Annals of the Sri Lanka Department of Agriculture. 2008.10:55-65.
IN VITRO PROPAGATION OF Lilium ASIATIC HYBRID “PRATO” THROUGH BULB SCALE CULTURE D.P. KARUNANANDA1, U.D.K. YAPA1, A.D. KULASINGHE2 and K.N. YAPA2 1 Horticulture Crops Research and Development Institute, Gannoruwa, Peradeniya 2 Department of National Botanic Gardens, Peradeniya
ABSTRACT Bulb scales of Lilium Asiatic hybrid “Prato” were cultured in M and G (Montezuma De Carvalho and Guimoraes) medium with different hormone concentrations (Kinetin 0, 0.1, 0.2, 0.3 and Indol Acetic Acid (IAA) 0, 0.5. 1, 2 mg/l) and their combinations. 0.2 mg/l kinetin and 2 mg/l IAA was identified as the best hormone combination for bulblet formation. Bulblets initiated were cultured in M and G medium supplemented with different combinations of Napthelic Acetic Acid (NAA; 0, 0.1, 0.2 mg/l) with (10 g/l) and without Activated Charcoal (AC) for root induction. Hormonefree medium with AC exhibited the best in vitro rooting. The survival rate of rooted plants was 100% when acclimatized in compost 1:sand 1 medium. More than 70% of the plants bloomed within 18 months after field planting. KEYWORDS: Activated charcoal, Bulb scales, Indol acetic acid, Kinetin, Lilium.
INTRODUCTION The Genus Lilium belongs to the Family Liliaceae and comprises about 85 species including many attractive ornamental species. Although the flowers of many Lilium species are very beautiful, hybrid lilies are the most popular in the world flower market due to their beautiful flowers, ideal shape of the inflorescence and long vase life (Mu Ding, 2005). All lily cut-flower varieties also fetch a very high demand in the local floriculture market. However, limited cultivation in higher elevated regions and unavailability of planting materials of good quality are the major reasons for the low supply of quality flowers to the market. Therefore, the introduction of new lily varieties and mass production of their planting materials are necessary. Lilium Asiatic hybrids are popular cut-lily varieties in the floriculture industry which have been produced by extensive breeding of Lilium tigrinum, L. cernuum, L. davidii, L. hollandicum, L. pummilum L. bulbiferum, L. concolar and L. maculatum (Okazaki et al., 1996). Less overlapping petals with straight margins, long lasting attractive flowers, different dark colours and seven to eight flowers per spike are the common characters of these hybrids. They are mainly adapted to the temperate regions of the world and require low temperature to bloom. Hence only the adaptable varieties to the local conditions should be introduced. The Lilium Asiatic
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hybrid variety, “Prato” has shown a very good adaptability to the local conditions. It has been observed that this variety performs well in Kothmale, Nuwara Eliya and Bandarawela regions. Hence, there is a high potential for rapid multiplication of this variety under local conditions and distribution of planting material. Lilium species are monocots and bulb scale plant production is the commercial practice that fulfills the demand of planting materials. The scale bulblets can be grown into larger bulbs after re-planting in the field or keeping in the field for next generation (Simmonds and Cumming, 1976). However, production of planting material through this procedure takes a long period in the field and requires 2-3 planting cycles. Therefore, there is a high risk of losing plant materials due to bad weather conditions. Furthermore, multiplication in the field can lead to virus infections which could completely devastate the cultivation. Therefore, to overcome these problems and to achieve rapid large scale clonal propagation, in vitro propagation techniques can be applied. The successful use of tissue culture techniques for the rapid propagation of some species of Lilium including Lilium longiflorum (Han et al., 2004), L. ledebournii (Azadi and Khui, 2007), L. Oriental hybrid (Lian et al., 2002), L. Asiatic hybrids (Sato and Miyoshi, 2007) etc has been reported. The in vitro propagation techniques for Lilium species using bulb scales as an explant source has been reported by Nhut et al. (2006). Even though lily is a popular cut-flower in Sri