Shoot induction and plant regeneration from receptacle tissues of Lilium longi orum

Scientia Horticulturae 87 (2001) 131±138 Shoot induction and plant regeneration from receptacle tissues of Lilium longi orum Duong Tan Nhut a,*, Bui Van Le b, Michio Tanaka c, K. Tran Thanh Van b a Institute of Biology in Dalat, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam b Institut de Biotechnologie des Plantes, UMR 0569, Universite de Paris-Sud, F-91405 Orsay Cedex, France c Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Accepted 12 April 2000 Abstract An ef cient system has been developed for the in vitro plant regeneration of Lilium longi orum Thunb. by culturing receptacle sections from ower buds. The sections were cultured on one-half MS medium plus 30 g l 1 sucrose, 8 g l 1 agar, 5.4 mm NAA or 4.9 mm IBA plus 2.2 mm BAP.A section size of 3±4 mm was found to be optimal. After 60 days an average of 41 shoots were formed per explant. More vigorous shoots were obtained by subculturing on hormone-free medium with 20 g l 1 sucrose. Rooting occurred on one-half MS medium with 1.1 mm NAA. Rooted plants were hardened-off in a greenhouse for two months, and normal owering plants were produced. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Flower bud; Section size; Thin cell layer; Vigorous shoot 1. Introduction Lilium longi orum has been the object of extensive studies for plant regeneration from bulb-scales (Gupta et al., 1978; Nightingale, 1979; Stimart Abbreviations: BAP, 6-benzyladenine; IAA, indole-3-acetic acid; IBA, indole butyric acid; MS, Murashige and Skoog; NAA, naphthaleneacetic acid * Corresponding author. Present address: Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. Correspondence address: Duong Tan Nhut, 761-0702, Kagawa-ken, Kitagun, Miki-cho, Hiragi 407-6, Japan. Tel./fax: 81-87-898-4995. E-mail addresses: duong@stmail.ag.kagawa-u.ac.jp, nhutduong@hotmail.com (D. Tan Nhut). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0304-4238(00)00163-1

132 D. Tan Nhut et al. / Scientia Horticulturae 87 (2001) 131±138 and Ascher, 1981), leaves (Stenberg et al., 1977), anthers (Qu et al., 1988), stem segments (Nhut, 1998) or embryos (McRae and McRae, 1979). In previous studies on the micropropagation of Lilium species, bulb-scales were shown to be the more favorable explant source for the production and formation of bulblets (Stimart and Ascher, 1978; Takayama and Misawa, 1980, 1983; Niimi, 1986; Gerrits and De Klerk, 1992; Priyadarshi and Sen, 1992; Wickremesinhe et al., 1994; Tanimoto and Matsubara, 1995). Flower stalk fragments of lily (Bigot, 1974) produced adventitious buds and bulblets originating from peripheral tissue. In the present report we describe factors in uencing the regeneration of shoots by using explants from receptacles of L. longi orum owers. 2. Materials and methods 2.1. Plant materials Closed ower buds, 8 cm in length, of L. longi orum Thunb. `nellie white' were washed thoroughly under running tap water for 30 min, soaked in detergent (Viso, Dong nai, Vietnam) for 5 min, rinsed six times with distilled water, and then with ethanol (70%) for 40 s. After being washed again with distilled water, the closed ower buds were disinfected with a 0.1% aqueous solution of HgCl 2 for 5 min, then rinsed six times in sterile distilled water. The ower stalk, receptacle, folded inner petal, outer petal sheath, ovary, stigma, stamen and anther of closed ower buds were separated out and cut into transversal sections (length is 5 mm). 2.2. Experimental design 2.2.1. Effect of all parts of ower on shoot regeneration All parts of ower buds (nˆ40) were cultured on one-half MS medium (Murashige and Shoog, 1962) containing 30 g l 1 sucrose, 8 g l 1 agar (Wako Pure Chemical Industries, Japan) and supplemented with auxin (0 or 5.4 mm NAA, 0 or 4.9 mm IBA, 0 or 5.7 mm IAA) and cytokinin (0, 0.9 or 2.2 mm BAP) for testing the possibility of regeneration of all parts of the ower bud. 2.2.2. Effect of explant thickness on shoot regeneration Receptacles 1±4, or 5 mm in length were used to test the effect of size on optimal regeneration. These explants were cultured on one-half MS medium containing 30 g l 1 sucrose, 8 g l 1 agar and supplemented with auxin (either 5.4 mm NAA, 4.9 mm IBA, or 5.7 mm IAA) and cytokinin (2.2 mm BAP). Initially, 3-mm long explants (nˆ40) were used. Explants yielding the highest number of shoots were subsequently used as source of material (shoot clusters) for these experiments.

