Assessment of genetic and biochemical fidelity of field-established Hedychium coronarium J. Koenig regenerated from axenic cotyledonary node on meta-topolin supplemented medium

Industrial Crops & Products 134 (2019) 206–215

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Assessment of genetic and biochemical fidelity of field-established Hedychium coronarium J. Koenig regenerated from axenic cotyledonary node on meta-topolin supplemented medium

T

Shashikanta Beheraa, Subrat K. Karb, Kedar K. Routc, Durga P. Barika, Pratap C. Pandab, ⁎ Soumendra K. Naika, a b c

Department of Botany, Ravenshaw University, Cuttack, 753003, Odisha, India Plant Taxonomy and Conservation Division, Regional Plant Resource Centre, Bhubaneswar, 751015, Odisha, India Gonasika Science College, Keonjhar, 758001, Odisha, India

ARTICLE INFO

ABSTRACT

Keywords: Cotyledonary node Hedychium coronarium HPTLC ISSR Micropropagation

Hedychium coronarium is an important medicinal plant of Zingiberaceae family and is widely used in cosmetics industry and perfumery. H. coronarium is also the source of a number of valuable secondary metabolites including coronarin D, a compound with immense potential to treat cancer. Therefore, there is an urgent need to ensure sustainable use of this plant. The current study reports an one-step in vitro plant regeneration (simultaneous production of shoot and root) protocol for H. coronarium using axenic cotyledonary nodes by optimizing the type and concentration of plant growth regulators. In this study, meta-topolin (mT) alone was found to be optimum, as compared with other individual cytokinins and combinations of growth regulators. The production of shoot/ plantlets was highest on MS medium supplemented with 3.0 mg/L mT. Axenic shoot segments isolated from these primary shoots/ plantlets were cultured in the aforesaid medium to produce about 4590 plantlets. Around 200 plantlets were used for acclimatization; approximately 93% plantlets were successfully acclimatized and established in field. The genetic fidelity of the micropropagated plants with that of the mother plant was ascertained using Inter Simple Sequence Repeats (ISSR) analysis. Quantitative biochemical and High Performance Thin Layer Chromatography (HPTLC) analysis of the micropropagated plant vis-à-vis the mother plant confirmed biochemical similarities. In summary, an efficient plant propagation system was developed for H. coronarium to enable sustainable use of the plant for commercial and conservation purposes.

1. Introduction Medicinal plants are under stress in their natural habitat due to urbanization, defragmentation, deforestation, pollution, climate change, natural calamities and indiscriminate harvesting (Chen et al., 2016). In India, for example, a number of medicinal plants with high trade value are collected from wild habitat unsustainably and nothing is being done to cultivate the plants (Ved and Goraya, 2007). As a result, these plants are at risk of becoming endangered. Such a threat can be addressed through biotechnological intervention such as plant tissue culture to produce, throughout the year, a large number of plants, which are both genetically and biochemically similar to the mother plant (Amoo et al., 2012). Hedychium coronarium J. Koenig (Family: Zingiberaceae) is an erect, rhizomatous herb. It is widely used in traditional systems of medicine,

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particularly in India and China (Behera et al., 2018a, b). Its attractive butterfly-shaped flower has a sweet smell and hence the plant is popular for ornamental use in tropical region (Yue et al., 2015). While the flower has volatile compounds, the rhizome is also known to have volatile oil; thus both the flowers and rhizome are used for perfumery (Chadha, 2005; Chan and Wong, 2015; Yue et al., 2015). In addition, different parts of H. coronarium including rhizomes, flowers, and tender shoots are consumed as vegetables in various parts of the world (Behera et al., 2018a). H. coronarium has already been overexploited in different parts of India. Loss of the plant’s natural habitat has pushed it into the list of threatened plants in various states of India (Chadha, 2005; Ved et al., 2008). The plant is usually propagated through rhizome or seed. However, propagation through its geophytic unit, rhizome, although time consuming, is most common as seed germination is very poor

Corresponding author. E-mail address: [email protected] (S.K. Naik).

https://doi.org/10.1016/j.indcrop.2019.03.051 Received 5 December 2018; Received in revised form 20 March 2019; Accepted 21 March 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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(Behera et al., 2018a). Regeneration of large number of plants round the year using tissue culture is a proven alternative to conventional methods of propagation for conservation and other uses of the plant without affecting the natural population. During the past few years, a number of reports presented various in vitro plant regeneration protocols of H. coronarium using different explants including rhizome, shoot tip derived from axenic seedling, rhizome with a sprouted bud and shoot base (Bisht et al., 2012; Parida et al., 2013; Mohanty et al., 2013; Verma and Bansal, 2013, 2014; Parida, 2018; Behera et al., 2018b). Huang and Tsai (2002) and Verma and Bansal (2012) reported somatic embryogenesis using sheath & leaves and rhizome explants respectively. However, most of these protocols are not quite efficient for production of large number of plants. Therefore, the present study was envisaged to develop a simple, single step in vitro plant regeneration protocol of H. coronarium for large scale production of plants via axillary shoot proliferation from axenic cotyledonary nodes. This study represents the first report on a plant regeneration method for a medicinal plant, H. coronarium using cotyledonary nodes. After large scale propagation of plant through tissue culture, it is necessary to assess the genetic as well as biochemical integrity of the regenerated plants vis-à-vis the mother plant. A number of factors during culture period may induce variations in the in vitro regenerated plants (Bose et al., 2016). To assess the clonal fidelity researchers have employed a number of molecular markers out of which inter simple sequence repeats (ISSR) is commonly used because it requires no prior sequence of genome. Other advantages of ISSR are: low cost, simplicity, requirement of small amount of DNA, non-radioactive nature, and quickness (Pathak and Dhawan, 2012). Similarly different biochemical parameters of the micropropagated plants have to be compared vis-àvis mother plants. Coronarin D is a labdane type diterpene compound present in H. coronarium, which has already shown various biological activities including anticancer properties (Chen et al., 2017; Lin et al., 2018). Coronarin D is very expensive and is sold at a rate of USD 260/ 5 mg (Parida, 2018). As mentioned earlier there are several reports on propagation of H. coronarium through tissue culture, but reports on the assessment of genetic and/ or biochemical fidelity of the micropropagated plants are scanty (Parida et al., 2013, 2015a, b; Behera et al., 2018b; Parida, 2018). Therefore, in this study the micropropagated plants were tested for genetic fidelity by ISSR markers and biochemical analysis was also carried out to estimate tannins, saponins, flavonoids, and phenolics. The potential of retention of coronarin D production ability of the micropropagated plant was also assessed using HPTLC analysis, prior to suggesting their use as substitute to natural mother plants.

