In vitro propagation of traditional medicinal and dye yielding plant Morinda coreia Buch.–Ham

South African Journal of Botany 100 (2015) 43–50

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In vitro propagation of traditional medicinal and dye yielding plant Morinda coreia Buch.–Ham M.S. Shekhawat a,⁎, N. Kannan b, M. Manokari a a b

Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C., Mahe, Pondicherry 673311, India Biotechnology Unit, K.M. Centre for Postgraduate Studies, Pondicherry 605 008, India

a r t i c l e

i n f o

Article history: Received 28 December 2014 Received in revised form 29 April 2015 Accepted 22 May 2015 Available online xxxx Edited by J Van Staden Keywords: Morinda coreia Micropropagation Semi-solid medium Ex vitro rooting Hardening

a b s t r a c t An efficient micropropagation protocol has been developed successfully for Morinda coreia Buch.–Ham. by culturing nodal segments. The explants were washed, sterilized with HgCl 2 and inoculated on semi-solid Murashige and Skoog (MS) medium containing various concentrations and combinations of plant growth regulators (PGRs). Shoot bud initiation was observed after one week and 8.6 ± 0.32 shoots (per explant) harvested after five weeks on MS medium with 4.0 mg l−1 concentration of 6-benzylaminopurine (BAP). The regenerated shoots were further multiplied on semi-solid MS medium augmented with 2.0 mg l− 1 BAP + 1.0 mg l− 1 Kinetin (Kin). Maximum 24.5 ± 0.34 shoots per explant was obtained after five weeks on this media combination. The long (4–5 cm) and healthy shoots were rooted in vitro with 100% success rate on half strength MS medium + 1.0 mg l− 1 indole-3 butyric acid (IBA). Rooting and acclimatization were achieved simultaneously by ex vitro rooting method using 200 mg l− 1 IBA for 5 min with very good success rate (28.67 ± 05.51 roots per shoot with 100% response). The rooted shoots were transferred to the greenhouse for acclimatization for 4–5 weeks. The hardened plantlets were finally shifted to the field for further growth in the natural conditions after another five weeks. This is the first report on micropropagation of M. coreia, which can be successfully used for the large-scale multiplication and conservation of germplasm of this important medicinal plant. © 2015 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Morinda coreia Buch.–Ham., which belongs to the family Rubiaceae, is an important traditional medicinal and dye yielding plant (synonyms; Morinda pubescens J.E. Smith, Morinda tinctoria Roxb.). It is native to India, South-East Asia and Polynesia (Lu-berck and Hannes, 2001), and widely distributed in the Indian Peninsular in natural conditions along with fences and road sides due to its wider adaptability to hard environmental conditions. This Morinda species is naturally growing in pastures, alien grass lands, open areas, forests, shorelines, coconut plantations, fallow areas and in waste lands. M. coreia is naturally growing in the Coromandel Coastal region of India which includes Thiruvallur, Kanchipuram, Villupuram, Cuddalore, Nagapattinam, Ramanathapuram, Tuticorin, Thanjavur and Kanyakumari districts of Tamil Nadu, and Puducherry and Karaikal districts of Puducherry. It is commonly known as Nuna in Tamil and Manjanuna and Manjanathi in Malayalam and Tamil in India (Mathivanan et al., 2006). M. coreia is an important ethno-medicinal plant, rich in metabolites like anthraquinones, phenolics, aucubin, scopoletin, asperuloside, ⁎ Corresponding author at: Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C. Mahe, Pondicherry 673311, India. Tel.: +91 8943157187. E-mail address: [email protected] (M.S. Shekhawat).

http://dx.doi.org/10.1016/j.sajb.2015.05.018 0254-6299/© 2015 SAAB. Published by Elsevier B.V. All rights reserved.

