Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant

G Model JARMAP-82; No. of Pages 6

ARTICLE IN PRESS Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Applied Research on Medicinal and Aromatic Plants journal homepage: www.elsevier.com/locate/jarmap

Micropropagation and genetic fidelity 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 b

G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora- 263 643, Uttarakhand, India Graphic Era (Deemed) University, Clement Town, Dehradun 248 002, Uttarakhand, India

a r t i c l e

i n f o

Article history: Received 4 February 2016 Received in revised form 4 July 2016 Accepted 6 July 2016 Available online xxx Keywords: Amomum subulatum Genetic fidelity RAPD Micropropagation Medicinal plants Himalaya

a b s t r a c t An efficient protocol for in vitro regeneration of a commercially important plant, Amomum subulatum Roxb., was developed using small pieces of rhizome explants taken from mature plants. Murashige and Skoog (MS) medium supplemented with 4.0 ␮M 6-benzylaminopurine (BAP) and 1.0 ␮M ␣-naphthalene acetic acid (NAA) resulted in maximum shoot numbers (32.6) with highest shoot length of 14.00 cm 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 plantlets could be obtained per culture cycle of three weeks. Ninety percent survival was recorded at the end of the 5 weeks of acclimatization, and cent percent survival was observed after 150 days of transfer of acclimatized plantlets into earthen pots, containing a mixture of soil and farmyard manure (3:1, v/v) when the pots were kept in the open nursery with partial shade. Random amplified polymorphic DNA (RAPD) marker analysis of ten randomly selected tissue culture raised plantlets confirmed their genetic fidelity with the mother plant. High multiplication rate associated with observed genetic stability clearly indicates the efficacy 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 Large cardamom (Amomum subulatum Roxb., family: Zingiberaceae, 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 2013–14 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 flavoring 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 amplified polymorphic DNA. ∗ Corresponding author. E-mail address: shyamal [email protected] (S.K. Nandi).

it is also used as preventive as well as curative agent for throat troubles, congestion of lungs, inflammation 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 subulin, aurone glycoside, cardamomin-␣-chalcone and alpinetin-␣ flavanone (Shankaracharya et al., 1990). In nature the plant propagates through seeds and rhizomes (vegetatively); however, low (29%) seed germination (Bisht et al., 2010) and slower rate of vegetative 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 problems, use of in vitro method of propagation is considered to offer an alternative approach for effective and rapid means of multiplication 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.

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003

G Model

ARTICLE IN PRESS

JARMAP-82; No. of Pages 6

S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

2

Fig. 1. In vitro regeneration of A. subulatum and establishment of plants in the soil (A–F). 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 0.0 2.0 2.0 3.0 3.0 4.0 4.0 5.0

3.20 ± 0.59c 4.00 ± 0.45c 3.20 ± 0.59c 6.40 ± 0.52c 11.8 ± 0.87b 12.8 ± 0.59b 32.6 ± 2.60a 4.20 ± 0.87c

4.80 ± 0.67d 7.80 ± 0.67bc 6.00 ± 1.16c 8.00 ± 2.99bc 8.08 ± 0.61b 4.00 ± 0.45d 14.00 ± 1.11a 8.40 ± 0.94bc

7.0 ± 0.72d 14.8 ± 1.49cd 14.6 ± 3.4cd 15.0 ± 3.23cd 35.0 ± 3.59b 11.6 ± 1.09cd 61.4 ± 3.36a 18.4 ± 2.42c

4.00 ± 0.72e 6.90 ± 0.69d 9.40 ± 1.23bc 7.30 ± 0.59cd 6.80 ± 0.86d 2.30 ± 0.26e 16.90 ± 1.05a 11.70 ± 0.89b

NAA 0.0 0.5 1.0 0.5 1.0 0.5 1.0 1.0

Note:- Values are mean ± standard error; Mean values with same letters in a column are not significantly 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 fidelity and survival of tissue culture raised plants in the field. The present study reports the development of an efficient 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 (courtesy 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 diameter) 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 identified pot, washed under running tap water for 15–20 min to remove adhering soil and other debris. The mother plant was labeled and kept in the green house (80% relative humidity, 25 ± 1 ◦ C, 18/6 h day/night, light- 50% of ambient) for further growth and subsequent use. While initiating the experiment the rhizomes (from mature plant) were washed and cleaned (as mentioned above) and then immersed in water containing liquid 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 flow cabinet (Thermadyne, India), followed by treatment with freshly prepared mercuric