2.2. Culture media and conditions for seed germination and shoot and plantlet regeneration The surface-sterilized seeds were inoculated for in vitro germination in culture vessels containing different strength of Murashige and Skoog (1962) (MS) medium i.e. MS or ½ MS either alone or supplemented with different concentrations of gibberellic acid (GA3) (0.5–3.0 mg/L). In this study, the primary shoots and roots were removed from the 15 days-old axenic seedlings and the cotyledonary nodes were used as explants for shoot proliferation or in vitro plantlet regeneration (one step; simultaneous shoot and root formation). The explants were inoculated in MS medium either alone or supplemented with different concentrations and combinations of growth regulators and additives including N6-benzyladenine (BA) (3.0 mg/L), Kinetin (KIN) (1.0–5.0 mg/L), Zeatin (Z) (1.0–5.0 mg/L), meta-topolin (mT) (1.0–5.0 mg/L), Thidiazuron (TDZ) (0.1–1.0 mg/L), α-Naphthalene acetic acid (NAA) (0.5–1.5 mg/L), Indole-3-butyric acid (IBA) (0.5–1.5 mg/L), Indole-3-acetic acid (IAA) (0.5–1.5 mg/L), and Adenine sulphate (ADS) (50–150 mg/L). These growth regulators and additives were used to find out the suitability of medium for in vitro shoot proliferation/plants regeneration. The pH of all the media was adjusted to 5.8 ± 0.1 prior to the addition of 0.7% (w/v) agar and autoclaved at 15 lb/cm2 pressure and 121 °C for 17 min. For production of large number of plants, primary shoots produced from cotyledonary nodes were excised and cut into small pieces. These shoot segments were cultured on MS supplemented with 3.0 mg/L mT, which was optimized as best culture medium for one step plant regeneration in the previous experiment. All the cultures were maintained in a culture room at 25 ± 1 °C with photoperiod of 16 h light /8 h dark under an illumination of 50 μmol m−2 s-1 photon flux density provided by cool white fluorescent tube. 2.3. Acclimatization of in vitro regenerated plantlets Multiple in vitro regenerated shoots with roots (clump of in vitro plantlets) was taken out of the culture medium and the roots were washed thoroughly, but with care, under running tap water to remove the agar. Each in vitro shoot with few roots was separated carefully from the clump. The plantlets were then transplanted in small pots (6.5 × 7.5 cm) containing autoclaved garden soil and sand (1:1). All pots were covered with polyethylene bags to maintain humidity. The potted plantlets were kept in the culture room under controlled environmental conditions as mentioned earlier for in vitro culture. After one week the polyethylene bags were removed and plantlets were kept in the culture room for another one week. During these two weeks the plantlets were watered at regular intervals as per need. The potted plantlets were taken out from culture room and kept in the shade for a week. Subsequently the plantlets were transferred to larger plastic pots (16 × 17 cm) containing the same potting mix and kept under shade for one week before being transferred to direct sunlight where they stayed for three more weeks. Finally, they were transferred to field conditions.

2. Materials and methods 2.1. Collection and surface sterilization of seeds The ripened fruits of H. coronarium were collected from the plants maintained in the garden of the department of Botany, Ravenshaw University in Odisha, India. The seeds were removed from the yellowish fleshy part of the fruits and healthy seeds were used for germination. The other seeds were dried and stored in air tight container to be used for viability testing for up to six months. For in vitro seed germination the seeds were washed under running tap water for about 25 min, treated for 15 min with 5% (v/v) aqueous solution of a liquid detergent ‘Teepol’ (Reckitt Benckiser Ltd., India) and then treated with 2% (w/v) fungicide ‘Bavistin’ (BASF, Mumbai, India) for 10 min. Finally the seeds were rinsed three to five times with double distilled water. Prior to inoculation of seeds, surface sterilization was carried out under aseptic condition with 0.1% (w/v) aqueous solution of mercuric chloride (HgCl2, Hi-media, India) for 6 min followed by 5 times rinse with sterile double distilled water. To check the ex vitro germination potential another set of seeds were sown in pots containing garden soil.

