Utilization of Embryogenic Cell Cultures for the Mass Production of Bulblets in Lilies

Utilization of Embryogenic Cell Cultures for the Mass Production of Bulblets in Lilies Sun Ki Kim and Byung Joon Ahn College of Bioresources Science Dankook University Cheonan 330-714 Korea Keywords: Oriental lily, callus, bioreactor, Lilium longiflorum, 2,4-D, dicamba, picloram Abstract To utilize a somatic embryogenic cell culture system for the mass production of bulblets in Oriental and Easter lilies, methods of embryogenic callus induction, multiplication and bulblet production were investigated. The sliced bulb scales of a Lilium longiflorum hybrid (L. longiflorum x L. fomolongi) and Oriental hybrids ( Marco Polo and Casa Blanca ) were cultured on MS solid medium (3% sucrose and 0.3% Phytagel) supplemented with 2,4-D, dicamba or picloram at the concentrations of 1, 3, 6 and 9 mg L -1. The most embryogenic calli were produced with 2 mg L -1 of dicamba in Lilium longiflorum hybrid and 6 mg L -1 of picloram in the Oriental hybrids. The embryogenic cells of the Oriental hybrid Marco Polo and Casa Blanca were cultured in an air-lift-type 5-L bioreactor using liquid MS medium (3% sucrose, ph 5.8) supplemented with 2 mg L -1 of picloram, and transferred to culture boxes (96 x 96 x 90 mm) containing 80 ml of hormone-free MS agar medium (0.5% Phytagel and 6% sucrose) for bulblet formation. Numerous bulblets larger than 5 mm in diameter were produced after four months and planted in greenhouse after 8 weeks of 4 C storage. Adult plants of L. longiflorum hybrid, regenerated from the cells maintained over 4 years, produced mostly morphological variants of flower deformities, but among the plants derived from 6-month-old callus of the Oriental hybrid Marco Polo and Casa Blanca morphological variants of flower deformities were not found. Therefore, bulblets of Oriental lilies having high quality and uniformity, can be produced in quantity in relatively short periods using embryogenic cell cultures. INTRODUCTION Since 1960 s, lilies have become a major flower crop and the world production area has increased for both cut flower and bulb production. Asiatic lilies were the initial major lily crop, and Oriental lily cultivars have been increasingly grown since the early 1990 s (Van Tuyl, 1997). For bulb production, bulblets are produced vegetatively from bulb scales and by tissue culture methods (Beattie and White, 1993). Instead of the in vitro method in which bulblets are formed directly from bulb scales, callus culture methods have been tried as an alternative method for bulblet production. In Easter lilies, calli have been readily induced from either bulb scales or floral tissues such as filaments, styles and flower pedicels in the presence of dicamba, picloram or NAA (Sundeep et al., 1992; Tribulato et al., 1997; Arzate-Fernandez et al., 1997; Xu et al., 1998). In some cultures, regenerated plants from callus were morphologically normal even after 3 years of subculture (Sundeep et al., 1992; Arzate-Fernandez et al., 1997). However, in Gelria, out of a total of 400 plants regenerated from 3 year-old callus, 14 were morphological variants (Van Harmelen et al., 1997). In Asiatic and Oriental lilies, callus cultures have been rarely reported. Shoot primordia were cultured to form embryogenic callus in an Oriental lily and mostly normal plants were produced (Sugiura et al., 2000). In this study, we investigated methods of embryogenic callus induction, multiplication and bulblet production to utilize an embryogenic cell culture for the mass production of bulblets in Oriental and Easter lilies. Proc. IX th Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 731

