Rapid in vitro Micropropagation of Alpinia officinarum Hance, An Important Medicinal Plant, Through Rhizome Bud Explants
Baddireddi Subhadra Lakshmi
An efficient multiple shoot has been developed for the medicinal plant Alpinia officinarum Hance (Zingiberaceae) using rhizome buds as explants. Multiple shoots originated when the rhizome buds were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of growth regulators. Optimum shoot multiplication was observed on MS medium containing 3% (W/V) sucrose and 3.0 mg L-1 kinetin (Kn) and 1.0 mg L-1 Naphthalene Acetic Acid (NAA). Each rhizome bud gave rise to an average of 11 shoots per explant. Rooting experiments with half-strength Murashige and Skoog medium revealed that 0.5 mg L-1 Indole-3-butyric acid (IBA) was more suitable for root induction when compared to IAA and NAA. Healthy in vitro rooting plantlets were transferred to pots containing a mixture of vermiculite and soil (1:1) for acclimatization for a period of three-four weeks and 93% of plantlets survived under field conditions.
Alpinia officinarum Hance (Lesser galangal) belonging to the family Zingiberaceae is an aromatic perennial herb distributed throughout the tropical and the subtropical Asian region. The name Alpinia officinarum was given to this herb, as the source of Galangal is less when compared to Alpinia galangal. It can grow up to 3-5 feet in height and flowers are small, white with red streaks. The rhizomes are reddish brown and about 2 cm in diameter. They are widely used in India, China and Asia for a wide range of diseases like respiratory tract infections, arthritis, Cancer, microbicidal and gastro intestinal disorders (Uehara and Yasuda, 1987; Sakai and Miyazaki, 1989; Lin and Hsu, 1998; Tewari and Pant, 1999; Wu and Larsen, 2000). It has been used in Europe as a spice for over a thousand years, having probably been introduced as a medicinal plant by the Arabian or the Greek physicians. The active molecule (5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone) was found to possess significant inhibitor of pancreatic lipase activity in hyperlipidemic mice (Shin et al., 2004). One of the active principles of the plant, diarylheptanoids have been reported to inhibit the pro inflammatory mediators like the Nuclear Factor kappa B (NF-κB), phosphorylation of MAPK, P44/42 and also potential inhibitors of leukotriene (LT), biosynthesis and prostaglandin synthase enzymatic activity and also suppression of inducible nitric oxide synthase expression level in RAW 264.7 cell line (Prem et al., 2003; Matsuda et al., 2006; Lee et al., 2006). However, there are many species of Zingiberaceae family having antioxidant properties (Vankar et al., 2001). Six diarylheptanoids were elucidated from the root material which was determined quantitatively by HPLC of Alpinia officinarum (Liu et al., 2005; An et al., 2006). Alpina officinarum is widely used in the pharmaceutical industry for therapeutic purposes and it provides the essential raw material for many ayurvedic preparations. Razing of Alpinia officinarum is a necessity in order to meet pharmaceutical needs and also to prevent the plants from becoming endangered or extinct (Peter, 2006). Several Zingiberaceae species have been investigated through in vitro rhizome bud multiplication which is an easy and safe method for obtaining uniformity and it also assures the consistent production of true-to-type plants within a short span of time (Balachandran et al., 1990; Borthakur et al., 1999; Rahman et al., 2004) To our knowledge there is no work reported on Alpinia officinarum which include multiple shoot formation from rhizome buds and micro propagation from seedlings. Hence the aim of the present study was to develop efficient protocols for Alpinia officinarum multiple shoot formation from rhizome bud explants. This technique would facilitate an alternative method for rapid large-scale clonal production and successful outdoor establishment of this medicinal species.
MATERIALS AND METHODS
Young rhizome buds of Alpinia officinarum were collected in October 2005 from TAMPCOL Medicinal garden (Tamil Nadu Medicinal Plant Corporation Limited), Kolli Hills, South India. The rhizome buds were initiated for micro propagation at Tissue Culture and Drug Discovery Laboratory, Centre for Biotechnology, Anna University, Chennai, India. The explants used for the study were washed with 5% (V/V) commercial bleach (Tween 20) and then rinsed in running tap water for 10 min. The cleaned buds were sterilized by soaking it with intermittent agitation in 0.2% (W/V) mercuric chloride for 5 min. The rhizome buds were subsequently washed three times with sterile double distilled water and were cut into appropriate sizes and inoculated onto the appropriate sterile media.
The shoot multiplication media was composed of MS (Murashige and Skoog, 1962)
basal medium containing myo-inositol (100 mg L-1) and sucrose (3%
W/V); this media was supplemented with different concentrations of kinetin (Kn)
and 6-benzylaminopurine (BAP) alone or in combinations with naphthalene acetic
acid (NAA). Individual regenerated shoots were excised and used for rooting.
Root induction was carried out on half-strength MS solid medium supplemented
with different auxins. The pH of the medium was adjusted to 5.8 with KOH before
adding agar (0.7%). Medium without plant growth regulators were used as a control.
