An efficient protocol has been established for clonal propagation of Alpinia calcarata, a commercially important medicinal plant on Murashige and Skooge medium usingrhizome bud explants. Of the different concentrations of 6-benzylaminopurine (BAP) andBAP in combination with different levels of kinetin, the best response of axillary shootproliferation was achieved in a combination of 1.5 mg/l of kinetin in combination with 0.5mg/l of BAP producing 13.6 shoots per explant in 6-8 weeks of culture followed by 2 mg/lkinetin and 0.5 mg/l BAP with an average of 6.2 shoot buds from each of the explants.Rooting of the shoots also occurred in the same medium in 3 weeks of subculture. Shootstransferred to half strength MS medium with 0.5 mg/l IBA was optimum for healthyrooting. The healthy in vitro rooted plants were hardened on plastic cups in sterile sand andwere transferred to pots containing potting mixture under green house conditions for 3-4weeks for acclimatization. The survival rate was 87-90% and the plants established well inthe field and developed rhizomes after 4-6 weeks of growth under shade house. Thisprotocol proves its utility for rapid propagation of A. calcarata, which can be exploited forpharmaceutical and commercial purpose

Devi, A. Indira

Dwivedi, N.K.

K.I., Asha

Nair, R. Asokan

English

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Research in Plant Biology, 2(5): 13-17, 2012                                                           ISSN : 2231-5101  www.resplantbiol.com   Received: 10.7.2012; Accepted: 20.9.2012  Regular Article  In vitro propagation of Lesser Galangal (Alpinia calcarata Rosc.) - a commercially important medicinal plant through rhizome bud culture  Asha* K.I., A. Indira Devi, N.K. Dwivedi and R. Asokan Nair   NBPGR Regional Station, KAU.P.O., Vellanikkara, Thrissur-680656, Kerala, India *Corresponding author: ashakarthy@gmail.com    An efficient protocol has been established for clonal propagation of Alpinia calcarata, a commercially important medicinal plant on Murashige and Skooge medium using rhizome bud explants. Of the different concentrations of 6-benzylaminopurine (BAP) and BAP in combination with different levels of kinetin, the best response of axillary shoot proliferation was achieved in a combination of 1.5 mg/l of kinetin in combination with 0.5 mg/l of BAP producing 13.6 shoots per explant in 6-8 weeks of culture followed by 2 mg/l kinetin and 0.5 mg/l BAP with an average of 6.2 shoot buds from each of the explants. Rooting of the shoots also occurred in the same medium in 3 weeks of subculture. Shoots transferred to half strength MS medium with 0.5 mg/l IBA was optimum for healthy rooting. The healthy in vitro rooted plants were hardened on plastic cups in sterile sand and were transferred to pots containing potting mixture under green house conditions for 3-4 weeks for acclimatization. The survival rate was 87-90% and the plants established well in the field and developed rhizomes after 4-6 weeks of growth under shade house. This protocol proves its utility for rapid propagation of A. calcarata, which can be exploited for pharmaceutical and commercial purpose.  Keywords: Alpinia calcarata, rhizome bud, shoot multiplication, regeneration    The commercially important aromatic medicinal plant, Alpinia calcarata Rosc. (Zingiberaceae), native to India and China (Mangaly and Sabu, 1992) commonly known as Cheriyarasna, Chittaratha and Lesser galangal, is extensively used in the indigenous systems of medicine in Asian countries. In India, the dried rhizome forms a major ingredient of several Ayurvedic drug formulations as Rasna in the preparation of Ashwagandharistam, Rasnadichurnam, Rasnadikashayam and Rasnadithailam (Sabu, 2006). Drug prepared by using rhizomes are used in the treatment of rheumatism, bronchial catarrh and asthma. It is used against infection of the skin and also possesses antibacterial activity. It is also used to stimulate digestion, purify blood, prevent bad breath, improve the voice and also to treat inflammation and arthritis (Husain et al. 