vitamins A and C, alkaloids, flavone glycosides, terpenoids, linoleic acid, saponins, tannins and phenols (Wang and Su, 2001). The plant is reported to be used as styptic, alexeteric, digestive, carminative, febrifuge and tonic; and used in gastropathy, dyspepsia, diarrhea, stomach ulcer, wounds, gout, inflammation, hernia, sarcocele fever etc. (Cimanga et al., 2003; Prajapati et al., 2003). It showed a wide spectrum of antimicrobial (Srilakshmi et al., 2012), antibacterial (Jayasinghe et al., 2002) and antihypertensive activities (Solomon, 1999). The plant is used in the treatment of hypertension, painful menstruation, arthritis, diabetes gout, gingivitis, sties and depression (Pawlus et al., 2005; Pattabiraman and Muthukumaran, 2011). In India, it is also used to make the morindone dye, which is sold under the trade name “Suranji”. Morindone is used for the dyeing of cotton, silk and wool in shades of red, chocolate or purple. The coloring matter is found principally in the root bark and is collected when the plant reaches three to four years old. The active substance extracted as glucoside is known as morindin, this upon hydrolysis produces the dye (Singh and Tiwari, 1976). It is mordant dye giving a yellowish-red color with an aluminium mordant, chocolate with a chromium mordant, and dull purple to black with an iron mordant. The fresh fruits of M. coreia are rich in protein, carbohydrate, vitamin and mineral contents and could be used as an alternate to Noni (Morinda citrifolia) fruits. These contain more amounts of ascorbic acid

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and niacin whereas the dry fruit showed the presence of riboflavin and thiamine in high concentration as compared to Noni fruits (Anuradha et al., 2013). Since, the roots only produce dye (anthraquinones) the well grown plants are uprooted to get maximum amount of pigments. Due to this, the entire plant population is facing threat in natural conditions (Sharma, 2003; Sujit and Rahman, 2011). Therefore, it is an urgent need to restore the natural population of this valuable plant. Plant biotechnology has been used for conservation of ecosystem and multiplication of plant biodiversity (Wawrosch et al., 2001; Joshi and Dhar, 2003; Martin, 2003). Plant tissue culture techniques could be applied for rapid and mass production of rare, medicinal and economic valuable plants (Martin, 2002). To the best of our knowledge no reports are available on cloning and tissue culture studies on M. coreia at national and international levels. Therefore, we planned to develop an efficient and highly stable regenerative protocol for M. coreia for conservation strategy and to provide continuous supply of a better source of elite plants to be used as standard material in the field of drug research as well as in manufacturing of dyes. 2. Materials and methods 2.1. Explants and sterilization of explants The candidate plus trees (CPTs) of M. coreia were identified (Fig. 1A) from the Coromandel Coast of India which includes Kanchipuram, Villupuram, Cuddalore, Nagapattinam, Ramanathapuram, Tuticorin and Kanyakumari districts of Tamil Nadu, and Puducherry and Karaikal districts of Puducherry. Fresh shoot sprouts (Fig. 1B) were collected from four to five year old mature plants during the months of February to December, 2012 and nodal shoot segments (2–3 cm long) were used as explants (minimum 2 nodes). These were dressed, washed and cleaned with the help of 0.1% (w/v) Tween® 20 (Sigma Aldrich, India) for 5 min and treated with 0.1% (w/v) Bavistin solution (Systemic Fungicide, BASF India Ltd.) about 8–10 min and then with 0.1% (w/v) HgCl2 for 5–6 min in laminar air flow cabinet (Technico Pvt. Ltd., Chennai, India). The treated explants were thoroughly washed with autoclaved distilled water (5–6 times) to remove the traces of HgCl2 from the surface of explants, and finally dipped in 90% ethyl alcohol about 30–40 s as pre-treatment before inoculation. 2.2. Medium and establishment of cultures MS basal medium (Murashige and Skoog, 1962) was used during the present study. Agar gelled MS medium (Agar 0.8%) supplemented with various concentrations of 6-benzylaminopurine (BAP) and Kinetin