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003

G Model

ARTICLE IN PRESS

JARMAP-82; No. of Pages 6

S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

3

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 2 3 4 5 6 7 8 9 10 Mother

60 58 47 64 43 42 56 45 32 41 32

32 30 22 34 20 22 27 28 24 23 24

4 6 8 8 4 5 4 4 5 5 5

4 7 6 6 2 5 4 2 5 4 4

10 10 9 9 8 9 9 8 8 8 8

Table 3 RAPD banding pattern observed in A. subulatum samples. S. No

Primer code

Primer sequence (5 –3 )

Annealing temperature

No of amplified Fragments

No of bands

Monomorphic Polymorphic 1 2 3 4 5 6 7 8 9 10 11 12 Total

OPA 1 OPA 4 OPA 9 OPA 20 OPJ 5 OPJ 8 OPJ 11 OPJ 14 OPJ 15 OPJ 16 OPJ 17 OPJ 18

CAGGCCCTTC AATCGGGCTG GGGTAACGCC CAGCACCCAC CTCCATGGGG CATACCGTGG ACTCCTGCGA CACCCGGATG TGTAGCAGGG CTGCTTAGGG ACGCCAGTTC TGGTCGCAGA

chloride (0.1%, w/v; 15 min, Hi media, Mumbai, India) with continuous shaking. The rhizome explants were finally 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 2.0–3.0 cm pieces containing buds, and inoculated in 250 ml conical flasks 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 flasks were incubated in a room at 25 ± 1 ◦ C in 16 h light/8 h dark photoperiod with an irradiance of 40 ␮ E m−2 s−1 by cool fluorescent tubes (Philips TI 40 W/54, India).

34.0 32.0 34.0 32.0 37.5 36.0 32.0 35.0 37.0 36.0 32.0 36.0

3 4 5 3 3 3 4 6 4 4 2 1 42

3 4 5 1 3 2 4 6 4 4 1 1 38

0 0 0 2 0 1 0 0 0 0 1 0 4

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 frequent watering using a mist sprayer. After five 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 (0.5–1.0 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 flasks containing 100 ml of MS medium supplemented with various concentrations of PGRs (auxin: ␣-naphthalene acetic acid, NAA, 0.5–1.0 ␮M; cytokinin: 6-benzylaminopurine, BAP, 2.0–5.0 ␮M, see Table 1 for details). The culture flasks were placed in the culture room (as mentioned above). Four replicates (flasks) were used for each treatment and 3 explants were placed in each flask, i.e. 12 explants/treatment. The data on responsive explants were recorded after 3 weeks of inoculation. Most of the PGR combinations 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 fidelity was carried out using tissue culture raised plantlets (10 nos.) along with the mother plant. DNA was isolated following the modified 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 aqueous 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 isopropanol was added to precipitate the DNA. Tubes were centrifuged (room temperature; 8000 rpm, 10 min), the supernatant discarded

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003

G Model JARMAP-82; No. of Pages 6

ARTICLE IN PRESS S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

4

and the pellet washed with 75% aq. ethanol, air-dried and finally suspended in Tris (10 mM) EDTA (1 mM) or sterile distilled water. The DNA was then quantified 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). Amplification was carried out in a thermal cycler (Biometra Master, 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 amplified products were analysed in 1.2% agarose gel. 2.5. Data analyses Mean values of all data were calculated using Microsoft office Excel 2007. All experiments were carried out in a completely randomized block design. The mean values of results obtained under various PGR treatments were subjected to analysis of variance (ANOVA), and the significance level was determined at P < 0.05 using Duncan’s 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 significant (P < 0.05) variation among treatments (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 combinations 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 conditions (95–100% 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 morphologically 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 combinations of BAP and NAA used in this study (Table 1). Fully developed

Fig. 2. RAPD profiles of mother plant (P) along with ten tissue culture raised plants (1–10) using two primes OPJ 18 and OPJ 15. Note:-A-Profile with OPJ 18; B- Profile with OPJ 15; 1–10- 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 performance 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 fidelity. 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 profiles were used to calculate similarity matrix (Table 4). Maximum similarity 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). Morphologically 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 efficient 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 multiplication 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-

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003

G Model

ARTICLE IN PRESS

JARMAP-82; No. of Pages 6

S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

5

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

T2

T3

T4

T5

T6

T7

T8

T9

T10

Mother

0.952 0.929 0.929 0.952 0.952 0.929 0.952 0.905 0.929 0.929 0.929

0.929 0.929 0.929 0.929 0.929 0.929 0.905 0.929 0.929 0.929

0.929 0.929 0.929 0.929 0.929 0.905 0.929 0.929 0.929

0.976 0.952 0.929 0.952 0.905 0.929 0.929 0.929

0.976 0.952 0.976 0.929 0.952 0.952 0.952

0.952 0.952 0.929 0.952 0.952 0.952

0.976 0.929 0.952 0.952 0.952

0.929 0.929 0.929 0.929

0.952 0.952 0.952

0.952 0.952

0.952

Cluster Tree

tions and reported in several plant species (Larkin and Scowcroft, 1981; Barwale and Wildholm, 1987; Kaeppler et al., 2000). The variants 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 assessing the genetic stability of the biological material along all phases of storage and growth must be considered in the context of large scale multiplication.