Lanka, sufficient attention has not been given to develop an efficient method to provide planting materials to growers. Therefore, the present study was conducted with the objective of developing a suitable protocol for in vitro propagation and introduction of Lilium Asiatic hybrid variety “Prato”, for field cultivation. MATERIALS AND METHODS Formation of bulblets in vitro using bulb scales as explants Imported bulbs of Lilium Asiatic hybrid “Prato” (Fig. 1a) from the Netherlands were used to obtain scales for in vitro culture. The bulbs were subjected to a thorough quarantine investigation at the National Plant Quarantine Service, Katunayake and reported to be free of diseases. In the laboratory, the bulbs were sprayed with 0.1% Topsin (thiopenate methyl) solution, 2 days before culturing. The middle scale of the bulbs (Fig. 1c) were separated and washed thoroughly under running tap water, followed by a 15 seconds dip in 70% alcohol. Then the bulb scales were surface sterilized with a 2% NaOCl solution for 20 min. This was
IN VITRO PROPAGATION OF Lilium 57
followed by 3-4 times washing with sterile distilled water. Thereafter, 1 cm height portions from basal part of the scales were placed in 22x180 mm test tubes containing 15 ml of culture medium. The Montezuma De Carvalho and Guimoraes (M and G) medium (Montezuma De Carvalho and Guimoraes, 1974) supplemented with different combinations of IAA (0, 0.5, 1, 2 mg/l) and kinetin (0, 0.1, 0.2, 0.3 mg/l) was used for culture initiation. The medium was supplemented with 3% sucrose and solidified with 8 g/l agar. pH of the medium was adjusted to 5.5. The cultures were incubated at 25 ± 2°C under warm florescent lights (900-1500 lux) with 16:8 hrs (day:night) photoperiod. Performance of the cultures were observed continuously during the incubation period (8 weeks) and success percentage, number of bulblets/scale, size (diameter) of bulblets were recorded after the 8th week of culture initiation. The experiment was set up in a completely randomized design. Ten replicates were used for each treatment and effect of treatments was analysed using one way ANOVA with SAS statistical software. Mean comparison was done using Duncan’s Multiple Range Test (DMRT). The effect of hormones and Activated Charcoal (AC) on root initiation and bulblets regeneration Bulblets obtained from the best treatment of the previous experiment were separated from scales and used for root induction. M and G medium supplemented with different concentrations of Napthelic Acitic Acid (NAA) (0, 0.1, 0.2 mg/l) with (10 g/l) and without AC were used as treatments. The cultures were incubated under the same conditions described above. Four weeks after culturing rooting medium, root number, root length and number of bulbs regenerated were recorded. The experiment was set up in a Completely Randomized Design. Eight replicates were used for each treatment and effect of treatments was analysed using one way ANOVA with SAS statistical software. Mean comparison was done using Duncan’s Multiple Range Test (DMRT). Acclimatization and field planting Compost and sand 1:1 (growing) medium was placed in jam jars (50 g/jar) and sterilized in an autoclave at 15 lbs pressure and 121 °C temperature for 15 min. Rooted bulblets were washed thoroughly with distilled water and directly transferred in to jam jars containing the growing medium. The Jars were covered with poly propylene and kept in the culture room under the condition described above. After 1 week, culture jars were
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transferred from the incubation room to the plant house and gradually the cover was opened throughout a week. Once the jars were fully opened plants were transferred to pots containing the same medium and sent to Nuwara Eliya for further hardening. Potted plants were maintained in a polytunnel at the President’s House, Nuwara Eliya for 10 months. Albert solution (liquid fertilizer mixture) was applied to the plants once a week (50 ml/plant). Field planting was done after 10 months of acclimatization. Staking was practiced to avoid fall and the plants were maintained in the field until blooming. RESULTS AND DISCUSSION Formation of bulblets in vitro The sterilization procedure