A

(Kin) (ranging from 1.0 to 6.0 mg l− 1), and indol-3 acetic acid, IAA (0.1 to 1.0 mg l−1) were used alone or in combination to induce shoots from the nodal meristems. Ten ml of medium (10 replicates) was poured in each culture tube. All the experiments were repeated thrice. Sterilized explants inoculated vertically on the medium and the cultures were incubated in culture shelves with illumination from white fluorescent tubes at incubation temperature of 25 ± 2 °C to 30 ± 2 °C under different light:dark photoregimes (12:12 h, 14:10 h, 10:14 h) for shoot bud induction. 2.3. Multiplication of shoots in vitro The cultures were maintained for five weeks in an incubation chamber. After five weeks the multiple shoots (3–4 cm long) regenerated from the nodal meristems were ready for subculturing on multiplication medium. These shoots were further multiplied by two approaches (i) the mother explants were repetitively transferred to fresh medium for 2–3 passages after harvesting in vitro raised shoots and (ii) the in vitro produced shoots were cut into 2–3 cm long segments (each with at least 1–2 nodes) and subcultured on fresh medium. MS medium augmented with different concentrations and combinations of BAP and Kin ranging from 0.1 to 3.0 mg l−1 and IAA (0.1–1.0 mg l−1) was used for multiplication. The cultures were maintained at 28 ± 2 °C temperature, 40–45 μmol m− 2 s− 1 spectral flux photon (SFP) light intensity under 10:14 h light:dark photoperiod for multiplication of shoots in vitro. These culture conditions supported the maximum growth of the shoots in vitro in the establishment of the cultures from the explants because M. coreia was naturally grown in the tropical and sub-tropical conditions. About 100 ml of medium (10 replicates) was poured in each culture flask. All the experiments were repeated thrice. The cultures were maintained for five weeks in the incubation chamber and the data were recorded. 2.4. In vitro rooting of the shoots The healthy and long (4–5 cm) multiple shoots were selected and excised at the time of subculturing of shoot clumps to the fresh multiplication medium. These shoots were inoculated to full strength, half strength and one-fourth strength MS basal salt medium fortified with various concentrations of indol-3 butyric acid (IBA) and IAA ranging from 0.5 to 5.0 mg l−1. Ten ml of this medium with 10 replicates was poured in each culture tube for root induction from the cut end of the shoots. These cultures were incubated at diffuse light (20– 25 μmol m−2 s−1 SFP intensity) conditions at 28 ± 2 °C temperature. After 4 weeks of culture, number and length of roots and significance of callus were measured.

B

Fig. 1. A: Morinda coreia growing in natural habitat. B: Branch of the plant used in explant preparation.

M.S. Shekhawat et al. / South African Journal of Botany 100 (2015) 43–50

A

B

45

C

Fig. 2. A: Bud breaking from the nodal meristems of explant on MS medium with BAP. B: Bud breaking from the nodal meristems of explant on MS medium with Kin. C: Multiple shoots after five weeks of inoculation.

2.5. Ex vitro rooting of regenerated shoots Experiments were conducted to achieve rooting and acclimatization simultaneously using ex vitro rooting method of in vitro regenerated shoots to save energy, cost of production and time. Healthy and tall shoots (4–5 cm) were selected from the in vitro multiplied shoots at the time of subculturing of shoot clumps to get the next crop of shoots (multiplication stage). The cut ends of the shoots were dipped in auxin solutions (IBA or IAA) for 5 min. Different concentrations of

auxins (50, 100, 200, 300, 400 and 500 mg l−1) were prepared with the help of distilled water. Finally, the pulse treated shoots were directly transferred to the eco-friendly plain paper cups (size 150 ml; Vandana Paper Products, Chennai, India) containing 55 g autoclaved soilrite® (a mixture of perlite, Irish peat moss and exfoliated vermiculite; KelPerlite, Bangalore, India), moistened with 10 ml aqueous 1/4th MS salt solution by the interval of one week and maintained in the greenhouse for five weeks. 2.6. Hardening of rooted plantlets in greenhouse and acclimatization

Table 1 Effect of cytokinins (BAP and Kin) and IAA on the induction of shoots from the nodal segments in M. coreia. Conc. of BAP (mg l−1)

Conc. of Kin (mg l−1)

Conc. of IAA (mg l−1)

Number of shoots/explants (Mean ± SD)

Response (%)

