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Micropropagation Scheme of Curcuma alismatifolia Gagnep. N. Toppoonyanont, S. Chongsang, S. Chujan, S. Somsueb and P. Nuamjaroen Department of Biology, Faculty of Science, Maejo University Chiang Mai 50290 Thailand Keywords: Curcuma alismatifolia Gagnep., thidiazuron, imazalil, retarded shoot,

micropropagation Abstract

This study was conducted to develop efficient and repeatable micropropagation scheme for Curcuma alismatifolia Gagnep., starting from initiation, multiplication, to rooting stage. During the initiation stage, Curcuma alismatifolia Gagnep. coflorescences were removed from each pouch of inflorescence, and used as starting materials. They were cultured in a modified MS (1962) containing 10 mg/L BA and 0.1 mg/L IAA. After 1 month in culture, they developed and reverted to vegetative shoots located at the same positions and arranged spirally within the bracteole, similarly to those in vivo. It was clear from anatomical analysis that shoots directly emerged from flower organs, not via callus formation. During multiplication stage factors such as BA and TDZ were evaluated at concentrations of 0, 0.1 and 0.5 mg/L with combinations of Imazalil (IMA) at 0, 2 and 4 mg/L. It was found that 0.5 mg/L TDZ in combination of 4 mg/L IMA was able to increase 30-40 new shoots from one explant with retarded shoot morphology. Before transferring to the greenhouse these retarded shoots elongated and rooted in medium containing 0.3 mg/L BA and 0.1 mg/L IAA, and 0.1 mg/L IAA, respectively. The micropropagation scheme is also presented. INTRODUCTION

Curcuma alismatifolia Gagnep. (Fig. 1A), known as Siam Tulip or summer tulip, is a native plant to the north-eastern part of Thailand. It belongs to a genus Zingiberaceae with about 70 species spread over the tropics from India, Burma, Indochina, Thailand, Malaysia to Queensland and the Pacific Islands (Chittenden and Synge, 1981). It is now becoming the promising both cut flower and pot plant in Europe.

The increasing demand for rhizomes for the export market has led to demand for continuity of supply over longer periods of the year. The plant tissue culture technology has greatly played a role in facilitating rhizome production. Normally, shoot tip from Curcuma alismatifolia Gagnep. rhizomes have been used as initial explants, however, this method is risky because of contamination and low multiplication rate problems. Thus several initial explants were considered. Successful shoot formation from inflorescence explants has been reported in different crops such as Gerbera jamesonii (Topoonyanont and Dillen, 1988), Limonium ‘Misty Blue’ (Topoonyanont et al., 2000). The succession of floral reversion depended on the developmental stage and the original position of the inflorescence.

Hamidah (1997) was successful in applying TDZ (thidiazuron) with IMA (imazalil) in propagating Anthuriums. The plant morphology showed resemblance of a complete plant except for the small size of retarded shoots. Aside from this, Werbrouck and Debergh (1996) reported that 1 µM TDZ was able to induce the production of Spathiphyllum floribundum shoot (50). However, the use of 1 µM TDZ with 34 µM IMA not only induced similar plants but there was an increased number of induced plants (77) than using TDZ alone. Moreover, using TDZ with IMA induced more plants when compared to other cytokinins such as BA, metatopolin and Zeatin.

Proc. IXth Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005

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MATERIALS AND METHODS Plant Material and Preparations of Initial Explant

Curcuma alismatifolia Gagnep. inflorescence grown in greenhouse conditions were selected and used as initial explants. After the lowest flower flowered the inflorescence were cut and all bracts were removed (Fig. 1B). The coflorescences of each pouch were taken and placed in 250 ml vessels containing 25 ml modified MS (1962) supplemented with 3% sucrose, 7.5 g/L agar, 10 mg/L BA and 0.1 mg/L IAA. The pH was adjusted to 5.8 prior to autoclaving at 121°C for 15 minutes. The vessels were then placed in the culture room at 25±2°C and 14 hours photoperiod of 40 µmol m-2 s-1 provided by fluorescent tubes. After 30 days, the cultures were observed. Histological Research

To investigate the origin of the newly formed shoots, initial explants were collected after 0 and 30 days on initiation medium. Ten explants were collected. Coflorescences were fixed overnight in FAA (3% formaldehyde, 5% acetic acid, 50% ethanol), dehydrated ethanol series, infiltrated and embedded in paraffin (Sass, 1966). Serial 14 µm longitudinal sections were stained with hematoxylin. Effect of BA, TDZ and IMA on Multiplication Rates

During the plant multiplication stage, shoots obtaining from floral reversion transferred to fresh medium, which is a modified MS (1962) supplemented with combination of IMA (0, 2 and 4 mg/L) with TDZ (0, 0.1 and 0.5 mg/L) or BA (0.1 and 0.5 mg/L) were tested. After 8 weeks the plants were evaluated.