used for the culture establishment of Lilium Asiatic hybrid “Prato” was successful and the success rate was 83%. In agreement with Montezuma De Carvalho and Guimoraes, (1974) it was observed that, the Lilium Asiatic hybrid “Prato” has an ability to produce bulblets and buds from bulb scales in M and G medium( Fig. 1d). Disinfected mature bulb scales are the most commonly used explant in tissue culture of Lilium species (Han et al., 2004; Azadi and Khui, 2007; Lian et al., 2002; Nhut et al., 2006; Niimi, 1985). According to Pelkonen and Kauppi (1999), multiplication by scales depends on the species, variety, size and arrangement of scales, bulb age and environment factors. The results of the present study also indicated that well matured scales of Lilium Asiatic hybrid “Prato” is a good source of explant for the production of bulblets under in vitro conditions. In this study, the highest number of successful cultures (produced at least one bulblet) were obtained in the medium with 0.2 mg/l kinetin and 0 mg/l IAA hormone combination (Fig. 1e). It was noted that addition of hormones significantly increased the formation of bulblets when compared to the control. Out of all the treatments tested, the highest number of bulblets was formed in the medium contained 0.2 mg/l kinetin and 2 mg/l IAA (Table 1). It was observed that the optimum kinetin level for bulblet production was 0.2 mg/l. Further, increase in kinetin concentration (0.3 mg/l) resulted in a decrease in bulblet production. Cytokinins are generally known to promote the formation of buds in many excised and in vitro culture organs. Among cytokinins, Benzyl Adenine (BA) has extensively been used for bulb formation in Lilium spp due to its pronounced effect on adventitious shoot formation (Han et al., 2004). However, Montezuma De Carvalho and Guimoraes (1974) and Nhut et al.
IN VITRO PROPAGATION OF Lilium 59
(2006) reported that kinetin gives more positive results in bulb production. Similarly, in this study, a high rate of bulb production could be observed in the presence of kinetin. Table 1. Effect of different combinations of IAA and Kinetin on shoot regeneration and bulb formation in Lilium Asiatic hybrid “Prato�. Treatmen IAA level Kinetin Success rate Bulblets/ Average bulb t mg/l level mg/l scale diameter (cm) 1 0 0 2.8c 1.398e 11.02a bc ed 2 0.5 0 3.8 1.744 10.44a bc ed 3 1 0 3.2 1.740 10.92a 4 2 0 3.4 bc 2.166cde 10.42a bc bc 5 0 0.1 3.4 2.732 10.6a bc bcd 6 0.5 0.1 4.4 2.230 10.52a ab bc 7 1 0.1 5.7 2.67 10.6a bc bc 8 2 0.1 3.8 2.764 10.16a a b 9 0 0.2 6.2 3.03 10.06a bc cde 10 0.5 0.2 4.2 2.158 10.46a bc bcd 11 1 0.2 4.0 2.502 9.86a bc a 12 2 0.2 4.0 4.592 10.34a 13 0 0.3 3.4 bc 2.7bc 10.18a bc c 14 0.5 0.3 3.2 2.72b 10.1a bc c 15 1 0.3 3.8 2.642b 9.92a bc c 16 2 0.3 3 2.712b 10.32a CV % 33.17 25.87 10.18 Values followed by the same letter in each column are not significantly different at p=0.05
Lian et al. (2002), Han et al. (2004) and Niimi (1985) have shown that formation of bulblets from scales was induced by IAA. However, according to Nhut et al. (2006) a higher number of bulblets are formed by scales in the media without IAA. In agreement with Lian et al. (2002), Han et al. (2004) and Niimi (1985), in this experiment higher number of bulbs were observed in 0.2 kinetin and 2 IAA contained medium. The results showed that there was no significant difference in the size of bulblets formed in different treatments. According to Azadi and Khui (2007) and Lian et al. (2002) different hormone and sugar levels, season of explant harvesting and method of media addition could affect the size of bulblets. The size of bulblets produced was comparable to the results obtained by Azadi and Khui (2007) and Nhut et al. (2006). Root induction and bulblet regeneration The bulblets formed in the best induction medium (0.2 mg/l kinetin and 2 mg/l IAA) were used for the rooting experiment. After 4 weeks of culture in the rooting medium, root number, root length and bulblet regeneration were recorded and the results are summarized in Table 2. A
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significantly higher number of roots were observed in the hormone free medium with 10 g/l AC, (Fig. 1f).
IN VITRO PROPAGATION OF Lilium 61 Table 2.