Cultures were incubated at 25±2°C under a 16/8 h (light/dark) photoperiod
with light supplied by white fluorescent tubes at 2,000 lux. Rooted plants were
removed from the culture medium, rinsed in water to remove media and transferred
to pots containing vermiculite and soil (1:1) then potting plants were transferred
to the greenhouse and subsequently to the field. Each experiment with 20 cultures
per treatment was repeated three times.
RESULTS AND DISCUSSION
The multiple shoots were cultured from rhizome bud explants of Alpinia officinarum
on MS solid medium supplemented with various concentrations and combinations
of auxin/cytokinins after 4-5 weeks of incubation (Table 1).
Hormone free MS medium was used as a control, which induced shoots at a rate
of 2.5 shoots per explant. Comparisons between the best individual concentrations
for each growth regulator (Kn/BAP) revealed an average of 8.5 and 5.6 shoots
and the length was 3.3 and 4.2 cm respectively at the rate of 3.0 mg L-1.
A lower number of shoot multiplication and elongation was observed at higher
concentration of 4.0 mg L-1 of kinetin and 4.0 mg L-1
of BAP (Table 1). However, the highest regeneration frequency
(100%) and a maximum of 11 shoots per explant were obtained after 4-5 weeks
on a medium containing 3.0 mg L-1 Kn in combination with 0.5 mg L-1
NAA which was most effective for rhizome bud multiplication (Fig.
1A). This was found to be considerably different from all other treatments.
Also shoot elongation was more prominent (7.5 cm) in the same treatment (Fig.
1B). Similar results were also observed in rhizome bud explants of Alpinia
galanga (Borthakur et al., 1999). The synergistic effect of cytokinins
in combination with a low concentration of auxin has been reported earlier for
several medicinal and aromatic plant species including Zingiber officinale
(Hosoki and Sagawa, 1977) Curcuma longa (Rahman et al., 2004)
Withania somnifera (Sen and Sharma, 1991).
||Effects of different concentrations of Kn/BAP alone or in
combination with NAA on shoot proliferation from rhizome bud explants of
A. officinarum. There were 20 explants in each treatment and data
(mean±SE) were recorded after 5 weeks of culture
||In vitro multiplication of Alpinia officinarum.
(A) Multiple shoot initiation from rhizome bud explant on MS medium with
Kn (3 mg L-1) and NAA (0.5 mg L-1), (B) Root formation
in shoot cuttings after 25 days in half strength MS medium containing, IBA
0.5 mg L-1 and (C) Rooted plantlets ready for hardening
Based on this information, the present study also exemplifies the positive
modification of shoot multiplication efficacy by low concentrations of an auxin
(NAA) in combination with a cytokinin (Kn). For further multiplication process,
the explants excised from sub-culturing shoots were placed on the same fresh
medium. During the 5-6 subculture passages the percentage of shoot development
as well as the number of shoots per explant retained the same value. Such type
of simultaneous production of multiple shoot was reported earlier for a few
medicinal plants species, such as Hemidesmus indicus (Sreekumar et
al., 2000) and Withania somnifera (Sen and Sharma, 1991).
Excised shoots failed to produce roots on half-strength or full-strength MS
medium without growth regulators even after 40 days of culture. The rate of
root multiplication of Alpinia officinarum, significantly differed based
on growth regulators (Table 2), when cultured on half-strength
MS solid medium. A maximum rate was achieved at 0.5 mg L-1 IBA, each
shoot developed an average of 7 roots and the length was 8.5 cm (Fig.
1C) after 20-25 days. The effectiveness of IBA in rooting has been reported
for medicinal plants like Hemidesmus indicus (Sreekumar et al.,
2000), Cunila galioides (Fracaro and Echeverrigary, 2001) and Aloe
polyphylla (Abrie and van Staden, 2001). The slow movement and slow degradation
of IBA facilitates its localization near the site of application and thus its
better function in inducing roots (Nickell and Kirk-othmer, 1982). Rooted shoots
of A. officinarum were transferred directly to small pots filled with
vermiculite and soil (1:1) and kept in green house for acclimatization.
||Effects of different concentrations of auxins on adventitious
root formation from in vitro shoots. Data represent the mean of 20
cultures. Growth period was 20-25 days (mean±SEM)
Whereas, 93% of plantlets survived their transfer into pots all of them survived
in the garden. Plants appeared to be morphologically uniform and were successfully
adapted to field conditions. It is known that plantlets obtained from organized
meristem shoot tissues of vegetative buds exhibit normally no signs of visible
morphological variation and are genetically identical (Bajaj et al.,
1988). The present procedure might be used to produce around 500-600 whole plants
per bottle with out any intermediate callus phase within 4-5 weeks. It is important
especially for micro propagation of medicinal plants.
Abrie, A.L. and J. Van Staden, 2001.
Micropropagation of the endangered Aloe polyphylla
. Plant Growth Regul., 33: 19-23.CrossRef | Direct Link |
An, N., L.Z. Xu, Z.M. Zou and S.L. Yang, 2006.
