INTRODUCTION

Curcuma amada (mango ginger) an under exploited, important medicinal plant is cultivated for its aromatic rhizome. The rhizome is carminative, stomachic and applied over contusions and sprains (Nayak, 2002). It is also used in the treatment of bronchitis, asthma, cough itching and skin diseases. The root is diuretic, emollient, expectorant, astringent, antipyretic, appetizer and is used in mouth, ear diseases, lumbago and diarrhoea (Kirtikar and Basu, 1984). Due to its mango like aroma, it is used in pickles and culinary preparations. Two novel bioactive compounds were isolated and characterized from mango ginger rhizome having antimicrobial, antioxidant, platelet aggregation inhibitory activity and antitubercular activity (Policegoudra et al., 2010; Singh et al., 2010). C. amada is a vegetatively propagated plant with very low multiplication rate. Lack of seed set in this plant discourages conventional breeding efforts. In addition, its susceptibility to various rhizome rot diseases cause huge loss during each year. Hence, large amount of fresh planting materials are required each year for plantation (Barthakur and Bordoloi, 1992). These problems necessitate an alternative technique for the production of true-to-type planting material for C. amada. Nevertheless, in vitro culture has the potential to increase the multiplication rate of elite genotypes and to produce improved cultivars when combined with other tools of biotechnology.

In vitro clonal multiplication of other Curcuma species through rhizome buds has been reported by many authors (Nadgauda et al., 1978; Balachandran et al., 1990; Salvi et al., 2002). Available reports are however limited only to micropropagation of C. amada (Barthakur and Bordoloi, 1992), regeneration from leaf sheath callus and microrhizome production (Nayak, 2002). There is no such report describing clonal fidelity of micropropagated plants using molecular markers. Somaclonal variation is the major problem associated with in vitro culture among sub-clones of one parental line, arising as a direct consequence of in vitro culture of plant cells, tissue or organs (Gould, 1986; Larkin and Scowcroft, 1981). Periodic monitoring of genetic stability of in vitro conserved plants is important for commercial utilization of true-to-type plants of the desired genotype. Hence, the assessment of the genetic integrity of in vitro grown regenerants at regular intervals can significantly reduce or eliminate the chance of occurrence of somaclonal variation (Larkin and Scowcroft, 1981; Rani et al., 1995) at the early or late phase of culture. Molecular techniques are at present powerful and valuable tools used in analysis of genetic fidelity of in vitro grown plants and are the subject of many publication and reviews. Of the various molecular markers used, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) analysis are the simplest and quickest tools for genetic stability assessment of in vitro grown plants as reported in many species (Bhatia et al., 2009; Modgil et al., 2005; Mohanty et al., 2010).

The aim of the present study was to report micropropagation, in vitro conservation and genetic stability assessment of in vitro conserved plantlets of medicinally important Curcuma amada. For this study, one clone has been derived and tested by two molecular markers, RAPD (Williams et al., 1990) and ISSR (Zeitkiewicz et al., 1994).

MATERIAL AND METHODS

Plant material: Healthy and sprouted rhizomes of C. amada, collected from High altitude research station, Pottangi, Koraput, Orissa, were washed properly in tap water followed by a liquid detergent (Extran, Merck, Mumbai, India) for 10 min and wash with sterilized water. These were then surface sterilized with 0.1% (w/v) mercuric chloride for 11-12 min. After rinsing with sterile distilled water three times, explants were used for culture initiation. Approximate size of the explants was 10-12 mm in length.

In vitro multiplication and culture conditions: Explants were inoculated in MS medium (Murashige and Skoog, 1962) containing 30 g L^-1 sucrose and different concentration of BA (0.5-5 m g L^-1), IAA (0.5-1 m g L^-1) and Kinetin (1-3 m g L^-1). Explants were first inoculated in the shoot induction media and then subcultured to another media for shoot multiplication following the protocol of Mohanty et al. (2010).