2.4. Assessment of genetic fidelity through ISSR markers Ten in vitro regenerated plants were selected to check the genetic fidelity to the mother plant. Fresh tender leaves of both the sources i.e. mother plant as well as of the field-established micropropagated plants were collected and washed thoroughly with distilled water. The genomic DNA was extracted from these leaves following the procedure described by Doyle and Doyle (1990), with minor modification. The quality as well as the quantity of DNA was checked on 0.8% agarose gel by comparing their intensities with uncut λ DNA band. TE buffer (T10E1) was used to dilute the DNA for PCR amplification. A total of fifteen ISSR primers were initially used for genetic fidelity analysis. Out of the fifteen, ten primers were selected based on 207

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Table 1 Evaluation of seed viability and media for germination of H. coronarium seeds. Basal media

Soil ½ MS ½ MS ½ MS ½ MS ½ MS MS MS MS MS MS

GA3 (mg/ L)

Fresh seeds

0.0 0.0 0.5 1.0 2.0 3.0 0.0 0.5 1.0 2.0 3.0

46.0 34.7 44.7 52.0 64.0 58.7 76.0 80.0 84.7 94.7 90.0

Seed germination (%) ± ± ± ± ± ± ± ± ± ± ±

1.1i 0.7 k 1.2 j 0.7 h 1.2 f 0.7 g 0.5 e 1.0 d 0.6 c 0.6 a 1.7 b

After one month of harvest

After three months of harvest

After six months of harvest

DRG

Seed germination (%)

Seed germination (%)

Seed germination (%)

29 ± 2.0 a 24 ± 1.0 b 21 ± 1.7 c 21 ± 1.7 c 18 ± 2.6 d 15 ± 1.0 e 18 ± 2.0 d 14 ± 0.0 ef 12 ± 1.7 fg 10 ± 1.7 gh 8 ± 1.0 hi

44.7 30.0 42.0 50.0 58.0 54.7 72.0 80.0 84.7 88.0 85.3

± ± ± ± ± ± ± ± ± ± ±

0.7 0.0 0.7 0.0 0.7 1.1 0.7 1.3 0.7 1.2 1.3

i k j h f g e d bc a b

DRG 35 ± 3.0 29 ± 2.0 22 ± 2.6 21 ± 1.7 20 ± 1.7 17 ± 2.0 18 ± 1.7 14 ± 1.0 14 ± 1.7 12 ± 2.0 8 ± 1.0 j

a b c cd c−e e−g d−f gh gh hi

40.0 24.0 34.7 44.0 48.7 48.0 66.0 74.7 78.0 84.0 80.0

± ± ± ± ± ± ± ± ± ± ±

0.7 0.0 1.2 0.7 1.2 0.0 1.3 0.7 1.1 1.7 0.7

i k j g f fg e d c a b

DRG 39 32 27 25 23 20 21 17 16 13 12

± ± ± ± ± ± ± ± ± ± ±

2.6 1.7 3.5 1.0 2.0 0.0 1.0 0.0 1.7 1.0 0.0

a b c cd de fg ef h hi j jk

24.7 19.3 26.7 37.3 43.3 38.0 40.0 52.0 62.7 66.0 64.7

± ± ± ± ± ± ± ± ± ± ±

0.0 0.6 0.7 1.1 0.6 0.0 0.0 1.0 1.2 0.5 0.7

j k i gh e g f d c a b

DRG 45 35 32 29 25 22 28 21 19 15 17

± ± ± ± ± ± ± ± ± ± ±

1.0 2.6 1.7 1.0 2.6 1.7 2.0 1.0 1.7 1.0 0.0

a b c d f g de gh i k j

Values represent means ± standard deviation (SD). In a column, different letters in superscripts indicate statistically significant difference between the means (P≤0.05; Duncan’s new multiple range test). GA3: Gibberellic acid; DRG: Days required for germination.

production of clear and scorable bands. PCR amplification of ISSR primers was carried out by using the procedure described previously by Behera et al. (2018b). The annealing temperature of the ISSR primers was set at 5 °C lower than that of the melting temperature. After PCR amplification, the PCR products were separated on 1.5% agarose gel. The gels were observed using gel documentation system (Bio-Rad, USA) and photographed. The size of the bands was compared with a DNA ladder (GeneRuler 100 bp plus DNA ladder (3 kb), Thermo Scientific, USA). Irrespective of the intensities, only reproducible bands with same migration were considered homologous bands and scored for analysis.

(2018b). A HPTLC system (Camag, Switzerland) used in this study was equipped with an automatic sample applicator Linomat 5 (100 μL syringe), TLC scanner 3 and integrated Win CATS software (version 1.4.2). Dry acetone extract of rhizome (20 mg) was dissolved in 1 mL of acetone and 6 μL of the sample was used on HPTLC plate as 6 mm band using Linomat 5 applicator. The n-hexane-ethyl acetate (8:2) was used as mobile phase for chromatography.

2.5. Biochemical analysis of micropropagated plants vis-à-vis mother plant

For seed germination experiment each treatment consisted of 10 culture vessels and 5 seeds per vessel. For shoot or plantlet regeneration experiments, each treatment consisted of 15 culture vessels and each vessel consisted of one cotyledonary node explant. Data recorded at regular intervals during the culture period include percentage of seed germination, mean number of shoots per explant, mean number of roots per explant, and mean length of shoot and root. All the experiments were carried out thrice. Analysis of variance (ANOVA) for a completely randomized design (CRD) was used for data analysis and, to separate the mean values for significant effect Duncan’s New Multiple Range Test (DMRT) (Gomez and Gomez, 1984) was used. t-test was used to carry out data analysis for quantitative estimation of tannin, saponin, flavonoid, and phenolic.

