 1b). In the "scoring" method, deep cuts to the base of the bulb are done in order to damage its basal plate and the apical meristem (Fig. 1c) . " C oring" is other of these methods, and consists in the removal of a cylindrical section of the basal plate, including the apical meristem (Fig. 1d). In other species it is used the met hod called "sectioning" , where the entire bulb is cut in two or more vertical sections in order to produce adventitious bulblets (Fig. 1e) (Hartmann et al. 1997;

Schiappacasse et al. 2002; Olate and Bri dgen 2005) .

Fig. 1 Artificial propagation techniques used in some bulb species . a) Intact bulb, b) S cooping, c) S coring, d) C oring , and e) " S ectioning". Adapted from Kumar et al. (2010) .

Studies in Leucocoryne coquimbensis have determined that sectioning a bulb in equal sections could produce up to 2.2 bulbs of different sizes, while in

Leucocoryne ixiodes up to 5 new bulbs have developed from a singular initial bulb

(Schiappacasse et al. 2002) . Nevertheless, i n a similar study, L. ixioides multiplication rates of intact bulbs w ere compared to bulbs multiplied using scooping and scoring, founding no significant differences between the natur al and artificial methods (Salazar 2001) . These results do not coincide with the succe ssful results observed in Hyacinthus and Scilla species , where scooping technique s produce much higher multiplication rates than natural propagation, and it is used for commercial production (Hartmann et al. 1997) .

3.3 I n vitro propagation

Micropropagation, or in vitro propagation, corresponds to the culture of explants under aseptic conditions, onto a nutritive medium , under controlled temperature, humidity and luminosity conditions, to produce high number of clonal plan ts in a

15 reduced period of time (Roca and Mroginski 1991; Hartmann et al. 1997; Kyte et al.

2013) .

In vitro shoot growth can be achieved using direct organogenesis from axillary or adventitious buds, or using indirec t organogenesis from cells , cell suspensions or calluses. However, i ndirect organogenesis has the disadvantage of increas ing the risk of producing genetically no n true - to - type plants (somaclonal variation) and thus affecting the homogeneity of the plants produced (George et al. 2008) .

Micropropagation has several stages to obtain new plants. Stage 0 includes the s election of the genotype and the initiation explant. Stage I corresponds to the disinfection and establishment of the explant in aseptic culture conditions. Once the explants are stabilized in vitro conditions, mass plant multiplication could be performed (Stage II ). Stage III of the micropropagation process is where the in vitro root ing is achieved. Finally, once the plants are fully developed, plants are transferred to ex vitro conditions to be culture into soil or potting soil in order to start their acclimatization (Stage IV). Duration and conditions for each of these stages must b e adapted to different plant genotypes (George et al. 2008; Bach and

S ochacki 2012; Kyte et al. 2013) .

In contrast with other ornamental geophyte species, only a few studies of in vitro culture have been done in Leucocoryne sp . One of the main barriers hindering a wide r use of th e s e technique s in geophyte species, is the high contamination occurring during the in vitro initiation of bulbs, corms, rhizomes and tubers , because their permanent contact with soil and high level of microorganisms , as fungi and bacteria (Pedersen and Brandt 1992; Slabbert et al. 1993; Kritzinger et

16 al. 1998; Smith et al. 1999; Chang et al. 2003; Sochacki and Orlikowska 2 005;

Paredes et al. 2014) . Previous works in Leucocoryne species have reported contamination rates between 42% and 100% during the in vitro initiat ion of bulbs

(Fuentevilla 2004) . In order to surpass this problem, previous studies involving

Leucocoryne sp. Bulbs have used seeds as initial explants with the consequent problem of uncertain genotypic homogeneity of the plants used (Ham 2002;

Briones 2003; Escobar et al. 2008) . In one of these studies different concentrations of MS (Murashige and Skoog 1962) were tested as germination media for L. purpurea seeds (i.e. 12.5%, 25 %, 37.5%, 50%, 75% and 100% ) resulting in higher germination rates when seeds were cultured onto 75% MS or lower (Ham 2002) .

Escobar et al. (2008) studied indirect organogenesis in L. purpurea by using MS medium supplemented with 30 g·L - 1 sucrose and 0.6% agar, pH 5.7 and a combination of different plant growth regulators: 2,4 - Dichlorophenoxyacetic (2,4 -

D), 6 - bencyladenine (BA), 1 - Phenyl - 3 - (1 - 2 - 3 - thiadiazol - 5 - yl) - urea (TDZ), α - naphthaleneacetic acid (ANA), 6 - y - y - (dimethylallyamino) - purine (2iP) and 4 - amino - 3,5,6 - trichloropicolinic acid (Pi cloram). Callus proliferation and shoot formation was achieved. Then, after seven months of culture , 3 - 4 bulbs/explant were obtained when subcultur e was done in absence of plant growth regulators.

Also, in previous studies (Olate and Bridgen 2005; Olate 2006) , using MS medium supplemented with 30 g·L - 1 sucrose and 0.7% agar and pH adjusted to 5.7, it was observed that ligh/darkness and temperature treatments does not affect bulb production in L. purpurea , L. coquimbensis and L. ixioides . In the same study , in vitro bulb - cutting methods were also tested . An average of almost 7 bulbs/explants

17 were obtained using a scoring method in L. coquimbensis bulbs , and over 8 bulbs/explant were produced by sectioning the bulbs in four, contrasting with scarce occurrence of new bulblets on intact bulb s .

4 Hypothesis and objectives.

In terms of commercial potential and native germplasm concerns the re is an obvious need to develop asexual propagat ion techniques to assist either plant breeding and conservation programs of Leucocoryne spp. One of the techniques that provide several advantages over sexual propagation is the clonal micropropagation. This system could avoid the l ong life cycle from seed to flowering bulb and the low vegetative propagation rates that naturally occur in these spe cies . In this sense, hybrid s or interest ing clonal lines would benefit from a n in vitro propagation system , using direct organogenesis for example, to propagate reliable and large populations without altering the genetic and phenotypic characteristics of t he original germplasm . Therefore, future studies regarding the in vitro multiplication of Leucocoryne spp. should focus on increasing clonal multiplication rates and on the optimal environmental conditions for the growth and development of the explants.

In this context, we formulated the following hypothesis:

4.1 Hypothesis.

Adjustment and optimization of the culture media, plant growth regulators, explant type and growing temperature produces a more effective initiation process and high er multiplication ra tes during the in vitro propagation of Leucocoryne species, thus improving breeding and conservation outcomes .

4.2 General objective.

To determine in vitro media culture, propagation techniques and

environmental conditions to increase the growth and propagation rate

of Leucocoryne species to support bre eding and conservation pro gram s.

4.3 Specific objectives.

1. To determine the effect of culture media and system on in vitro bulb propagation rates of Leucocoryne sp.

2. To establish the effect of plant growth regulators on in vitro bulb propagation of Leucocoryne sp. bulbs

3. To evaluate the effect of bulb - cutting methods on in vitro bulb propagation rates of Leucocoryne sp.

4. To determine the effect of culture temperature on in vitro seed germination of Leucocoryne sp.

References.

Araneda L, Salas P, Mansur L (2004) Chromosome numbers in the Chilean

endemic genus Leucocoryne (Huilli). J Am Soc Hortic Sci 129:77 – 80.

Bach A, Sochacki D (2012) Propagation of Ornamental Geophytes. In: Ornamental

Geophytes. CRC Press, pp 261 – 286

Bridgen M (2000) Flowering geophytes of Chile have ornamental potential. In:

Seeds: Trade, Production and Technology. pp 218 – 230

Briones C (2003) Leucocoryne sp. Pontificia Universidad Católica de Chile

Chang HS, Chakrabarty E, Hahn EJ, Paek KY (2003) Micropropaga tion of Calla lily

(Zantedeschia albomaculata) via in vitro shoot tip proliferation. Vitr Cell Dev

Biol — Plant 39:129 – 134. doi: 10.1079/IVP2002362

De la Cuadra C, Mansur L (2004) DESCRIPCIÓN DE LA PRIMERA ETAPA DEL

CICLO DE VIDA DE TRES GENOTIPOS DE Leucoco ryne sp.: SEMILLA A

BULBO. Agric Técnica. doi: 10.4067/S0365 - 28072004000200009

De la Cuadra C, Vidal A, Lefimil S, Mansur L (2016) Temperature Effect on Seed

Germination in the Genus Leucocoryne (Amaryllidaceae ). HortScience

51:412 – 415.

Escobar L, Jordan M, Olate E, et al (2008) Direct and indirect in vitro

organogenesis of leucocoryne pupurea (Alliaceae) a chilean ornamental

geophyte. Propag Ornam Plants 8:59 – 64.

Fuentevilla C (2004) Propagación in vitro de algunas especies de Leucocoryne.

Pontificia Univ ersidad Católica de Valparaíso

George EF, Hall MA, Klerk G - J De (2008) Plant Propagation by Tissue Culture.

Springer Netherlands, Dordrecht

Ham F (2002) Evaluación de la germinación in vitro de Leucocoryne coquimbensis

F. Phil., geófita enémica con potencial ornamental. Pontificia Universidad

Católica de Chile

Hartmann HT, Kester DE, Davies FT, Geneve RL (1997) Plant Propagation:

Principles and Practices., Sixth edit. Prentice - Hall, Inc., New Jersey, USA

Jara P, Arancio G, Moreno R, Carmona M (2006) Factores abióticos que

influencian la germinación de seis especies herbáceas de la zona árida de

Chile. Rev Chil Hist Nat 79:309 – 319.

Jorquera C, Moreno R, Arancio G (2007) Domesticación de recursos nativos con

fines comerciales y de conservación: potencia l de uso múltiple de una

bullbosa endémica de chile, Leucocoryne spp. Rev Bras Agroecol 2:1512 –

1517.

Kim HH, Ohkawa K, Nitta E (1998) EFFECTS OF BULB WEIGHT ON THE

GROWTH AND FLOWERING OF LEUCOCORYNE COQUIMBENSIS F.

PHILL. Acta Hortic 341 – 346. doi: 10.1766 0/ActaHortic.1998.454.40

Kritzinger EM, Vuuren RJ Van, Woodward B, et al (1998) Elimimation of external

and internal contaminants in rhizomes of Zantedeschia aethiopica with

commercial fungicides and antibiotics. Plant Cell 61 – 65. doi:

10.1023/A:1005937016 338

Kumar GNM, Guse WE, Larsen FE (2010) Propagation of Plants from Specialized

Structures. Pacific Northwest Ext. Publ. 12.

Kyte L, John K, Scoggins H, Bridgen M (2013) Plants from test tubes: An

Introduction to Micro - Propagation, Fourth. Timber press, Lo ndon

Lancaster JE, Shaw ML, Walton EF (2000) S - Alk(en)yl - l - cysteine sulfoxides,

alliinase and aroma in Leucocoryne. Phytochemistry 55:127 – 130. doi:

10.1016/S0031 - 9422(00)00245 - 4

Mansur L (2002) Ciclo de vida, autoincompatibilidad y plasticidad genética en

diseño y color de flores. In: Leucocoryne un género nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chile, pp 9 – 15

Mansur L, Cisternas M (2005) Leucocoryne talin ensis ( Alliaceae ), a New Species

from Chile. Novon 15:324 – 326.

Mansur L, Gonzalez M, Rojas I, Salas P (2004) Self - incompatibility in the Chilean

Endemic Genus. J Amer Soc Hort Sci 129:836 – 838.

