Improvement of Breeding Efficiency for Interspecific Hybridization of ...

Improvement of Breeding Efficiency for Interspecific Hybridization of 

Lilies in Korea 

Hye Kyung Rhee, Jin Hee Lim 

J.M. van Tuyl 

and Young Jin Kim 

Plant Research International 

National Horticultural Research Institute Wageningen University and Research Centre 

RDA, Suwon 440-310 P.O. Box 16 

Korea 

6700 AA Wageningen 

The Netherlands 

Keywords: Lilium formolongi, pollen sterility, FDA, polyploidization, Fusarium 

oxysporum 

Abstract 

Interspecific hybridization of lily in Korea is focused on increasing breeding 

efficiency especially under Korean climate conditions. Cultivars derived from L. 

formolongi were propagated by seed and were sensitive to day length. These 

cultivars differ from flowering time with the Asiatic and Oriental hybrids. Therefore, 

to breed interspecific hybrids by using L. formolongi, the use of stored viable pollen 

is a prerequisite. 

Lily pollen was stored at four different temperature conditions for one year 

and pollen viability was investigated using three different methods. As storage 

temperatures increased, pollen viability was drastically reduced. Viability of lily 

pollen stored was maintained effectively at temperatures lower than -20°C. FDA test 

was the most effective and convenient method to ascertain pollen viability. 

To overcome pre-fertilization barriers in interspecific hybridization of lilies, 

pollen tube growth in the stigma or style, and the penetration of pollen tubes into 

the ovules were observed. OA (Oriental Asiatic), OL (Oriental Longiflorum), FA 

(Formolongi Asiatic), and FO (Formolongi Oriental) hybrids were obtained through 

embryo rescue and stigmatic and CSM pollination, while AO (Asiatic Oriental), AL 

(Asiatic Longiflorum), LA (Longiflorum Asiatic) and LO (Longiflorum Oriental) 

hybrids were not obtained through these methods. 

Backcrosses were used in order to reduce scattered petals in interspecific 

hybrids. It was possible to obtain BC 107 hybrids between FA (Fomolongi Asiatic) and 

Asiatic hybrids as male parent. But, it was difficult to obtain BC 1 hybrids between 

FA hybrid and L. formolongi as female parent. Normal pollination was better than 

CSM (Cut style pollination method) to get BC 1 hybrids. 

In order to overcome the sterility of interspecific hybrids pollen, low 

concentration of oryzalin was more effective than high concentration of colchicine to 

obtain tetraploid. The fertility of pollen increased by about 40% in tetraploid level. 

Fusarium oxysporum screening test was carried out. ‘Hae-wool’, ‘Sinavro’ 

and FA 96-16 were classified as highly disease resistant and ‘Supia’ and ‘Doran’ as 

moderately resistant to Fusarium bulb-rot. The FO-hybrid lilies from NHRI were 

susceptible. 

INTRODUCTION 

Lily is one of the most economically important flower bulb crops in Korea. Korea 

exported lily cut flowers to Japan worth about 10 million dollars and we imported bulbs 

from the Netherlands worth about 4 million dollars in 2003. 

There are more than 10 native lily species in Korea. Commonly cultivated lilies 

include the Asiatic, Oriental, and Longiflorum hybrids. Asiatic hybrids have no fragrance 

but have diverse flower color. Oriental hybrids are susceptible to virus and Fusarium 

disease but have strong fragrance and they take a very long time to produce bulb. Recently, 

the cultivation of L. formolongi hybrids, such as ‘Raizan’, ‘F 1 Augusta’ has increased. 

These hybrids are F 1 progeny obtained by crossing L. longiflorum and L. formosanum. 

Proc. IX th Intl. Symp. on Flower Bulbs 

Eds.: H. Okubo, W.B. Miller and G.A. Chastagner 

Acta Hort. 673, ISHS 2005 

107

The advantages of L. formolongi hybrids are seed propagation, shorter generation, upward 

flowering type, convenience of packing and transport of cut flower, and long stem. Since 

the early 1990s interspecific hybridization of lily has been carried out in NHRI of Korea 

(Rhee, 2002). 