Control 1.0 2.0 3.0 4.0 5.0 6.0 – – – – – – 1.0 2.0 3.0 4.0 5.0 6.0 – – – – – –

– – – – – – – 1.0 2.0 3.0 4.0 5.0 6.0 – – – – – – 1.0 2.0 3.0 4.0 5.0 6.0

– – – – – – – – – – –

0.0 ± 0.00 3.4 ± 0.89c 5.3 ± 0.13gh 6.5 ± 0.56jk 8.6 ± 0.32m 7.4 ± 0.47l 6.2 ± 0.87ij 2.8 ± 0.54b 3.7 ± 0.19cd 4.8 ± 0.36fg 6.5 ± 0.43jk 5.7 ± 0.26hi 4.3 ± 0.79ef 3.5 ± 0.35cd 5.2 ± 0.57gh 6.8 ± 0.35jk 7.1 ± 0.44kl 7.5 ± 0.21l 6.2 ± 0.73ij 2.2 ± 0.81a 3.6 ± 0.64cd 4.1 ± 0.88de 4.8 ± 0.24fg 4.4 ± 0.91ef 2.4 ± 0.68ab

0 43 67 83 88 74 68 32 57 63 72 61 49 54 61 73 81 70 59 41 59 63 68 46 39

– 0.1 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.4 0.6 0.8 1.0

Note: Experiments were conducted three times and ten replicates were used. Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance variation between the concentration was studied using DMRT at 0.5% level. Superscript letters denote the highest/lowest significant value within the concentrations/groups in this study. The same superscript letters are not significantly different according to DMRT at P b 0.05.

The in vitro rooted plantlets were washed with sterilized distilled water to remove adhered nutrient agar from the roots. These plantlets were kept on a moist paper and transplanted immediately to the ecofriendly plain paper cups containing soilrite® and moistened with aqueous solution of 1/4th strength of MS basal salts. These cups were covered by the disposable transparent plastic cups (size 200 ml; Swastik Table 2 Effect of cytokinins (BAP and Kin) and auxin (IAA) on multiplication of shoots of M. coreia on semi-solid MS medium. Conc. of BAP (mg l−1)

Conc. of Kin (mg l−1)

Conc. of IAA (mg l−1)

Number of multiple shoots (Mean ± SD)

Length of shoots (cm) (Mean ± SD)

Control 0.1 0.5 1.0 1.5 2.0 2.5 3.0 0.1 0.5 1.0 1.5 2.0 2.5 3.0

– 0.1 0.4 0.6 0.8 1.0 2.0 3.0 0.1 0.4 0.6 0.8 1.0 2.0 3.0

– – – – – – – – 0.1 0.2 0.3 0.4 0.6 0.8 1.0

0.0 ± 0.00 11.1 ± 0.53a 13.8 ± 0.11bc 17.4 ± 0.76g 20.2 ± 0.93i 24.5 ± 0.34j 22.7 ± 0.35k 19.1 ± 0.56g 11.5 ± 0.86a 13.6 ± 0.81b 14.8 ± 0.47de 16.2 ± 0.82f 17.4 ± 0.18g 15.1 ± 0.52c 14.3 ± 0.69cd

0.0 ± 0.00 5.7 ± 0.86ab 6.4 ± 0.51cd 6.8 ± 0.22de 7.2 ± 0.41e 7.1 ± 0.43e 6.8 ± 0.97de 5.3 ± 0.19a 5.1 ± 0.33a 5.3 ± 0.91a 6.7 ± 0.42cde 6.8 ± 0.32de 7.0 ± 0.89de 6.6 ± 0.65cde 6.1 ± 0.37bc

Note: Experiments were conducted three times and ten replicates were used. Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance variation between the concentration was studied using DMRT at 0.5% level. Superscript letters denote the highest/lowest significant value within the concentrations/groups in this study. In each column, the same superscript letters are not significantly different according to DMRT at P b 0.05.

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A

B

C

Fig. 3. A: Multiplication of shoots after two weeks with agar gelled MS medium. B: Multiplication of shoots after five weeks in culture flasks. C: Multiplied shoots outside of culture vessel after five weeks.