2.2.3. Effect of sucrose concentration on shoot regeneration The effect of sugar was examined on the development of vigorous, clonal shoots. Shoot clusters (nˆ40) were subcultured on one-half MS medium containing different sucrose concentrations (0, 10, 20 or 30 g l 1 ). 2.3. Culture condition All multiplication experiments were carried out in borosilicate test tubes (25 mm150 mm), while for rooting, vigorous shoots were cultured in 150-ml asks on one-half MS medium plus 1.1 mm NAA for 45 days. Each test tube and ask contained 15 or 20 ml, respectively, of medium and was capped with double aluminum foil. Media were adjusted to ph 5.8 before autoclaving at 1218C at 105 KPa for 15 min. All cultures were incubated at 2518C with a 12 h photoperiod per day at quantum ux density of 40 mmol m 2 s 1 from uorescent lamps. For each treatment, at least 40 explants were used and the experiment repeated three times. Data were recorded at 45, 60 and 90 days after culture. The resulting data from treatments are presented as means with standard errors. 2.4. Transplantation D. Tan Nhut et al. / Scientia Horticulturae 87 (2001) 131±138 133 Fifty shoots were rooted as described previously by culturing on one-half MS medium with 1.1 mm NAA (Nhut, 1998). Plantlets with well-developed roots were removed from the culture vessels and after having their roots washed in running tap water, were transferred to pots containing soil with N/P/K (10:10:10). A plastic cover was inverted over each plantlet to ensure high humidity during the rst few days after transfer. Subsequently, the plants were transferred to eld conditions. 3. Results The objective of such a series of experiments is to obtain uniform and vigorous shoots. Since no shoots or roots occurred from explants on media without hormones, and since shooting and rooting occurred together in media containing a single auxin or cytokinin, data for these are not shown since the latter was not desired. Explants were excised from several parts of L. longi orum ower buds and placed on one-half MS medium supplemented with various combinations of auxins and cytokinin in order to examine which part of the ower bud might form shoots. Of all the explants tested, receptacles had the highest rate of shoot production with 100% survival rate (Table 1). Most explants, except those from

134 D. Tan Nhut et al. / Scientia Horticulturae 87 (2001) 131±138 Table 1 The response of L. longi orum receptacle sections (nˆ40) 5 mm long when cultured on medium containing NAA, IAA, IBA and BAP, measured at 90 days Growth regulator (mm) NAA IBA IAA BAP Number of shoots Number of roots 5.4 0 0 2.2 17.4 3.9 0.4 0.1 5.4 0 0 0.9 14.6 3.3 3.7 0.8 0 4.9 0 2.2 17.1 3.8 2.5 0.6 0 4.9 0 0.9 14.4 3.2 4.0 0.9 0 0 5.7 2.2 16.1 3.6 1.7 0.4 0 0 5.7 0.9 12.9 2.8 1.0 0.6 ovaries, died within 45 days culture (data not shown). Ovary explant tissues enlarged slightly, but did not form shoots on the culture medium (Fig. 1D). In the presence of 4.9 mm IBA or 5.4 mm NAA, the number of shoots per explant was slightly higher than that in media with 5.7 mm IAA and 2.2 mm BAP. The data on rooting shown in Table 1 indicate that NAA and IBA are equally good in inducing roots while the use of IAA is less ef cient. While these experiments provided data on the optimum conditions of media, another set of Table 2 Effects of explant size (nˆ40) on the regeneration of receptacle sections of L. longi orum ower buds on three separate media: I, II, III Medium containing 2.2 mm BAP Size of explants (mm) Time (days) Survival rate (%) Number of shoots per surviving explant 5.4 mm NAA (Medium I) 1 45 15 10.6 0.7 2 45 85 30.6 1.7 3 60 100 41.4 2.3 4 60 100 35.1 1.7 5 90 100 18.3 0.9 4.9 mm IAA (Medium II) 1 45 15 9.2 0.4 2 45 80 25.0 1.6 3 60 100 38.6 1.9 4 60 100 33.8 1.5 5 90 100 17.6 0.8 5.7 mm IAA (Medium III) 1 45 15 7.9 0.6 2 45 80 19.9 1.3 3 60 100 31.9 2.1 4 60 100 25.0 1.0 5 90 100 17.2 0.7

Fig. 1. The growth and development of shoots derived from receptacle section culture: (A) top view of shoots regenerated from receptacle culture (3 mm in length) on medium containing 5.4 mmnaa and 2.2 mm BAP after 60 days culture (bar: 1 mm); (B) side view of shoots regenerated from receptacle culture (3 mm in length) on medium containing 5.4 mm NAA and 2.2 mm BAP after 60 days culture (bar: 1 mm); (C) formation of shoots, aerial roots and calli on 3 mm receptacle sections on various media (bar: 1 mm) (Table 1); (D) ovary section explant showing enlargement on various media (bar: 1 cm) (Table 1); (E) shoots derived from receptacle culture (3 mm in length) induced from medium containing 5.4 mm NAA and 2.2 mm BAP before subdivision (bar: 1 cm) (60 days culture); (F) plantlets formed on rooting medium containing 1.1 mm NAA after 45 days culture (bar: 1 cm).