In lily breeding, interspecific hybridization and polyploidization have greatly 

contributed to the breeding of commercial LA hybrids, which are becoming more 

important in the market (Van Tuyl et al., 2003). 

Interspecific hybridization of lily in Korea has focused on increasing breeding 

efficiency by using L. formolongi as female parent especially in Korean climate conditions. 

To enhance breeding efficiency, several experiments were conducted in this research, the 

objectives of which included the following: 1) To search for adequate storage condition 

and to sustain the viability of the pollen stored; 2) To identify pre-fertilization barriers in 

incongruent crosses; 3) To improve the percentage of interspecific hybrids obtained, 

through in vitro culture such as embryo rescue in incongruent crosses; 4) To overcome the 

sterility of F 1 hybrid obtained from interspecific hybridization by in vitro chromosome 

doubling; and 5) To screen disease such as Fusarium oxysporum. 

MATERIALS AND METHODS 

Plant Material 

Lily plants were cultivated in greenhouse and maintained at an average night and 

day temperature of 20 ± 5°C and 40 ± 5°C respectively. Normal culture practices for pest 

control, watering, ventilation, and fertilization were conducted. 

Fluorescein Diacetate (FDA) 

Pollen viability was evaluated using fluorochromatic reaction with FDA (0.002% 

with 20% sucrose) by fluorescence microscopy. 

In Vitro Pollen Germination 

Pollen germination test was performed in a medium containing 100 g sucrose, 5 g 

agar, and 20 mg boric acid per 1 L by light microscopy. 

Pollen Tube Growth in Style 

Pollen tube growth in stigma and style was observed using aniline blue staining 

and UV-fluorescence. Fixing was conducted in a solution of ethanol 3:glacial acetic acid 1 

(V/V) in 4°C refrigerator for 24 hours. Softening was done in the solutions of 2N NaOH 

for 4 hours in Asiatic hybrids as female parents, 3N NaOH for 4 hours in Oriental hybrids, 

and 2N NaOH for 6 hours in Formolongi hybrids. Samples were stained with 0.1% aniline 

blue (W/V) for 24 hours and observed using UV-microscope. 

Penetration of the Pollen Tubes to the Ovule 

To determine the penetration of pollen tube in the micropyle, ovules which are 14 

days after pollination were cleared in a mixture of lactic acid, glycerol and water (1:2:1) at 

80°C for 1 hour, stained for 2 minutes in this solution supplemented with 1% aniline blue 

at the same temperature and destained again in the clearing solution at 100°C 

(modification of Gerlach, 1977; Janson et al., 1994). The number of ovules penetrated by a 

pollen tube was observed using a bright field microscope. 

In Vitro Chromosome Doubling and Ploidy Analyzer 

The media were composed of MS (Murashige and Skoog, 1962), supplemented 

with 6% (w/v) sucrose, and adjusted to pH 5.8. Colchicine and oryzalin were treated for 3 

hours, with 0.1-0.001% concentration in vitro lily scales. Ploidy level was detected with 

the ploidy analyzer-II (Partec Co. Munster). 

108

Screening for Fusarium oxysporum 

Fusarium oxysporum f. sp. lilii (CPRO-Fol 4 and CPRO-Fol 11) were used 

(Straathof and Loffler 1994a.b). For soil infestation, the fungi were incubated for two 

weeks at 23°C in an autoclaved (120°C, 2h) 1 oatmeal: 4 soil mixture (w/w). Fully-grown 

cultures were ground and mixed in a 1:100 ratio with non-sterilized potting soil. The 

disease severity was observed visually using a disease rating according to the following 

ordinal scale: 1 = ‘healthy’, 2 = slightly rotten’, 3 = ‘moderately rotten’, 4 = ‘heavily 

rotten’, 5 = ‘very heavily rotten’ and 6 = ‘completely decayed’. 