Before transferring to soil, shoots were subcultured to the same medium except adding 0.3 mg/L BA and 0.1 mg/L IAA for elongation for 4 weeks before transferring to rooting medium, which is the same medium but only 0.5 mg/L IAA was added. After 3 weeks, the rooted plants were then transferred to greenhouse. RESULTS Shoot Initiation from Inflorescence

In this experiment, the coflorescence of each pouch was studied under a stereo-type microscope (Fig. 2A-D). Results indicated that coflorescence consisted of florets that developed into 2 fully developed flowers namely: floret 1 (f1) and floret 2 (f2) and into remaining undeveloped florets (Fig. 2B). Floret 1 (f1) is the most developed and no bracteole. On the other hand, floret 2 (f2) has a floret 1 bracteole (bt1) wrapping around the base of floret 2 and the remaining florets (Fig. 2B). When floret 1 bracteole (bt1) was removed, it showed the presence of floret 3 (f3) (Fig. 2B-C). When the bracteole of floret 3 was also removed, florets were found in each bracteole and had similar structure with floret 2 and 3. When the said bract was further removed, it revealed the appearance of floret 4 and 5, respectively. Finally, the removal of the bract of the floret 5 revealed the presence of the floral primordia (fp) (Fig. 2D). From the study, it was found that the florets on coflorescence had spiral arrangement. Except floret 1 and floret 2, the remaining parts were cultured in vitro for 6 weeks, it was observed that the new shoots have emerged from the bracteoles (bt1, bt2, bt3) (Fig. 3A). This allowed the separation of new shoots based on their position in the bracteoles.

The emergence of new shoots was due to direct organogenesis since there was no evidence of callus induction. Interestingly, new shoots from each bracteole positions (Fig. 3A) showed different development which resembled the development of the florets (Fig. 2C). New shoots that emerged from bracteole (Fig. 3B) appeared similar to florets in Fig. 2A. It was also found that shoots were arranged spirally as previously mentioned in the coflorescence.

The most interesting part of the study was floral primordia (Fig. 4A). When they were cultured in initiation medium, they grew and developed into numerous multiple

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shoots at various developmental stages (Fig. 4B). Histological Analysis

Anatomical analysis of longitudinal serial sections of initial explant (Fig. 5A-D), showed the reversion of florets (f1, f2) into shoots (vg) in bracteole. Each floret did not develop uniformly. Florets 1 and 2 (f1, f2) (Fig. 5A) were observed to bear complete flower parts. At the same time, parts of floret 3 (f3) (Fig. 5B) showed vegetative meristem and leaf primordia (lf) (Fig. 5B), as well as floret 4 (f4). Some parts have developed as flower while the floral primordia revert into leaf primordia. Moreover leaf primordia of floret 4 appeared an axillary shoot (ax) with leaf primordia (Fig. 5B-D). Interestingly, floret 5 that was not yet a fully developed flower grew distinctively into a complete shoot (Fig. 5C). On the other hand, the floral primordia (fp) (Fig. 5D) was able to completely revert into shoots. Effect of BA, TDZ and IMA on Multiplication Rates

Results presented in Fig. 6 showed that shoots cultured on medium without both BA, TDZ or IMA did not proliferate. When BA or TDZ together with IMA were added, shoot proliferation enhanced. TDZ was found to be the most effective at 0.5 mg/L (9.33 plants), 5-6 times higher than BA at the same concentration (1.5 plants) (Fig. 6A). It was observed that TDZ brought about different morphological appearance; shorter and narrower leaves. Meanwhile, plants that grew from BA were higher and with more longer and wider leaves.

No direct effect was observed in plants cultured with IMA alone. But when supplemented with BA or TDZ, these enhanced the production of plants, in particular when 4 mg/L IMA was added to 0.5 mg/L TDZ, the highest number of new plants (11.3 plants), which was more than when IMA was added to BA.

During second subculture, with TDZ in combination of IMA, growth became very distinct. The number of shoots totaled 32.14 plants (Fig. 6B). The explants appeared ball-like cluster which were referred to as retarded shoots (Fig. 7A) while plants receiving BA appeared normal and resembled to the control plants.