2.6. Statistical analysis of data

2.5.1. Preparation of sample for biochemical analysis The leaf, rhizome, and root sample of both mother plant and in vitro regenerated plants of H. coronarium were collected and washed with tap water and finally rinsed with distilled water. The plant parts were cut into small pieces and dried at room temperature under shade till a constant weight was achieved. Finally they were powdered and stored in air tight container for use in biochemical experiments. However, only rhizome sample was used for HPTLC work. The sample for HPTLC was prepared by Soxhlet’s apparatus using 10 g powder with 250 mL of acetone. The acetone extract was collected, dried and stored at 4 °C for HPTLC analysis of coronarin D. 2.5.2. Estimation of flavonoid, saponin, phenolic, and tannin content The rhizome, leaf, and root samples of both mother plant and in vitro regenerated plants were used for biochemical analysis. The samples were analyzed for saponin content as described by Bankole et al. (2017), with minor modification. At the same time the estimation of flavonoids, phenolics, and tannins was carried out as described by Bankole et al. (2017); Kamtekar et al. (2014) and Tambe and Bhambar (2014), respectively. The saponin and flavonoid content were expressed as mg/g in dry weight of plant sample. Quantification for phenolics and tannins was carried out on the basis of gallic acid (GAE) and tannic acid (TAE), standard curve was prepared and finally the results were presented in mg standard equivalent weight/g of dry weight of plant powder.

3. Results 3.1. Evaluation of media for seed germination In vitro and ex vitro seed germination experiment was conducted on different culture media and soil, respectively (Table 1). The highest in vitro seed germination (94.7%) was achieved on MS augmented with 2.0 mg/L GA3 within 10 days, whereas lowest seed germination rate (34.7%) was observed on ½ MS within 24 days of culture of fresh seeds. About 76.0% of seeds were germinated on growth regulator free MS medium and an average of 18 days was required for the same. In vitro seed germination was found to be significantly higher than ex vitro seed germination in soil. Seeds sown on soil exhibited poor response of ex vitro seed germination (46.0%). Furthermore, it was observed that the seed germination rate gradually decreased with increase in the days of seed storage irrespective of the media tested. About 88.0%, 84.0% and 66.0% seeds were found to have germinated after one month, three months and six months of storage respectively compared with 94.7% germination of fresh seeds on MS + 2.0 mg/L GA3 (Table 1).

2.5.3. HPTLC analysis of micropropagated plant vis-à-vis mother plant for coronarin D Rhizome samples of mother and micropropagated plants were used for HPTLC analysis of coronarin D. HPTLC analysis was performed following the protocol as described by Ray et al. (2017) and Behera et al 208

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Fig. 1. (A) Axenic seedlings of Hedychium coronarium; (B) Axenic cotyledonary node explants after excision of shoot tip and roots; (C) One step plantlet production by simultaneous shoot and roots formation on MS medium; (D) One step multiple shoot and root formation on MS + 3.0 mg/L mT; (E) One step formation of multiple shoot and root on MS + 3.0 mg/L BA; (F) Simultaneous proliferation of multiple shoot and root on MS + 3.0 mg/L BA + 75 mg/L ADS; (G) Plantlets production from axenic stem segments on MS + 3.0 mg/L mT; (H) In vitro regenerated plantlets after separation from the cluster of shoots having roots, prior to acclimatization; (I) Acclimatized plantlets in small pots containing autoclaved garden soil and sand (1:1); (J) Acclimatized plants in the larger plastic pots. Bars: 2 cm.

3.2. Influence of growth regulators and additives on plantlet regeneration

concentration of BA (3.0 mg/L) in combinations with either ADS or NAA or IBA or IAA was also tested. ADS showed synergistic effect on regeneration of shoot when augmented with MS + 3.0 mg/L BA. Maximum number of shoots (22.3) and roots (42.5) having average length of 4.2 cm and 5.0 cm respectively were recorded on MS + 3.0 mg/L BA + 75 mg/L ADS (Table 2, Fig. 1F). To obtain large number of shoots, the primary axenic shoots were excised and cut into small pieces and used as explants for further proliferation of shoots from their pre-existing meristem. An average of three shoot segments (explant) was obtained per primary shoot. Thus, approximately 144 explants were obtained starting from a single cotyledonary node explant cultured on MS augmented with 3.0 mg/L mT medium. About 95.1% of these explants responded when cultured on the aforesaid medium. An average of 33.5 shoots with 64.5 roots with an average shoot and root length of 5.5 cm and 5.0 cm respectively, were obtained after ten weeks of culture (Fig. 1G). Thus, in a span of twenty weeks, it was possible to obtain approximately 4590 plantlets starting from a single cotyledonary node.

Fifteen-days old axenic cotyledonary nodes were used for shoot proliferation/ one- step plant regeneration (Fig. 1A, B). MS medium either alone or supplemented with different types and concentrations of growth regulators singularly or in combinations were tested and varied response was observed (Table 2). Shoot proliferation was observed from the axenic cotyledonary node explants after 7 days of culture, irrespective of the media tested. It was interesting to note that shoot proliferation was accompanied with simultaneous formation of sufficient roots (one-step plant regeneration) in H. coronarium. Thus there was no need of an additional step of rooting. Each shoot with enough roots (required for successful acclimatization) was easily separated from the shoot clumps formed on cotyledonary node explants. Only 53.3% of explants responded on growth regulator free MS medium. On this medium one shoot with 2.5 roots i.e. one plantlet was observed (Fig. 1C). mT (3.0 mg/L) exhibited highest regeneration frequency (97.8%) compared with all other individual or combinations of plant growth regulators where a maximum of 48.5 number of shoots and 94.5 roots were obtained. On this optimal medium the average shoot and root length was recorded to be 5.8 cm and 5.0 cm, respectively after ten week of culture (Fig. 1D). The number as well as length of shoot and roots decreased beyond this optimum concentration of mT. BA at an optimum concentration of 3.0 mg/L exhibited about 95.5% regeneration, which was not significantly different from the regeneration potential of 3.0 mg/L mT. However, MS supplemented with 3.0 mg/L BA exhibited inferior results, in terms of regeneration of shoot and root, compared with similar concentration of mT. About 6.5 shoots with an average of 18.0 roots were recorded per cotyledonary node on 3.0 mg/L BA (Fig. 1E). Although TDZ, as compared with BA, was able to cause proliferation of more shoots, shoots failed to grow in length on this medium. Thus, it was necessary to transfer the shoot clumps to MS augmented GA3 medium. Other cytokinins including KIN and Z were also found to be inefficient for in vitro shoot multiplication. Optimum