RESULTS AND DISCUSSIONS 

Pollen Storage and Viability 

Lily pollen was stored at four different temperature conditions for one year and 

pollen viability was investigated using three different methods. As storage temperature 

was high, pollen viability was drastically reduced. Viability of lily pollen stored was 

maintained effectively at temperatures lower than -20°C. Pollen viability by FDA was 

significantly (p

ovules by stigmatic and CSM pollination, respectively. In case of FA96-24 x A95-14, 10 

ovules were obtained at only CSM pollination. The number of FAF (BC 1 ) hybrids 

obtained in crossing between FA96-4 (F 1 ) and F 1 ‘Augusta’, Formolongi hybrid, were 1 

and 13 ovules by stigmatic and CSM pollination. In the case of backcrosses with FO97-9 

(F 1 progeny of F x O) x F 1 Augusta (F) and FA96-24 (F 1 ) x F 1 Augusta (F), no ovule was 

obtained by both pollination methods. Results of backcrosses can be divided into two 

groups according to the pollen grains used. In one group pollen grains were obtained from 

the female parent, and in the other group, from the male parent, in the crosses made for 

obtaining F 1 hybrids. Obviously, if interspecific hybrids are backcrossed with the parent’s 

pollen, the crosses are more successful when the male parent was used again as a pollen 

source. 

Characteristics of FA (F x A) Hybrids 

FA progenies, FA97-9, FA96-10 (Fig. 4C ‘Supia’), FA96-29, FA96-9 and FA99-1 

were obtained from a cross between ‘Raizan’ (Fig. 4A) and A95-14 (Fig. 4B). As to the 

flower color of these progenies, the rate of white and pink was 1:4. If the flower color of 

female parent was white and that of male parent was red, intermediate color of parents, 

pink color was dominant color. Flower characteristics such as trumpet shape and unspotted 

petal of the FA progenies were similar to those of their parents. Plant height, number of 

flowers, flower width, and flowering time of FA progenies were distributed more diversely 

than those of royjrt parents. Among the FA progenies, ‘Supia’ (FA96-10) was bred as a 

new cultivar at NHRI in 2000. This cultivar had characteristics such as unspotted petal, 

light pink color, small size and trumpet-shaped flower, upward-facing, early flowering, 

high plant height and sweet fragrance. 

Characteristics of FO (F x O) Hybrids 

FO progenies, FO97-3, FO97-12, FO97-4 (Fig. 4D), FO97-1, FO-97-10, and 

FO97-16 were obtained from cross combination between ‘Raizan’ (F) and ‘O54’ (O). 

These progenies flowered in 1997. The characteristics of FO progenies were similar to 

those of the oriental hybrids. These include the creamy ivory color of the female flower, 

deep pink color of the male flower, big flower, broad leaf shape, and strong fragrance. The 

advantage of FO hybrids compared to Oriental hybrids is short generation. Originally, 

breeding problems of Oriental hybrids are long generation time from seed to commercial 

bulb, long bulb production and their susceptibility to virus and Fusarium disease. We have 

worked this study to reduce breeding duration and bulb production of Oriental hybrids, 

using Formolongi hybrids as female parent, which is propagated by seeds and has short 

generation. In breeding program and bulb production, FO hybrids are more effective than 

Oriental hybrids in Korea. Among the FO hybrids, ‘Hanuri’ (FO97-4) was bred as new a 

cultivar at NHRI in 2001. It had characteristics such as unspotted petal, light pink and 

white color, big and Oriental-shaped flower, outward facing, early flowering and sweet 

fragrance. 

Characteristics of FAA (FA x A) Hybrids 

FAA progenies, FAA00-22, FAA01-7, FAA01-3, FAA00-4, FAA00-8 and 

FAA00-24 were obtained from backcrosses between FA hybrids and other Asiatic hybrids. 

The flower color of FAA progenies became deeper and flower shape was more stable than 

FA hybrids. As the flower type of pollen parent was dominant, flower type of most FAA 

progenies was similar to the Asiatic flower type. Also, pollen fertility of FAA lines was 

less than 1%. Some FAA hybrids were triploid. 

