Genetic Stability Assessment of Micropropagated Mango Ginger (Curcuma amada Roxb.) Through RAPD and ISSR Markers
Raj Kumar Joshi,
Curcuma amada Roxb., popularly known as mango ginger is an important
spice and medicinal plant of family Zingiberaceae. In this attempt, amplified
polymorphic DNA (RAPD) and Inter Simple Sequence repeats (ISSR) markers were
used to determine the genetic stability of micropropagated Curcuma amada.
Fifty regenerants were analyzed each at an interval of six months up to two
years in culture. Out of 25 RAPD and 10 ISSR primers screened, 19 RAPD primers
and 8 ISSR primers gave 3100 and 3300 bands, respectively. RAPD and ISSR analysis
revealed monomorphic bands showing the absence of polymorphism in all regenerants
analyzed, confirming their genetic uniformity. These results suggest that the
micropropagation protocol developed by us for rapid in vitro multiplication
is appropriate and applicable for clonal mass propagation of Curcuma amada.
September 20, 2011; Accepted: March 31, 2012;
Published: June 21, 2012
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.,
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)
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).
|| (a) Curcuma amada explants, (b, c) Shoot and root
multiplication, (d) Potted plant and (e) Tissue cultured plants in greenhouse
|| 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,
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
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.
|| RAPD banding pattern of micropropagated and field-grown mother
plants of C. amada
|| (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)
|| 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).
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.
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.
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