3.3. Acclimatization of in vitro generated plantlets Individual plantlets (shoot with few roots) were separated carefully from the shoot clump (culture) (Fig. 1H). Of the 200 plantlets used for acclimatization, about 93% of these plantlets survived during acclimatization in small pots having a mixture of garden soil: sand (1:1), after three weeks from the beginning of acclimatization process (Fig. 1I). All these plantlets continued to thrive when transferred to larger pots containing the same potting mixture (Fig. 1J) and subsequent field transfer under direct sunlight. 3.4. Genetic fidelity analysis Genetic fidelity of the micropropagated plants with that of the mother plant was assessed by ISSR markers. Monomorphic banding 209

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Table 2 In vitro shoot and one step plantlet production using axenic cotyledonary nodes of H. coronarium on MS basal medium augmented with various plant growth regulators. MS basal medium with growth regulators (mg/L)

% response

BA

Z

KIN

mT

TDZa

ADS

IBA

NAA

IAA

0.0 3.0 – – – – – – – – – – – – – – – – – – – – 3.0

– – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – 1.0 2.0 3.0 4.0 5.0 – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – 0.1 0.3 0.5 0.8 1.0 – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – 50 75 100 125 150 – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – 0.5 1.0 1.5 – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.5 1.0 1.5 – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.5 1.0 1.5

53.3 95.5 86.7 93.3 88.9 82.2 80.0 68.9 73.3 77.8 80.0 77.8 88.9 93.3 97.8 91.1 88.9 77.8 82.2 88.9 86.7 80.0 95.5 95.5 93.3 88.9 88.9 95.5 93.3 84.4 93.3 91.1 91.1 95.5 95.5 93.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 m 2.2 ab 2.2 ef 2.2 bc 2.2 de 2.2 gh 2.0 hi 2.2 l 1.3 k 1.0 ij 1.7 hi 0.0 ij 2.2 de 2.2 bc 3.8a 1.9 cd 1.7 de 0.8 ij 0.3 gh 1.5 de 1.0 ef 0.5 hi 2.2 ab 1.3 ab 0.5 bc 0.4 de 0.5 de 1.8 ab 2.2 bc 3.3 fg 1.2 bc 1.1 cd 1.9 cd 0.9 ab 3.9 ab 2.2 bc

Shoots/explant

Roots/ explant

Shoot length (cm)

Root length (cm)

1.0 ± 0.0 x 6.5 ± 0.5 lm 3.5 ± 0.2 rst 3.8 ± 0.2 rs 3.5 ± 0.5 r−t 3.0 ± 0.2 s−u 2.5 ± 0.0 t−v 2.0 ± 0.0 vw 2.0 ± 0.0 vw 2.5 ± 0.5 t−v 3.0 ± 0.1 s−u 2.0 ± 0.0 vw 23.5 ± 1.3 d 37.8 ± 1.4 b 48.5 ± 1.8a 35.0 ± 1.0 c 22.3 ± 0.5 e 5.6 ± 0.3 m−p 8.9 ± 0.4 i−k 11.8 ± 0.3 gh 9.5 ± 0.5 ij 7.3 ± 0.2 l 12.5 ± 0.4 g 22.3 ± 0.3 e 14.8 ± 0.8 f 9.8 ± 0.3 i 6.4 ± 0.3 l−n 6.5 ± 1.0 lm 6.4 ± 0.4 l−n 4.5 ± 0.2 qr 6.0 ± 0.5 m−o 5.4 ± 0.2 n−q 4.5 ± 0.1 qr 7.3 ± 0.5 l 6.5 ± 0.5 lm 6.0 ± 0.2 m−o

2.5 ± 0.5 xy 18.0 ± 1.7 mn 11.0 ± 1.0 r−t 15.4 ± 0.7 p 11.5 ± 0.5 rs 8.5 ± 0.4 uv 6.5 ± 0.2 w 3.0 ± 0.0 x 6.5 ± 0.5 w 9.5 ± 0.9 tu 12.5 ± 0.5 qr 9.5 ± 0.2 tu 54.6 ± 2.9 d 62.5 ± 1.7 c 94.5 ± 3.1a 77.8 ± 1.8 b 49.3 ± 0.7 e 13.3 ± 0.5 q 21.7 ± 0.2 kl 30.5 ± 1.0 h 17.9 ± 0.9 m−o 15.4 ± 0.4 p 26.3 ± 0.3 i 42.5 ± 0.6 f 38.4 ± 0.7 g 23.2 ± 0.3 jk 19.5 ± 0.3 m 24.6 ± 0.6 ij 30.5 ± 1.1 h 11.5 ± 0.2 rs 21.7 ± 0.5 kl 19.5 ± 0.5 m 15.4 ± 0.5 p 26.30 ± 1.5 i 21.7 ± 0.6 kl 15.4 ± 1.0 p