Muñoz M, Moreira A (2000) Los Géneros Endémicos de Monocotile dóneas de

Chile Continental.

http://www.chlorischile.cl/monocotiledoneas/leucocoryne.htm. Accessed 8 Nov

2016

Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays

with Tobacco Tissue Cultures. Physiol Plant 15:473 – 497. doi: 10.1111/ j.1399 -

3054.1962.tb08052.x

Olate E (2006) Chilean Geophytes: Micropropagation and Cut Flower Production.

University of Connecticut

Olate E, Bridgen M (2005) Techniques for the In Vitro Propagation of Rhodophiala

and Leucocoryne spp. Acta Hortic 673:335 – 342 . doi:

10.17660/ActaHortic.2005.673.42

Olate E, Schiappacasse F (2013) Geophyte Research and Production in Chile. In:

Ornamental Geophytes. CRC Press, pp 449 – 470

Paredes K, Delaveau C, Carrasco P, et al (2014) In vitro bulbing for the

propagation of Traubia modesta (Amaryllidaceae), a threatened plant endemic

to Chile. Cienc e Investig Agrar 41:13 – 14. doi: 10.4067/S0718 -

16202014000200007

Pedersen C, Brandt K (1992) A METHOD FOR DISINFECTION OF

UNDERGROUND RHIZOME TIPS OF ALSTROEMERIA AND HELICONIA.

Ac ta Hortic 499 – 504. doi: 10.17660/ActaHortic.1992.325.69

Riedemannn P, Aldunate G (2001) Flora Nativa de valor Ornamental Chile Zona

Centro, Identificación y Propagación, 1st edn. Editorial Andrés Bello, Santiago,

Chile

Roca W, Mroginski L (1991) Cultivo de Tejidos en la Agricultura. Ciat 969.

Salas P, Mansur L (2004) Gene Flow between Parents with Different Ploidy Levels

in a Natural Population of Leucocoryne. J Amer Soc Hort Sci 129:833 – 835.

Salazar R (2001) Efecto de la estratificación de semillas de Leuc ocoryne ixioides,

L. coquimbensis, L purpurea y de distintas técnicas de Propagación

Vegetativa en Leucocoryne ixioides. Universidad de Talca

Schiappacasse F, Peñailillo P, Yáñez P (2002) Propagación de Bulbosas Chilenas

Ornamentales. Editorial Universidad de Talca, Talca, Chile

Slabbert MM, de Bruyn MH, Ferreira DI, Pretorius J (1993) Regeneration of

bulblets from twin scales of Crinum macowanii in vitro. Plant Cell Tissue

Organ Cult 33:133 – 141. doi: 10.1007/BF01983226

Smith RH, Burrows J, Kurten K (1999) Challenges associated with

micropropagation of Zephyranthes and Hippesatrum sp. (Amaryllidaceae). Vitr

Cell Dev Biol - Plant 35:281 – 282. doi: 10.1007/s11627 - 999 - 0032 - y

Sochacki D, Orlikowska T (2005) The obtaining of narcissus plants free from

potyviruses via adventitious shoot regeneration in vitro from infected bulbs. Sci

Hortic (Amsterdam) 103:219 – 225. doi: 10.1016/j.scienta.2004.04.018

Verdugo G (2013) FLOWER BREEDING OF NATIVE PLANTS: THE CHILEAN

EXPERIENCE. Acta Hortic 1000:401 – 405. doi:

10.17660/Acta Hortic.2013.1000.55

Verdugo G, Teixeira JA (2006) From Wild to the Table : Leucocoryne and From

Wild to the Table : Leucocoryne and Chloraea. In: Floriculture, Ornamental

and Plant Biotechnology Volume IV. Global Science Books, pp 356 – 360

Walton EF, Wu RM, Sutherland P (2008) Bulb and inflorescence development in

Leucocoryne ixioides (Hook.) Lind. J Hortic Sci Biotechnol 83:574 – 580.

Zoellner O (2002) Descripción del género Leucocoryne (Alliaceae) su forma de

propagación y distribución. In: Leucocoryne un gé nero nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chile, pp 23 – 27

Zoellner O (1972) El género Leucocoryne. An del Mus Hist Nat Valparaíso 5:9 – 83.

Zuloaga F, M orrone O, Belgrano M (2009) Catálogo de las plantas vasculares del

cono sur (Argentina, southern Brazil, Chile, Paraguay y Uruguay). In: Inst.

Botánica Darwinion, CONICET.

http://www2.darwin.edu.ar/Proyectos/FloraArgentina/fa.htm. Accessed 8 Nov

2016

CHAPTER 2

AN EFFICIENT CLONAL MICROPROPAGATION PRO TOCOL FOR

Leucocoryne , A GEOPHYTE GENUS END EMIC TO CHILE

Alejandro Altamira 1 , Eduardo Olate 1 , Marlene Gebauer 1 , Levi Mansur 2 and Gloria

Montenegro 1

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería

Forestal, Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul,

Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaí so, Avda. San

Fran cisco s/n, La Palma , Quillota, Chile

This chapter was sent to Plant Cell, Tissue and Organ Culture (PCTOC) - Journal of Plant Biotechnology – Springer ( December 2016 )

An efficient clonal micropropagation protocol for Leucocoryne, a geophyte

genus endemic to Chile

Alejandro Altamira 1* , Eduardo Olate 1 , Marlene Gebauer 1 , Levi Mansur 2 and Gloria

Montenegro 1 .

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,

Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul, Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San

Francisco s/n, La Palma, casilla 4 - D, Quillota, Chile

*Corresponding author:

Name: Alejandro Altamira

Address: Departamento de Ciencias Vegetales, Facultad de Agronomía e I ngeniería

Forestal, Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul,

Santiago, Chile

Telephone: +56 9 90915256

E - Mail: aealtami @uc.cl

Keywords: Amaryllidaceae, bulb, plant tissue culture, cytokinin, culture media, cut flower.

Abstract

Leucocoryne (Amaryllidaceae) is a geophyte genus endemic to Chile with exceptional characteristics to use it as cut flower, pot or garden plant. The objective of this work is to develop and improve an in vitro protocol for mass clonal propagation to assist breeding and conservation efforts. We evaluated different in vitro culture systems (agar, cotton), nutrient media (MS, LS, B5), BAP concentrations (0.5, 1.0, 1.5 and 2.0 mg  L - 1 ) and different type of explants (intact bulb, scoring and sectioning) in four Leucocoryne genotypes ( Leucocoryne purpurea, L. vittata, L. ixioides and L. sp. Pichicuy). Using an improved disinfection process it was possible to initiate bulbs in vitro with a low contamination rate of 7 %.

High bulb sprouting percentages were achieved, with values between 80 – 100%.

There was a higher rate of bulb multiplicat ion and higher shoot production when

BAP was added to the growing media . Bulb - cutting methods resulted in the highest multiplication r ates reported to date, obtaining 11 - 16 bulblets per se ctioned bulb, depending on the genotype used.

Abbreviations: BAP - N 6 - benzylaminopurine; IAA - indole - 3 - acetic acid ; MS –

Murashige and Skoog medium (1962); LS - Linsmaier and Skoog medium (1965);

B5 – Gamborg et al. medium (1968) ; Benomyl - Methyl - 1 - (b utylcarbamoyl) - 2 - benzimidazole carbamate ; Captan - N - (triclorometiltio) ciclohex - 4 - eno - 1,2 - dicarboximida .

Introduction

Leucocoryne is a geophyte genus endemic to Chile that belongs to

Amaryllidaceae. It is commonly known as "Huilli" or "Glory - Of - The - Sun " (Mansur

2002; Zoellner 2002; Sassone et al. 2 014) .

These p lants present exceptional characteristics to be used as cut flowers , potted or landscap e plants due to their long life in vase and wide variety of shapes, designs and colors (Mansur 2002; De la Cuadra et al. 2016) .

There are 17 taxa (15 species and 2 subspecies) in the genus . However, classification of Leucocoryne can be very confusing due to its high varia bility in morphology and the occurrence of natural hybrids (Zoellner 1972; Muñoz and

Moreira 2000; Mansur and Cisternas 2005; Olate and Schiappacasse 2013;

Sassone et al. 2014; Jara - Arancio et al. 2014) . P lants develop a small tunicate bulb 1.5 - 2.5 cm in diameter, thin leaves and a 1 - 3 floral scape 30 - 80 cm in height arranged in a terminal umbel . Its colorful flowers are actinomorphic and trimerous

(6 tepals ) varying in quantity from 3 - 4 to 8 - 12 depending on the species colors ranging from white to sky - blue, to purple and violet , forming different patterns and designs depending on the species . A distinctive characteristic of Leucocoryne flowers is the presence of three prominent staminodes ( sterile stamens) that protrude from the central part of the corolla (Mansur 2002; Schiappacasse et al.

2002; Zoellner 2002; Olate and Bridgen 2005; Hoffmann et al. 2015) .

Although Leucocoryne plants ha ve been cultivated in European gardens since the

19 th century (Muñoz and Moreira 2000) , in the 1990’s the ornamental industry started to show interest to produce these species commercially , in order to satisfy the constant demand for new species and cultivars with new shapes and colors.

Th e latter has le d to several research efforts, looking for the domestication and commercialization of these species, in countries like Japan, Israel, The

Netherlands and New Zealand (Kim et al. 1998; Lancaster et al. 2000; Catley

2003; Walton et al. 2008; Olate and Schiappacasse 2013; Hoffmann et al. 2015) .

In the same way, several studies have been conducted in Chile on Leucocoryne sp . , including botanical identification; chromosomal studies; self - incompatibility processes; life cycl e ; plant propagation and breeding techniques. These Chilean studies have been carried out primarily by the Pontificia Universidad Católica de

Valparaíso that has been able to register three new cultivars in addition to the ones offer ed from The Netherlands (Bridgen et al. 2002; De la Cuadra and Mansur

2004; Verdugo an d Teixeira 2006; Verdugo 2013; Olate and Schiappacasse 2013;

De la Cuadra et al. 2016) .

S eeds are usually used for Leucocoryne propagation. However, as in other geophytes species, such as Narcissus and Tulipa , these species have a very long life cycle and juvenile period, requiring between 3 and 4 years to form a floral size bulb from seed (Riedemannn and Aldunate 2001; Mansur 2002; Escobar et al.

2008; Bach and Sochacki 2012) . Because it is self - incompatible (Mansur 2002;

Mansur et al. 2004) , this genus presents a wide range of genetic variability and hence, natural populations with individuals showing segregation can be easily

30 found . T his fact offers a great potential for breeding purposes but at the same time, it makes difficult to propagate heterozygous lines having traits of interest by sexual reproduction (Mansur 2002) . Seed propagation, as the most important technique used for breeding and conservational purposes, has been largely studied in aspects like germination requirements , and other aspects related to the growth and development of the plants (De la Cuadra et al. 2002; De la Cuadra and Mansur

2004; De la Cuadra et al. 2016) .

Until now v egetative propagation is the best way to mai ntain over time the genetic characteristics of Leucocoryne plants, either naturally inherited or produced by controlled crosses. This technique s allow to produce new individuals with identical genotype to the original plant from which the propagule was obt ained (Mansur

2002; Schiappacasse et al. 2002) . It has been reported tha t natural vegetative propagation occurs in Leucocoryne species, from either a two or three - year old plant , which will produce lateral bulblets or dropper s (a type of bulblet that grows deep in the ground ) develop ed from the base of the mother bulb (Riedemannn and

Aldunate 2001; Mansur 2002) . Currently, bulblet separation is the propagation technique commercially used , in spite of its low multiplication rates and its dependence with the plant genotype ( Escobar et al. 2008) . On the other hand, there are artificial techniques that enhance and accelerate the vegetative propagation capacity in geophytes . These are essentially bulb - cutting methods consisting in different levels and types of controlled mech anical damages depending on the genotype to propagate . In the case of the tunicate bulbs, sectioning, scoring, scooping and coring have been used (Schiappacasse et al.

