Journal of Applied Research on Medicinal and Aromatic Plants 4 (2017) 2126

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Journal of Applied Research on Medicinal and

Aromatic Plants
journal homepage: www.elsevier.com/locate/jarmap

Micropropagation and genetic delity analysis in Amomum subulatum

Roxb.: A commercially important Himalayan plant
Sumit Purohit a , Shyamal K. Nandi a, , Shilpi Paul a , Mohd. Tariq a , Lok Man S. Palni a,b
a
G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora- 263 643, Uttarakhand, India
b
Graphic Era (Deemed) University, Clement Town, Dehradun 248 002, Uttarakhand, India

a r t i c l e i n f o a b s t r a c t

Article history: An efcient protocol for in vitro regeneration of a commercially important plant, Amomum subulatum
Received 4 February 2016 Roxb., was developed using small pieces of rhizome explants taken from mature plants. Murashige and
Received in revised form 4 July 2016 Skoog (MS) medium supplemented with 4.0 M 6-benzylaminopurine (BAP) and 1.0 M -naphthalene
Accepted 6 July 2016
acetic acid (NAA) resulted in maximum shoot numbers (32.6) with highest shoot length of 14.00 cm
Available online 21 July 2016
and on an average 61.4 roots and with 16.90 cm root length per explant. This could be repeated when
individually separated shoots were transferred again on the same medium. Thus, on an average, about 30
Keywords:
plantlets could be obtained per culture cycle of three weeks. Ninety percent survival was recorded at the
Amomum subulatum
Genetic delity
end of the 5 weeks of acclimatization, and cent percent survival was observed after 150 days of transfer
RAPD of acclimatized plantlets into earthen pots, containing a mixture of soil and farmyard manure (3:1, v/v)
Micropropagation when the pots were kept in the open nursery with partial shade. Random amplied polymorphic DNA
Medicinal plants (RAPD) marker analysis of ten randomly selected tissue culture raised plantlets conrmed their genetic
Himalaya delity with the mother plant. High multiplication rate associated with observed genetic stability clearly
indicates the efcacy of the present in vitro clonal propagation protocol of this important medicinal plant
of high commercial value.
2016 Elsevier GmbH. All rights reserved.

1. Introduction it is also used as preventive as well as curative agent for throat

Large cardamom (Amomum subulatum Roxb., family: Zingiber-
aceae, Hindi: Bari Elaichi) is a tall perennial rhizomatous herb and
an important cash crop cultivated between 600 and 2000 m asl. It
is mainly used as a spice and India is the largest producer of large
cardamom with production of 4465 metric tons in 201314 and
exported 1110 metric tons during the same year (Anon, 2015).
In India, it is cultivated in the states of Sikkim, West Bengal,
Arunachal Pradesh, Nagaland, Mizoram, Manipur and Uttarakhand
(Bisht et al., 2010). It is also cultivated in neighboring countries like
Nepal and Bhutan. The seed has a pleasant aromatic odour due to
which it is extensively used for avoring food preparations, and has
also been used in Ayurvedic medicines (Sharma et al., 2000). Seeds
are also considered as an antidote to snake and scorpion venom;
Abbreviations: IBA, Indole-3-butyric acid; BAP, 6-benzylaminopurine; NAA, -
naphthalene acetic acid; MS, Murashige and Skoog medium; PGRs, plant growth
regulators; PCR, polymerase chain reaction; RAPD, random amplied polymorphic
DNA.
Corresponding author.
E-mail address: shyamal nandi@rediffmail.com (S.K. Nandi).
troubles, congestion of lungs, inammation of eye lids, digestive
disorders and in the treatment of pulmonary tuberculosis (Verma
et al., 2010; Bisht et al., 2011).
The volatile oil present in the seeds of large cardamom is a major
constituent responsible for the typical odour. The seeds contain 3%
essential oil (Gupta et al., 1984), which is dominated by 1, 8-cineole
(Bal Krishnan et al., 1984; Gurudutt et al., 1996; Bhandari et al.,
2013); seeds also contain of a number glycosides, namely petunidin
3, 5-diglucoside, leucocyanidin 3-o--d-glucopyranoside subu-
lin, aurone glycoside, cardamomin--chalcone and alpinetin-
avanone (Shankaracharya et al., 1990). In nature the plant prop-
agates through seeds and rhizomes (vegetatively); however, low
(29%) seed germination (Bisht et al., 2010) and slower rate of vege-
tative multiplication fail to cope up with ever increasing demand of
plant propagules for expanding cultivation. One of the important
problems associated with this crop is the occurrence of viral and
fungal diseases (Sharma et al., 2009) which affect productivity and
also result in high plant mortality. In order to overcome these prob-
lems, use of in vitro method of propagation is considered to offer
an alternative approach for effective and rapid means of multipli-
cation of elite genotypes. Although in vitro regeneration protocol of
A. subulatum has been reported earlier (Sajina et al., 1997), data are