MATERIALS AND METHODS Bulb scales of Oriental hybrids ( Marco Polo and Casa Blanca ) were excised and surface-sterilized in 0.5% NaClO solution for 10 min and rinsed thoroughly in sterile distilled water. They were sliced into 1 x 1 cm fragments and cultured on MS solid medium (3% sucrose and 0.3% Phytagel) supplemented with 2,4-D, dicamba or picloram at the concentrations of 1, 3, 6 and 9 mg L -1. Induced calli were subcultured every 4 weeks using the same medium, and were characterized by their morphologies and regenerability upon transfer to hormone-free MS medium. Induced embryogenic calli were also cultured in liquid agitation cultures of 250-ml flasks containing 50 ml of liquid MS media (5% sucrose) supplemented with either picloram or zeatin of various concentrations (0, 0.3, 1 and 3 mg L -1 ). For mass production of the embryogenic calli of Marco Polo Oriental hybrid, ten grams of suspension cells were inoculated in a 5-L air-lift-type bioreactor. Liquid MS medium (5% sucrose, 2 mg L -1 of picloram) was added gradually to maintain the optical density of the medium inside approximately 0.6 at 600 nm. One gram of the suspension cell clumps were placed on 100 ml of hormone-free MS solid medium (0.3% Phytagel and 9% sucrose) in culture boxes (96 x 96 x 90 mm) for bulblet formation. Bulblets larger than 5 mm in diameter were harvested after four months and stored over 8 weeks at 4 C. They were planted in trays (45 x 60 cm) filled with bedding soil (peat moss: pearlite: vermiculite = 1:1:1) and grown in a greenhouse with fertilizing Osmocote (Scotts CO., Heerlen) at the rate of 40 g/m 2 every 9 weeks. Calli of L. longiflorum hybrid (L. longiflorum x L. fomolongi) were also induced using the same method as the Oriental lilies except the plant growth regulator treatment. For the Easter lily, medium was supplemented with 2 mg L -1 dicamba. The induced calli were maintained in liquid cultures by subculturing every week using the same medium. Plants were regenerated from calli maintained for four years, were kept at 4 C for 8 weeks, and grown to mature plants in greenhouse to examine the rate of somaclonal variation. Also, in the Oriental hybrid Marco Polo, plants were regenerated from 6-month-old calli and grown for two years to bloom. RESULTS AND DISCUSSION Since somaclonal variations often occur in lilies (Van Harmelen et al., 1997), media supplemented with picloram at lower concentrations or zeatin were compared for the multiplication of the cells in liquid cultures using 250-ml flasks. All cultures in media supplemented with 0.3, 1.0, 3.0 mg L -1 zeatin and 1 mg L -1 picloram proliferated with little difference in growth rate over 8 weeks (Fig. 1). However, since the cultures in the zeatin treatments regenerated bulblets, the embryogenic cells needed to be cultured in the presence of picloram at 1 mg L -1. Bulb scale explants of the Oriental lilies were cultured on MS agar medium supplemented with either 2,4-D, dicamba or picloram for 12 weeks. The scales, cultured on the medium supplemented with 2,4-D at the concentrations up to 9 mg L -1, produced little calli and mostly formed bulblets and roots in both Marco Polo and Casa Blanca (Fig. 2B and 2D). This indicates that 2,4-D is not an efficient auxin for Oriental lilies unlike in Easter lilies which formed callus at an even lower concentration of NAA (Sundeep et al., 1992; Tribulato et al., 1997; Arzate-Fernandez et al., 1997; Xu et al., 1998). However, in the media containing dicamba or picloram, most scales produced calli, but embryogenic calli were produced more with picloram. For all cultivars, the best embryogenic calli were obtained with the treatment of picloram at a concentration of 3 mg L -1 (Fig. 2B and 2D). Embryogenic calli were determined by their rapid regeneration on hormone-free MS medium and appeared as a globular type hard callus (Fig. 3B). When the calli were cultured in suspension cultures using liquid media with 2 mg L -1 of picloram, the embryogenic cells proliferated rapidly as globular clumps (Fig. 3C). The embryogenic cells of the Oriental hybrid Marco Polo were cultured in a continuous type of 5-L bioreactor. The 10 grams of the original inoculum increased to 250 g during 20 weeks of the culture (Fig. 3C). When the cell clumps were cultured on 100 ml of hormone-free MS agar medium (0.3% Phytagel and 9% sucrose) in culture boxes, 732