2002) . Schiappacasse et al (2002) conducted studies in Leucocoryne coquimbensis and Leucocoryne ixioides , where the use of bulb sectioning made it possible to increase the bulb - multiplication rates.

In Leucocoryne , as in other geophytes, natural bulblet multiplication rates are very low due to the reduced number of axillary meristems existing in modified stems as bulbs, corms and rhizomes (Bach and Sochacki 2012) . In addition to bulb - cutting methods, there are more sophisticated technique s like in vitro propagation, also known as micropropagation, by which p otentially higher multiplication rates can be achieved (mass clonal propagation). Micropropagation is composed by several techniques by which the totipotency of plant cells is used. During micropropagation small plant pieces or explants are cultured under aseptic conditions onto a media supplemented with nutrients and plant growth regulators . The explants are then cultured in an environment with controlled light, temperature and humidity. Using micropropagation, plant growers are capable to supply stocks for commercialization by multiplying elite cultivars in large scale without altering the genotype (Rout et al. 2006; George et al. 2008; Sharma and Agrawal 2012 ; Bach and Sochacki 2012) . This technique has five stages : Stage 0, which corresponds to the selection of the genotype and the explant selection and preparation . Stage I involves the disinfection of the explant s and their in vitro establishment. During

Stage II the explants are mass propagated by different means and techniques.

During Stage III the in vitro rooting of the explants is achieved. Finally, in Stage IV fully developed plants are acclimatized to ex vitro conditions. Duration and

32 conditions for each of these stages must be adapted to each plant genotype

(George e t al. 2008; Bach and Sochacki 2012; Kyte et al. 2013) .

In vitro culture has been widely used in several ornamental plants as Narcissus,

Lilium, Gladiolus, Freesia, Zantedeshia, Crocus, Hippeastrum, Allium, Tulipa and

Hyacinthus, among others (Bach and Sochacki 2012) . In the case of Leucocoryne only a few studies have been conducted on the in vitro propagation of these species and specific conditions for a successful in vitro culture are still poorly understood (Olate and Bridgen 2005; Verdugo and Teixeira 2006; Escobar et al.

2008) .

In this context, th e objective of this research is to improve the in vitro propagation techniques for Leucocoryne species and cultivars, to obtain a more effic ient method of mass clonal propagation.

Materials and methods.

Plant material.

Dormant bulbs of Leucocoryne purpurea, L. ixioides, L. vittata and the ecotype

‘Pichicuy’ ( reported as L. aff. v ittata ) were harvested from the Leucocoryne breeding program of the Pontificia Universidad Católica de Valparaiso (Fig.1a - d) .

Bulbs of 1.0 ± 0.2 g in weight of each genotype were selected and stored at room temperature under dry conditions until their in vitro initiation .

Disinfection and in vitro establishment.

In order to establish the plant material under in vitro conditions, the dry protective tunics of the bulbs were eliminated and bulbs were washed under running tap water for 5 min. Then, the bulbs were submerged in a fungicide solution of 1g  L - 1

B enomyl ® and 1g  L - 1 C aptan ® for 30 min, rinsed with distilled water and washed with ethanol 95% for 1 min. Subsequently , the explants were disinfected under a laminar flow chamber using a solution of 50 g·L - 1 NaOCl with addition of 2 drops of

Tween® 20 , during 30 min and constant agitation. B ulbs were rinsed three times in sterile distilled water.

After the sterilization all the bulb storage scales and the outer basal plate section were completely remove from the explant until exposing the vegetative growing point. Thus, each explant consisted by the apical growing point and a section of the basal plate (Fig.1e)

The explants were established in culture tubes containing culture media supplemented with 30 g  L - 1 sucrose and pH adjusted to 5.7, and previously sterilized in autoclave at 12 1 °C for 15 min. Macro and micronutrient composition of the media and the use of a gelling agent varied depending on the experiment.

E xplants were cultured under growing chamber conditions at 2 0±1°C, 16 h photoperiod and luminous intensity of 125 µmol·m - 2 ·s - 1 .

Experiment 1: Effect of the basal media and BAP on the in vitro growth and multiplication of Leucocoryne .

T o determine a proper culture media for the in vitro propagation of L eucocoryne purpurea, L. ixioides and L. vittata explants of each of these species were established in 20 mL of three different basal culture media: MS (Murashige and

Skoog 1962) , LS (Linsmaier and Skoog 1965) y B5 (Gamborg et al. 1968) . The media cultures were solidified by add ing 0. 6% agar . Half of the treatments were supplemented with BAP 1.0 mg  L - 1 . Each treatment consisted of fifteen individual explants as repetitions. We evaluated the percentage of bulb sprouting as the bulbs that developed sprouts, percentage of single shoot as the bulbs that developed only one shoot, percentage of multiple shoots as the bulbs that developed two or more shoots, percent age of bulb multiplication as the bulbs that developed lateral bulblets, bulb production as the number of bulbs obtained per an initial bulb and bulb fresh weight as the average final weight obtained per explant.

Experiment 2: Effect of the BAP concentrat ion on the in vitro growth and multiplication of Leucocoryne .

In order to determine an adequate BAP concentration for the in vitro growth and multiplication of L eucocoryne twenty explants of L. purpurea, L. ixioides and L. vittata were established onto 20 mL of MS medium solidified with 0,6% agar and supplemented with 0 (control), 0.5, 1.0, 1.5 and 2.0 mg·L - 1 of BAP. Percentage of bulb sprouting, percentage of single shoot, percentage of multiple shoots,

35 percentage of bulb multiplication, bulb productio n and bulb fresh weight were evaluated as described above.

Experiment 3: Effect of bulb - cutting methods, culture system and the addition of

BAP on the in vitro growth and propagation of Leucocoryne vittata .

L. vittata in vitro cultured - bulbs were used as explants and cultured in MS medium.

T o evaluate two different in vitro bulb - cutting methods : scoring and sectioning.

Scoring consisted in vertical incisions in the basal plate of the bulbs in order to damage the main growing po int and thus to break the apical dominance (Fig. 1g).

S ectioning consisted in to divide each bulb longitudinally into four isolated identical parts (Fig. 1h). Cutting treatments were compared to intact bulbs as controls (Fig.

1f). In addition to the bulb - c utting methods two different culture system s were compared: semi - solid ( 0.6% agar ) and liquid media imbibed in a cotton pad. Bulbs were cultured onto MS medium, and half of the treatments were supplemented with

1.0 mg  L - 1 BAP. Five bulbs of 0.8 + 0.1 g were used in each treatment as replicates.

Percentage of bulb sprouting, percentage of bulb multiplication and bulb production were evaluated as described above.

Experiment 4: Effect of BAP on the in vitro growth and multiplication of

Leucocoryne sectioned bul b s

In vitro bulbs of L . purpurea, L. ixioides, L. vittata and L. sp . Pichicuy of 0.8 + 0.1 g in weight were sectioned in four parts and used as explants (Fig. 1h). Five

36 replicates per treatment were used, each replicate consisted of the four section s that were obtained from the cut of the same bulb. Each of these four sections were cultured in the same container onto MS medium solidified with 0.6% agar , and half of the treatments were supplemented with 1.0 mg  L - 1 BAP . Percentage of bulb sprouting, percentag e of bulb multiplication, bulb production and bulb fresh weight were evaluated as described above.

Statistical analysis.

Results of shooting and multiplication were statistically analyzed using Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) (Zar 2010) . In the case of bulb production and bulb fresh weight data were analyzed performing an analysis of variance (ANOVA) followe d by Bonferroni's multiple comparison test

(P ≤0.05) using GraphPad Prism 5 software (GraphPad Software Inc., San Diego,

California, USA).

Results.

Experiment 1: Effect of the basal media and BAP on the in vitro growth and multiplication of Leucocoryne .

The methods of d isinfection and establishment in vitro resulted in a successful initiation of the explants , with a contamination level of only 7% after 4 weeks of culture.

Bulbs with vegetative s prou ting w ere in average 91.9% of the total, varying between 80 and 100% depending on the treatment. L. purpurea and L. ixioides explants cultivated onto any of the three culture media assayed presented 100% bulb sprouting when they were supplemented with 1.0 mg  L - 1 BAP . I n the case of L. vittata that level of vegetative sprouting was only achieved when MS, B5 and B5 +

1.0 mg  L - 1 BAP media was used (Table 1). A fter 13 weeks of culture the addition of

BAP produced the highest bulb sprouting with multiple shoots per bulb . This difference between the control s and the media supplemented with BAP was even clearer in L. ixiodes bulbs but not in the other genotypes (Table 1). After 30 weeks of culture different multiplication rates were observed among the genotypes. In general, L. ixioides bulbs showed the highest multiplication rate, reaching up to

100% when cultivated onto MS + 1.0 mg  L - 1 BAP medium (Fig. 2a). In this treatment it was produced a n average of 5.3 bulbs/explant, which was higher than in MS without BAP , which produced a multiplication rate of 30.8% and only 1.9 bulbs/explant (Fig.2b).

In most of the genotype/medium combinations bulb multiplication was produced except in the case of L. purpurea , where no multiplication of the bulbs cultured onto

B5 + 1.0 mg  L - 1 BAP medium was observed (Fig. 2 a) . The latter treatment also produced the lowest bulb fresh weight . In the case of L. ixioides the highest values of bulb fresh weight occurred in MS + 1.0 mg  L - 1 BAP, while in L. vittata no difference among treatments was observed (Fig. 2c).

Experiment 2: Effect of the BAP concentration on the in vitro growth and multiplication of Leucocoryne .

In this experiment the in vitro establishment protocol also resulted in a very low contamination rate (7.7%) after 4 weeks of culture.

A high bulb sprouting rate (98.2% average ) was produced , varying between 90 and

100% depending on the treatment (Table 2). L . purpurea and L. vittata sprouting rates did not show any significant differences when different BAP concentrations were added to the culture media . On the other hand, L. ixioides showed the lowest sprouting rate (90%) when 1.0 mg·L - 1 BAP was added to the medium (Table 2).

This genotype also showed a significant higher rate of multiple shoots by the addition of BAP to the media compared to the contro l without BAP . However, n o difference was observed between the different BAP concentrations used (Table 2).

In the other two genotypes no clear tendency was observed between treatments in terms of single or multiple shoot production after 13 weeks of cultu re (Table 2).

Multiplication varied according to the genotype and BAP concentration. The highest multiplication rates were obtained in L. ixioides with values between 47.9%

(control plants) and 94.4% when bulbs were cultured onto MS + 2.0 mg·L - 1 BAP

(Fig. 3a; Fig. 4). In this genotype it was also observed the highest bulb production per explant, with 6.2 and 5. 7 bulbs/explant when cultured onto MS plus 1.0 or 2.0 mg·L - 1 BAP respectively . These results were significantly higher than the ones measured i n the control treatment, with only 2.9 bulbs/explant produced . In L. purpurea and L. vittata no differences were observed between the different treatments (Fig. 3b).

The highest bulb fresh weight was produced in L. purpurea and L. ixioides when

BAP was ad ded to the culture media. I n the case of L. vittata no significant difference w as observed among treatments (Fig. 3c).

Experiment 3: Effect of bulb - cutting methods, culture system and the addition of

BAP on the in vitro growth and propagation of Leucocoryne vittata .

