237 Note Plant Biotechnology, 21 (3), 237 241 (2004) In Vitro Propagation and Storage ofBrugmansia versicolor Lagerheim Indra Dutt BHATT, Jung In CHANGa and Noboru HIRAOKA* Niigata University of Pharmacy and Applied Llfe Sciences, 265 IHigash jima. Niitsushi, Niigata 956‑8603, Japan Welfare, 5, "Present address.' Central PharmaceuticalAffairs Council. Ministry ofHealth Nokbun Dong, Eunpyung ‑ Gu, Seoul 122 704. Korea *Corresponding author Email address: hiraoka@niigatapharm.ac.jp & Received I April 2004; aceepted I June 2004 Abstract Brugmansia versicolor Lagerheim of the family Solanaceae was propagated through shoot tip culture and shoots were cold stored in vitro at 5, 10 or 15 C under light or dark condition. All the shoots died after cold storage for 6 months at 5 C irrespective of light condition. When shoots were stored for 12 months at 15 iC under light illumination, the best 100% survival rate was obtained. The plants regenerated from shoots stored for 6 or 12 months retained the ability to accumulate scopolamine as much as the control plants which were raised from shoots maintained under normal culture condition without cold ‑ storage. These findings show that slow growth storage of in vitro B, versicolor shoots at 15 C can be used as a germplasm conservation system for short ‑ or medium term duration without deterioration of the ability to accumulate the secondary metabolites. Key words: germplasm conservation, micropropagation, refrigeration, seopolamme Solailaceae tropane alkaloid. Abbreviations BA, 6benzyladenine; IAA, indole3acetic IBA, indole3butyric acid; LS, Linsmaier and Skoog (1965) medium. acid; The genus Brugmansia (tree datura; angel's native to South America and widely all distributed over the world in the temperate and tropical regions. Several species are known within the genus and all are cultivated as ornamentals trumpet) is because of their decorative flowers (Preissel and Preissel, 2002). the 10ng time under refrigeration remains to be inves‑ We previously reported successful slow growth storage ofAtractylodes lancea (Hiraoka and Kagoshima, 1993), A. ovata (Hiraoka et al., 2003), and Scopolia japonica (Hiraoka et al., 2004) shoot cultures under refrigeration for two years without deterioration of the biochemical characteristics of regenerated plants. As a continuation of the investi‑ gation along this line, we now present the results on the clonal propagation through shoot tip culture and biochemical assessment of the plants regenerated from cold ‑ stored shoot cultures of B. versicolor. The plant material used in the present study has been maintained in greenhouse condition at the Medicinal Plant Garden, Niigata University of Pharmacy and Applied Life Sciences in Niigata City (plant registration number: #477). It is a small tree (4 m high) possessing elliptic to oblong leaves covered with short soft hairs. Pendulous flowers are about 30 cm long and have a spathe Iike calyx with tigated. Brugmansia species exhibit much same type of alkaloid spectrum including scopolamine and hyoscyamine as principal alka‑ loidal components. Since these alkaloids are of clinical importance, Brugmansia plants have been cultivated in South America for the production of the alkaloids. Tissue culture techniques were applied for the clonal propagation of useful culti‑ vars (Griffin and Lin, 2000). Although progress has been made on in vitro propagation technology as well as conservation methods (Fletcher, 1994; Hiraoka et al., 1995; Bonnier and Tuyl, 1997; Bekheet, 2000; Reed et al., 2003), biochemical stability of plants preserved for The corolla is trumpetshaped and light green when emerged from a calyx and it turns first white then becomes light orange with time. The a single split. constricted near the calyx leaving a between parts of the flower. Corolla teeth two space long about and recurved. The flower has are 3 cm corolla tube is 238 A ftuit is fusiform and strong scent in the evening. about 20 cm long. The plant was introduced to the These morphological features, however, suggest it to be B. versicolor Lagerheim. Its habitat is restricted to the tropical regiGns of Ecuador (Preissel and Preissel, 2002), Throughout the present experiments, a basal medium was LS supplemented with 3% sucrose, O.8% agar, and pH was adjusted