Poly Pack, Chennai, India) in inverted position to maintain moisture. These plantlets were maintained in the greenhouse for acclimatization. After 4–5 weeks, plantlets were transferred to perforated nursery bags containing garden soil, soilrite®, manure and vermi compost in 1:1:1:1 ratio and allowed for one and half month in the greenhouse for hardening. Finally, the hardened plantlets were planted to the natural field from the greenhouse.

respectively) was regenerated from the nodal meristem of the explants. Gajakosh et al. (2010) also observed callus, during shoot initiation experiments in M. citrifolia. The physiological status of explants and their size, quality, collection period and other environmental factors play important roles in the establishment of cultures (Smith, 2000). Less number of shoots (7.41 and 7.73 shoots per explant) was induced from the nodal meristem of M. coreia explants under 12:12 and 14:10 light:dark photoregimes at 28 ± 2 °C.

2.7. Experimental design, data collection and statistical analysis 3.2. Multiplication of shoots in vitro in agar gelled medium The experiments followed in this study were laid down according to completely randomized block design (RBD) in the case of single factor experiments (Compton and Mize, 1999), with a minimum of 10 replicates per treatment and were repeated thrice. Data were subjected to analysis of variance by ANOVA and the significance of differences was calculated by Duncan's multiple range test using SPSS software (version 16.0). 3. Results and discussion 3.1. Induction of multiple shoots from the nodal meristems Shoot bud initiation was observed from the nodal meristems of explants within one week of inoculation due to the presence of cytokinins (BAP or Kin) in the medium. Shoot buds were not induced from the nodal meristems of the explants inoculated on MS medium without cytokinins (control). The meristematic cells of the nodal region were induced to produce fresh shoots in many plant species (Rathore et al., 2013a,b; Phulwaria et al., 2013; Patel et al., 2014). Cytokinins were proved responsible for cell division, cell elongation and to induce shoots from the nodal meristem of the explants. The agar gelled MS medium supplemented with 4.0 mg l−1 BAP was found as the best medium combination for shoot induction, where 88% explants responded and maximum 8.6 ± 0.32 shoots were regenerated from each explant (Fig. 2A and C, Table 1). On the other hand less number of shoots (6.5 ± 0.43) was regenerated on MS medium + 4.0 mg l− 1 Kin (Fig. 2B). The rate of shoot initiation from the nodal explant was higher in the present study than the earlier studies on M. citrifolia with MS medium augmented with BAP (Sreeranjini and Siril, 2014). Explants collected during the months of March to June were responded earlier (within week) as compared to the explants collected from July to December. The superiority of BAP over Kin in bud initiation from the explants has been proved in many plant species such as Holarrhena antidysenterica, Arnebia hispidissima, Randia dumetorum, Tectona grandis, M. citrifolia and Turnera ulmifolia (Ahmed et al., 2001; Ferdousi et al., 2003; Kumar et al., 2005; Shirin and Rana, 2005; Shekhawat and Shekhawat, 2011; Shekhawat et al., 2014; Sreeranjini and Siril, 2014). MS medium with BAP/Kin + IAA induced callus from the base of the explants and less number of shoots (7.1 and 4.8 shoots

The rate of shoot multiplication after the 3rd and 4th transfers was over 24.5 ± 0.34 shoots when the semi-solid MS medium was supplemented with 2.0 mg l−1 BAP and 1.0 mg l−1 Kin from single explant (Table 2 and Fig. 3A to C). Healthy shoots with very good length (7.1 ± 0.43 cm) were regenerated on this medium combination. Less numbers of shoots (17.4 ± 0.18 shoots) was observed when the MS medium was combined with BAP + Kin + IAA and callus was induced with this combination from the basal part of the shoot clumps. Hyperhydration or vitrification was not observed at any stage in this study. Maximum 4–5 shoots were regenerated from the nodal explants of seedlings of Noni on BAP and Kin supplemented medium by Subramani et al. (2007). Wei et al. (2006) studied the varying effects of cytokinin and auxin combinations on M. citrifolia for in vitro induction of shoots. Gajakosh et al. (2010) reported organogenesis from shoot tips and leaf explants of M. citrifolia, but they were unable to regenerate complete plantlets from the in vitro methods. Sreeranjini and Siril (2014) multiplied M. citrifolia shoots (5.3 shoots per culture vessel) and observed callus when the cultures were inoculated on MS medium supplemented with BAP and NAA. Our results are better (in terms of number of shoots multiplied) than the previous work on different plant species of the family Rubiaceae (Kai et al., 2008; Jimenez et al., 2011). The higher concentration of cytokinin than auxin in the culture Table 3 Effect of strength of MS medium augmented with 1.0 mg l−1 IBA on in vitro root initiation from shoots of M. coreia. Strength of MS medium Full strength Half strength 1/4th strength