1992). Rhizomes of A. calcarata yield essential oil that contains 1,8-ciniole and α-fenchyl acetate which are useful as commercial herbicide and pesticide in addition to pharmaceutical use. The plant is propagated vegetatively through rhizomes due to its rare flowering and lack of seed set which seems insufficient to meet the present day demand especially in the northern districts of Kerala where approximately 1.70 tons of dried rhizome is required annually (Sasidharan and Muraleedhara, 2000). Considering the high demand and greater economic and medicinal values of A. calcarata, it is Asha et al. / Research in Plant Biology, 2(5): 07-11, 2012  14  necessary to develop a suitable protocol for in vitro production of disease free stocks through tissue culture techniques. Protocol for in vitro propagation of A. calcarata and evaluation of their chemical fidelity through comparison of volatile compounds has been reported (Sudha et al., 2012).  Materials and Methods Healthy and actively growing young axillary shoot buds derived from the rhizomes of the field harvested 12 month old stock plants from the accession IC373610 (Fig.1) maintained in the herbal garden of NBPGR Regional Station, Thrissur, Kerala served as the source of explants for the present experiments. The explants were cleaned thoroughly in running tap water after removing the roots and scale leaves. The trimmed out rhizome bud explants were initially wiped with 70% alcohol and were taken in a conical flask and surface sterilized using 2% extran detergent (v/v) for 10 minutes with vigorous shaking and then rinsed repeatedly under tap water and then rinsed thrice in distilled water. This was followed by treating the explants with 70% alcohol for one minute and then treated with 0.1% HgCl2 for 10 minutes followed by rinsing with sterile water 3-5 times under the laminar air flow chamber. The surface sterilized explants were trimmed to appropriate sizes (0.6-1.0 cm) by the removal of exposed scale leaves and cut base of the buds and were inoculated on to MS (Murashige & Skooge, 1962) basal solid medium, supplemented with myo-inositol (100 mg/l) and 3% (w/v) sucrose and different concentrations of 6-benzylaminopurine (BAP) (0.5-3.0 mg/l) and combinations of BAP (0.5 mg/l) and kinetin (0.5-3 mg/l) for shoot multiplication. Individual regenerated shoots were excised and used for rooting. Root induction was carried out on subculture to the same medium and on to full and half-strength solid medium supplemented with 0.5 mg/l IBA. The pH of the media was adjusted to 5.8 prior to gelling with 0.7% agar and dispensed into the culture tubes and sterilized by autoclaving at 1210C and 108 Pa for 18 min. The well prepared explants were inoculated into the sterile media. The cultures were incubated in the culture room at 24±20C under 16/8 h (light/dark) photoperiod with light provided by white fluorescent tubes at 2000 lux. The Rooted individual shoots were removed from the culture medium, rinsed in water to remove the media and transferred to plastic cups with sterile soil for hardening and were then transferred to pots containing potting mixture to green house conditions for 3-4 weeks for acclimatization and subsequently to the field.    Results and Discussion Experiments were conducted with a view to find out the optimum concentration of BAP and also combination of BAP (0.5 mg/l) with different concentrations of kinetin for maximum shoot multiplication from the culture of rhizome bud explants (Fig.2). The multiple shoots were cultured from rhizome bud explants of Alpinia calcarata on MS solid medium supplemented with different concentrations of BAP and 0.5 mg/l BAP in combination with different levels of kinetin after 4-8 weeks of incubation (Table-1). Among the different treatments, the best response was obtained in a combination of 1.5 mg/l of kinetin with 0.5 mg/l of BAP (Table-1). In this combination almost 90% of the inoculated explants showed shoot bud regeneration within 4-6 weeks of inoculation and the average number of shoots per explant was 13.6 (Fig.3). The second