A fter 8 weeks of culture 100% of sprouting was observed in either intact (control) or treated bulbs with one of the cutting methods , when they were cultured onto liquid media imbibed in cotton pads and supple mented with 1.0 mg·L - 1 BAP (Table

3). The latter was also observed in sectioned bulbs and cultured onto MS media solidified with agar and supplemented with 1.0 mg·L - 1 BAP. The lowest sprouting rates (40%) were produced in intact bulbs , when they were cultu red onto media supplemented with BAP, independently of the system used (Table 3).

A tendency to increase the multiplication rate was observed when bulb - cutting treatments were used , especially in those including sectioning (Figure 5). I ntact bulb treatments showed multiplication rates below 20% (Table 3). The highest bulb production occurred in sectioned bulbs cultured onto media solidified with agar either supplemented or not with BAP, producing 13 and 9.2 bulbs/explant respectively. Independentl y of the culture system used and the addition or not of

BAP, the lowest bulb production was clearly observed in intact bulbs, showing values between 0 y 0.6 bulbs/explant (Table 3). In terms of culture system, differences between treatments were observed o nly when sectioned bulbs were cultured onto media without BAP . In this case, bulbs cultured on media with agar produced significantly higher number of bulbs than the ones cultured on cotton pads imbibed with liquid media, with values of 13 and 4.4 bulbs/ex plant respectively (Table 3).

Experiment 4: Effect of BAP on the in vitro growth and multiplication of

Leucocoryne sectioned bulb s.

Considering the results from experiment 3, sectioning was used o n four

Leucocoryne genotypes cultured onto growing media either supplemented or not with BAP. In L. purpurea sprouting rates of 66.6% were observed in control plants and 50% when BAP was added to the media , but showing no statistical difference between those groups of plants . On the o ther hand, L. ixioides and L. vittata plants showed high er sprouting rates reaching 80% and 100% but there was no influence of BAP addition to the growing media . In the case of the plants belonging to the L.

41 sp . Pichicuy only 40% of them sprouted in the ab sence of BA, but 80% of them did sprout when BAP was added to the media .

In terms of multiplication , after 6 months of culture it was possible to observe different rates between genotypes, although no statistical difference was founded when data were analy zed (Fig. 6a). Thus b ulb production varied considerably among genotypes from 2.6 to 16 bulbs/explant (Fig. 7). In L. sp . Pichicuy bulb production was significantly larger in BAP treatments compared to the controls , showing values previously mentioned. In L. purpurea and L vittata differences were also observed but they all remained below the threshold for statistical difference . In the last genotype, L. ixioides, no differences in bulb production were also observed among treatments , with values between 12. 4 and 14 bulbs/explant for the control s and BAP treatments, respectively (Fig. 6b).

The addition of BAP to the growing media did not affect the fresh weight of the bulbs . Values varied between 0.07 and 0.19 g /bulb with no significant differences between tr eatments (Fig 6c).

Discussion.

We developed an effective disinfection and in vitro establishment protocol for

Leucocoryne bulbs (Stage 0 and 1) . Explant disinfection is one of the most challenging steps in the process of the in vitro clonal propagation . The protocol to use w ill depend on the type and origin of the explant and its sensitivity to

42 disinfectant agents (Smith et al. 1999; Kyte et al. 2013) . In addition, the combination of explant , culture media and controlled environment gives the perfect conditions for the proliferation of any pathogenic and non - pathogenic microorganism. Therefore , a proper disinfection system must be implemented to avoid both explant contamination and extensive tissue damage (Mroginski and

Roca 1991) . The disinfection protocol developed in this work resulted in a very effective and safe protocol for the in vitro establishment of Leucocoryne bulbs, sh owing in average only 7% of contamination . This is quite remarkable knowing the major difficulty that is faced when establishing vegetative tissues that grow on the ground or even underground in direct contact with soil, such as bulbs, corms, rhizomes and tubers. In such cases, disinfection protocols should be labor - intensive and more aggressive for the explant, using combinations of agents and/or longer disinfections periods (Pedersen and Brandt 1992; Slabbert et al. 1993;

Kritzinger et al. 1998; Smith et al. 1999; Chang et al. 2003; Sochacki and

Orlikowska 2005; Paredes et al. 2014 ) . Studies in other geophytes species have also reported the difficulty and importance of the disinfection process. In vitro establishment of Zantedeschia sp. has evidenced low success when using conventional disinfection techniques (Kritzinger et al. 1998; Chang et al. 2003) . In the case of Narcissus sp. , which possess a tunicate bulb similar to Leucocoryne , problems during the in vitro initiation have been also reported, with high bacterial and fungal contamination rates, surpassed in some cases only by using hot water treatments plus use of fungicides (Sochacki and Orlikowska 2005) . High contamination rates have been also reported in other bulb species: 20 - 40% in

Crinum macowanni (Slabbert et al. 1993) , 90% in Eucomis autumnalis (Ault 1995) ,

20 - 90% in Hippeastrum sp. (Smith et al. 1999) and 50 - 80% in Traubia modesta , another endemic species to Ch ile with ornamental potential (Paredes et al. 2014) .

The d isinfec tion protocol developed in this work resulted in low contamination rates , even though it is a more aggressive and laborious procedure than the conventional ones , survival of the explants was not negatively affected given the high sprouting rates obtained ( 80% ) . In previous studies including Leucocoryne bulbs, in vitro contamination has been the main limitation for further advance in a suitable protocol, with values between 40 - 100% (Fuentevilla, unpublished). Due to this problem, the few in vitro studies on Leucocoryne micropropagation ha ve started their plant material from seeds (Escobar et al. 2008) .

The different experiments of this work resulted in the development of a suitable protocol for mass clonal propagation for the Leucocoryne genus. Among the different media tested, basal MS medium was the most suitable for in vitro growth and multiplication . It was observed high levels of sprouting and a normal growth and development of the explants. Previous works on the in vitro propagation of

Leucocoryne have only used MS medium (Escobar et al., 2008; Olate & Br idgen,

2005) . The o ther two media used in this work, LS and B5, also showed to be suitable for Leucocoryne in vitro culture but in the case of B5 media a lower fresh weight of the bulbs was obtained. These three media have previously been used for the micropropagation of other geophyte species. For instance , MS has resulted in better results than B5 in terms of embryo germination in Alstroemeria (Lu and

Bridgen 1996) and also on callus formation and shoot d ifferentiation in Lilium longiflorum (Ramsay et al. 2003) . Besides, MS medium has been widely used in

44 different ornamental geophytes species, for example, Zhephyra elegans (Vidal et al. 2012) , Narcissus sp. (Sochacki and Orlikowska 2005) , Hippeastrum sp . (Smith et al. 1999) , Mus cari muscarimi (Ozel et al. 2015) and Zeph y ranthes sp. (Smith et al. 1999) . B5 medium has been successfully used i n other geophytes from the

Allium genus (Dunstan and Short 1977; Shahidu l Haque et al . 1997 ; Barandiaran et al . 1999 ; Martıń ez et al. 2000) in the same way as LS medium have been used in Allium sp . (Nagakubo et al. 1993; Ayabe an d Sumi 1998) , Crocus sp. (Mir et al.

2011) and Narcissus sp. (Stone 1973) .

The addition of BAP to the culture media contributed to a higher multiplication rate like the one observed o n L. ixioides . However, no significant differences were observed over the sprouting and multiplication rates when using different BAP concentrations. Bulb production values of 6.2 and 5.7 bulbs/explants were possible to obtain when plants were established o n MS medium supplemented with 1 or 2 mg·L - 1 BAP respectively, which contrast with the much lower values observed in natural populations. In nature Leucocoryne bulbs are able to produce 1 - 2 bulblets per plant every other year but only when they have reached their mature stage

(Riedemannn and Aldunate 2001) . A bulb would reach their maturity from seed only after 2 or 4 years of growth (Riedemannn and Aldunate 2001; Mansur 2002) .

We have also concluded that the addition of BAP to the growing media produced a larger explant fresh weight in L. purpurea and L. ixioides than the controls . In previous studies Escobar et al. (2008) reported obtaining 3 - 4 bulbs/explant after seven months of culture of Leucocoryne plants onto MS medium without any pl ant growth regulators . That particular study produced the in vitro bulbs via indirect

45 organogenesis and further induction of the bulbs on MS supplemented with 1.0 mg·L - 1 BAP. In Allium cepa micropropagation , it has been reported that 1.0 mg·L - 1

BAP added to the growing media resulted in the best treatment , however higher

BAP concentration s resulted in a lower production of new plantlet s (Kamstaityte and Stanys 2004) . In Muscari azureum 1.0 mg·L - 1 BAP plus 0.25 mg·L - 1 IAA resulted in a larger shoot and bulblet production (Uranbey 2011) . Bridgen et al.

(2009) also reported the use of MS medium supplemented with 2.0 mg·L - 1 BAP for an adequate Alstroemeria sp. micropropagation.

Bulb - cutting methods allow accelerating or increasing the efficiency of vegetative propagation of some geophyte species. These methods include bulb division or

"sectioning", "scoring", "scooping" and "coring". All these methods are focused in a controlled damage of the main growing point and basal plate and thus favor new bulblet production (Hartmann et al. 1997; Kumar et al. 2010; Schiappacasse et al.

2002) . Hyacinthus is an important plant genus in which these techniques have been commercially ad opted, producing up to 60 bulblets from a single bulb using scooping, although these bulblets will require four to five years of growth before flowering (Hartmann et al. 1997) . In many other bulb species it has been a lso reported successful use of this kind of technique. van Leeuwen and van der

Weijden ( 1997) using sectioning was able to increase the natural propagation rates not only in Chionodoxa ( from 1.9 to 6.5 ) , but also in Gelanthus ( 2.7 to 6.5 ) , Muscari

( 2.7 to 10.0 ) and Scilla ( 1 .1 to 8.0 ) . Species with larger bulbs like those belonging to Hippeastrum , allow a more intensive sectioning obtaining up to 28.5 bulbs when the initial bulb has been divided in 24 sections (Sandler - Ziv et al. 1997) . Scoring

46 has been reported to be a successful technique in Nerine (Mori et al. 1997) and in

Crinum x powellii (Knippels 2012) . In the present work we observed a higher multiplication rate when bulb - cutting methods were applied compared to intact bulbs, however by using scoring we obtained a lower bulb multiplication and production rates than sectioning. Similar results were observed by Solgi et al.

( 2015) in Fritillaria imperialis where the number of bulbs obtained from an initial bulb was greater by using sectioning compared to scoring. Schiappacasse et al.

(2002) working with traditional propagation reported the production of 2.2 bulblets per initial bulb in L. coquimbensis and 5 bulblets in L. ixioides by using sectioning in two vertical sections. By using in vitro bulb sectioning we obtained up to 11.3 bulbs/explant in L. purpurea and 14 bulbs/ explant in L. vittata and L.ixioides . In the case of L. sp. Pichicuy, the addition of BAP significantly increased bulb production from 2.6 to 16 bulbs/explant. Previous ly Olate and Bridgen (2005) reported on

Leucocoryne coquimbensis 7 bulbs/explant using scoring, 8 bulbs using a 4 - sectioned bulb and almost no bulblets from intact bulbs, but in that case no BA P was added to the media .

When comparing bulb - cutting methods and culture system i n L. vittata , a higher bulb production was achieved using MS medium solidified with agar than using liquid MS media imbibed in cotton pads . This result contradicts the potential advantages of the liquid media in terms of producing a higher multiplication rate reported by Kim et al. ( 2003) in Allium sativum .