2.3 5.3 3.6 4.5 4.1 3.3 3.0 3.0 3.5 4.3 4.8 4.0 4.2 4.8 5.8 5.3 4.5 5.0 5.0 5.3 4.8 4.0 3.5 4.2 4.0 3.2 2.0 4.0 4.5 2.0 3.5 3.2 3.0 3.5 3.5 3.2

1.5 4.5 3.1 4.3 4.0 3.6 2.9 2.4 4.0 4.5 4.5 4.3 3.0 4.5 5.0 4.0 3.8 4.0 4.5 4.8 4.3 3.5 2.5 5.0 4.3 2.0 2.0 3.0 2.9 1.5 3.0 2.0 2.5 3.0 2.5 2.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 ° 0.3 b 0.2 j 0.5 de 0.2 f−h 0.2 j−l 0.3 l−n 0.0 l−n 0.2 jk 0.3 ef 0.3 cd 0.0 f−i 0.2 e−g 0.2 cd 0.3a 0.0 b 0.4 de 0.2 bc 0.0 bc 0.1 b 0.2 cd 0.4 f−i 0.1 jk 0.1 e−g 0.0 f−i 0.1 k−m 0.2 op 0.3 f−i 0.5 de 0.3 op 0.0 jk 0.3 k−m 0.1 l−n 0.3 jk 0.3 jk 0.2 k−m

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 m−o 0.5 a−c 0.3 g−i 0.2 b−d 0.0 c−e 0.3 e−g 0.3 i−k 0.1 k−m 0.2 c−e 0.3 a−c 0.0 a−c 0.3 b−d 0.0 h−j 0.5 a−c 0.5 a 0.2 c−e 0.2 d−f 0.3 c−e 0.3 a−c 0.8 ab 0.4 b−d 0.2 e−h 0.2 j−l 0.0a 0.2 b−d 0.0 l−n 0.2 l−n 0.2 h−j 0.4 i−k 0.0 m−o 0.2 h−j 0.4 l−n 0.2 j−l 0.5 h−j 0.3 j−l 0.3 l−n

Values represent means ± standard deviation (SD). In a column, different letters in superscripts indicate statistically significant difference between the means (P≤0.05; Duncan’s new multiple range test). BA: N6-benzyladenine, KIN: kinetin, Z: zeatin, mT: meta-Topolin, TDZ: thidiazuron, NAA: α-naphthaleneacetic acid, IAA: indole-3-acetic acid, IBA: indole-3-butyric acid, ADS: adenine sulphate. a Data (shoots/ explant, roots/ explant, shoot length, root length) was recorded following sub-culture on MS + 1.0 mg/L GA3.

profile was obtained in all the ten ISSR primers. Highest numbers of bands (8) ranging from 475 bp to 1225 bp were amplified by ISK 35 primer (Fig. 2A). At the same time 7 bands were amplified by ISK 25 primer where the range of amplicons was recorded to be 750 bp to 1950 bp (Fig. 2B). A minimum of two bands were amplified by ISK 12 and ISK 31 primers (Table 3). An average of 4.7 scorable bands were produced per primer.

rhizome of the mother plant was found to be more than that of the micropropagated plant. Similarly, the tannin content in rhizome and root of mother plant was found to be slightly higher than in the micropropagated plant (Table 4). 3.5.2. HPTLC for coronarin D Coronarin D content of rhizome of both mother plant and micropropagated plants of H. coronarium was analyzed by HPTLC. On the basis of peak area the level of coronarin D in both the sources was compared. HPTLC analysis not only confirmed the presence of coronarin D in both mother as well as micropropagated plants but also showed that the content of coronarin D was comparatively higher in the micropropagated plants than that of the mother plant (Fig. 3A, B).

3.5. Biochemical analysis 3.5.1. Estimation of flavonoid, saponin, phenolic, and tannin content Biochemical analysis was carried out to estimate flavonoid, saponin, phenolic, and tannin contents in different plant parts including rhizome, leaf, and root sample of both field established micropropagated plant and mother plant. The content of these phytochemicals varied if different plant parts which were being tested. But the content of most of these phytochemicals in the mother plant was at par with that of the micropropagated plant (Table 4). Results revealed that the content of flavonoids was approximately the same in the rhizome and leaf samples of both the mother and the in vitro regenerated plant. No significant difference was observed in the saponins content of root and leaf samples of both the sources. At the same time the saponins content of the

4. Discussion 4.1. Seed germination The rate of seed germination for H. coronarium is normally low (Bisht et al., 2012), which is in agreement with results of the present study of ex vitro seed germination on soil. However, Bisht et al. (2012) reported 80% of in vitro seed germination on MS medium within 10–15 210

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Fig. 2. ISSR amplification profiles of mother plant (Lane 1) and ten randomly selected micropropagated plants of H. coronarium (Lane 2–11) using ISSR primers: (A) ISK 35 and (B) ISK 25; M is the marker (3 kb). Table 3 Details of ISSR primers and banding pattern obtained during assessment of genetic fidelity of the micropropagated plant of H. coronarium vis-à-vis mother plant. Primers

ISK ISK ISK ISK ISK ISK ISK ISK ISK ISK

12 25 26 28 31 33 35 36 38 39

Primer sequences (5’-3’)

Melting temperature (ºC)

Annealing temperature (ºC)

No. of scorable bands

Approximate range of amplifications (bp)

(GATA)4C GTGC(TC)7 (GT)6CG (AGC)4C (CT)8GAC (GCA)8AG (ACG)5C (AGA)5GC (TCG)5G (CA)8T

47.5 59.9 52.6 54.8 60.2 72.5 61.8 52.4 61.8 54.8

42.5 54.9 47.6 49.8 55.2 67.5 56.8 47.4 56.8 49.8

2 7 4 4 2 7 8 3 4 6

500–1450 750–1950 550–2800 1000–1600 700–1100 650–1350 475–1225 600–1175 600–1500 550–2000