50-100 bulblets larger than 5 mm in diameter were produced within 16 weeks from 0.5 g of cell clumps (Fig. 3D). After 8 weeks of storage at 4 C, the bulblets sprouted to give 2 or 3 leaves and produced small bulbs with a circumference of 6 to 9 cm in 8 months (Fig. 3E, 3H). Plants regenerated from 4-year-old calli of L. longiflorum hybrid (L. longiflorum x L. fomolongi), grew to bloom in a greenhouse. A total of 89 plants produced 302 shoots and most of them formed flower buds. However, 163 flower buds aborted before anthesis and 77 flowers showed deformities such as malformed flowers, and abnormal stamens and pistils, a rather high proportion as compared to the plants propagated by seed (Table 1, Fig. 4D, 4F). In the Oriental hybrids Marco Polo and Casa Blanca, bulblets were produced from 6-month-old calli and grown to bloom in three years. Most flowers showed normal form blooming (Fig. 4A, 4B). This result indicates that in lilies, a long-term callus culture might cause high rates of somaclonal variation, but calli cultured for a short period like 6 months can produce bulbets without severe occurrence of variants. In conclusion, we found that bulblets of Easter and Oriental lilies can be produced in quantity in a relatively short period using embryogenic cell cultures and grown to make flower bulbs. ACKNOWLEDGEMENTS The research was conducted by the research fund of Dankook University in 2002. Literature Cited Arzate Fernandez, A.M., Nakazaki, T., Okumoto, Y. and Tanisaka, T. 1997. Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorium Thunb.). Plant Cell Reports. 16:836-840. Beattie, D.J. and White, J.W. 1993. Lilium hybrids and species. p.423-454. In: A. De Hertogh and M. Le Nard (eds.), The physiology of flowering bulbs, Elsevier, Amsterdam. Sugiura, H., Agong, S.G., Enami, A., Kaneko, H. and Honma, T. 2000. Comparison between embryoid and shoot primordia methods of plantlet production for Asiatic Hybrid Lily and Oriental Hybrid Lily. African Crop Sci. J. 8:117-127. Sundeep, P., Sumitra, S., Priyadarshi, S. and Sen, S. 1992. A revised scheme for mass propagation of Easter lily. Plant Cell Tiss. Org. Cult. 30:193-197. Tribulato, A., Remotti, P.C., Loffler, H.J.M. and van Tuyl, J.M. 1997. Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb. Plant Cell Reports. 17:113-118. Van Harmelen, M.J., Loffler, H.J.M., van Tuyl, J.M. and Halevy, A.H. 1997. Somaclonal variation in lily after in vitro cultivation. Acta Hort. 430:347-350. Van Tuyl, J.M. 1997. Lily production and breeding in the Netherlands. Lecture in RDA, Suwon, Korea 26 June. (http://www.gironet.nl/home/lilium/korea.htm) Xu, P.S., Niimi, Y. and Araki, H. 2000. Production of virus-free bulblets from callus induced from scale culture of Lilium longiflorum Georgia. J. Jpn. Soc. Hort. Sci. 69:97-102. Tables Table 1. Occurrence of morphological variants among plants derived from embryogenic callus in Lilium longiflorum hybrid L. longiflorum x L. fomolongi. Origin No. No. No. Flower Flower Morphological variation of of of of length diameter Aborted Malformation bulb plants shoot flower (cm) (cm) flower Flower Stamen Pistil Seed 45 91 91 13.8±1.6 1 10.7±1.1 27 2 0 2 0 SEC 3 89 302 299 11.6±1.5 9.6±1.3 163 4 55 18 1 Mean±Standard error. 2 Number of flower. 3 Somatic embryogenic callus. 733

Figures Fig. 1. Fresh weight increase of embryogenic callus in liquid agitation cultures of 250 ml flasks with liquid MS media containing either picloram or zeatin. Fig. 2. Effect of 2,4-D, Picloram and Dicamba of various concentrations on callus formation and organogenesis from bulb scales of Lilium Oriental Hybrids. 734

Fig. 3. Mass production of bulblet through embryogenic callus culture in Oriental lily Marco Polo. (A) non embryogenic callus, (B) globular type hard callus of embryogenic callus (C) embryogenic callus culture in 5-L air-lift bioreactor using liquid MS medium supplemented 1 mg L -1 picloram and 5% sucrose, (D) embryogenic callus-derived bulblets formed on hormone-free MS agar medium supplemented with 0.3% phytagel and 9% sucrose, (E) sprouted bulblets in bed soil 1 month after planting in a greenhouse, (F) bulblet-derived plants growing in a greenhouse, (G) mass production of small bulblets harvested after culture for 4 months in vitro and (H) bulbs harvested after growing for 8 months in a greenhouse. 735

Fig. 4. Flowers of plants derived from embryogenic calli of Oriental lily and L. longiflorium hybrids. Normal flower of Oriental lily Casa Blanca (A) and Marco Polo (B), regenerated from six-month-old callus, normal flower of L. longiflorium hybrid (C), flowers with morphological variation in L. longiflorium hybrid, regenerated from the callus maintained over for years. Malformation of stamens (D) and aborted flower (E) and flower (F). 736