In conclusion, in this work we have developed a highly efficient mass clonal micropropagation of Leucocoryne . Our protocol also includes an effective

47 disinfection and explant preparation method for a successful in vitro establishment.

By using different bulb - cutting methods, culture media and growth regulator addition, we also have been able to culture four different Leucocoryne genotypes, achieving the highest multiplication rates reported to date. Th ese results represe nt an important set of tools to be used by conservational or commercial purposes, such as breeding programs or nurseries . These techniques may also contribute to the advance in the knowledge of the propagation of other native and ornamental geophyte specie s.

References.

Ault JR (1995) In vitro propagation of Eucomis autumnalis , E . comosa , and E .

zambesiaca by twin - scaling. HortScience 30:1441 – 1442.

Ayabe M, Sumi S (1998) Establishment of a novel tissue culture method, stem - disc

culture, and its practical application to micropropagation of garlic ( Allium

sativum L.). Plant Cell Rep 17:773 – 779. doi: 10.1007/s002990050481

Bach A, Sochacki D (2012) Propagation of Ornamental Geophytes. In: Ornamental

G eophytes. CRC Press, pp 261 – 286

Barandiaran X, Martín N, Rodríguez - Conde MF, et al (1999) Genetic variability in

callus formation and regeneration of garlic ( Allium sativum L. ). Plant Cell Rep

18:434 – 437. doi: 10.1007/s002990050599

Bridgen M, Kollman E, Ch unsheng Lu (2009) Interspecific hybridation of

Alstroemeria for the development of new ornamental plants. Acta Hortic

836:73 – 78. doi: 10.17660/ActaHortic.2009.836.9

Bridgen MP, Olate E, Schiappacasse F (2002) Flowering geophytes from Chile.

Acta Hortic 570 :75 – 80. doi: 10.17660/ActaHortic.2002.570.6

Catley JL (2003) Temperature and Irradiance Effects on Flowering of Two Species

of Leucocoryne. J Am Soc Hortic Sci 128:809 – 814.

Chang HS, Chakrabarty E, Hahn EJ, Paek KY (2003) Micropropagation of Calla lily

( Za ntedeschia albomaculata ) via in vitro shoot tip proliferation. Vitr Cell Dev

Biol — Plant 39:129 – 134. doi: 10.1079/IVP2002362

De la Cuadra C, Mansur L (2004) Descripción de la primera etapa del ciclo de vida

de tres genotipos de Leucocoryne sp .: semilla a bu lbo. Agric Técnica. doi:

10.4067/S0365 - 28072004000200009

De la Cuadra C, Mansur L, Verdugo G, Arriagada L (2002) Deterioro de las

semillas de Leucocoryne spp. en función del tiempo de almacenaje. Agric

Técnica 62:3 – 5. doi: 10.4067/S0365 - 28072002000100005

D e la Cuadra C, Vidal A, Lefimil S, Mansur L (2016) Temperature Effect on Seed

Germination in the Genus Leucocoryne (Amaryllidaceae ). HortScience

51:412 – 415.

Dunstan DI, Short KC (1977) Improved Growth of Tissue Cultures of the Onion ,

Allium cepa . Physiol Plant 41:70 – 72. doi: 10.1111/j.1399 - 3054.1977.tb01525.x

Escobar L, Jordan M, Olate E, et al (2008) Direct and indirect in vitro

organogenesis of Leucocoryne pupurea (Alliaceae) a chilean ornamental

geophyte. Propag Ornam Plants 8:59 – 64.

Gamborg OL, Miller R a., Ojima K (1968) Nutrient requirements of suspension

cultures of soybean root cells. Exp Cell Res 50:151 – 158. doi: 10.1016/0014 -

4827(68)90403 - 5

George EF, Hall MA, Klerk G - J De (2008) Plant Propagation by Tissue Culture.

Springer Netherlands, Dordrech t

Hartmann HT, Kester DE, Davies FT, Geneve RL (1997) Plant Propagation:

Principles and Practices., Sixth edit. Prentice - Hall, Inc., New Jersey, USA

Hoffmann A, Watson J, Flores AR (2015) Flora Silvestre de Chile, Cuando el

Desierto Florece Vol.I. Monocoti ledóneas, 1st edn. Ediciones Fundación

Claudio Gay, Zapallar, Chile

Jara - Arancio P, Arroyo MTK, Guerrero PC, et al (2014) Phylogenetic perspectives

on biome shifts in Leucocoryne (Alliaceae) in relation to climatic niche

evolution in western South America. J Biogeogr 41:328 – 338. doi:

10.1111/jbi.12186

Kamstaityte D, Stanys V (2004) Micropropagation of onion ( Allium Allium cepa L

.). Acta Univ Latv Biol 676:173 – 176.

Kim E, Hahn E, Murthy H, Paek K (2003) High frequency of shoot multiplication

and bulblet fo rmation of garlic in liquid cultures. Plant Cell Tissue Organ Cult

73:231 – 236. doi: 10.1023/A:1023029302462

Kim HH, Ohkawa K, Nitta E (1998) Fall flowering of Leucocoryne coquimbensis F.

Phil. after long - term bulb storage treatments. HortScience 33:18 – 20.

Knippels PJM (2012) Advanced in Vivo Propagation Techniques for Specialty

Bulbs. Floric Ornam Biotechnol 6:154 – 157.

Kritzinger EM, Vuuren RJ Van, Woodward B, et al (1998) Elimimation of external

and internal contaminants in rhizomes of Zantedeschia aethiop ica with

commercial fungicides and antibiotics. Plant Cell 61 – 65. doi:

10.1023/A:1005937016338

Kumar GNM, Guse WE, Larsen FE (2010) Propagation of Plants from Specialized

Structures. Pacific Northwest Ext. Publ. 12.

Kyte L, John K, Scoggins H, Bridgen M (2 013) Plants from test tubes: An

Introduction to Micro - Propagation, Fourth. Timber press, London

Lancaster JE, Shaw ML, Walton EF (2000) S - Alk(en)yl - l - cysteine sulfoxides,

alliinase and aroma in Leucocoryne . Phytochemistry 55:127 – 130. doi:

10.1016/S0031 - 942 2(00)00245 - 4

Linsmaier EM, Skoog F (1965) Organic Growth Factor Requirements of Tobacco

Tissue Cultures. Physiol Plant 18:100 – 127. doi: 10.1111/j.1399 -

3054.1965.tb06874.x

Lu C, Bridgen M (1996) Effects of genotype, culture medium and embryo

developmental stage on the in vitro responses from ovule cultures of

interspecific hybrids of Alstroemeria . Plant Sci 116:205 – 212. doi:

10.1016/0168 - 9452(96)04385 - 3

Mansur L (2002) Ciclo de vida, autoincompatibilidad y plasticidad genética en

diseño y color de flores. In: Leucocoryne un género nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chil e, pp 9 – 15

Mansur L, Cisternas M (2005) Leucocoryne talinensis ( Alliaceae ), a New Species

from Chile. Novon 15:324 – 326.

Mansur L, Gonzalez M, Rojas I, Salas P (2004) Self - incompatibility in the Chilean

Endemic Genus Leucocoryne Lindley. J Am Soc Hortic S ci 129:836 – 838.

Martıń ez LE, Agüero CB, López ME, Galmarini CR (2000) Improvement of in vitro

gynogenesis induction in onion ( Allium cepa L .) using polyamines. Plant Sci

156:221 – 226. doi: 10.1016/S0168 - 9452(00)00263 - 6

Mir JI, Ahmed N, Wani SH, et al (2011 ) In vitro development of microcorms and

stigma like structures in saffron ( Crocus sativus L.). Physiol Mol Biol Plants

16:369 – 373. doi: 10.1007/s12298 - 010 - 0044 - 4

Mori G, Hirai H, Imanishi H (1997) Vegetative propagation of Nerine bulbs by

cross - cutting. A cta Hortic 377 – 382. doi: 10.17660/ActaHortic.1997.430.58

Mroginski L, Roca WM (1991) Establecimiento de cultivos de tejidos vegetales in

vitro. Cultiv. tejidos en la Agric. Fundam. y Apl. 20 – 40.

Muñoz M, Moreira A (2000) Los Géneros Endémicos de Monocotile dóneas de

Chile Continental.

http://www.chlorischile.cl/monocotiledoneas/leucocoryne.htm. Accessed 8 Nov

2016

Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays

with Tobacco Tissue Cultures. Physiol Plant 15:473 – 497. doi: 10.1111/j.1399 -

3054.1962.tb08052.x

Nagakubo T, Nagasawa A, Ohkawa H (1993) Micropropagation of garlic through in

vitro bulblet formation. Plant Cell Tissue Organ Cult 32:175 – 183. doi:

10.1007/BF00029840

Olate E, Bridgen M (2005) Techniques for the In Vitro Propagation of Rhodophiala

and Leucocoryne spp. Acta Hortic 673:335 – 342. doi:

10.17660/ActaHortic.2005.673.42

Olate E, Schiappacasse F (2013) Geophyte Research and Production in Chile. In:

Ornamental Geophytes. CRC Press, pp 449 – 470

Ozel CA, Khawar KM, Un al F (2015) Factors affecting efficient in vitro

micropropagation of Muscari muscarimi Medikus using twin bulb scale. Saudi

J Biol Sci 22:132 – 138. doi: 10.1016/j.sjbs.2014.09.007

Paredes K, Delaveau C, Carrasco P, et al (2014) In vitro bulbing for the

prop agation of Traubia modesta (Amaryllidaceae), a threatened plant endemic

to Chile. Cienc e Investig Agrar 41:13 – 14. doi: 10.4067/S0718 -

16202014000200007

Pedersen C, Brandt K (1992) A method for desinfection of underground rhizome

tips of Alstroemeria and He liconia. Acta Hortic 499 – 504. doi:

10.17660/ActaHortic.1992.325.69

Ramsay JL, Galitz DS, Lee CW (2003) Basal Medium and Sucrose Concentration

Influence Regeneration of Easter Lily in Ovary Culture. HortScience 38:404 –

406.

Riedemannn P, Aldunate G (2001) Fl ora Nativa de valor Ornamental Chile Zona

Centro, Identificación y Propagación, 1st edn. Editorial Andrés Bello, Santiago,

Chile

Rout GR, Mohapatra A, Jain SM (2006) Tissue culture of ornamental pot plant: A

critical review on present scenario and future p rospects. Biotechnol Adv

24:531 – 560. doi: 10.1016/j.biotechadv.2006.05.001

Sandler - Ziv D, Cohen A, Ion A, et al (1997) Improving Hippeastrum propagation

and bulbil yield by cutting and incubation techniques. Acta Hortic 355 – 360.

doi: 10.17660/ActaHortic.19 97.430.55

Sassone AB, Arroyo - Leuenberger SC, Giussani LM (2014) Nueva circunscripción

de la tribu Leucocoryneae (Amaryllidaceae, Allioideae). Darwiniana 2:197 –

206. doi: 10.14522/darwiniana.2014.22.584

Schiappacasse F, Peñailillo P, Yáñez P (2002) Propagaci ón de Bulbosas Chilenas

Ornamentales. Editorial Universidad de Talca, Talca, Chile

Shahidul Haque M, Wada T, Hattori K (1997) High frequency shoot regeneration

and plantlet formation fromroot tip of garlic. Plant Cell Tissue Organ Cult

50:83 – 89. doi: 10.10 23/A:1005973929862

Sharma A, Agrawal V (2012) Tissue culture aspects of ornamental plants. CIBTech

J Biotechnol 1:40 – 48.