Effect of NAA and activated charcoal on root induction and bulblet regeneration in Lilium Asiatc hybrid “Prato”. With AC
0 NAA mg/ l CV%
Root No. 3.75a
0.1
3
ab
0.2
2.5bc 35.67
Root length 0.95a
Without AC Regeneration % 80 a
1.25d
Root length 1.45a
1.325
60
b
1.5cd
1.5a
50bc
1.025a
60b
1.5cd
1.125a
60b
a
52.46
14.03
Root No.
35.67
52.46
Regeneration % 70ab
14.03
Values followed by the same letter in each column are not significantly different at p=0.05
Although Nhut et al. (2006) have given 0.2 mg/l NAA as the best treatment for root production, Azadi and Khui (2007) have shown endogenous hormones may also be one of the most important factors determining the root production in Lilium in vitro cultures. They have reported that higher root production of winter harvested Lilium ledebourii bulb scale explants was due to the accumulation of IAA during the cold season. However, results of this experiment (Table 2) revealed that root production of in vitro cultures of Lilium Asiatic hybrid is not affected by exogenic hormone treatments. In agreement with Han et al. (2004), the results of the experiment revealed a higher root formation in the hormone free medium, containing AC. Nhut et al. (2006) have reported that though AC has an important role in inducing cell development and differentiation in some plants such as orchids, onion and carrot, growth of roots and bulbs of Lilium sp. was not affected by AC. In contrast to these results, in the present study, root production in the hormone free medium with AC was three folds higher than that of hormone-free medium without AC. Activated charcoal might absorb phenolic and other toxic substances in the culture medium and stimulate root formation and normal regeneration of bulblets. Han et al. (2004) indicated that AC adsorbs not only inhibitory substances accumulating in the culture medium but also cytokinin and auxins, thus altering the ratios of medium components and subsequently influencing regeneration. Root development and higher rate of bulblet regeneration were observed in this medium (Table 2). Therefore, hormone free M and G medium with AC was identified as the suitable medium for regenerating in vitro produced bulblets of Lilium Asiatic hybrid “Prato” due to the higher root development and higher rate of bulblet regeneration (Fig. 1g).
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Figure 1. Micro propagation of Lilium Asiatic hybrid “Prato”. 1a. Field cultivation of Prato,1b. Harvested bulbs of “Prato”, 1c. Lilium bulb and scales, 1d. Establishment of scale in vitro culture, 1e.Bulblet initiation, 1f. Root induction, 1g. Regenaration of bulblets in rooting medium, 1h. acclimatization, 1i. Hardening in plant house, 1j. blooming micro propagated plants in the field, 1k. Flowers in floral arrangements
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Acclimatization and field planting Transferring of rooted bulblets to sterilized compost medium under in vitro conditions helped to reduce possible fungal infections caused by direct exposure of plantlets to outer environment. Maintenance of these cultures in culture room with the same environmental conditions as previous cultures facilitated roots to grow into the media. According to previous experiences, opening of these cultures within the culture room caused plants to desiccate quickly due to low relative humidity created by air conditioners. In this experiment, hundred percent plant survival was observed when cultures were gradually exposed to external environment under plant house conditions (Fig. 1h). Gradual opening of culture bottles throughout a week facilitated the regenerated bulblets to adapt to outer environment easily. According to personal communication with local tissue culture technicians, survival rate of most ornamentals during acclimatization is low as 30% in anthurium, 45% in orchids 15% in gerbera and 75% in gladiolus. Transferring of plantlets to pots was done carefully with minimum physical damage and transferring plants along with the medium minimized root damage and transfer stress. Since the climatic condition in Nuwara Eliya is ideal for Lilium cultivation, hardening of potted plants was practiced in that location. According to Mu Ding (2005), ideal day and night temperatures for Lilium Asiatic hybrids are 25째C and 15-13째C respectively. The location selected satisfies this requirement as well. In the initial stage, growth of plants was very slow and plants were maintained for ten months in the same pots under polytunnel conditions (Fig. 1i). According to Grassotti et al. (1996) normally the plants obtained from bulblets need 3 years to develop to flowering size. Usually growers in Europe, lift the bulb after 1 year of cultivation (autumn) and store in refrigerator till the next summer in order to avoid loss of the bulblets due to low temperature during winter. Since the temperature of Nuwara Eliya does not lower below about 8째C and plants were grown in a greenhouse, harvesting and replanting practice was not required in this experiment. The plants started to grow rapidly after 10 months and then they were transferred to the field. Generally lilies require cold treatment or climatic phase change to generate flowering stems from bulbs (Mu Ding, 2005). Although special cold treatment or seasonal change was not provided in this experiment, 94% plants started to generate flowering stems and 70% plants initiated blooming four month after field planting (Fig. 1 j and k).