http://dx.doi.org/10.1016/j.jarmap.2016.07.003
2214-7861/ 2016 Elsevier GmbH. All rights reserved.
22 S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants 4 (2017) 2126

Fig. 1. In vitro regeneration of A. subulatum and establishment of plants in the soil (AF).
A: Mother plants of A. subulatum, the source of explants,
B: Rhizome explants cultured on MS medium,
C: Profuse shoot multiplication in a 3-week old culture; 1 bar = 1 cm,
D: A close view of plantlets along with roots from a 3 week old culture; 1 bar = 1 cm,
E: Acclimatization of tissue culture raised plantlets after transfer of such plantlets in to thermocole trays kept for 3 weeks in a green house,
F: Established tissue culture raised plants after transfer of acclimatized plants in clay pots.

Table 1
Effect of different PGRs on shoot multiplication, elongation and rooting in A. subulatum.

Plant growth regulators (Concentration, M) Number of shoots Shoot length (cm) Number of roots Root length (cm)

BAP NAA
0.0 0.0 3.20 0.59c 4.80 0.67d 7.0 0.72d 4.00 0.72e
2.0 0.5 4.00 0.45c 7.80 0.67bc 14.8 1.49cd 6.90 0.69d
2.0 1.0 3.20 0.59c 6.00 1.16c 14.6 3.4cd 9.40 1.23bc
3.0 0.5 6.40 0.52c 8.00 2.99bc 15.0 3.23cd 7.30 0.59cd
3.0 1.0 11.8 0.87b 8.08 0.61b 35.0 3.59b 6.80 0.86d
4.0 0.5 12.8 0.59b 4.00 0.45d 11.6 1.09cd 2.30 0.26e
4.0 1.0 32.6 2.60a 14.00 1.11a 61.4 3.36a 16.90 1.05a
5.0 1.0 4.20 0.87c 8.40 0.94bc 18.4 2.42c 11.70 0.89b

Note:- Values are mean standard error; Mean values with same letters in a column are not signicantly different (P < 0.05; DMRT). The data have been recorded on per
explant basis and recorded after 3 weeks of inoculation.

missing on the clonal delity and survival of tissue culture raised
plants in the eld. The present study reports the development of an
efcient in vitro propagation protocol, using rhizome pieces with
bud as explants, and assessment of genetic stability of regenerated
plants and their subsequent cent percent survival 5 month after
transfer to pots.
2. Materials and methods
2.1. Plant material and tissue culture
Plants of Amomum subulatum cv. Sawney were collected (cour-
tesy Dr. K.K. Singh, Senior Scientist at Sikkim unit of GBPIHED
located at Tadong near Gangtok) from Kabi area (altitude 1500 m
asl; 27 15 05 N; 88 39 24.27 E) in Sikkim during July 2007,
and these were brought to the Institute at Kosi Katarmal,
Almora (altitude 1150 m asl; 29 38 22.54 N; 79 37 24.87 E).
These were grown in earthen pots (20 cm height and 18 cm diame-
ter) containing potting mixture (soil and farmyard manure; 3:1,
v/v) under open nursery conditions with partial sun light using
green shade nets (Fig. 1A). After several months, the rhizomes
were removed from an identied pot, washed under running tap
water for 1520 min to remove adhering soil and other debris. The
mother plant was labeled and kept in the green house (80% rel-
ative humidity, 25 1 C, 18/6 h day/night, light- 50% of ambient)
for further growth and subsequent use. While initiating the exper-
iment the rhizomes (from mature plant) were washed and cleaned
(as mentioned above) and then immersed in water containing liq-
uid detergent (0.2% Tween-20, v/v; Hi Media, India) for 20 min, and
washed with sterile distilled water (x4). The explants were then
treated with Bavistin solution (2%, w/v; a systemic fungicide; BASF,
Mumbai, India) on a shaker for 30 min, and then rinsed again with
sterile distilled water (x4) in a Laminar air ow cabinet (Therma-
dyne, India), followed by treatment with freshly prepared mercuric
 S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants 4 (2017) 2126 23

Table 2
Growth performance of tissue culture raised plants of A. subulatum 180 days after transfer in pot.