Field transfer: After acclimatization in the greenhouse, the plants were transferred to normal atmospheric conditions and were grown to maturity. All the experiments were repeated three times with a minimum of ten replicates.

DNA extraction: Healthy and young leaves of C. amada, were taken both from in vitro and ex vitro grown mother plants. Leaf samples were taken in every six months interval up to two years for RAPD and ISSR analysis. DNA extraction was done by following Doyle and Doyle (1987) method.

RAPD and ISSR analysis: RAPD and ISSR analysis was done up to 2 years with an interval of six months. For RAPD analysis, 30 random primers were used, out of which 19 random decamer primers (Operon Tech, Almeda, USA) were selected as responded well. In case of ISSR out of 10 primers 8 were selected. The RAPD analysis was performed as per the method of Williams et al. (1990) and for ISSR analysis the method of Zeitkiewicz et al. (1994) was followed.

Statistical analysis: Data were subjected to analysis of variance for a factorial experiment. Critical Differences (CD) were calculated to determine the statistical significance of different treatment means.

RESULT AND DISCUSSION

In vitro multiplication and conservation: In C. amada, sprouted buds of rhizome were taken as explants (Fig. 1a) for the initiation of in vitro culture. Various combinations of BA (1-5 mg L^-1), IAA (0.5-2 mg L^-1) and kinetin (0.1-1 mg L^-1) were tried for shoot initiation and multiplication (Table 1). MS media with 3 mg L^-1 BA showed maximum percentage of shoot initiation i.e., 87.0±0.5 after 20-25 days of culture (Fig. 1b).

Fig. 1(a-e):	(a) Curcuma amada explants, (b, c) Shoot and root multiplication, (d) Potted plant and (e) Tissue cultured plants in greenhouse

Table 1:	In vitro shoot multiplication of C. amada on MS medium fortified with different growth regulators
Mean having same letter in a column were not significantly different at p<0.005 level, Data represent the mean of 15 replicates for each treatment, Kn: Kinetin, BA: Butyric acid, IAA: Indde acetic acid, Kn: Kinetin

When in vitro grown shoots were transferred to media with 2 mg L^-1 BA and 0.5 mg L^-1 IAA, 3.8±0.2 number of shoots was formed with 2.4±0.2 numbers of roots (Fig. 1c, d). At low concentration of BA fewer shoots were obtained. This report is in agreement with Prakash et al. (2004) who reported maximum number of shoots in media containing MS and BAP. BA with NAA showed negligible response in both shoot initiation and multiplication which is in contrast to the report of Barthakur and Bordoloi (1992). Shoots and roots developed in the same media. In vitro conservation could be done by keeping the plants on the half MS media with 2 mg L^-1 BA, 30 g L^-1 sucrose and 10 g L^-1 mannitol by subculturing at an interval of 10 months. In vitro grown cultures were then transferred to multiplication media and 90% of the plants resumed normal growth. Cultured plants were successfully established in field after 30 days of acclimatization in green house (Fig. 1e).

RAPD and ISSR analysis: In C. amada, 19 primers were selected, out of 30 RAPD primers tried on the basis of good resolution and reproducibility. 62 scorable bands were formed ranging from 320-3000 bp (Table 2). Average number of bands per primer was 3.2, highest number of band was 6 in primer OPC5 (ranging from 1031-2500 bp) and lowest number of bands i.e., 1 in OPA9 (1400 bp) and OPD12 (1031 bp). 3100 number of bands produced [(number of plantlets analyzed) x (number of bands with all primers)] by RAPD techniques were all monomorphic (Fig. 2a) without having any polymorphism in all 50 plants analyzed. ISSR analysis in C. amada with 10 primers was done up to 2 years with 6 months interval. Out of ten primers, eight were selected due to their clarity. 66 bands were produced by 8 ISSR primers ranging from 250-2900 bp, with an average of 8.3 bands per primer. All bands are monomorphic in nature (Fig. 2b). Highest number of monomorphic band was found to be 14 in primer (GGA)4 (ranging from 300-1500 bp) and lowest of 4 in primer (GTGC)4 and (GA)9T (ranging from 500-1200 bp). A total of 3300 bands [(number of plantlets analyzed)x(number of bands with all primers)] were generated by the ISSR techniques (Table 3). All 50 in vitro conserved plantlets analyzed were true-to-type showing no variation through out the period of study.