Diarylheptanoids from Alpinia officinarum
. J. Asian Nat. Prod. Res., 8: 637-641.Direct Link |
Bajaj, Y.P.S., M. Furmanowa and O. Olszowska, 1988.
Biotechnology of the Micropropagation of Medicinal and Aromatic Plants. In: Biotechnology in Agriculture and Forestry, Bajaj Y.P.S. (Ed.). Springer, Berlin, Heidelberg, New York, pp: 60-103
Balachandran, S.M., S.R. Bhat and K.P.S. Chandel, 1990. In vitro
clonal multiplication of turmeric (Curcuma
spp.) and ginger (Zingiber officinale
Rosc.). Plant Cell Rep., 8: 521-524.CrossRef | Direct Link |
Borthakur, M., J. Hazarika and R.S. Singh, 1999.
A protocol for micro propagation of Alpinia galangal
. Plant Cell Tissue Organ Cult., 55: 231-233.Direct Link |
Fracaro, F. and S. Echeverrigaray, 2001.
Micropropagation of Cunila galioides
, a popular medicinal plant of South Brazil. Plant Cell Tissue Organ Cult., 64: 1-4.CrossRef |
Hosoki, T. and Y. Sagawa, 1977.
Clonal propagation of ginger (Zingiber officinale
Rosc.) throught tissue culture. HortScience, 12: 451-452.
Lee, H.J., J.S. Kim and J.H. Ryu, 2006.
Suppression of inducible nitric oxide synthase expression by diarylheptanoids from Alpinia officinarum
. Planta Med., 72: 68-71.Direct Link |
Lin, M. and H.S.Y. Hsu, 1998.
Studies of antiulcer Chinese herbs (1). Chin. Pharm. J. Taipei, 50: 55-66.
Liu, Z., S. Sang, G. Thomas, Hartman, Chi-Tang Ho and T. Robert, 2005.
Determination of diarylheptanoids from Alpinia officinarum
(lesser galangal) by HPLC with photodiode array and electrochemical detection. Phytochem. Anal., 16: 252-256.Direct Link |
Matsuda, H., S. Ando, T. Kato, T. Morikawa and M. Yoshikawa, 2006.
Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity. Bioorganic Med. Chem., 14: 138-142.Direct Link |
Murashige, T. and F. Skoog, 1962.
A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.CrossRef | Direct Link |
Nickell, G.L. and Kirk-othmer, 1982.
Encyclopaedia of Chemical Technology. Vol. 18, Wiley, New York
Peter, K.V., 2001.
Handbook of Herbs and Spices. Vol. 3, Woodhead publishing Limited, Abington, ISBN: 185573 5628
Yadav, P.N., Z. Liu and M.M. Rafi, 2003.
A diarylheptanoid from lesser galangal (Alpinia officinarum
) inhibits proinflammatory mediators via inhibition of mitogen-activated protein kinase, p44/42 and transcription factor nuclear factor-kB. J. Pharm. Exp. Therap., 302: 925-931.Direct Link |
Rahman, M.M., M.N. Amin, H.S. Jahan and R. Ahmed, 2004. In vitro
regeneration of plantlets of Curcuma longa
Linn. A valuable spice plant in Bangladesh. Asian J. Plant Sci., 3: 306-309.CrossRef | Direct Link |
Sakai, K. and Y. Miyazaki, 1989.
Effect of extracts of Zingiberaceae herbs on gastric secretion in rabbits. Chem. Pharm. Bull., 37: 215-217.
Sen, J. and A.K. Sharma, 1991.
Micropropagation of Withania somnifera
from germinating seeds and shoot tips. Plant Cell Tissue Organ Cult., 26: 71-73.Direct Link |
Shin, J.E., M.J. Han, M.C. Song, N.I. Baek and D.H. Kim, 2004.
5-Hydroxy-7-(4'-hydroxy-3'-methoxyphenyl)-1-phenyl-3-heptanone: A pancreatic lipase inhibitor isolated from Alpinia officinarum
. Biol. Pharm. Bull., 27: 138-140.Direct Link |
Sreekumar, S., S. Seeni and P. Pushpangadan, 2000.
Micropropagation of Hemidesmus indicus
for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde. Plant Cell. Tissue Organ Cult., 62: 211-218.Direct Link |
Tewari, A. and A.K. Pant, 1999.
A review on Alpinia
species: Chemical, biocidal and pharmacological aspects. J. Med. Aromatic Plant Sci., 21: 1155-1168.
Uehara, S.I. and I. Yasuda, 1987.
Diarylheptanoids from the rhizomes of Curcuma xanthorrhiza
and Alpinia officinarum
. Chem. Pharm. Bull., 35: 3298-3304.
Padma, S.V., T. Vandana, S.T. Warjeet and S. Ningombam, 2004.
Antioxidant properties of some exclusive species of Zingiberaceae family of Manipur. Elect. J. Environ. Agric. Food Chem., 5: 1318-1322.Direct Link |
Wu, T.L. and K. Larsen, 2000.
Family Zingiberaceae. In: Flora of China, Wu, Z.G. and P.H. Raven (Eds.). Science Press, St. Louis, Missouri, USA., pp: 322-377