to 5.6 prior to autoclaving at 121 C for 20 min. A culture tube was capped with a silicone sponge plug. Cultures were maintained at 25 ) in culture room condition under sl light a 16h photoperiod of 50 flmol provided by eool white fluorescent tubes whenever Garden as Datura arborea L. m2 A otherwise stated. meristematic part of about I diameter was aseptically removed from each of culture three shoot tips and placed in a 13 x 130 tube containing 20 ml medium supplemented with 1 f!M Inaphthaleneacetic acid and 10 ftM BA. Out of three shoot tips, one initiated a shoot at the primary culture stage. Since initiation medium enhanced callus formation at the base of shoots, other hormonal combinations were tried to find out better conditions for shoot multiplication. Among tested combinations, 0.1, I or 10 f4M BA/kinetin alone or in combination with 0.1 ftM IAA, I /lM kinetin alone gave the best result, 2.4 i 1.3 shoots per culture. Therefore, shoots were routinely sub‑ cultured on shoot proliferation medium containing 1 mm mm fLM kinetin at bimonthly intervals. In a later experi‑ ment, however, we have found that I / M BA in combination with 1‑ 10 ftM gibberellic acid gave a better result on shoot multiplication rate, 4 ‑ 5 shoots per culture. Rooting medium included I flM IBA, since it was the best hormonal condition for rooting of shoots among hormonefree, IAA (1 ftM), and IBA (0.1, 1.0 and 10 l!M) by a preliminary experiment. Experiments were designed to evaluate the re‑ sponse of shoots to cold storage and its effect on their viability after reculturing on freshly prepared medium. Shoots were precultured on shoot prolif‑ eration medium under normal culture condition for 18 days, and then 15 cultures were stored in each under cold room condition of 5, 10 and 15"C darkness or of 5, 10 and 15･C under a 16h photo‑ s 1 Iight provided by cool period of 10 ,Lmol a white fluorescent tube. After 6, 12 and 18 months of shoots from each storage treatment cold storage, taken out and recultured on freshly prepared were shoot proliferation medium. The control shoots were maintained on the same medium at bimonthly intervals under normal culture condition without cold storage. The criterion of the survival per‑ centage was the initiation of new growth from m2 5 recultured shoots maintained under normal culture condition. Data on shoot number and shoot length were recorded after the third passage of reculturing process. At the 4th passage of shoot reculturing, shoots from each group were transferred to rooting medium. [n vitro plantlets were harvested after 30 days of incubation on rooting medium for biological and chemical assessment. To determine the appro‑ priate growth stage for the assessment of scopol‑ amine accumulation, plant materials at different growth stages were harvested and dried at 6(rC in a hotair oven. Those included four cultures of in vitro shoots, Ieaves of each three in vitro plantlets grown on three kinds of media ccntaining different concentrations of IBA, Ieaves of three potted plants, and six fully expanded leaves collected from a mature or mother plant. Two flowers were also harvested from the mother tree for alkaloid analysis. The scopolamine content was determined by gas chromatography following the procedure of Hiraoka et al. (1996). The percentage of scopolamine was expressed on a dry weight basis. The experiments were repeated twice excluding coldstorage under darkness. Values were expressed as mean stan‑ dard deviation. The significance of the difference was assessed by applying Student's t‑test or Dun‑ can's multiple range test (p Shoots stored for 6 and 12 months at different light and dark conditions showed variation in their response. At low temperature (5 C) all the shoots died in both the light and dark conditions. However, 100% shoots survived at 10 and 15'C in the light after 6 months storage. Shoots stored for 12 months showed 100% viability at 15 C in the light. The reduction in percentage survival was observed in the dark at 10 and 15 C. In these conditions (cold storage for and 12 months) 40 809; o shoots could survive. The shoot number hardly increased during storage for or 12 months under either condition. The shoot length increased nearly four times after 12 months of storage. Leaf color remained