Response Number of roots (%) (Mean ± SD) 86.3 100 83.6

31.8 ± 9.51a 43.20 ± 11.20 26.4 ± 9.66b

c

Length of roots (cm) (Mean ± SD)

Intensity of callus

1.76 ± 0.34a

Negligible callus Negligible callus Negligible callus

2.84 ± 0.48

b

2.27 ± 0.81ab

Note: All the experiments were conducted three times and ten replicates were used. Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance variation between the concentration was studied using DMRT at 0.5% level. Superscript letters denote the highest/lowest significant value within the concentrations/groups in this study. In each column, the same superscript letters are not significantly different according to DMRT at P b 0.05.

M.S. Shekhawat et al. / South African Journal of Botany 100 (2015) 43–50

A

B

C

D

E

47

F

G

Fig. 4. In vitro rooting of the shoots; A: control, B: roots with IBA 0.5 mg l−1, C: roots with IBA 1.0 mg l−1, D: roots with IBA 2.0 mg l−1, E: roots with IBA 3.0 mg l−1, F: roots with IBA 4.0 mg l−1, G: roots with IBA 5.0 mg l−1.

medium could induce shoot organogenesis (Rout and Das, 1997; Sharma and Singh, 1997; Shekhawat et al., 2011). 3.3. Rooting of in vitro cultured shoots Half strength MS medium was proved most effective for in vitro root induction and all the replicates responded 100% with IBA. Full and onefourth strength MS medium with 1.0 mg l−1 IBA induced 31.8 ± 9.51 and 26.4 ± 9.66 roots per shoot respectively with negligible callus (Table 3). The intensity of callus formation was simultaneously increased from lower to higher concentrations of IBA. Maximum 243.60 ± 78.72 roots (Fig. 4G) were observed with the highest amount of callus on 5.0 mg l−1 IBA. Plantlets with more callus and compact roots were not responding well during hardening of the plantlets in the greenhouse. Healthy roots without callus or negligible callus were observed on 0.5 and 1.0 mg l− 1 IBA with 39.20 ± 13.81 and 43.20 ± 11.20 numbers of roots respectively (Fig. 4B, C and Table 4). There was no callus and root formation with the control experiment (Fig. 4A). Moderate incidence of callus was reported with 2.0 and 3.0 mg l−1 IBA (Fig. 4D and E) and compact roots with higher intensity of callus were induced with 4.0 mg l−1 IBA (Fig. 4F). The response of IAA in initiation of roots was poor as compared to IBA with less intensity of callus. Maximum 21.60 ± 2.84 roots were induced from the shoot at 2.0 mg l−1 IAA with moderated intensity of callus whereas 13.61 ± 2.33 and 17.11 ± 4.56 roots were induced from the shoot on 0.5 and 1.0 mg l−1 IAA respectively without callus or negligible callus intensity. There were no roots observed in the control experiments but Sreeranjini and Siril (2014) reported roots

from the shoots without any auxins in M. citrifolia. Subramani et al. (2007) reported rhizogenic calli on MS media with the combination of cytokinin and auxin in the case of M. citrifolia. Even trace amount of cytokinin in MS medium cannot enhance any root system from the cut ends of the shoots (Lui and Li, 2001). Misic et al. (2006) and Arikat et al. (2004) reported that auxins play important roles in the induction of roots from the cut ends of in vitro raised shoots of Salvia brachydon and Salvia fruticosa respectively. Biswas et al. (2011) observed 4.70 roots per shoot on half strength MS medium fortified with 0.5 mg l−1 NAA in Stemona tuberosa. It was observed in the present study that the cultures were multiplied maximum when these incubated at 28 ± 2 °C temperature with 14:10 h light:dark photoregime per day, because the mother plant (M. coreia) was naturally growing in the tropical and sub-tropical environmental conditions. 3.4. Ex vitro root induction from the cut ends of the shoots The excised shoots produced roots when these were pulse treated with auxin solutions for ex vitro rooting experiments. Maximum response (100%) with 28.67 ± 05.51 number of roots per shoot and root length (2.84 ± 0.84) was reported with IBA at 200 mg l−1 concentration (Table 5 and Fig. 5B). Ex vitro roots were not observed with 50 and 500 mg l− 1 IAA. Higher concentration of IAA (200 mg l−1) responded but less numbers of roots (3.20 ± 2.91) reported (Fig. 5E) as compared to all concentrations of IBA (Fig. 5A to D). Sreeranjini and Siril (2014) also reported ex vitro rooting in M. citrifolia with 26 roots when the micro-shoots were treated with IBA. Plantlets rooted under