Slabbert MM, de Bruyn MH, Ferreira DI, Pretorius J (1993) Regeneration of

bulblets from twin scales of Crinum macowanii in vitro. Plant Cell Tissue

Organ Cult 33:133 – 141. doi: 10.1007/BF01983226

Smith RH, Burrows J, Kurten K (1999) Challenges associated with

micropropagation of Zephyranthes and Hippesatrum sp . (Amaryllidaceae). Vitr

Cell Dev Biol - Plant 35:281 – 282. doi: 10.1007/s11627 - 999 - 0032 - y

Sochacki D, Orlikowska T (2005) The obtaining of narcissus plants free from

potyviruses via adventitious shoot regeneration in vitro from infected bulbs. Sci

Hortic (Amsterdam) 103:219 – 225. doi: 10.1016/j.scienta.2004.04.018

Solgi M, Dastyari K, Hadavi E (2015) The Evaluation Effects of Some Vegetative

Propagation Methods and Plant Growth Regulators on Bulblet Production Rate

in Crown Imperial ( Fritillaria imperialis L .). J Hortic For ans Biotechnol 19:1 –

Stone OM (1973) The el imination of viruses from Narcissus tazetta cv. Grand

Soleil d’Or, and rapid multiplication of virus - free clones. Ann Appl Biol 73:45 –

52. doi: 10.1111/j.1744 - 7348.1973.tb01308.x

Uranbey S (2011) In vitro bulblet regeneration from immature embryos of

endang ered and endemic Muscari azureum. Arch Biol Sci 63:209 – 215. doi:

10.2298/ABS1101209U van Leeuwen PJ, van der Weijden JA (1997) Propagation of specialty bulbs by

chipping. Acta Hortic 351 – 354. doi: 10.17660/ActaHortic.1997.430.54

Verdugo G (2013) Flower bre eding of native plants: the Chilean Experience. Acta

Hortic 1000:401 – 405. doi: 10.17660/ActaHortic.2013.1000.55

Verdugo G, Teixeira JA (2006) From Wild to the Table : Leucocoryne and From

Wild to the Table : Leucocoryne and Chloraea. In: Floriculture, Ornamental

and Plant Biotechnology Volume IV. Global Science Books, pp 356 – 360

Vidal AK, Han D, Nakano M, Niimi Y (2012) Decreased time from seed to flowering

corm size in Zephyra el egansvia in vitro cultivation. Cienc e Investig Agrar

39:577 – 584. doi: 10.4067/S0718 - 16202012000300017

Walton EF, Wu RM, Sutherland P (2008) Bulb and inflorescence development in

Leucocoryne ixioides (Hook.) Lind. J Hortic Sci Biotechnol 83:574 – 580.

Zar J (2010) Biostatistical Analysis, Fifth. Prentice Hall Inc., New Jersey, USA

Zoellner O (2002) Descripción del género Leucocoryne (Alliaceae) su forma de

propagación y distribución. In: Leucocoryne un género nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chile, pp 23 – 27

Zoellner O (1972) El género Leucocoryne . An del Mus Hist Nat Valparaíso 5:9 – 83.

Table 1 Effect of basal media and BAP addition on the in vitro sprouting of three Leucocoryne genotypes. Basal BAP Sprouting Single shoot Multiple shoots Genotype - 1 medium (mg*L ) (%) (%) (%) 0.0 86.7 b 86.7 b 0.0 b MS 1.0 100.0 a 71.4 b 28.6 a 0.0 83.3 b 75.0 b 8.3 ab L. purpurea LS 1.0 100 .0 a 92.9 ab 7.1 b 0.0 91.7 ab 91.7 ab 0.0 b B5 1.0 100 .0 a 100.0 a 0.0 b 0.0 92.3 ab 92.3 a 0.0 b MS 1.0 100.0 a 53.3 b 46.7 a 0.0 85.7 b 85.7 ab 0.0 b L. ixioides LS 1.0 100 .0 a 64.3 b 35.7 a 0.0 85.7 b 85.7 ab 0.0 b B5 1.0 100.0 a 53.3 b 46.7 a 0.0 100.0 a 93.3 ab 6.7 bc MS 1.0 86.7 b 73.3 b 13.3 b 0.0 93.3 ab 93.3 ab 0.0 c L. vittata LS 1.0 86. 7 b 40.0 c 46.7 a 0.0 100.0 a 100.0 a 0.0 c B5 1.0 100 .0 a 80.0 b 20.0 b Sprouting indicates the percentage of bulbs that developed sprouts. Single shoot indicates the percentage of bulbs that developed only one shoot. Multiple shoots indicates the number of bulbs that developed two or more shoots. Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) .

Table 2 Effect of different BAP concentration s added to the growing media on the in vitro sprouting of three Leucocoryne genotypes. BAP Sprouting Single shoot Multiple shoots Genotype - 1 (mg*L ) (%) (%) (%) 0.0 100.0 a 94.7 ab 5.3 bc 0.5 100.0 a 65.0 c 35.0 a L. purpurea 1.0 100.0 a 100.0 a 0.0 c 1.5 100.0 a 87.5 b 12.5 b 2.0 100.0 a 55.0 c 45.0 a 0.0 100.0 a 89.5 a 10.5 b 0.5 100.0 a 47.4 bc 52.6 a L. ixioides 1.0 90.0 b 30.0 c 60.0 a 1.5 94.4 ab 50.0 bc 44.4 a 2.0 100.0 a 61.1 b 38.9 a 0.0 93.3 a 80.0 b 13.3 a 0.5 100.0 a 100.0 a 0.0 b L. vittata 1.0 100.0 a 85.0 b 15.0 a 1.5 100.0 a 82.4 b 17.6 a 2.0 93.8 a 68.8 b 25.0 a Sprouting indicates the percentage of bulbs that developed sprouts. Single shoot indicates the percentage of bulbs that developed only one shoot. Multiple shoots indicates the number of bulbs that developed two or more shoots. Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) .

Table 3 Effect of the in vitro cutting method, culture system and the addition of BAP to the growing media on sprouting , bulb multiplicati on and bulb production in Leucocoryne vittata . Bulb Bulb BAP Sprouting Cutting Culture multiplication production method system (mg*L - 1 ) (%) (%) ( bulbs/explant)

0.0 40.0 b 20.0 cd 0.6 c Agar Intact bulb 1.0 80.0 ab 20.0 cd 0.2 c (control) Liquid 0.0 40.0 b 0.0 d 0.0 c ( Cotton pads) 1.0 100.0 a 20.0 cd 0.6 c 0.0 80.0 ab 60.0 bc 2.8 bc Agar 1.0 80.0 ab 60.0 bc 3.2 bc Scoring Liquid 0.0 60.0 b 40.0 bc 0.4 c ( Cotton pads) 1.0 100.0 a 80.0 ab 2.6 bc 0.0 100.0 a 100.0 a 13.0 a Agar 1.0 80.0 ab 80.0 ab 9.2 ab Sectioning Liquid 0.0 80.0 ab 80.0 ab 4.4 bc ( Cotton pads) 1.0 100.0 a 100.0 a 7.8 ab Sprouting indicates the percentage of bulbs that developed sprouts. Bulb multiplication indicates the percentage of bulbs that developed lateral bulblets. Bulb production indicates the number of bulbs obtained per an initial bulb. Different letters in the same column indicate statistical differences based on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) and analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

Fig. 1 Leucocoryne genotypes and explant types . (a) Leucocoryne purpurea ; (b) Leucocoryne ixioides ; (c) Leucocoryne vittata ; (d) Leucocoryne sp. Pichicuy; (e) Initiation e xplant composed by the inner basal plate section plus the vegetative growing point; (f) Intact bulb; (g ) Scoring and (h) Sectioning done to in vitro bulb s .

Fig. 2 Effect of basal media and the addition of BAP on three Leucocoryne genotypes. (a) Percentage of bulbs that developed lateral bulblets ; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant. Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

Fig. 3 Effect of BAP concentration on three Leucocoryne genotypes . (a) Percentage of bulbs that developed lateral bulblets ; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant . Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

Fig. 4 Effect of BAP concentration on in vitro Leucocoryne ixioides : (a) MS medium (control) ; (b) MS+ 0.5mg·L - 1 BAP; (c) MS+ 1.0mg·L - 1 BAP d) MS+ 1.5mg·L - 1 BAP and (e) MS+ 2.0mg·L - 1 BAP .

Fig. 5 Leucocoryne bulb production in response to different bulb - cutting methods . (a) Intact bulb (control) ; (b) Scoring ; and (c) Sectioning .

Fig. 6 Effect of sectioning technique and BAP addition on four Leucocoryne genotypes. (a) Percentage of bulbs that developed lateral bulblets ; (b) Number of bulbs obtained per an initial bulb; and (c) Average final fresh weight obtained per explant. Different letters indicate statistical differences for each genotype based, in the case of bulb multiplication, on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) and for bulb production and bulb fresh weight, on analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

Fig. 7 Visual appearance of a Leucocoryne bulb and adventitious bulblet produced after in vitro sectioning. (a) Sectioned original explant; (b) Adventitious new bulblets.

CHAPTER 3

AN EFFICIENT IN VITRO PRODUCTION SYSTEM FO R

Leucocoryne s p p. PLANTS INITIATED FRO M SEEDS

Alejandro Altamira 1 , Eduardo Olate 1 , Marlene Gebauer 1 , Levi Mansur 2 , Carlos De

la Cuadra 2 and Gloria Montenegro 1* .

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería

Forestal, Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul,

Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaí so, Avda. San

Francisco s/n, La Palma, casilla 4 - D, Quillota, Chile

This chapter will be sent to Propagation of Ornamental Plants (POP) – International Journal (March 2017)

An efficient in vitro production system for Leucocoryne spp. plants initiated

from seeds

Alejandro Altamira 1* , Eduardo Olate 1 , Marlene Gebauer 1 , Levi Mansur 2 , Carlos De la

Cuadra 2 and Gloria Montenegro 1 .

1 Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería Forestal,

Pontificia Universidad Católica de Chile. Vicuña Mackenna 4860, Macul, Santiago, Chile.

2 Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Avda. San

Francisco s/n, La Palma, casilla 4 - D, Quillota, Chile

*Corresponding author:

Name: Alejandro Altamira

Address: Departamento de Ciencias Vegetales, Facultad de Agronomía e Ingeniería

Forestal, Pontificia Universidad Católica de Chi le. Vicuña Mackenna 4860, Macul,

Santiago, Chile

Telephone: +56 9 90915256

E - Mail: [email protected]

Keywords: Amaryllidaceae, in vitro germination, geophyte, sprouting, bulb, optimal temperature

Abstract

Leucocoryne spp. are geophyte plants that belongs to Amaryllidaceae, which ha ve a great potential in the ornamental plant industry due to its colorful flowers, distinctive shape and long vase life . The plants are suitable to be used as cut flowers, potted plants and also as landscap e plants . This research focused on optimizing an in vitro seed germination protocol and the subsequent bulb growth and development of three Leucocoryne genotypes. Culture temperatures of 15 and

20 °C were used from seed initiation until bulblet formation . Seeds were in vitro ini tiated on 12.5% MS medium and subsequently transferred to 50% MS media optionally supplemented with BAP. Germination rates varied between 85 - 98% at

15 ºC and 48 - 83% at 20°C, depending on the genotype. In all the genotypes, the highest bulb growth and devel opment occurred at 15°C. Use of 50% MS supplemented with BAP broke down bulb dormancy and stimulated lateral bulblets.

Final fresh weight of the bulbs was higher at 15°C in two of the genotypes and varied between 0.04 and 0.10 g per bulb .