T4 T3 T2 T1 T7 T5 T10 T6

5. Conclusions

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 (T1–T10).

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 8–12 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 supplemented 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 cardamom, 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 fidelity 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 molecular 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-

This study appears to be the first report of cent percent survival of tissue culture raised plants after 5 month of transfer to pots containing soil and FYM mixtures, along with genetic fidelity assessment of the regenerates. Conventionally A. subulatum is propagated 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 propagules for expanding cultivation of this important cash crop; but it needs to be tested for large scale multiplication and field plantation. Confirmation of genetic fidelity of micropropagated plants is important and necessary for the production of uniform regenerates for use in commercial plantations. Thus this investigation has tremendous commercial implications as large cardamom is used as spice worldwide, as flavoring agents and for pharmaceutical purposes by different industries. Conflicts of interest The authors declare no conflicts of interest, financial or otherwise. Acknowledgements The authors thank Director, G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora for facilities and encouragement; Dr. K.K. Singh, Senior Scientist at Sikkim unit of GBPIHED, Tadong, near Gangtok, Sikkim is thanked for providing the plant material. All colleagues of the Group are thanked for cooperation and help. Financial support in the form of an Institute project (no. 19) is duly acknowledged. References Agnihotri, R.K., Mishra, J., Nandi, S.K., 2009. Improved in vitro shoot multiplication and rooting of Dendrocalamus hamiltonii Nees et Arn. ex Munro: production of genetically uniform plants and field evaluation. Acta Physiologiae Plantarum 31, 961–967. Anonymous, 2015. Annual Report 2014–15 Spices Board. Ministry of Commerce & Industry, Government of India, 137 p. http://www.indian spices.com. Bal Krishnan, K.V., George, K.M., Mathulla, T., Narayana Pillai, O.G., Chandaran, C.V., Verghese, J., 1984. Studies in cardamom 1. Focus on oil of. Indian Spices, 21., pp. 9–12.

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003

G Model JARMAP-82; No. of Pages 6 6

ARTICLE IN PRESS S. Purohit et al. / Journal of Applied Research on Medicinal and Aromatic Plants xxx (2016) xxx–xxx

Barwale, U.B., Wildholm, J.M., 1987. Somaclonal variation in plants regenerated from cultures of soybean. Plant Cell Reports 6 (5), 365–368. Bhagyalakshmi, Singh, N.S., 1988. Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin. Journal of Horticultural Science and Biotechnology 63, 321–327. Bhandari, A.K., Bisht, V.K., Negi, J.S., Baunthiyal, M., 2013. 1-8, Cineole a predominant component in the essential oil of large cardamom (Amomum subulatum Roxb.). Journal of Medicinal Plants Research 26, 1957–1960. Bisht, V.K., Purohit, V., Negi, J.S., Bhandari, A.K., 2010. Introduction and advancement in cultivation of large cardamom (Amomum subulatum Roxb.) in Uttarakhand, India. Research Journal of Agricultural Sciences 1 (3), 205–208. Bisht, V.K., Negi, J.S., Bhandari, A.K., Sundriyal, R.C., 2011. Amomum subulatum Roxb.: traditional, phytochemical and biological activities- An overview. African Journal of Agricultural Research 6 (24), 5386–5390. Gupta, P.N., Naqvi, A.N., Mishra, L.N., Sen, T., Nigam, M.C., 1984. Gas chromatographic evaluation of the essential oils of different strains of Amomum subulatum Roxb. growing wild in Sikkim. Perfum Kosmet 65, 528–529. Gurudutt, K.N., Naik, J.P., Srinivas, P., Ravindranath, B., 1996. Volatile constituents of large cardamom (Amomum subulatum). Flavour and Fragrance Journal 11, 7–9. Hosoki, T., Sagawa, Y., 1977. Clonal propagation of ginger Zingiber officinal Rosc.) through tissue culture. HortScience 12, 451–452. Ilahi, I., Jabeen, M., 1987. Micropropagation of Zingiber officinale Rosc. Pakistan Journal of Botany 19, 61–65. Islam, M.A., Kloppstech, K., Jacobsen, H.J., 2004. Efficient procedure for in vitro microrhizome induction in Curcuma longa L. (Zingiberaceae)–a medicinal plant of tropical Asia. Plant Tissue Culture 14 (2), 123–134. Kaeppler, S.M., Kaeppler, H.F., Rhee, Y., 2000. Epigenetic aspects of somaclonal variation in plants. Plant Molecular Biology 43, 179–188. Khanuja, S.P.S., Shasany, A., Darokar, M.P., Kumar, S., 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Molecular Biology Reporter 17, 1–7. Kumar, K.B., Kumar, P.P., Singh, P., Balachandran, S.M., Iyer, R.D., 1985. Development of clonal plantlets from immature panicles of cardamom. Journal of Plantation Crops 13, 31–34. Larkin, P.J., Scowcroft, W.R., 1981. Somaclonal variation- a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics 60 (4), 197–214. Mohanty, S., Panda, M.K., Sahoo, S., Nayak, S., 2011. Micropropagation of Zingiber rubens and assessment of genetic stability through RAPD and ISSR markers. Biologia Plantarum 55, 16–20. Mukhopadhyay, M., Bantawa, P., Mondal, T.K., Nandi, S.K., 2016. Biological and phylogenetic advancements of Gaultheria fragrantissima: Economically important oil bearing medicinal plant. Industrial Crops and Products 81, 91–99. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia plantarum 15, 473–497.