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CONCLUSIONS Bulblets could be initiated from bulb scales of Lilium by culturing on M and G medium supplemented with 0.2 mg/l kinetin and 2 mg/l Indol Acetic Acid (IAA). Bulblets initiated could be rooted successfully on hormone free medium with activated charcoal (10 g/l) and the survival rate of rooted plants was 100% during acclimatization in compost and sand (1:1) medium. More than 70% of the plants bloomed within 18 months after field establishment. ACKNOWLEDGEMENTS The Authors express their gratitude to the Director General and the staff of Department of National Botanic Gardens for the support and facilities provided to conduct the research and special thanks to Mr. A. Gunasena, Vanila International private limited, Kothmale, for providing planting materials. REFERENCES Azadi, P. and M.K. Khui. 2007. Micro propagation of Lilium ledebourii (Baker) Boiss as affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments. Electronic journal of Biotechnology 10(4):582-591. Grassotti, A., J. Lee and M.S. Roh. 1996. Economics and culture techniques of Lilium production in Italy. Acta Horticulturae 414:25-34. Han, B.H., H.J. Yu, B.W. Yae and K.Y. Peak. 2004. In vitro micropropagation of Lilium longiflorum “Georgia” by shoot formation as influenced by addition of liquid medium. Scientia Horticulturae 103:39-49. Lian, M., D. Chakrabarthy and K.Y. Paek. 2002. Growth and uptake of sucrose and mineral ions by bulblets of Lilium oriental hybrid ‘Casablanca' during bioreactor culture. The Journal of Horticultural Science and Biotechnology 77(3):253257. Montezuma de Carvalho, J. and M.L.L. Guimaraes, 1974. Production of plantlets from the stamen’s filament of Lilium regale cultivated in vitro. Biologia plantarum 10:472-473 Mu Ding, 2005. Technical aspects of cut flower production-Lilium and Chrysanthemum. Hand book of International Training Workshop on Cultivation Technology of Ornamental Plants. Institute of vegetables and flowers, Chinese Academy of Agricultural Science, Beijing, China. 101-108 pp. Niimi,Y. 1985. Factors affecting the regeneration and growth of bulblets in bulb-scale cultures of Lilium rubellum Baker. Journal of the Japanese Society for Horticultural Science 54(1):82-86. Nhut, D.T, N.D.T. Tam, V.Q. Luan, N.Q. Thien, N.T Minh, T.S. Du, and B.V. Le. 2006. Standerization of in vitro lily (Lilium spp.) Plantlets for propagation and bulb formation. In Proceedings of International Workshop on Biotechnology
IN VITRO PROPAGATION OF Lilium 65 in Agriculture, Nong Lam University, Ho Chi Minh City, Vietnam. 134137p. Okazaki, K., J. Lee and M.S. Roh, 1996. Lilium species native to Japan, and breeding and production of Lilium in Japan. Acta Horticulturae 414:81-92. Pelkonen, V.P. and A. Kauppi, 1999. The effect of light and auxins on the regeneration of lily (Lilium regale Wil.) cells by somatic embryogenesis and organogenesis. International Journal of Plant Sciences 160(3)483-490. Simmonds, J.A. and B.G. Cumming, 1976. Propagation of Lilium hybrids. I. Dependence of bulbet production on time of scale removal and growth substances. Scientia Horticulturae 5(1):77-83. Sato, T. and K. Miyoshi. 2007. Restoration of intact anthers in a thermo sensitive, antherless, Male sterile cultivar of Asiatic hybrid lily in response to high temperature. Journal of Horticultural Science and Biotechnology 82(5) 791-797.