136 D. Tan Nhut et al. / Scientia Horticulturae 87 (2001) 131±138 Table 3 Effects of sucrose on the development of shoots from shoot clusters (nˆ40), measured at 60 days Sucrose (g l 1 ) Shoot height (cm) Shoot width (mm) 30 2.5 0.6 2.1 0.5 20 3.2 0.7 3.1 0.7 10 3.3 0.7 2.5 0.6 tests was designed to optimize the size of explants for shoot production. In tests with receptacle explant of varying sizes (1±5 mm), higher number of shoots were obtained in the 3 and 4 mm range (Table 2, Fig. 1A and B). After 60 days in culture the survival rate of the shoots was 100%. To induce a further development of shoots, shoot clusters were cultured on onehalf MS media containing 10, 20 and 30 g l 1 of sucrose, respectively. The media contained no hormones. The culture period was 60 days. Data obtained (Table 3) show that shoots were slightly sturdier when 20 g l 1 was used. When no sugar was used, the shoots became necrotic (100%). Subculturing of the initial shoot clusters on rooting medium enhanced root formation (Fig. 1E as compared to Fig. 1F). In the presence of 30 g l 1 there was slight callusing while at 10 g l 1 the shoots tended to become necrotic, indicating that 20 g l 1 would be the optimum for shoot development. 4. Discussion Verron et al. (1995) showed that NAA is the most effective auxin for inducing shoot formation in vitro from ower buds and that a cytokinin is essential. From the data presented in this report, it can be observed that the in vitro shoot regeneration of L. longi orum is shown to occur with a high ef ciency using receptacle explants on media with NAA or IBA and without passing through a callus phase. The results also suggested that the tissue might have had a relatively high endogenous concentration of cytokinins, since shoot formation occurred in the presence of NAA and IBA and appeared to be independent of the addition of BAP (0 mm, data not shown). High endogenous cytokinin activity has also been shown to be expressed in the induction process of adventitious shoots in transverse thin cell layer explants of Amaranthus edulis (Bui et al., 1998). The stimulating effect of sucrose on shoot formation has also been shown in other lily species (Takayama and Misawa, 1980; Gerrits and De Klerk, 1992; Bonnier and van Tuyl, 1997). However an increase in sucrose concentrations from 6 to 9% reduced shoot growth in L. longi orum (Bonnier and van Tuyl, 1997).

D. Tan Nhut et al. / Scientia Horticulturae 87 (2001) 131±138 137 The importance of explant size is in accordance with the results of Silvertand et al. (1992) who showed that 5 mm segments excised from 10 cm long ower stalks of Allium ampeloprasum L. formed more than 40 shoots per explant without callus formation. In our studies with L. longi orum, receptacle sections, 3 mm in length, excised from ower buds produced up to 41 shoots per explant, thus indicating that shoot formation does not only depend on hormone type but also on explant size. In conclusion we have established a method for direct shoot regeneration in L. longi orum Thunb. without an intermediate callus phase using receptacle explants, thus limiting the possibility of obtaining somaclonal variation. This method has already been successfully applied to L. longi orum Thunb. `ace' and Lilium auratum Lindl. Acknowledgements The authors thank Institute of Biology in Dalat for their support, Dr. Oluf L. Gamborg for critical reading of the manuscript and many valuable suggestions and Mr. Jaime Teixeira da Silva for critical reading of the manuscript and discussion. The authors also would like to express their appreciation to Dr. Wayne Parrott, Department of Crop and Soil Sciences, University of Georgia, Athens, GA, USA, for reviewing and providing valuable commentaries in the preparation of the manuscript. References Bigot, C., 1974. Comparaison des aptitudes pour le bourgeonnemant de tissus super ciels et de tissus profonds cultives in vitro. CR Acad. Sci. Serie D. 278, 1027±1030. Bonnier, F.J.M., van Tuyl, J.M., 1997. Long term in vitro storage of lily: effect of temperature and concentration of nutrients and sucrose. Plant Cell Tiss. Organ. Cult. 49, 81±87. Bui, V.L., Do My, N.T., Jeanneau, M., Sadik, S., Shanjun, T., Vidal, J., Tran Thanh Van, K., 1998. Rapid plant regeneration on a C 4 dicot species: Amaranthus edulis. Plant Sci. 132, 45±54. Gerrits, M.M., De Klerk, G.J., 1992. Dry-matter partitioning between bulb and leaves in plantlets of Lilium speciosum regenerated in vitro. Acta Bot. Neerl. 41, 461±468. Gupta, P., Sharma, A.K., Charturvedi, H.C., 1978. Multiplication of Lilium longi orum Thunb. by asceptic culture of bulb-scales and their segments. Indian J. Exp. Biol. 16, 940±942. McRae, E.A., McRae, J.F., 1979. Eight years of adventure in embryo culture. Lily Yearb N. Am. Lily Soc. 32, 74±81. Murashige, T., Shoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473±497. Nhut, D.T., 1998. Micropropagation of lily (Lilium longi orum) via in vitro stem node and pseudobulblet culture. Plant Cell Report 17, 913±916. Nightingale, A.E., 1979. Bulblet formation on Lilium longi orum Thunb. `Nellie white' by foliar spray applications of PBA. Hort. Sci. 14, 67±68.

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