Table 4 Quantitative phytochemical analysis of field established in vitro regenerated plant of H. coronarium vis-à-vis mother plant. Phytochemicals Flavonoids (mg/g DW) Saponin (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW) Flavonoids (mg/g DW) Saponin (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW) Flavonoids (mg/g DW) Saponin (mg/g DW) Tannins (mg TAE/g DW) Phenolics (mg GAE/g DW)

Mother Rhizome 5.8 ± 0.4a 27.0 ± 1.0a 163.0 ± 3.0a 12.1 ± 1.0a Root 9.0 ± 0.3a 21.2 ± 1.0a 121.5 ± 1.8a 5.4 ± 0.5a Leaf 5.4 ± 0.4a 22.5 ± 0.8a 201.0 ± 1.0a 20.7 ± 0.5a

Micropropagated 5.9 ± 0.5a 24.9 ± 1.0b 143.3 ± 1.3b 11.8 ± 0.6a 7.3 ± 0.5b 20.3 ± 0.6a 117.1 ± 0.9b 5.5 ± 0.4a 5.12 ± 0.4a 23.7 ± 1.5a 200.3 ± 2.0a 20.5 ± 0.8a

Values represent means ± standard deviation (SD). Different letters in a row in superscripts indicate statistically significant difference between the means (P≤0.05; t-test). DW: dry weight, GAE: gallic acid equivalent, TAE: tannic acid equivalent.

days. In contrast, in this study 76% seed germination was observed on MS medium devoid of any growth regulators. Addition of GA3 has been proved beneficial for promoting in vitro seed germination in a number of plants including Pongamia pinnata (Sugla et al., 2007), Acacia nilotica, Albizzia lebbeck and Prosopis cineraria (Dhupper, 2013). In the quest to enhance the rate of seed germination, GA3 augmented MS medium was also tested in this study. It was observed that addition of GA3

significantly increased the percentage of seed germination in H. coronarium and highest rate of seed germination (94.7%) was achieved on MS medium augmented with 2.0 mg/L GA3. 211

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Fig. 3. HPTLC chromatogram of coronarin D; (A) rhizome of mother plant, (B) rhizome of field established micropropagated plant.

4.2. Influence of growth regulators and additives on in vitro plantlet regeneration

been demonstrated in a number of species of Zingiberaceae including H. coronarium (Borthakur et al., 1999; Mohanty et al., 2013; Purohit et al., 2017; Jena et al., 2018; Behera et al., 2018b). The same feature i.e. one step production of plantlets was also observed in this study using axenic cotyledonary node explant. Simultaneous rooting and shooting in micropropagation protocol does not need a separate rooting stage, thus reducing time and resources required for developing plant regeneration protocol (Mohanty et al., 2013). The plant growth regulator-free MS medium also exhibited both shoot and roots regeneration simultaneously but failed to produce multiple shoots. However, when plant growth regulators were added to the basal medium multiple shoot proliferation was observed. Plant growth regulators for tissue culture mediated plant regeneration should be selected based on their efficacy in formation of sufficient numbers of normal shoot and roots to be acclimatized successfully (Bairu et al., 2007). In a previous study, among the different concentrations of BA tested, MS supplemented with 3.0 mg/L BA was optimum (Behera, 2014). However in a number of

In the last few decades the cotyledonary node derived from axenic seedling has been widely used as an explant for large scale propagation and conservation of a number of medicinal plant species (Naik et al., 2000; Faisal et al., 2006; Nayak et al., 2013; Moharana et al., 2017). Although rhizome has been the preferred explants in the plants belonging to Zingiberaceae family, a few plant regeneration protocols using axenic seedling have also been reported for Zingiber petiolatum (Prathanturarug et al., 2004) and Hedychium spicatum (Badoni et al., 2010; Giri and Tatma, 2011). It was assumed that being a juvenile explant the shoot regeneration potential of cotyledonary node would be higher than the rhizome explants. Thus, axenic cotyledonary node was chosen as the starting plant material in this experiment. Simultaneous production of shoot and roots (one-step production of plant without an additional rooting step) from rhizome explant has 212

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plant species of Zingiberaceae family including Amomum subulatum (Pradhan et al., 2014) and Curcuma longa (Ghosh et al., 2013) inclusion of an auxin to a BA supplemented media was found to be beneficial. No such synergistic influence was observed in this study. However, an optimum concentration of BA in combination with ADS, stimulated the proliferation of more number of shoots. ADS is known to instantly provide nitrogen content to the plant cells, which is better than the inorganic nitrogen content that activates cell development and promotes shoot proliferation (Murashige, 1974; Sivanandhan et al., 2015). In this study, mT (3.0 mg/L) evoked the best response and produced highest numbers of both shoot and root. It was also observed that the efficiency of 3.0 mg/L mT for shoot proliferation and one-step plant regeneration was higher than all the other protocols reported for H. coronarium. mT was initially isolated and identified from leaves of poplar (Horgan et al., 1973, 1975). This natural aromatic cytokinin was also subsequently isolated from mature leaves of Populus × canadensis Moench, cv. Robusta by Strnad et al. (1997). A number of studies indicated that mT is a potential alternative to BA (one of the most preferred cytokinins for micropropagation) (Werrouck, 2010). In agreement to the present study, mT was also shown to stimulate shoot proliferation in a number of plant species (Gentile et al., 2014) including Curcuma longa, a member of Zingiberaceae family (Salvi et al., 2002). At the same time, there are reports on unfavorable response of mT for shoot proliferation in some plant species including Sorbus torminalis (Malá et al., 2009) and a Citrus hybrid (Niedz and Evens, 2011). The production of more number of shoots, plantlets and, finally, successful acclimatization with negligible mortality determine the success of a tissue culture protocol. In this study about 48–49 shoots or plantlets from a single cotyledonary node were obtained using MS + 3.0 mg/L mT. It is common to use axenic explants derived from in vitro formed primary shoot for further proliferation of multiple shoots. Using this technique about 4590 plantlets were generated from a single cotyledonary node in 20 weeks. In vitro regenerated plants on being transferred to outer environment of the culture room are expected to expose to stress conditions including altered temperature, light intensity and water stress (Chandra et al., 2010; Kumar and Rao, 2012). Still in this study about 93% plantlets were acclimatized in field conditions, thus demonstrating that the plant propagation protocol developed in this study is successful.

