J. Hort. Sci. Vol. 1 (1): 24-27, 2006 Low cost strategy for micropropagation of Lilium Asiatic hybrid cv. Toscana R. K a u r , Neetu Thakur and D. R. Sharma Department of Biotechnology University of Horticulture & Forestry Nauni, Solan 173230 (H.P.), India E-mail: rkaur uhf@rediffmail.com ABSTRACT A low cost protocol for in vitro propagation of Lilium cv. Toscana has been developed through incorporation of cost-effective media components. MS medium supplemented with 0.75 mg I* BAP (6-benzylaminopurine) and 0.5 mg 1' NAA (a-naphthalene-acetic acid) was prepared with tapioca granules, table sugar and tap water in different combinations in place of agar-agar, sucrose and distilled water, respectively. Culture medium containing all the cost effective components was found to be the best for in vitro establishment of cultures yielding 6.00 bulblets per explant and medium supplemented with tapioca granules as cost effective component was found to be the best for in vitro multiplication of bulblets giving 3.70 bulblets per in vitro formed bulblet five weeks from third subculture. Tapioca supplemented MS medium containing 1 mg I' NAA was significantly better than all the other modified media giving 86.62% in vitro rooting, 2.86 average root number and 4.60 cm average root length. For hardening of in vitro rooted bulblets, coco peat, peat moss and coco peat in combination with peat moss were found to be at par giving 100% survival. Key words: Lilium, micropropagation, low-cost strategy INTRODUCTION Lilium, a monocot belonging to the family Liliaceae, is one of the leading cut flowers of the world. It has become commercially important due to its bold, beautiful and fascinating form of flowers, long vase life and capacity to rehydrate after long transportation. Lilium is native to the Northern hemisphere and is widely distributed over China, South Canada, Siberia and extends upto Florida in USA. In India, Lilium is found in Nilgiri hills and in the Himalayan region. Lilium can be propagated by both sexual and asexual means. Most commercially grown cultivars are propagated through scaling, a technique which produces 3-4 bulbs from each bulbscale depending upon bulbscale size and variety. Though a bulb under ideal conditions may yield anywhere between 50 to 100 bulblets, this rate is far too low to meet the present day demand for planting material. Also, reduced vigour of bulbs with repeated cycles of vegetative propagation is reported which may be due to accumulation of soil borne diseases (Van Aartrijk and Blom-Bamhoom, 1983). Thus, mass propagation through tissue culture is needed for research and development of the Lilium industry. Cost effective micropropagation would facilitate commercialization of the technology. In this paper, we describe a rapid and low-cost protocol for micropropagation of Lilium using cheaper medium components. MATERIAL AND METHODS Bulbs of Lilium cv. Toscana were collected from a private nursery at Darang, Palampur (HP), India. Bulbscales were excised from mother bulbs of 12cm -14 cm diameter stored in saw dust at 5°C for six weeks after harvest. The scales were surface sterilized in 0.2% solution of bavistin (carbendazim) for 8-9 min., washed with sterile water and treated with 0.1% solution of HgCl^ for 3 min., followed by thorough washing with sterile water. For in vitro establishment of cultures, basal segment each of about Icm^ was excised from surface sterilized scales and inoculated onto standard MS medium (Murashige and Skoog, 1962) and MS medium modified by replacing sucrose, distilled water and agar-agar with table sugar, tap water (potable drinking water) and tapioca granules (Manihot esculentum), respectively (Table 1). MS medium (standard and modified) was supplemented with BAP (0.75 mg 1') and NAA (0.5 mgl-'). On formation of in vitro bulblets, these were separated and individually subcultured both on standard as mailto:uhf@rediffmail.com Kaur et al well as modified media (Table 1). After 3-4 subcultures each of 4-5 weeks, in vitro induction of rooting was attempted in in vitro formed bulblets on MS standard medium and MS modified media supplemented with NAA (Table 2). All the media compositions shown in Tables 1 & 2 were