Plant number Plant height (cm) Leaf length (cm) Leaf width (cm) No. of rhizomes No. of leaf

1 60 32 4 4 10
2 58 30 6 7 10
3 47 22 8 6 9
4 64 34 8 6 9
5 43 20 4 2 8
6 42 22 5 5 9
7 56 27 4 4 9
8 45 28 4 2 8
9 32 24 5 5 8
10 41 23 5 4 8
Mother 32 24 5 4 8

Table 3
RAPD banding pattern observed in A. subulatum samples.

S. No Primer code Primer sequence Annealing No of amplied No of bands

(5 3 ) temperature Fragments

Monomorphic
Polymorphic

1 OPA 1 CAGGCCCTTC 34.0 3 3 0

2 OPA 4 AATCGGGCTG 32.0 4 4 0
3 OPA 9 GGGTAACGCC 34.0 5 5 0
4 OPA 20 CAGCACCCAC 32.0 3 1 2
5 OPJ 5 CTCCATGGGG 37.5 3 3 0
6 OPJ 8 CATACCGTGG 36.0 3 2 1
7 OPJ 11 ACTCCTGCGA 32.0 4 4 0
8 OPJ 14 CACCCGGATG 35.0 6 6 0
9 OPJ 15 TGTAGCAGGG 37.0 4 4 0
10 OPJ 16 CTGCTTAGGG 36.0 4 4 0
11 OPJ 17 ACGCCAGTTC 32.0 2 1 1
12 OPJ 18 TGGTCGCAGA 36.0 1 1 0
Total 42 38 4

chloride (0.1%, w/v; 15 min, Hi media, Mumbai, India) with contin-
uous shaking. The rhizome explants were nally rinsed with sterile
distilled water (x4) to remove traces of disinfectant and detergent.
The scales (leaves) from the explants were carefully removed; the
explants were then excised into 2.03.0 cm pieces containing buds,
and inoculated in 250 ml conical asks on Murashige and Skoog
(1962) medium, without plant growth regulators (PGRs). The pH
of the medium was adjusted to 5.8 0.2 with 1 N NaOH or 1 N HCl
prior to autoclaving (1.05 kg/cm2 , 121 C, 20 min). The culture asks
were incubated in a room at 25 1 C in 16 h light/8 h dark photope-
riod with an irradiance of 40 E m2 s1 by cool uorescent tubes
(Philips TI 40 W/54, India).
2.3. Acclimatization and hardening
After ten weeks, in vitro raised plantlets were taken out from the
culture medium, their basal portions washed thoroughly in running
tap water to remove agar and traces of medium, and then rinsed
with distilled water. These plantlets were then treated with 0.5%
(w/v) Bavistin solution (10 min) to prevent fungal contamination,
and transferred to thermocole trays [32 cm length, 32 cm width,
and 10 cm height; 16 holes (depth 9 cm; diameter 7 cm) per tray; 1
explant per hole] containing soil and farmyard manure (FYM; 3:1,
v/v) and maintained in the green house. These trays required fre-
quent watering using a mist sprayer. After ve weeks, the hardened
plants were transferred to earthen pots (20 cm height and 18 cm
diameter) containing soil and FYM as reported above, and shifted
in the open nursery under partial sunlight (50% shading).