The plants that appeared as retarded shoots were able to develop as complete plants when subcultured to fresh medium except adding 0.3 mg/L BA and 0.1 mg/L IAA for elongation (Fig. 7B). Before transferring to greenhouse, these elongated shoots were transferred again to rooting medium containing 0.1 mg/L IAA for 3 weeks. A production scheme of Curcuma alismatifolia Gagnep. at the industrial level is presented in Fig. 8. DISCUSSION

When Curcuma alismatifolia Gagnep. inflorescence was cultured in vitro, a reversion into a shoot was observed. However, the position and appearance of reversion were not similar. Floral reversion of Limonium hybrid ‘Misty Blue’ (Topoonyanont et al., 2000) occurred due to the reversion of inflorescence meristem. While in Curcuma alismatifolia Gagnep. the reversion was from flower organs. Nevertheless, the similarity between these two plants was that the gradients along the axis were found. Moreover, floral primordia at the distal end of coflorescence can revert into numerous complete shoots, which indicated that it can be the most appropriate initial explant sources for successful Curcuma micropropagation scheme.

Proliferation of retarded shoot and its morphology of Curcuma alismatifolia Gagnep. in vitro depended on the type and concentration of BA, TDZ and IMA. This special morphology had better results because they resulted in a new method of production system which helps in reduction of labor cost.

The IMA substance has a role in production of retarded shoots of Curcuma alismatifolia Gagnep. Similarly, Werbrouck and Debergh (1996) have reported that IMA, together with TDZ, produced Spathiphyllum floribundum shoots up to 77 per cluster, and was more than TDZ alone (50 shoots). The role of IMA was concerned with assisting the TDZ to increase its effects but no further study. However, Werbrouck and Debergh (1996)

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demonstrated that IMA might be involved in different parts of the complex interaction among different hormones. Literature Cited Chittenden, F.J. and Synge, P.M. 1981. The Royal Horticutural Society of Gardening vol.

2. Oxford University Press, London. p.513-1088. Hamidah, M. 1997. Optimisation of micropropagation systems in Anthurtium spp. Med.

Fac. Landbouwn. Univ. Gent. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with

tobacco tissue culture. Physiol. Plant. 15:473-497. Sass, J.E. 1966. Botanical microtechnique. The Iowa State University Press, Iowa. Topoonyanont, N. and Dillen, W. 1988. Capitulum explant as a start for micropropagation

of gerbera: culture technique and applicability. Med. Fac. Landbouw. RUG. 53:169-173.

Topoonyanont, N., Ampawan, R. and Debergh, P.C. 2000. Reversion of Limonium hybrid ‘Misty Blue’ inflorescence development and its applicability in micropropagation. Scientia Hort. 83:283-299.

Werbrouck, S.P.O. and Debergh, P.C. 1996. Imidazole fungicides and paclobutrazol enhance cytokinin induced adventitious shoot proliferation in Araceae. J. Plant Growth Regul. 15:81-85.

Figures

Fig. 1. Curcuma alismatifolia Gagnep inflorescence (A) showing spike bearing prominent

spiral bracts which laterally fuse to form pouches. Each pouch subtends a coflorescence of 4-5 flowers (B), arranged spirally with floral primordia at the distal end. The terminal bracts from a sterile cluster called a coma, often brightly and attractively colored. The tips of the bracts have chlorophyll, which give them a green tinger at the tip. (bar = 1 cm)

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Fig. 2. A flower cluster from a pouch of Curcuma alismatifolia Gagnep. showing different

developmental flowers (f1, f2, f3, f4, f5) arranged within bracteoles (bt1, bt2) and floral primordia (fp) at the most distal end. (bar = 2 mm)

Fig. 3. Shoot proliferated from bracteoles (bt1, bt2, bt3) when cultured in vitro, showing

front view (A) and back view (B) of shoots emerged from bracteoles. (bar = 5 mm)

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Fig. 4. Loral primordium (A) (bar = 1 mm) further developed into numerous shoots (B)

(bar = 1 cm) 6 weeks cultured in vitro.

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Fig. 5. Longitudinal serial sections of cinninus of Curcuma alismatifolia Gagnep.

showing different flowers at different developmental stages. (bar = 1 mm)

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Fig. 6. Interaction between 0, 2 and 4 mg/L IMA and 0, 0.1 and 0.5 mg/L TDZ or BA on

the number of new plantlets per cluster of Curcuma alismatifolia Gagnep. In the first (A) and second subcultures (B) after a 6 weeks incubation.

Fig. 7. Growth and development characteristics of retarded shoot clusters (A), and

elongations of retarded shoots when transferred to modified MS (1962) supplemented with 0.3 mg/L BA and 0.1 mg/L IAA for six weeks (B). (bar=0.5 cm)

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Fig. 8. Micropropagation scheme of Curcuma alismatifolia Gagnep. at the industrial level

showing the different stages from initiation, multiplication, rooting, until transferring to greenhouse.