highest response was observed in a combination of 2 mg/l Kinetin and 0.5 mg/l BAP where an average of 8.2 shoot buds were regenerated from each of the explants (Fig.4). These results are in agreement with the report of shoot induction and multiplication in A. zerumbet in a combination of BAP (1.5 mg/l) and kinetin (0.5 mg/l) (Rikkimuthu et al., 2011). Among the different concentrations of BAP, maximum shoot induction was recorded in 0.5 mg/l BAP where 6.4 shoots/explant Asha et al. / Research in Plant Biology, 2(5): 07-11, 2012  15  were produced (Fig.5) followed by 1mg/l with the production of 5.2 shoots/explant (Fig.6) compared to the control MS solid basal medium without any growth hormones producing only 1.87 shoots/explants (Fig.7). As the concentration of BAP was increased to 3 mg/l, the number of shoots induced showed a reduction to 2.8 per explant (Fig.8), suggesting that lower concentration of BAP as best for shoot proliferation in the species. A gradual reduction in the BAP concentration facilitating rapid induction of normal plants during subsequent subculture was reported by Sudha et al., 2012 in A. calcarata. An efficient regeneration protocol from rhizome bud explants in MS medium with BAP and kinetin was reported in A. purpurata (Kochuthressia et al., 2010) and Kaempferia galanga (Kochuthressia et al., 2012). Multiplication can be continued by transferring each of the separated shoot explants to the same media or to a medium with BAP at 0.5 mg/l (Fig.9) in jam bottles. Individual shoots on subculture to the same shoot initiation medium after 3-4 weeks initiated roots spontaneously (Fig.10). Similar result supporting spontaneous rooting of shoots in the same concentration of shoot induction media with cytokinins BAP and kinetin was reported in A. purpurata (Kochuthressia et al., 2010) and simultaneous production of shoot and roots in MS medium with kinetin 3 mg/l from rhizome bud explants was reported in A. galanga (Borthakur et al., 1999). Rhizome bud as potent explants for root induction from shoots in shoot inducing medium itself was reported in Curcuma longa and Zingiber officinale (Balachandran et al., 1990), Alpinia calcarata (Agretious et al., 1996) and A. galanga (Borthakur et al., 1999). Healthy and higher rooting (95%) was obtained in the shoots sub-cultured to half strength MS solid medium with 0.5 mg/l IBA (Table-1; Fig.11). Optimum shoot production in half-strength MS medium with 0.2 mg/l IBA was reported in A. calcarata (Sudha et al., 2012), in 0.5 mg/l IBA in A. officinarum (Selvakumar et al., 2007) and A. zerumbet (Rikkimuthu et al., 2011) and in 1 mg/l IBA in Kaempferia galanga (Kalpana and Anbazhagan,  2009). The rooted plants thus obtained were washed well in running tap water to make it free from agar gel and were separated (Fig.12) and transferred to plastic cups with sterile sand for hardening (Fig.13). After 3 weeks, these plants were transferred to pots with potting mixture in greenhouse (Fig.14) and were then transferred to the field and the survival rate was recorded.  Table 1: Effect of different concentrations of BAP alone and BAP (0.5 mg/l) in combination with kinetin (0.5-3 mg/l) in MS basal solid medium on shoot multiplication from rhizome bud explants and rooting of the induced shoot buds in Alpinia calcarata  Concentration of BAP (mg/l) Concentration of Kinetin (mg/l) Number of shoots/ explant Percentage of  regeneration Rooting Percentage MS Basal -- 1.87 80 58 0.5 -- 6.4 87 65 1.0 -- 5.2 76 78 2.0 -- 4.2 82 79 3.0 -- 2.8 79 79 0.5 0.5 3.58 85 85 0.5 1.0 5.32 92 80 0.5 1.5 13.6 90 82 0.5 2.0 8.2 85 75 0.5 3.0 3.4 78 80 Shoots on transfer to MS basal medium with 0.5 mg/l IBA 85 Shoots on transfer to ½ MS basal medium with 0.5 mg/l IBA 95  Asha et al. / Research in Plant Biology, 2(5): 07-11, 2012  16                                                      Figure 1. IC373610, the accession of Alpinia calcarata maintained in the herbal garden of NBPGR Regional Station, Thrissur, Kerala, mother plant for the rhizome bud explants. 2. Surface sterilized rhizome bud inoculated on to the medium for shoot initiation. 