2.2. Shoot regeneration and rooting formation 2.4. DNA isolation and RAPD analysis

The sprouted buds (0.51.0 cm) on rhizome explants were
removed and following excision of the basal part (from aseptically
grown cultures mentioned above), were again inoculated in 250 ml
Erlenmeyer asks containing 100 ml of MS medium supplemented
with various concentrations of PGRs (auxin: -naphthalene acetic
acid, NAA, 0.51.0 M; cytokinin: 6-benzylaminopurine, BAP,
2.05.0 M, see Table 1 for details). The culture asks were placed
in the culture room (as mentioned above). Four replicates (asks)
were used for each treatment and 3 explants were placed in each
ask, i.e. 12 explants/treatment. The data on responsive explants
were recorded after 3 weeks of inoculation. Most of the PGR com-
binations showed shoot formation along with root initiation, but
maximum roots along with shoots were observed in the medium
supplemented with 4.0 M BAP and 1.0 M NAA. The length of
shoots and roots were measured using a ruler scale.
The analysis of genetic delity was carried out using tissue cul-
ture raised plantlets (10 nos.) along with the mother plant. DNA
was isolated following the modied procedure of Khanuja et al.
(1999). Leaf tissue (1 g) was powdered using liquid nitrogen and
transferred to polypropylene tubes containing 10 ml of extraction
buffer. The tubes were vortexed and kept at 65 C for lysis (1 h). After
lysis equal volume of chloroform: isoamyl alcohol (1:1, v/v) was
added, centrifuged (10,000 rpm, 22 C, 15 min), and the upper aque-
ous layer was transferred into fresh tubes. RNase was added and
the tubes were incubated for 1 h (37 C). A mixture of chloroform:
isoamyl alcohol (1:1, v/v) was then added to this RNase treated
material. This mixture was subsequently centrifuged (10,000 rpm,
22 C, 15 min), the supernatant retained and 0.6 vol of chilled iso-
propanol was added to precipitate the DNA. Tubes were centrifuged
(room temperature; 8000 rpm, 10 min), the supernatant discarded
24 S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants 4 (2017) 2126

and the pellet washed with 75% aq. ethanol, air-dried and nally
suspended in Tris (10 mM) EDTA (1 mM) or sterile distilled water.
The DNA was then quantied and further used for PCR. The PCR
reaction was carried out in 25-l volume containing 50 ng genomic
DNA, 0.3U Taq DNA polymerase, 1x buffer, 15 mM MgCl2 , 100 mM
dNTPs and 5 pmol of RAPD primers (OPA & OPJ kit of Operon; USA).
Amplication was carried out in a thermal cycler (Biometra Mas-
ter, Germany) using 94 C for 5 min, 94 C for 1 min, 35 C for 1 min,
72 C for 2 min for 35 cycles program. The amplied products were
analysed in 1.2% agarose gel.

2.5. Data analyses

Mean values of all data were calculated using Microsoft ofce

Excel 2007. All experiments were carried out in a completely ran-
domized block design. The mean values of results obtained under
various PGR treatments were subjected to analysis of variance
(ANOVA), and the signicance level was determined at P < 0.05
using Duncans multiple range test (DMRT) using SPSS (version 7.5).
Genetic similarity matrix was calculated using Nei and Li (1979),
and the dendrogram was constructed through SYSTAT software
using unweighted pair-group method with the arithmetic averages
(UPGMA) method.