Nadgauda, R.S., Mascrenhas, A.F., Hendre, R.R., Jagannathan, V., 1978. Rapid clonal multiplication of turmeric Curuma longa L. plants by tissue culture. Indian Journal of Experimental Biology 16, 120–122. Nadgauda, R.S., Mascarenhas, A.F., Madhusoodanan, K.J., 1983. Clonal multiplication of Elettaria cardamomum Maton. by tissue culture. Journal of Plantation crops 11, 60–64. Nei, M.G., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonuclease. PNAS USA 76, 5269–5273. Nirmal Babu, K., Samsudeen, K., Ratnambal, M.J., 1992. In vitro plant regeneration from leaf-derived callus in ginger (Zingiber officinale Rosc.). Plant Cell Tissue and Organ Culture 29 (2), 71–74. Paul, S., Gupta, M.M., Khanuja, S.P.S., 2012. Maintaining the artemisinin content through direct and indirect in vitro regeneration and their assessment of variations with the field grown mother plants of Artemisia annua L. Natural Products and Bioprospecting 2 (5), 194–199. Pillai, S.K., Kumar, K.B., 1982. Clonal multiplication of ginger in vitro. Indian Journal of Agricultural Science 6, 397–399. Purohit, S., Rawat, V., Jugran, A.K., Singh, R.V., Bhatt, I.D., Nandi, S.K., 2015. Micropropagation and genetic fidelity analysis in Valeriana jatamansi Jones. Journal of Applied Research on Medicinal and Aromatic Plants 2, 15–20. Rout, G.R., Das, P., Goel, S., Raina, S.N., 1998. Determination of genetic stability of micropropagated plants of ginger using Random Amplified Polymorphic DNA (RAPD) markers. Botanical Bulletin of Academia Sinica 39, 23–27. Sajina, A., Mini, P.M., John, C.Z., Babu, K.N., Ravindran, P.N., Peter, K.V., 1997. Micro-propagation of large cardamom Amomum subulatum Roxb. Journal of Spices and Aromatic Crops 6, 145–148. Salvi, N.D., George, L., Eapen, S., 2002. Micropropagation and field evaluation of micropropagated plants of turmeric. Plant Cell Tissue and Organ Culture 68, 143–151. Shankaracharya, N.B., Raghavan, B., Abraham, K.O., Shankarayanarayana, M.L., 1990. Large cardamom chemistry, technology and uses. Spice India 3 (8), 17–25. Sharma, E., Sharma, R., Singh, K.K., 2000. A boon for mountain populations: large cardamom farming in the Sikkim Himalaya. Mountain Research and Development 20, 108–111. Sharma, G., Sharma, R., Sharma, E., 2009. Traditional Knowledge Systems in large cardamom farming: biophysical and management diversity in Indian mountainous regions. Indian Journal of Traditional Knowledge 8, 18–21. Verma, S.K., Rajeevan, V., Bordia, A., Jain, V., 2010. Greater cardamom (Amomum subulatum Roxb.), a cardio-adaptogen against physical stress. Journal of Herbal Medicine and Toxicology 4, 55–58. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphism amplified by arbitrary primers is useful as genetic markers. Nucleic Acid Research 18, 6531–6535. Wondyifraw, T., Wannakrairoj, S., 2004. Micropropagation of Krawan (Amomum krervanh Pierre ex Gagnep). ScienceAsia 30, 9–15.

Please cite this article in press as: Purohit, S., et al., Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: A commercially important Himalayan plant. J. Appl. Res. Med. Aromat. Plants (2016), http://dx.doi.org/10.1016/j.jarmap.2016.07.003