4.4. Biochemical fidelity analysis H. coronarium is known for the presence of various secondary metabolites. Tannins, saponins, flavonoids, and phenolics are some of the representatives of diverse array of plant secondary metabolites known for their bio-activities in a number of plant species (Bose et al., 2016). Coronarin D is also an important bioactive compound found in H. coronarium and is known for anti-allergy, antimicrobial, anti-inflammatory, and anticancer properties (Chen et al., 2017; Behera et al., 2018b). Thus it was necessary to assess the biochemical fidelity of the micropropagated plant so that it can be used for commercial purposes without disturbing the natural population in wild. In fact not only genetic fidelity but also the biochemical fidelity of the tissue culture raised plants defines the success of a micropropagation protocol. Thus in the present study the biochemical analysis of tannins, saponins, flavonoids, and phenolics in rhizome, root, and leaf was carried out. Similarly analysis of coronarin D in rhizome was undertaken by HPTLC. Results showed the retention of biosynthetic ability of all the phytochemicals including coronarin D in the micropropagated plants. Behera et al. (2018b) reported the retention of such biosynthetic ability in micropropagated H. coronarium plants obtained from rhizome segment. Similar studies of ascertaining biochemical fidelity of several medicinal plants using HPTLC have been documented. These plants include Aloe vera (Pandey et al., 2016), Celastrus paniculatus (Sasidharan et al., 2017) and Lawsonia inermis (Moharana et al., 2018). 5. Conclusion This paper reports a highly efficient plant regeneration protocol for H. coronarium. For the first time cotyledonary node derived from axenic seedling was used as the explant for propagation of H.coronarium. Optimum result for one-step plant regeneration i.e. production of both shoot and root simultaneously was observed when cotyledonary nodes were cultured on MS supplemented 3.0 mg/L mT medium. Up scaling of shoots/ plantlets was also achieved by culturing the axenic shoot segments derived from primary shoots/ plantlets on MS medium augmented with mT (3.0 mg/L). Using this protocol it was possible to obtain 4590 plantlets from a single cotyledonary node in twenty weeks. Most importantly these regenerated plants were genetic and biochemical clones of the mother plant as ascertained by ISSR analysis and HPTLC analysis respectively. Being an industrially important but overexploited plant, the present micropropagation protocol of H. coronarium may prove helpful for production of large number of clonal plants to be used for ex situ conservation, reintroduction in wild habitat as well as commercial use in pharmaceutical and cosmetic industries.

4.3. Genetic fidelity analysis In this study no morphological variation was observed in the micropropagated plants as compared with the mother plant. But various factors including culture media, types, and concentrations of plant growth regulators, type, age, and source of explants, culture conditions as well as duration of culture may induce variation in in vitro regenerated plant at genetic level which is not sufficient to change the phenotype in a detectable manner (Palombi and Damiano, 2002; Singh et al., 2013; Bose et al., 2016). For commercial utilization, it is vitally important to assess the genetic fidelity of the in vitro regenerated plants. Several techniques are available to screen the genetic fidelity of the tissue culture raised plants, of which PCR-based molecular markers are preferred the most. In this study PCR-based markers namely ISSR was used as these markers are stringent and thus reproducible (Chhajer and Kalia, 2017). Besides, it is obvious by now that ISSR markers are quick, reliable, cost-effective, and have the ability to screen the complete genome randomly and quickly (Nayak et al., 2013; Chhajer and Kalia, 2017). Genetic fidelity study of tissue culture-raised plants has been confirmed by ISSR markers in a number of plant species (Parida et al., 2016; Baradwaj et al., 2017; Behera et al., 2018b, c; Jena et al., 2018). Consequently, in the present study, ISSR markers were used for clonal fidelity testing. Similar ISSR banding profile of the mother and micropropagated plants ascertained the clonal fidelity of the regenerated plants.

Author contributions SB and SKN conceptualized the study. SB conducted all the experiments and wrote the first draft of the manuscript. SKK helped in ISSR experiment. KD helped in HPTLC data analysis. SB, along with DPB, carried out the data analysis of tissue culture. PCP supervised the ISSR experiment. SKN corrected the manuscript and supervised the entire research. All authors read and approved the final manuscript. Acknowledgements Science and Technology Department, Government of Odisha is gratefully acknowledged for funding this research. We also acknowledge the DST-FIST programme of the Department of Botany, Ravenshaw University, Cuttack, Odisha for infrastructural facilities. We thank Central Laboratory, Orissa University of Agriculture and Technology, Bhubaneswar for HPTLC facility. We also thank Dr. Sashi Kanta Dash and Gouri Sankar Acharya for their help in conducting HPTLC work. 213

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