supplemented with 3 mg/1 thiamine-HCl, 0.1 g/1 inositol, 3% sucrose; pH was adjusted to 5.8 before autoclaving at 121 °C and 15 psi for 15 min. Cultures were maintained at 25±2°C under 16 h photoperiod provided by cool, white, fluorescent lamps (40 |a,mole m'^s'). Survival and establishment of in vitro rooted bulblets was studied after transplanting the bulblets into pots containing different soil mixtures (Table 3). These were kept at 25 "C under 16 h photoperiod of 3000 lux intensity and 75% relative humidity. All experiments were repeated thrice with 72 replicates. Data recorded for different parameters were subjected to completely randomized design (CRD) (Gomez and Gomez, 1984). Statistical analysis based on mean values per treatment was made using analysis of variance (ANOVA) technique of CRD. RESULTS AND DISCUSSION Pre-treatment of bulbscale segments with 0.2 % carbendazim solution for 8-9 min. followed by 0.1 % solution of HgCl2 for 3 min. yielded 94% cultures free from contamination. In vitro induction of bulblets and in vitro multiplication of bulblets on standard MS medium supplemented with 0.75 mg 1 ' BAP and 0.5 mg 1' NAA was carried out to standardize a general protocol for micropropagation of Lilium cv. Toscana. The same protocol was modified by adding different but cheaper components into the medium. There were significant differences among different media in terms of number of bulblets per explant as well as the rate of multiplication of bulblets. Among these media, the maximum number of bulblets per explant (8.0) was produced on M^ medium (Table 1) having all the cost effective components such as tapioca granules, table sugar and tap water. The least effective modified medium was M^ having tapioca alone as the cost effective component,which produced 1.24 bulblets per explant. . In vitro induced bulblets when multiplied on modifified M^ medium gave the maximum multiplication rate of 3.70 bulblets per in vitro bulblet. The lowest multiplication rate of 1.46 bulblets per bulblet was obtained on M^ medium (Table 1). For induction of rooting, in vitro formed bulblets were separated and cultured singly on various rooting media (Table 2). Out of four modified media, R̂ having tap water as the cost effective component was the best, yielding 86.62% rooting, 2.86 average root number and 4.6 cm average root length. It was followed by R̂ with 74.25 % rooting, average root number 2.19 and average root length 1.35 cm. Out of all the cost effective media, the least effective medium for in vitro induction of rooting was R̂ Table 1. In vitro establishment of scale segments and in vitro bulblet multiplication ot Lilium cv. Toscana on standard MS medium (1962) and modified MS medium MS basal medium +BAP (0.75 mgl') + NAA (0.5 mgl') Mj (standard medium) Mj (modified medium) Mj (modified medium M^ (modified medium) M, (modified medium) Type of gelling agent Agar-agar (8g/l) Agar-agar (8g/l) Agar-agar (8g/l) Tapioca granules (125/1) Tapioca granules (125/1) Type of sugar used (30 gl') Sucrose Table sugar Sucrose Sucrose Table Sugar Type of water used Distilled water Distilled water Tap water Distilled water Tap water No. of bulblets per basal segment (at! from seven weeks inoculation) 11.20 3.20 4.40 1.24 8.00 Rate of multiplication of bulblets (at six weeks from subculture) 5.75 3.20 3.70 1.95 1.46 Table 2. In vitro induction of rooting in in vitro bulblets of Lilium cv. Toscana on standard MS medium (1962) and modified MS at six weeks from culture MS basal medium +NAA(lmgl-') R, (standard medium) Rj (modified medium Rj (modified medium R̂ (modified medium Rj (modified medium) CD at 5% Type of gelling agent Agar-agar (8g/l) Agar-agar (8g/l) Agar-agar (8g/l) Tapioca granules (125/1) Tapioca granules (125/1) Type of sugar used (20 gl') Sucrose Table Sugar Sucrose Sucrose Table Sugar Type of water used Distilled water Distilled water Tap water Distilled water Tap water Rooting percentage 100.00 74.25 86.62 59.58 72.41 4.94 No. of roots per bulblet 3.42 2.19 2.86 1.65 2.02 0.62 Root length (cm) 4.42 1.35 4.60 0.45 0.86 - J. Hon. Sci. Vol. 1(1): 24-27, 2006 25 Low cost strategy for micropropagation of Lilium having tapioca as the cost effective component, yielding 59.58% rooting, average root number 1.65 and average root length 0.45 cm. R3 medium was significantly better than the other modified media. Rj and R̂ were statistically at par with respect to rooting percentage and number of roots per bulblet. In vitro rooted plantlets, generated on different modified media, were transferred to different potting mixtures (Table 3). A total of 600 plantlets were transferred. 