green at months of storage at 10 and 15 ) in the light, but it became yellowish after 12 months of storage. Shoots partially died under dark conditions and remained healthy in the light at 10 and 15 C. Shoots recultured after and 12 months of storage did not in their multiplication poten‑ differences show any tial when compared to respective control (Table l). On rooting medium, 80% or more shoots rooted in months storage groups, but rooting ratio decreased 6 6 6 6 6 50 70% in 12 months storage groups (Table 2). Regenerated plants did not show any apparent abnormality in morphology. Biomass production was not affected by cold storage for 6 months, but it decreased significantly by storage for 12 months to 239 Table 1 Multiplication potential of Brugmansia versicolor shoots after three passage of reculture 6months storage Treatmentl) No 5 Control } Shoot No./culture of samples . LIO L15 10 DIO D15 2 4 12 months storage 2.4 2.7 8 3.8 Shoot No./culture 1.1 17 1.7 0.8 d: 1.6 14 1.6 17 1.7 0.5 a O.8 a 3.5 3.5 No, of samples 2 i i a a l.2 a 0.7 a 1.9 a 9 13 a 1.5 i O.5 a 1.4 O.7 a L and D stand on light and dark conditions, respectively, and figures indicate temperature during storage. Mean values with standard deviations. significantly different Means by Duncan's multiple range within a column followed by the test (p Table 2 Biomass and scopolamine production of Brugmansia Yersicolor same plantlets regenerated letter are not ftom cold stored shoots in vitro Duration and n2 condition of storage Fresh weight of aerial part (g) No. of leaves Fresh weight of leaf (g) Scopolamine content (%) 6Months a3 8.0 i 2.0 a 0.22 a O.35 a 9.1 1.1 ab 0.63 9.2 1.0 ab O.62 O.35 a O.45 a 10.3 9.8 i: O.19b O.88 :!: O.74 i Control 20/20 1.26 LIO*) 20/25 1.26 L15 17/19 1.25 DIO D15 617 1.06 :!: 13/14 1.29 dl 10/16 1.35 LIO L15 7114 8/14 DIO D15 7114 O.85 10/14 0.86 O.46 i 0.72 i b O.72 i 1.7b 14.8 O.38 a O.29 a O.32 a O.32 a 11.6 i O.33 a 0.21 0.16 a O.27 a O.22 i O.OI a i O.02 a O.25 O.20 i 0.76 : 0.25 a 0.32 a O.25 i: i 5.1 b 0.79 i O.16 b O.10 11.7 :!: 2.9 ab 0.52 : 11.1 i 1.9 0.53 0.21 a 0.14 a O.15 1.4 0.03 a O.07 a 0.03 a 12 Months Control l) 2) 3 11.0 i a 2.6 ab O.59 :!: 1.8 O.54 :!: a O.19 0.17 a 0.13 0.16 O.20 a i O.OI a O.OI c O.03 bc O.OI ab : 0.05 c See Table 1. No. of rooted shoots/No. of inoculated shoots. Mean values with standard deviations. Means within a column of each duration followed by the same letter are not significantly different by Duncan's multiple range test lp irrespective of storage condition compared with the No significant differences in biomass pro‑ B, versicolor plants have been developed. Shoot cultures could be stored in the light at 15 C for 12 duction were recorded for plantlets raised from shoot cultures that had been stored under condi‑ tions differing in light illumination, temperature, and storage period. None of the shoots were alive when coldstored for 18 months at or 1(fC in both the light and dark conditions. However, 2 and out cultures withstood the storage at 15･C in the of light and in the dark, respectively. The foliage of those shoots was mostly damaged, but the shoots grew on shoot multiplication medium under normal culture condition and rooted on rooting medium. The in vitro plantlets accumulated as much scopol‑ months. This ccntrol. 8 5 5 3 amine as control ones. In the present study, a simple shoot multiplication protocol and a medium term preservation method of agreement with data on other tropical species which could be stored at the same temperature for longer periods (Banerjee and de Langhe, 1985; Sharma and Chandel, 1992). A11 the B. versicolor shoots stored for 12 months at 15"C in the light grew well when recultured under normal culture condition, but shoots stored at 5 C could not survive. This could be attributed to the fact that the species growing in the tropical region resulted in the is in death of shoots the highest survival percentage was obtained at 15'C after 12 months of storage, considering the minimum of monthly average temperatures, 23'C, at the natural habitat regions of the species (Legates and Wilmott, at 5 C. It is likely that 1990; Preissel and Preissel, 2002). Light seemed to 240 Table 3 Relationship between growth stage and alkaloid accumulation Growth Stage No. ofsamples Hormone