3. Multiple shoot induction (13.6) from rhizome bud explant in MS medium with 0.5 mg/l BAP and 1.5 mg/l kinetin. 4. Induction of 8.2 shoots from rhizome bud explant in MS medium with 0.5 mg/l BAP and 2 mg/l kinetin. 5. Multiple shoot induction (6.4) from rhizome bud explant in MS medium with 0.5 mg/l BAP. 6. Induction of 5.2 shoots from rhizome bud explant in MS medium with 1 mg/l BAP. 7. Initiation of 1.87 shoots from rhizome bud explants in MS basal medium. 8. Reduction in the number of shoots initiated to 3 in MS medium with 3 mg/l BAP. 9. Multiplication of shoots on transfer to fresh media in jam bottles. 10. Spontaneous rooting of shoots in shoot induction medium itself. 11. Vigorous rooting of shoots on transfer to MS solid basal medium supplemented with 0.5 mg/l IBA. 12. Well rooted shoots separated out. 13. Shoots transferred to plastic cups with sterile sand for hardening. 14. Hardened plants in pots in the greenhouse.                 8 4 5 6 7 9 3 2 11 12 13 14 1 10 Asha et al. / Research in Plant Biology, 2(5): 07-11, 2012  17   Almost 90% of the regenerated plants survived and showed vigorous growth of rhizome and roots without showing any morphological variations. Successful establishment of regenerated plantlets with 95% survival rate was reported in A. calcarata (Sudha et al., 2012), 93% in A. officinarum (Selvakumar et al., 2007) and 80% survival within 10-12 weeks in A. galanga (Borthakur et al., 1999). The present protocol on in vitro propagation of A. calcarata proved viable to be exploited for mass multiplication of the species for pharmaceutical and commercial purpose.  Acknowledgements Authors are thankful to Dr. K.C. Bansal, Director and Dr. M. Dutta, Head, Germplasm Evaluation Division, NBPGR, New Delhi for their support and for providing the necessary research facilities to carry out the present work. The financial assistance provided by ICAR is greatly acknowledged.   References Agretious, T.K., K.P. Martin and M. Hariharan. 1996. In vitro clonal multiplication of Alpinia calcarata Roscoe. Phytomorphology, 46:133-138. Balachandran, S.M., S.R. Bhat and K.P.S. Chandel. 1990. In vitro clonal multiplication of Turmeric (Curcuma spp.) and Ginger (Zingiber officinale Roscoe). Plant Cell Reports, 8(9): 521-524. Borthakur, M., J. Hazarika and R.S. Singh. 1999. A protocol for micropropagation of Alpinia galanga. Plant Cell, Tissue and Organ Culture, 55(3):231-233. Husain, A., Virmani, O. P., Popli, S. P., Misra, L. N., Gupta, M. M., Srivastava, G. N. Abraham, Z. and A.K. Singh. 1992. Dictionary of Indian Medicinal Plants. CIMAP, Lucknow, India.546p.  Kalpana, M. and M. Anbazhagan. 2009. In vitro production of Kaempferia galanga (L.) - an endangered medicinal plant. Journal of Phytology, 1(1):56-61. Kochuthressia, K.P., S. John Britto, L. Joelri Michael Raj, M.O. Jaseentha and S.R. 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Sasidharan, N. and P. K. Muraleedhara, 2000. Survey on the commercial exploitation and consumption of medicinal plants by the drug industry in northern Kerala. Research Report No. 193, ISBN 0970-8103, Kerala Forest Research Institute, Thrissur, Kerala 2000. Selvakumar, C., A. Balakrishnan and B.S. Lakshmi. 2007. Rapid in vitro Micropropagation of Alpinia officinarum Hance., An Important Medicinal Plant, Through Rhizome Bud Explants. Asian Journal of Plant Sciences, 6(8):1251-1255. Sudha, C., M. George, K. Rameshkumar and G. M. Nair, 2012. Improved Clonal Propagation of Alpinia calcarata Rosc., a Commercially Important Medicinal Plant and Evaluation of Chemical Fidelity through Comparison of Volatile Compounds. American Journal of Plant Sciences, 3(7): 930-940. 

In vitro propagation of Lesser Galangal (Alpinia calcarata Rosc.) - a commercially important medicinal plant through rhizome bud culture

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In vitro propagation of Lesser Galangal (Alpinia calcarata Rosc.) - a commercially important medicinal plant through rhizome bud culture

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