100% survival rate was achieved on Pj, P^ and P̂ and only 61.88% of in vitro rooted bulblets survived on P3 at one month from transplantation. Tissue culture has a number of advantages in Lilium propagation. Though the advantages of micropropagation are tremendous, cost is a limiting factor. Attempts have been made in the present investigation to explore the possibility of cost reduction in micropropagation of Lilium. In the present study 94% cultures free from contamination were obtained by treating the explants with 0.2% carbendazim solution for 8-9 min. and 0.1% solution of HgCI^ for 3 min. Priyadarshi and Sen (1992) achieved a high rate of sterilization using Bavistin (0.2%). Novak and Petru (1981) Rybczynski and Gomolinska (1989) and Dabrowaski et al (1992) recommended the use of sodium hypochlorite for successful sterilization of bulbscales of Lilium for in vitro culture. Mj medium consisting of table sugar, tapioca and tap water gave reasonably high number of in vitro bulblets per explant (Table 1). Earlier attempts by Sharma et al (1992) in 'Colt'- a rootstock of cherry, Ganapathi et al (1992) in banana and Okuno et al (1996) in Brassica campestris, tried to bring down the cost of in vitro muliplication on MS medium containing tap water and table sugar as cost effective components of culture medium. The present results are also supported by some earlier findings in Lilium cultivars by Jeong et al (1996). In the present investigation, out of four modified media, R3 medium containing tap water was more effective in in vitro induction of rooting. Sharma and Singh (1995) in ginger and Kaul (1998) in kiwifruit suggested the use of Table 3. effect of various potting mixtures used for Iiardening of in vitro rooted bulblets of Lilium cv. Toscana Medium P, P. P. P4 Potting mixture Coco peat Peat moss Sand •. Soil: FYM Coco peat: Peat moss 1:1:1 1:1 % Survival at one month from transplantation to field 100.00 100.00 61.86 100.00 commercial grade sugar and tap water over sucrose and distilled water, respectively, for in vitro shoot multiplication Chandra et al (1990) used table sugar for microtuberization and micropropagation of potato, and, glucose and fructose were found to be better than sucrose in stimulating the formation of embryos in anther culture of many genotypes of Triticum aestivum (Chu et al, 1990). Last and Brettell (1990) Orshinky et al (1990). Nene et al (1996) suggested that agar agar can be successfully replaced by tapioca in chickpea micropropagation and tobacco regeneration. Sorvari et al (1997) used starch as the gelling agent in in vitro germination of encapsulated somatic embryos of carrot (Daucus carota). From the above studies, it may be concluded that table sugar, tapioca and tap water in culture medium can be effectively used at different stages of micropropagation of Lilium Asiatic hybrid cv. Toscana. The use of commercial grade sugar in place of the more expensive sucrose, tapioca granules in place of agar-agar and tap water instead of distilled water, could reduce the cost of in vitro raised plants thus making micropropagation in this variety a viable proposition for commercialization. Further, this can be also tried for other commercially important cultivars of Lilium and ornamental crops. REFERENCES Chandra, R., Upadhyay, M. D. and Chaudhary D. R. 1990. A simplified low cost medium for potato micropropagation. In: Proc. Second Int'l. Conf. Biotech. lARI, New Delhi. Chu, C. C , Hill, R. D. and Brule-Babel, A. L. 1990. 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Adventitious bud formation from bulbscale explants of Lilium speciosum Thunb: In vitro effects of wounding, TIBA and temperature. Pflanzenphysiol, 110:353-63. (MS Received 29 March, 2006 Revised 17 July, 2006) J. Hort. Sci. Vol. 1 (1); 24-27,2006 27