Abbreviations: BAP - N 6 - benzylaminopurine; MS – Murashige and Skoog medium

(1962)

Introduction .

The o rnamental crop industry constantly demands for new cultivars or species with new shapes and color s (Olate and Bridgen 2005) . Leucocoryne is a geophyte genus endemic to Chile which belongs to Amaryllidaceae and possess exceptional qualities to be used as cut flower and potted pla nt, as well as in landscaping, due to its long vase life and wide flower shape, design and color variety (Bridgen 2000;

Mansur 2002; Sassone et al. 2014; De la Cuadra et al. 2016) . There are 15 to 20 species distributed throughout northern and central Chile. Its major diversity center is located between the Coquimbo and Valparaiso regions (Zoellner 1972; Muñoz and Moreira 2000; Mansur and Cisternas 2005; Olate and Schiappacasse 2013;

Jara - Arancio et al. 2014) . Plants belonging to this genus have a small tunicate bulb and a single flor al scape 30 - 40 cm in height , which ends in an umbel carrying 3 - 12 flowers depending on the species. One of the main characteristics are its flower pattern and color, varying between white, blue and purple (Mansur 2002;

Schiappacasse et al. 2002; Zoellner 2002; Olate and Bridgen 2005; Hoffmann et al. 2015) .

The ornamental value of Leucocoryne has brought special interest in the knowledge and domestication of the species , with several studies and commercial efforts done in Japan, The Netherlands, Israel, New Zealand and Chile (Kim et al.

1998b; Bridgen 2000; La ncaster et al. 2000; Schiappacasse et al. 2002; Catley

2003; Walton et al. 2008; Olate and Schiappacasse 2013) . This particular interest and the anthropic and climate threat s that natural populations face , have led to the

71 development of a research and b reeding program in Chile for the conservation and commercialization of this genus. This particular program has already registered three new cultivars in addition to the ones already developed in The Netherlands

(De la Cuadra et al. 2016) .

Few studies on the propagation of Leucocoryne have been published to date.

It is therefore necessary to make progress in these techniques in order to help commercial and conservation advances . has a slo w life cycle that takes up to three or four years from seed to floral bulb (Mansur 2002) . It has been reported a high genetic variation among species and ecotypes , which implies a great potential to do breeding but, at the same time, it is a sign of the difficulties to maintain interesting characteristics using sexual propagation (Mansur 2002; Mansur et al.

2004) . Leucocoryne , as other geophyte genus, has the natural capacity of vegetative propagation through the development of one or two lateral bulblets a t the bulb base , or by the production of “droppers”, a type of bulblet that come out as an additional stem from the center of the bulb but instead grows deep er into the ground , also from the second or third year of growth (Riedemannn and Aldunate

2001; Mansur 2002; Schiappacasse et al. 2002) . Bulb - cutting methods have been studie d in order to increase propagation efficiency (Schiappacasse et al. 2002) .

Some of these studies include s pecific in vitro culture research on Leucocoryne species (Olate and Bridgen 2005; Escobar et al. 2008) .

S exual propagation of Leucocoryne spp. can be an interesting tool of propagation for breeding purposes or when genetic stability of the plants is needed .

Nevertheless, seed propagation is still the easiest and inexpensive way to

72 propagate Leucocoryne plants in big quantities, either for c onservation purposes or during the first stages of plant breeding. The in vitro propagation from seeds adds the extra advantage of a more reliable system under a controlled environment, and independent from seasonal environmental conditions. It is importan t to fully understand the s eed propagation process and all the factors that control the different phases of a particular methodology . In that sense, germination and growing temperature is one of the main factors to determine. It has been reported that diff erent Leucocoryne species show different optimal temperatures for seed germination measured either in natural growing habitats (Jara et al. 2006) or under artificial conditions (De la Cuadra et al. 2016) .

In this context, the objectives of this study are to develop and to optimize the protocol for the in vitro seed germination of three Le ucocoryne genotypes and to determine the effect of the temperature on the in vitro plant and bulb production.

Materials and methods.

Plant material.

Seed belonging to three Leucocoryne species were used as explants: Leucocoryne purpurea (Fig. 1a) , Leucocoryne vittata (Fig. 1b) and ecotype Leucocoryne sp.

Pichicuy (Fig. 1c). Seeds were collected from plants under greenhouse conditions in the germplasm bank of the Leucocoryne Breeding Program at the Pontificia

Universidad Católica de Valparaíso Experimental Station , located in Quillota (lat.

32°53´ S; long. 71°12’ W). Seeds were stored up to six months in paper bags at

20±2°C in dark conditions , until they were use d .

The in vitro studies were conducted in the laboratories of the Plant Science

De partment of the Pontificia Universidad Católica de Chile, in Santiago, Chile

(33°29’ S; long. 70° 36’W).

Disinfection and in vitro establishment.

To disinfect the seeds were submerged in a 1 0 g·L - 1 hydric solution of sodium hypochlorite for 15 min under a laminar flow chamber and rinsed 3 times with sterile distilled water. S eeds were then imbibed in sterile distilled water for 96 h at

8°C in dark conditions (Ha m 2002; De la Cuadra and Mansur 2004; Jara et al.

2006) . After imbibition, seeds were sown in 200 mL glass culture vessels containing 30 mL of 12.5% MS medium supplemented with 30 g·L - 1 sucrose, 6 g·L -

1 agar and pH were adjusted to 5.7 (Murashige and Skoog 1962; Ham 2002) .

In vitro seed germination.

A total of 300 seeds per Leucocoryne genotype were sown in vitro conditions .

Seeds were cultured under two temperature treatments: 15±1°C and 20 ±1°C, in a growth chamber with a 16h photoperiod. Treatments consisted in six replicates of five vessels each, containing five seeds/ vessel . After 13 weeks of in vitro culture, germination rate , bulbing rate , dormancy rate and bulb fresh weight were evaluated.

Bulb development and multiplication.

After 13 weeks of in vitro culture, bulbs from seeds were transferred on to 25% MS medium , and after 10 additional weeks they were transferred onto 50% MS supplemented with 1 mg·L - 1 BAP and 30 g·L - 1 sucrose, 6 g·L - 1 agar and pH adjusted to 5.7. After 24 weeks , bulbing rate , bulb sprouting, bulblet production, rooting rate and bulb fresh weight were evaluated.

Statistical analysis.

B ulb fresh weight data were analyzed by analysis of variance (ANOVA) and

Bonferroni's multiple comparison test (P ≤0.05) using GraphPad Prism 5 software

(GraphPad Software Inc., San Diego, California, USA). In the case of germination, bulbing, dormancy, sprouting , bulblet production and rooting data were analyzed using Sheffé's multiple comparison for proportions test (P ≤0.05) (Zar 2010) .

Results and discussion.

Disinfection and in vitro establishment protocols for Leucocoryne seeds were highly effective, showing less than 1% contamination and high germination rates in all the genotypes used. This indicates that disinfection method used did not damage seeds.

In terms of germination rate after 13 weeks of in vitro culture, the best results were obtained at 15°C in the three genotypes, with values of 85.3% in L. purpurea ,

96.7% in L. vittata and 98.7% in L. sp. Pichicuy, significantly higher than germination rat es at 20°C (Table 1). However, w e observed that seed germination occurred earlier at 20°C than at 15°C (data not showed). Ham (2002) reported 89% germination in L. coquimbensis using the same culture medium but using 12°C and darkness conditions , also reporting that 12.5% MS worked as the best MS concentration for seed ger mination compared to higher concentrations (25%,

37.5%, 50% and 100%). Our results are also in accordance with reports of De la

Cuadra et al. (2016) that us ed the same three seed genotypes , but moist chromatographic paper and darkness as germination conditions. They found that germination rates were significantly higher at 10 ºC and 15°C than at 20°C, reporting also no seed germination at 25°C. As mentioned by Jara et al. (2006) , these optimal low temperatures for Leucocoryne seed germination and the inhibitory effect caused by high temperatures under laboratory conditions coincide with the natural desert and Mediterranean environments in which these species are found .

We also evaluated the occurrence of bulbs (bul bing) from the seedlings produced.

The highest bulbing percentage occurred in seedlings cultured at 15°C (Table 1;

Fig. 2), thus repeating the positive effect of the lower temperature treatment for seed germination . Bulbs obtained in both treatments were c learly differe nt in size ; therefore bulb fresh weight was also evaluated. In the same way , better results were obtained at the lowest culture temperature with higher bulb fresh weight at

15°C than at 20°C (Fig. 2; Fig. 3).

A fter seed germination, during bulb and plant development, we observed the occurrence of premature senescence of the seedlings. Apparent dormancy started to show up after 13 weeks of in vitro culture, thus the effect of temperature culture on dormancy of the bulbs was also evaluated. Bu lbs were considered as dormant when more than 2/3 of the shoot showed senescence before transferring them to fresh media . In average, L. purpurea showed the lowest dormancy rates between

28% and 53%, while L. vittata and L. sp. Pichicuy showed over 73% of dormant bulbs (Table 1). Culture temperature affected dormancy rates, occurring lower dormancy rates at 15°C in all genotypes . De la Cuadra and Mansur (2004) reported seedling senescence 11 weeks after germination, and completely senescent plants after 13 week s of growth.

After transferring the bulbs onto 25% MS medium, we were able confirm that most of the bulbs were dormant. After 10 weeks, the bulbs achieved a sprouting rate of only 9.1% (Fig. 4a). During this period, bulb fresh weight did not significantly change , showing 0.0 3 g /bulb in L. purpurea in both culture temperature s

77 treatments, and 0.04 and 0.02 g /bulb in L. vittata and L. sp. Pichicuy at 15 ºC and

20°C respectively.

Transferring the bulbs onto 50% MS medium supplemented with 1.0 mg*L - 1 BAP triggered dormancy break . The interruption of the bulb dormancy could be verified with the appearance of vegetative shoots from the explants, regardless of the culture temperature (Table 2). This would confirm that BAP produces an effect on bulb dormancy break in Leucocoryne specie s under in vitro conditions . BAP addition to in vitro culture media has been used before for stimulating cell division, shoot formation and growth from axillary buds in other geophyte species (Jha

2005; Maślanka and Bach 2014) . We could also observe the appearance of adventitious bulblet s between 4.9% and 12.6% of the explants, independently of the culture temperature (Table 2; Fig. 4b). Therefore, BAP could also promote and accelerate adventitious bulb let formation in Leucocoryne species under in vitro conditions . Although the adventitious bulb l et formation observed in this study was low, it is remarkable that naturally bulb formation in Leucocoryne species does n ot occur until the second or third year of growth (Riedemannn and Ald unate 2001;

Mansur 2002) . Therefore, the use of in vitro propagation and BAP could delay or avoid bulbs entering in the natural early dormancy and shorten the time from seed to adventitious bulblet appearance , thus allowing a faster and more efficient propagation system . In terms of bulb rooting, statistical differences were observed between temperature treatments in both L. purpurea and L. sp. Pichicuy , with higher bulb rooting at 15°C (Table 2).

We would also like to highlight the fact that gradual increase of MS concentration to the growing media ( 12.5% to 25% and then to 50% ) allowed seedlings to continue growing by gradually adapting to increasing osmotic levels produced by higher salt content s of the culture media. Previously, Vidal et al. (2012) reported that Zephyra elegans plants, another geo phyte species endemic to Chile with ornamental potential, were unable to grow in vitro when seedlings germinated on water agar were directly subculture d onto 100% MS medium.