Numerous studies on detection of somaclonal variations have been done using PCR-based techniques such as RAPD, ISSR, SSR and AFLP and RAPD being one of the most used.

Table 2:	RAPD banding pattern of micropropagated and field-grown mother plants of C. amada

Fig. 2(a-b):	(a, b) RAPD and ISSR banding pattern in both micropropagated and field grown mother plants of C. amada (Lane 1: Mother plant; Lane 2-12: Micropropagated plants)

Table 3:	ISSR banding pattern of micropropagated and field-grown mother plants of C. amada

In our study two PCR based molecular markers i.e., RAPD and ISSR were used to show the genetic integrity in micropropagated C. amada because of their cost effectiveness and simplicity. The use of two types of markers which amplify different regions of the genome, allow better analysis of genetic stability/variation of the plantlets (Martins et al., 2004; Venkatachalam et al., 2007). Palombi and Damiano (2002) also suggested the use of more than one DNA amplification technique as advantageous in evaluating somaclonal variation. In C. amada, the duration plantlets were kept in culture (two years) did not seem to affect their genetic integrity. Martins et al. (2004), Angel et al. (1996) and Mohanty et al. (2010) also found no variation in regenerants kept in in vitro culture for more than 2 years. Some authors however have reported that the time in in vitro culture could promote somaclonal variation (Hartmann et al., 1989; Orton, 1985). According to Gould (1986) culture time does not seem to be the only parameter affecting genetic stability. Vendrame et al. (1999) reported that genetic variation in a culture line could be affected more by the genotype than by the period in culture. Genotype and the nature of the explants could influence the phenotypic stability of the plant obtained (Hammerschlag et al., 1987).

Mode of regeneration also affects the genetic stability of micropropagated plants. Micropropagation through explants containing an organized meristem is generally regarded as having a lower risk of genetic instability (Shenoy and Vasil, 1992). Our study, in close agreement to Shenoy and Vasil (1992) reveals that the relative stability of micropropagated C. amada, could be due to the direct mode of plant regeneration through multiplication of sprouted bud of rhizome.

Among species of Zingiberaceae, molecular marker based assessment of genetic stability of micropropagated plantlets are limited to cultivated species of C. longa and Z. officinale using RAPD analysis only (Mohanty et al., 2008; Panda et al., 2007) lacking any report on stability analysis using ISSR markers. In the present study RAPD and ISSR analysis of in vitro conserved C. amada showed a profile similar to that of the control indicating that no genetic variation had occurred in vitro, confirming their genetic integrity. RAPD and ISSR analysis of in vitro grown plants has been reported earlier in many species (Bhatia et al., 2009; Joshi and Dhawan, 2007; Martins et al., 2004; Mohanty et al., 2008; Panda et al., 2007; Rout and Das, 2002; Salvi et al., 2002; Venkatachalam et al., 2007).

CONCLUSION

An efficient protocol on micropropagation of C. amada has been developed for the first time with genetic integrity. Our results demonstrate that RAPD and ISSR analysis can be applied to asses the genetic integrity of in vitro conserved plantlets of C. amada, on a large scale, there by facilitating the crop improvement programme in Curcuma species.

ACKNOWLEDGMENTS

The authors are grateful to Dr. S.C. Si, Dean, Centre of Biotechnology and Dr. M.R. Nayak, President, Siksha O Anusandhan University for providing facilities and encouraging throughout. Financial assistance from Department of Biotechnology, New Delhi, India is also duly acknowledged.