Pollen Fertility and Pollen Morphology after In Vitro Chromosome Doubling 

Most interspecific lily hybrids have pollen sterility. To restore the pollen sterility of 

interspecific hybrids, we conducted in vitro chromosome doubling with colchicines and 

oryzalin to obtain tetraploid hybrids. Low concentration (about 0.001-0.003%) of oryzalin 

was more effective to obtain tetraploid hybrids than high concentration (about 0.1%) of 

110

colchicines. 

Pollen fertility of FA96-18 was 0%, while that of tetraploid FA96-18 was 42.3% in 

in vitro germination test. 

Most sterile pollen grains were small empty ‘shell’ shape and fertile pollen of 

tetraploid FA96-18 was normal ‘full oval’ shape (Fig. 3). 

Fusarium oxysporum Screening 

‘Hae-wool’, ‘Sinavro’ and FA 96-16 were classified as highly resistant cultivars 

and ‘Supia’ and ‘Doran’ as moderately resistant to Fusarium bulb-rot disease. 

In interspecific crosses between cultivars of different sections, if Oriental and 

Formolongi hybrids were used as female parents, interspecific hybrids could be obtained 

successfully. However, Asiatic and Longiflorum hybrids as female parent could not 

produce any interspecific hybrids. Most interspecific hybrids of lilies were obtained when 

Longiflorum hybrids were used as female parent. However, we suggest that cultivars 

derived from L. formolongi are more effective than those derived from L. longiflorum as 

female parent for interspecific hybridization (Okazaki et al., 1992). 

Literature Cited 

Gerlach, D. 1977. Botanische Mikrotechnik. Eine Einfuhrung. Georg Thieme Verlag, 

Stuttgart. 311 

Janson, J., Reinders, M.C., Valkering, A.G.M., van Tuyl, J.M. and Keijzer, C.J. 1994. Pistil 

exudates production and pollen tube growth in Lilium longiflorum Thunb. Annals of 

Botany 73:437-446. 

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with 

tabacco tissue cultures. Physiol. Plant. 15:473-497. 

Okazaki, K., Umada, Y., Urashima, O. and Kawada, J. 1992. Interspecific hybrids of 

Lilium longiflorum and L. x formolongi with L. rubellum and L. japonicum through 

embryo culture. J. Japan. Soc. Hort. Sci. 60:997-1002. 

Rhee, H.K. 2002. Improvement of breeding efficiency in interspecific hybridization of 

lilies. Ph.D thesis, Seoul National University. 118p. 

Straathof, Th.P. and Loffler, H.J.M. 1994a. Resistance to Fusarium oxysporum at different 

developmental stages of Asiatic hybrid lilies. J. Amer. Soc. Hort. Sci. 119:1068-1072. 

Straathof, Th.P. and Loffler, H.J.M. 1994b. Screening for Fusarium resistance in seedling 

populations of Asiatic hybrid lily. Euphytica 78:43-51. 

Van Tuyl, J.M. and Lim, K.B. 2003. Interspecific hybridization and polyploidisation as 

tools in ornamental plant breeding. Acta Hort. 612:13-22. 

Figures 

Fig. 1. Many pollen tubes grew straight along the style in compatible (A x A) cross 

combination (left). The bundle of pollen tubes turned back to the style and stopped 

in incompatible (A x O) cross combination (right). 

111

Fig. 2. Pollen tube penetration into the micropyle of lilies observed under light 

microscope at 14 days after pollination (left). Pollen tube penetration was failed 

into the micropyle (right). 

Fig. 3. The sterile pollen of FA96-18 was empty ‘shells’ shape (left) and the fertile pollen 

of doubled FA96-18 were normal ‘full oval’shape (right). 

A 

B C D 

Fig. 4. Flowers of parents and interspecific FA and FO hybrid lilies. A) ‘Raizan’ 

(Formolongi hybrid, mother parent), B) A95-14 (Asiatic hybrid, father parent), C) 

‘Supia’ (interspecific FA hybrid), D) ‘Hanuri’ (interspecific FO hybrid). 

112

Improvement of Breeding Efficiency for Interspecific Hybridization of ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?