Table 4 Effect of auxins (IBA and IAA) on induction of roots from the shoots of M. coreia on half strength MS medium. Conc. of IBA (mg l−1)

Conc. of IAA (mg l−1)

Response (%)

Number of roots (Mean ± SD)

Length of roots (Mean ± SD) (cm)

Intensity of callus

Control 0.5 1.0 2.0 3.0 4.0 5.0 – – – – – –

0 – – – – – – 0.5 1.0 2.0 3.0 4.0 5.0

0 100 100 100 100 100 100 100 100 100 100 100 100

0.0 ± 0.0 39.20 ± 13.81g 43.20 ± 11.20h 181.71 ± 61.79i 190.67 ± 65.30j 210.50 ± 66.51k 243.60 ± 8.72l 13.61 ± 2.33b 17.11 ± 4.56d 21.60 ± 2.84f 17.80 ± 3.22e 15.36 ± 1.65c 11.44 ± 2.51a

0.0 ± 0.0 2.43 ± 0.40bcd 2.84 ± 0.48d 4.63 ± 0.68f 4.68 ± 0.85f 4.06 ± 0.95e 4.77 ± 0.66f 2.18 ± 0.46bc 2.06 ± 0.92abc 2.82 ± 0.37d 1.85 ± 0.63ab 2.63 ± 0.44cd 1.49 ± 0.50a

No callus No callus Negligible callus Moderate callus Moderate callus High callus High callus No callus Negligible callus Moderate callus Moderate callus Negligible callus Negligible callus

Note: All the experiments were conducted three times and ten replicates were used. Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance variation between the concentration was studied using DMRT at 0.5% level. Superscript letters denote the highest/lowest significant value within the concentrations/groups in this study. In each column, the same superscript letters are not significantly different according to DMRT at P b 0.05.

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Table 5 Effect of auxins (IBA and IAA) on ex vitro root induction from the shoots of M. coreia in the greenhouse. Auxins

Conc. of auxins (mg l−1)

Response (%)

Number of roots (Mean ± SD)

Length of roots (Mean ± SD)

Control IAA

0 50 100 200 300 400 500 50 100 200 300 400 500

0 0 17 26 13 13 0 100 100 100 100 100 100

0.0 ± 0.0 0.00 ± 0.00a 1.00 ± 2.24b 3.20 ± 2.91d 1.60 ± 1.34c 0.20 ± 0.45a 0.00 ± 0.00a 14.75 ± 08.62e 20.40 ± 08.47f 28.67 ± 05.51j 25.40 ± 11.86 i 23.6 ± 10.74h 21.80 ± 18.96g

0.0 ± 0.0 0.00 ± 0.00a 0.42 ± 0.94b 0.12 ± 0.18ab 0.50 ± 0.22b 0.10 ± 0.22ab 0.00 ± 0.00a 2.75 ± 1.87d 2.81 ± 1.42d 2.84 ± 0.84d 1.60 ± 0.98c 2.77 ± 1.84d 2.64 ± 0.74d

IBA

Note: Experiments were conducted three times and ten replicates were used. Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance variation between the concentration was studied using DMRT at 0.5% level. Superscript letters denote the highest/lowest significant value within the concentrations/groups in this study. In each column, the same superscript letters are not significantly different according to DMRT at P b 0.05.