concentration In vitro shoot 4 1 ft M kinetin Mature plant O.16 0.09 O.15 i O.08 0.20 O.05 0.17 i 0.02 O.11 O.03 i standard deviation affect the survival percentage of stored shoots. All the shoots 6 months old (100%) survived in the 10 and 15 C, but this value reduced shoot The 40‑80% appearance and light condition, leaf color were also affected by light in in the condi‑ the yellowish dark and green tion. Light affected shoot survival percentage, but scarcely seemed to affect post‑storage vigor of shoots or in vitro plantlets. Irrespective of the conditions (light and dark), recultured shoots multi‑ plied as vigorously as the control and did not show any variation in their multiplication potential. These results are in agreement on data obtained with other species such as poplar (Hausman et al., 1994), Glehnia littoralis (Hiraoka et al., 1995), Atrac‑ tylodes ovata (Hiraoka et al., 2003) where storage condition did not affect the proliferation rate of recultured shoots. Post storage vigor depends on the storage condition and may affect the morphological characteristics (Hausman et al., 1994). In the case of Brugmansia, no significant differences were ob‑ served between in vitro plantlets derived from cold stored shoots and those of control set except the number of leaves. This could be attributed to the fact that the storage condition was favorable to this species and no carryover effect on the morphology of the plants was detected. As for the possible duration of cold storage, 12 months seemed to be the limit under the present storage conditions, because shoot survival percentage was low and shoot appearance was deteriorated to a considerable light condition at to 0.001 ) O.032 flM IBA /lM 1 IBA 10 /lM IBA 3 3 3 6 Potted plant Mean Scopolamine content (% of dry weight) O.1 In vitro plantlet l in the leaf of Brugmansia versicolor in the dark. moths after storage. A whole plant of B. versicolor contains scopol‑ amine as a major tropane alkaloid, but hyoscyamine is hardly detected in both the leaf and flower. Its extent 18 quantity in the leaf varied with different develop‑ mental stages from an in vitro shoot to a mature plant (Table 3). Shoots in multiplication stage produced a lower level of scopolamine. The highest scopolamine content. O.28%, was recorded in the flower. The scopolamine content in the in vitro plantlets is comparable to that of potted plants, demonstrating that assessment of alkaloid accumu‑ lation in plants regenerated from coldstored shoots can be carried out at their in vitro plantlet stage instead of a pot or mature stage. The regenerated plants of all the shoot storage regimes accumulated scopolamine not less than the control crable 2). Their alkaloid composition was same with the parent plant, comprising scopolamine as a sole major alkaloid and the amount of other alkaloids was negligible. concluded that B. versicolor shoots in vitro can be preserved at 15 C under the weak light illumination for 12 months and that the plantlets regenerated from cold stored shoots show neither It is any detectable variation in their morphology nor any reduced potential in the scopolamine accumu‑ lation in the leaf. Acknowledgements The authors wish to thank Ms.'s M. Hiro'oka, K. Utashiro and S. Kobayashi for their assistance in experiments. Funding for this study was provided by the Ministry of Education, Science, Culture, and Sports of Japan (grant No. 11672232 to and and IDB). IDB also grant No. P02820 to thanks to the Japan Society for the Promotion of Science for being awarded the JSPS Postdoctoral Fellowship for Foreign Researchers (P‑ 02820). NH NH References Banenjee, N., de Langhe. E., 1985. A tissue culture technique and storage under minimal conditions of and plantain ). Plant (banana growth Musa Cell Rep., 4: 351 354. for rapid clonal propagation Bekheet. S. A., 2000. In itro preservation of officinalis. Biol. Plant., 43: Bonnier, F. J. M., Tuyl, storage of lily: J. M. 179‑ Asparagus 183. V., 1997. Long term effect of ternperature in vitro and concentration of nutrients and sucrose. Plant Cell Tissue Organ Cult., 49: 81 87. Fletcher. P. J., 1994. In vitro long term storage ofAsparagus. New Zealand J. Crop Hort. Sci., 22: 351 359. Griffin, W. J.. Lin, D, D., 2000. 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