3. Results
3.1. Establishments of shoot cultures and hardening
Varied morphological responses were observed with different
PGR (auxin and cytokinin) combinations and their concentrations
on rhizome explants (Table 1; Fig. 1B). All the combinations used
in this study showed good response for both shoot, leaf and root
induction after three weeks of inoculation (Fig. 1C & D). The
DMRT test revealed signicant (P < 0.05) variation among treat-
ments (Table 1). The response of rhizome explants on medium
devoid of any PGR (control) was moderate (average shoot length:
4.80 0.67 cm, shoot number: 3.20 0.59, average root number:
7.0 0.72 and root length: 4.00 0.72 cm). Out of various PGR com-
binations tried, MS medium supplemented with 4.0 M BAP and
1.0 M NAA exhibited the overall best response (average shoot
length: 14.00 1.11 cm; shoot number: 32.6 2.60; root number:
61.4 3.36 and root length: 16.90 1.05 cm Table 1). The second
best response was obtained in medium supplemented with 3.0 M
BAP and 1.0 M NAA (average shoot length: 8.08 0.61 cm; average
shoot number: 11.8 0.87; root number: 35.0 3.59 and average
root length: 6.80 0.86 cm; Table 1).
Based on the best observed response about 30 plantlets could be
obtained after 3 weeks of culture, and this could be repeated with
similar results when individually separated shoots were planted
again on the same medium. Thus on an average 30 plantlets could
be produced per shoot per culture cycle of three weeks.
In this study, the combinations of BAP and NAA used were found
to be highly suitable (Table 1) not only for shoot multiplication
but also for root formation, and hence a separate constitution for
root initiation was not required as is often the case. Complete and
well rooted plantlets could be produced after ten weeks of culture,
and the individual plantlets (Fig. 1D) were subsequently transferred
into thermocole trays (Fig. 1E) and maintained under humid condi-
tions (95100% RH) in greenhouse for the initial 10 days and then
gradually acclimatized for another 20 days. All the regenerated and
hardened plantlets (90% survival) were found to be morphologi-
cally uniform and exhibited morphological characteristics similar
to those of the mother plant (Fig. 1A). It is worth mentioning that
well-developed plantlets could be obtained across all the combina-
tions of BAP and NAA used in this study (Table 1). Fully developed
Fig. 2. RAPD proles of mother plant (P) along with ten tissue culture raised plants
(110) using two primes OPJ 18 and OPJ 15.
Note:-A-Prole with OPJ 18; B- Prole with OPJ 15; 110- tissue culture raised
plants; P- Mother plant; M- 100 bp ladder marker.
and hardened plantlets were subsequently transferred to large
earthen pots (Fig. 1F) and 100% survival was recorded after 150 days
of transfer of these hardened plants into pots. The growth perfor-
mance of tissue culture raised plants of A. subulatum 180 days after
transfer to pot is summarized in Table 2.
3.2. Analysis of genetic stability by RAPD
Ten tissue culture raised plants were randomly selected along
with the mother plant for the analyses of genetic delity. A total
of 40 RAPD primers were used and amongst them 12 were found
to produce clear (Figs. 2A and B) and reproducible bands. A total
of 42 bands were observed and only 3 primers (OPA 20, OPJ 8 and
OPJ 17; Table 3) produced polymorphic bands. These RAPD proles
were used to calculate similarity matrix (Table 4). Maximum simi-
larity was observed between T5 to T6 and T8 (97%) and minimum
between T8, T6, T1, T2, T3, T4 (90%); based on this similarity matrix
a dendrogram was constructed (Fig. 3). The dendrogram showed
two separate groups (Group I & II); Group I comprised of T4 plant,
and the group II comprised of all other tissue culture raised plants
(T1 to T3 , T5 to T10 ) along with the mother plant (M). Morpholog-
ically T4 plant was found to be taller with longer leaves than the
other plants (180 days after transfer in pots; Table 2), this could be
a somaclonal variant and hence it formed a separate group.
4. Discussion
The objectives of the study reported in this paper were to
establish a reproducible and efcient in vitro propagation protocol
that could be used for commercial purpose. The use of auxin and
cytokinin combination is well known for in vitro shoot multiplica-
tion as has been reported for several species, including A. subulatum.
In a previous study on A. subulatum (Sajina et al., 1997), when
rhizome segments were cultured an MS medium supplemented
with BAP or NAA alone or in combination of BAP + IBA (Indole-3-
 S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants 4 (2017) 2126 25

Table 4
Similarity matrix of mother plant and randomly selected ten tissue culture raised plants of A. subulatum.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Mother

T1 0.952
T2 0.929 0.929
T3 0.929 0.929 0.929
T4 0.952 0.929 0.929 0.976
T5 0.952 0.929 0.929 0.952 0.976
T6 0.929 0.929 0.929 0.929 0.952 0.952
T7 0.952 0.929 0.929 0.952 0.976 0.952 0.976
T8 0.905 0.905 0.905 0.905 0.929 0.929 0.929 0.929
T9 0.929 0.929 0.929 0.929 0.952 0.952 0.952 0.929 0.952
T10 0.929 0.929 0.929 0.929 0.952 0.952 0.952 0.929 0.952 0.952
Mother 0.929 0.929 0.929 0.929 0.952 0.952 0.952 0.929 0.952 0.952 0.952