an ex vitro environment are better suited/adapted to the natural climate and easy to harden (Yan et al., 2010). These have more vigor to tolerate stresses experienced during hardening stage. It has been reported that ex vitro rooted plants are better suited to tolerate environmental stresses (Baskaran and Van Staden, 2013). Ex vitro root induction was successfully proved by many researchers in Melothria maderaspatana (Baskaran et al., 2009), Ceropegia bulbosa (Phulwaria et al., 2013), Caralluma edulis (Patel et al., 2014) etc. 3.5. Hardening of the plantlets Hardening is the most important task in the acclimatization of in vitro propagated plantlets. The rooted plantlets were transferred to paper cups containing autoclaved soilrite® (Fig. 6A) and covered with the help of plastic cups in inverted position. After 4–5 weeks the micropropagated plantlets were transferred to nursery bags (Fig. 6B) containing garden soil, soilrite®, manure and vermi compost (1:1:1:1) in the greenhouse. Rooting and acclimatization could be achieved simultaneously using ex vitro rooting method (Baskaran and Van Staden, 2013). It reduced time and cost of plantlet production. Finally, hardened M. coreia plantlets were planted in the garden in natural conditions (Fig. 6C and D) where, flowering and fruit setting were observed (Fig. 6E) in the tissue culture raised plants after six months of transplanting with 100% survival rate. Tissue culture raised plants have been reported to show better growth in height, branching, and an early flowering and fruiting cycle

A

B

(El-Siddig et al., 2006; Hiwale, 2015). In the case of M. coreia treelets the flowering was started at an average height of 72.4 cm after six months of transplantation in the field. This compared with seedling raised plants which generally flowered when the seedlings had reached at the age of four to five years and average height of 4 to 5 m. Te-Chato and Lim (2004) reported more branches in tissue culture raised mangosteen trees which started blooming early with more number of flowers and fruits as compared to the seed-derived trees. Flower morphogenesis in tissue culture raised plantlets depends upon various physical and chemical factors and the intrinsic and extrinsic stimuli (Zeng et al., 2013). Early flowering and fruiting are possibly due to the presence of plant growth regulators in the medium. The medium containing growth regulators, especially cytokinin for a long time, the juvenile phase of the plant may be reduced (Te-Chato and Lim, 2004; Ziv and Naor, 2006; Ishimori et al., 2009; Jana and Shekhawat, 2011). This allows the plant to flower in a shorter time in comparison with seedlings and conventionally propagated trees. According to Taylor et al. (2005) plant growth regulators play key roles in the process of flowering by bringing about changes such as initiation of mitosis and regeneration of cells and organ formation due to accumulation of cytokinin in the cells of tissue of cultured plants. Since, the explants were collected from the mature trees; all the phenotypic characters were expressed at this stage of the plant. This may also induce in vitro flowering and fruiting in some plant species (Nizam and Te-Chato, 2012; Rathore et al., 2013a,b). The in vitro cultured plantlets expressed similar physical characters as the mother plant in the case of shape and size of leaves, flowers and fruits.

4. Conclusion The present study describes an efficient in vitro regeneration protocol for micropropagation of M. coreia. Promising plant regeneration from nodal explants was influenced markedly by combinations of BAP and Kinetin. All the in vitro regenerated shoots were rooted successfully by ex vitro rooting methods with 28.6 roots per shoot; this could reduce the time, energy, labor and cost of production of plantlets. The protocol can be used for drug research, genetic transformation, adventitious root cultures for in vitro dye production and the commercial cultivation of M. coreia.

Acknowledgments Authors are grateful to the University Grants Commission's grant No. 42-203/2013(SR), New Delhi, Government of India and Department of Science, Technology and Environment's grant No. 10/DSTE/GIA/RP/ JSA-I/2013/13, Government of Puducherry for providing financial support as major research project and grant-in-aid scheme respectively.

C

D

E

Fig. 5. Ex vitro rooting experiments; A: rooting with IBA 100 mg l−1, B: rooting with IBA 200 mg l−1, C: rooting with IBA 300 mg l−1, D: rooting with IBA 400 mg l−1, E: rooting with IAA 200 mg l−1.

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A

C

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B

D

E

Fig. 6. Hardening of plantlets; A: in vitro raised plantlets in paper cups with soilrite, B: plantlets hardened in nursery bags, C: plantlet transferred to natural habitat, D: plantlet in natural conditions (after two months), E: flowering and fruiting in in vitro raised plant within six months.

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