F inal fresh weight of the bulbs varied between 0.04 and 0.1 g depending on both genotype and culture temperature, but without statistical differences (Fig. 5) . It has been reported that Leucocoryne bulbs must have a fresh weight over 0.3 g in order to produce quality cut flowers (Kim et al. 1998a) Nevertheless, it has been also reported that bulbs 0.1 - 0.2 g in weight are able to increase their weight up to 1 g afte r one growing season under greenhouse conditions , producing high quality cut flowers in the subsequent season (Kim et al. 1998a) .

Future works in Leucocoryne sexual propagation under in vitro conditions should consider increasing the macro and micronutrient concentration of the media gradually until reaching 100% MS . In that way bulbs will be able to increase and accelerate their weight constantly . By maintaining this continuous in vitro bulb growth, and avoiding dormancy, it should be possible to reduce the time needed t o obtain flowering size bulbs.

In conclusion, in this work we determined that 15ºC as culture temperature produced a very efficient Leucocoryne plant production using in vitro seed germination. In all the genotypes tested high germination and bulbing rates were

79 achieved at 15°C. Culture temperatures did not produce statistical differences on bulb fresh weight, but temperatures lower than 15ºC could produce an incre ase on this parameter . With the addition of BAP it was possible to break the dormancy of the bulbs, thus favoring sprouting and the multiplication of the explants. The protocol developed in this work leads to high in vitro plant production efficiency and may reduce the time needed to produce flowering size bulbs.

Referenc es.

Bridgen M (2000) Flowering geophytes of Chile have ornamental potential. In:

Seeds: Trade, Production and Technology. pp 218 – 230

Catley JL (2003) Temperature and Irradiance Effects on Vegetative Growth of Two

Sp ecies of Leucocoryne. J Am Soc Hortic Sci 128:815 – 820.

De la Cuadra C, Mansur L (2004) Descripción de la primera etapa del ciclo de vida

de tres genotipos de Leucocoryne sp.: semilla a bulbo. Agric Técnica. doi:

10.4067/S0365 - 28072004000200009

De la Cuadra C, Vidal A, Lefimil S, Mansur L (2016) Temperature Effect on Seed

Germination in the Genus Leucocoryne (Amaryllidaceae ). HortScience

51:412 – 415.

Escobar L, Jordan M, Olate E, et al (2008) Direct and indirect in vitro

organogenesis of leucocoryne pupurea (Alliaceae) a chilean ornamental

geophyte. Propag Ornam Plants 8:59 – 64.

Ham F (2002) Evaluación de la germinación in vitro de Leucocoryne coquimbensis

F. Phil., geófita enémica con potencial ornamental. Pontificia Universidad

Católica de Chile

Hoffmann A, Watson J, Flores AR (2015) Flora Silvestre de Chile, Cuando el

Desierto Florece Vol.I. Monocotiledóneas, 1st edn. Ediciones Fundación

Claudio Gay, Zapallar, Chile

Jara - Arancio P, Arroyo MTK, Guerrero PC, et al (2014) Phylogenetic perspectives

on biome shif ts in Leucocoryne (Alliaceae) in relation to climatic niche

evolution in western South America. J Biogeogr 41:328 – 338. doi:

10.1111/jbi.12186

Jara P, Arancio G, Moreno R, Carmona M (2006) Factores abióticos que

influencian la germinación de seis especies h erbáceas de la zona árida de

Chile. Rev Chil Hist Nat 79:309 – 319.

Jha TB (2005) Plant Tissue Culture: Basic and Applied. Universities Press

Kim HH, Ohkawa K, Nitta E (1998a) EFFECTS OF BULB WEIGHT ON THE

GROWTH AND FLOWERING OF LEUCOCORYNE COQUIMBENSIS F.

PHILL. Acta Hortic 341 – 346. doi: 10.17660/ActaHortic.1998.454.40

Kim HH, Ohkawa K, Nitta E (1998b) Fall flowering of Leucocoryne coquimbensis F.

Phil. after long - term bulb storage treatments. HortScience 33:18 – 20.

Lancaster JE, Shaw ML, Walton EF (2000) S - Alk(en)yl - l - cysteine sulfoxides,

alliinase and aroma in Leucocoryne. Phytochemistry 55:127 – 130. doi:

10.1016/S0031 - 9422(00)00245 - 4

Mansur L (2002) Ciclo de vida, autoincompatibilidad y plasticidad genética en

diseño y color de flores. In: Leucocoryne un gé nero nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chile, pp 9 – 15

Mansur L, Cisternas M (2005) Leucocoryne talinensis ( Alliaceae ), a New Species

from Chile. Novon 15:324 – 326.

Mansur L, Gonzalez M, Rojas I, Salas P (2004) Self - incompatibility in the Chilean

Endemic Genus. J Amer Soc Hort Sci 129:836 – 838.

Maślanka M, Bach A (2014) Induction of bulb organogenesis in in vitro cultures of

tarda tulip (Tulipa tarda Stapf.) from seed - derived explants. Vitr Cell Dev Biol -

Plant 50:712 – 721. doi: 10.1007/s11627 - 014 - 9641 - 1

Muñoz M, Moreira A (2000) Los Géneros Endémicos de Monocotiledóneas de

Chile Continental.

http://www.chlorischile.cl/monocotiledoneas/leucocoryne.htm. Accessed 8 Nov

2016

Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays

with Tobacco Tissue Cultures. Physiol Plant 15:473 – 497. doi: 10.1111/j.1399 -

3054.1962.tb08052.x

Olate E, Bridgen M (2005) Techniques for the In Vitro Propagat ion of Rhodophiala

and Leucocoryne spp. Acta Hortic 673:335 – 342. doi:

10.17660/ActaHortic.2005.673.42

Olate E, Schiappacasse F (2013) Geophyte Research and Production in Chile. In:

Ornamental Geophytes. CRC Press, pp 449 – 470

Riedemannn P, Aldunate G (2001) Flora Nativa de valor Ornamental Chile Zona

Centro, Identificación y Propagación, 1st edn. Editorial Andrés Bello, Santiago,

Chile

Sassone AB, Arroyo - Leuenberger SC, Giussani LM (2014) Nueva circunscripción

de la tribu Leucocoryneae (Amaryllidaceae, Allio ideae). Darwiniana 2:197 –

206. doi: 10.14522/darwiniana.2014.22.584

Schiappacasse F, Peñailillo P, Yáñez P (2002) Propagación de Bulbosas Chilenas

Ornamentales. Editorial Universidad de Talca, Talca, Chile

Vidal AK, Han D, Nakano M, Niimi Y (2012) Decreased time from seed to flowering

corm size in Zephyra elegansvia in vitro cultivation. Cienc e Investig Agrar

39:577 – 584. doi: 10.4067/S0718 - 16202012000300017

Walton EF, Wu RM, Sutherland P (2008) Bulb and inflorescence development in

Leucocoryne ixioides (Hoo k.) Lind. J Hortic Sci Biotechnol 83:574 – 580.

Zar J (2010) Biostatistical Analysis, Fifth. Prentice Hall Inc., New Jersey, USA

Zoellner O (1972) El género Leucocoryne. An del Mus Hist Nat Valparaíso 5:9 – 83.

Zoellner O (2002) Descripción del género Leucocoryne (Alliaceae) su forma de

propagación y distribución. In: Leucocoryne un género nativo chileno y su uso

como planta de jardín, 1st edn. Oficina de transferencia tecnológica

Universidad Católica de Valparaíso, Viña del Mar, Chile, pp 23 – 27

Table 1 Effect of temperature on the in vitro germination, bulbing and dormancy of three Leucocoryne genotypes after 13 weeks of culture from initiation . Temperature Seeds Germination Bulbing Dormancy Genotype (°C) (N°) (N°) (%) (N°) (%) (N°) (%) 15 150 128 85.3 a 118 92.2 a 37 28.9 b L. purpurea 20 150 73 48.7 b 60 82.2 b 39 53.4 a 15 150 145 96.7 a 144 99.3 a 108 74.5 b L. vittata 20 150 118 78.7 b 114 96.6 b 106 89.8 a 15 150 148 98.7 a 140 94.6 a 108 73.0 b L. sp. Pichicuy 20 150 125 83.3 b 108 86.4 b 104 83.2 a Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) .

Table 2 Effect of temperature on bulb occurrence bulbing, bulb sprouting, bulblet production and rooting after 24 weeks in MS 50% + 1.0 mg*L - 1 BAP of three Leucocoryne genotypes . Genotype Temperature Bulbing Bulb Bulblet Rooting sprouting production (°C) (N°) (N°) (%) (N°) (%) (N°) (%) 15 107 71 66.4 a 7 6.5 a 33 30.8 a L. purpurea 20 53 36 67.9 a 4 7.5 a 11 20.8 b 15 143 94 65.7 a 7 4.9 a 44 30.8 a L. vittata 20 107 76 71.0 a 7 6.5 a 28 26.2 a 15 135 97 71.9 a 17 12.6 a 66 48.9 a L. sp. Pichicuy 20 85 63 74.1 a 7 8.2 a 28 32.9 b Different letters in the same column indicate statistical differences for each genotype based on Scheffé´s procedure for multiple comparison of proportions (P ≤0.05) .

Fig. 1 Leucocoryne genotypes . (a) Leucocoryne purpurea ; (b) Leucocoryne vittata ; and (c) Leucocoryne sp. Pichicuy.

Fig. 2 Effect of temperature on the in vitro bulb production of three Leucocoryne genotypes after 13 weeks of culture from seed initiation .

Fig. 3 Effe ct of temperature on the bulb fresh weight of three Leucocoryne genotypes after 13 weeks from seed in vitro initiation. Different letters indicate statistical differences for each genotype according to analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

Fig. 4 Different v isual appearance of Leucocoryne bulbs on in vitro conditions: (a) Dormant bulbs on MS 25% ; (b) Active bulb s with shoot and bulblet producti on on MS 50% + 1.0 mg*L - 1 BAP.

Fig. 5 Effe ct of culture temperature on bulb fresh weight of three Leucocoryne genotypes after 24 weeks of in vitro culture on MS 50% + 1.0 mg*L - 1 BAP. Different letters indicate statistical differences for each genotype according to analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (P ≤0.05) .

CHAPTER 4

CONCLUSION S

In this study we have developed an efficient protocol for the in vitro propagation of the Leucocoryne genus using different culture techniques.

Different c ulture medi a , supplemental addition of BAP, different culture systems and type of explants were evaluated in order to develop mass clonal bulb propagation. We are also report ing an effective disinfection method as part of the in vitro establishment protocol, by which we have surpassed one of the main barriers for the Leucocoryne in vitro culture.

By studying the effect of culture media and BAP addition, higher bulb prod uction rates than the naturally occurred were achieved . A high er bulb fresh weight was also achieved, which allows the use of such larger bulbs in further propagation techniques.

We have achieved the highest rate of propagation of Leucocoryne bulbs report ed to date, considering both in vitro and ex vitro conditions, when bulbs were subjected to bulb - cutting methods.

The effects of the culture t emperature BAP addition on the in vitro seed germination and bulb development , was also established. Results regar ding both factors ha ve great potential of use in obtaining active and large plant populations from sexual reproduction.

P rotocols developed in this study will help to overcome the need of a reliable mass propagation method, which is one of the most important pending challenge s for the

Leucocoryne genus . An efficient mass propagation method is an essential and valuable advance and an important contribution to the domestication and

93 sustainable use of this plant genetic resource. Therefore, the use of t hese protocols will assure a high production of plant s to be used for conservation, breeding or commercial purposes. This will greatly increase the value of

Leucocoryne genus in the ornamental crop industry.