Cluster Tree
T4
T3
T2
T1
T7
T5
T10
T6
T9
MOTHER
T8
0.0 0.1 0.2 0.3
Distances
Fig. 3. Dendrogram illustrating the genetic relationship among the mother plant
and randomly selected 10 tissue culture raised plants (T1T10).
butyric acid, as auxin), BAP + NAA or IBA + NAA produced shoots
and roots simultaneously. However, best response was obtained in
a combination of MS medium with BAP (1.0 mg/l) and IBA (0.5 mg/l)
producing 812 shoots/culture and 5 roots/shoot. These workers
have reported 90% survival of plants after transfer to soil (Sajina
et al., 1997). Similarly in the present investigation MS medium
supplemented with 4.0 M BAP and 1.0 M NAA was found to be
suitable for large scale multiplication. Thus MS medium supple-
mented with natural cytokinin (BAP) and synthetic auxin (NAA)
is favorable for micropropagation of A. subulatum. Earlier reports
on micropropagation of other Zingiberaceous plants like small car-
damom, ginger and turmeric indicated similar results (Hosoki and
Sagawa, 1977; Nadgauda et al., 1978, 1983; Pillai and Kumar, 1982;
Kumar et al., 1985; Ilahi and Jabeen, 1987; Bhagyalakshmi and
Singh, 1988; Nirmal Babu et al., 1992; Salvi et al., 2002).
Micropropagation and genetic delity assessment has been
reported using rhizome, nodal and leaf explants in several other
species (Agnihotri et al., 2009; Purohit et al., 2015; Mukhopadhyay
et al., 2016). In general, the use of rhizome explants ensures clonal
propagation of true-to-type plants. RAPD markers have been used
successfully to assess the genetic stability among species belonging
to Zingiberaceae (Rout et al., 1998; Islam et al., 2004; Wondyifraw
and Wannakrairoj, 2004; Mohanty et al., 2011). In recent years,
systematic sampling of germplasm and analysis of their molecu-
lar status through the use of DNA markers has become a common
practice (Williams et al., 1990). Among different techniques used
to be detect DNA polymorphism (e.g., RFLP, AFLP, ISSR), RAPD has
been widely used to study clonal integrity, detect genetic and
somaclonal variations (Agnihotri et al., 2009; Paul et al., 2012;
Purohit et al., 2015; Mukhopadhyay et al., 2016). These results
were well supported by the present investigations in A. subulatum.
However, phenotypic and genetic variations have been reported to
occur during in vitro regeneration resulting in somaclonal varia-
tions and reported in several plant species (Larkin and Scowcroft,
1981; Barwale and Wildholm, 1987; Kaeppler et al., 2000). The vari-
ants thus obtained are useful and can be exploited further by the
breeders for commercial purposes. Obviously, the risks of genetic
changes induced by tissue culture and the importance of assess-
ing the genetic stability of the biological material along all phases
of storage and growth must be considered in the context of large
scale multiplication.
5. Conclusions
This study appears to be the rst report of cent percent sur-
vival of tissue culture raised plants after 5 month of transfer to
pots containing soil and FYM mixtures, along with genetic delity
assessment of the regenerates. Conventionally A. subulatum is prop-
agated through seeds and rhizomes (vegetatively) which is slow.
Therefore, the in vitro propagation protocol developed can be very
effective method for multiplication of elite and/or high yielding
plants to cope up with ever increasing demand of plant propag-
ules for expanding cultivation of this important cash crop; but it
needs to be tested for large scale multiplication and eld planta-
tion. Conrmation of genetic delity of micropropagated plants is
important and necessary for the production of uniform regener-
ates for use in commercial plantations. Thus this investigation has
tremendous commercial implications as large cardamom is used
as spice worldwide, as avoring agents and for pharmaceutical
purposes by different industries.
Conicts of interest
The authors declare no conicts of interest, nancial or other-
wise.
Acknowledgements
The authors thank Director, G.B. Pant Institute of Himalayan
Environment and Development, Kosi-Katarmal, Almora for facili-
ties and encouragement; Dr. K.K. Singh, Senior Scientist at Sikkim
unit of GBPIHED, Tadong, near Gangtok, Sikkim is thanked for pro-
viding the plant material. All colleagues of the Group are thanked
for cooperation and help. Financial support in the form of an Insti-
tute project (no. 19) is duly acknowledged.
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