Indian Journal of Science RESEARCH · supplemented with 2.0 mg/l BAP and 5 g/l AC. Rooted plants were grown in the Polyhouse and acclimatized successfully with 70% survival rate

Hemant Sharma and Vashistha,In vitro plant regeneration through callus in Giloy (Tinospora cordifolia (Willd.) Miers ex Hook. f & Thoms.),Indian journal of Science, 2015, 12(34), 59-68, www.discovery.org.inhttp://www.discovery.org.in/ijs.htm © 2015 Discovery Publication. All Rights Reserved

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In vitro plant regeneration through callus in Giloy(Tinospora cordifolia (Willd.) Miers ex Hook. f & Thoms.)

Hemant Sharma1, Vashistha BD2

1. Department of Botany, Dayanand P.G. College, Hisar-125001, Haryana, India2. Department of Botany, Kurukshetra University, Kurukshetra-136119, Haryana, India

҉Corresponding author: Hemant Sharma; Department of Botany, Dayanand P.G. College, Hisar-125001, Haryana, India; Email:[email protected]

Publication HistoryReceived: 20 November 2014Accepted: 28 December 2014Published: 21 January 2015

CitationHemant Sharma, Vashistha BD. In vitro plant regeneration through callus in Giloy (Tinospora cordifolia (Willd.) Miers ex Hook. f & Thoms.).Indian Journal of Science, 2015, 12(34), 59-68

ABSTRACTA simple and efficient plant regeneration system was developed for Giloy (Tinospora cordifolia) through callus development. Leaf explants wereinoculated on MS medium and woody plant medium supplemented with different concentrations (0.5, 1.0, 2.0 and 4.0 mg/l) of growthregulators IAA, NAA; 2, 4-D, BAP and Kn. Activated charcoal (5 g/l) was also added in the medium. Leaf segments did not give any response incontrol cultures and cultures supplemented with BAP and Kn. Explants produced callus at all the four concentrations of IAA, NAA and 2, 4-D onboth the media. Maximum average fresh and dry weights were obtained from the callus at 4.0 mg/l 2,4-D on WPM. The leaf derived callus wastransferred to media with 0.5, 1.0, 2.0 and 4.0 mg/l of BAP and Kn. The maximum shoots were developed on the callus cultured on WPMsupplemented with 2.0 mg/l BAP and 5 g/l AC. Rooted plants were grown in the Polyhouse and acclimatized successfully with 70% survival rate.

Key words: Callus, Leaf explants, WPM, Giloy

AbbreviationsMS- Murashige and Skoog, WPM- Woody plant medium, AC- Activated charcoal, BAP- 6 benzylaminopurine, Kn- Kinetin, IBA- Indole-3-butyricacid, IAA- Indole-3-acetic acid, NAA- a-Naphthalene acetic acid; 2,4-D- 2,4-Dichloro phenoxy acetic acid, ANOVA- Analysis of Variance.

1. INTRODUCTIONMicropropagation technique offers a viable tool for mass multiplication of plants. The technique involves plant regeneration via direct andindirect organogenesis. There are few reports of direct organogenesis of Tinospora cordifolia from nodal explants (Kumar et al., 2003; Raghu et

Indian Journal of Science RESEARCH

International Journal for ScienceISSN 2319 – 7730 EISSN 2319 – 7749


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al., 2006; Gururaj et al., 2007). We have developed a procedure for whole plant regeneration via indirect organogenesis. This is the report ofsuccessful callus induction and plant regeneration through leaf explants of Tinospora cordifolia.

Tinospora cordifolia is a large, woody climbing shrub belongs to the family Menispermaceae. It is commonly known as Giloy. It is a well-known medicinal plant, used in Ayurvedic and other systems of medicine. The drug obtained from stem and leaves have anti-pyretic, anti-inflammatory, anti-allergic and anti-diabetic properties (Anonymus, 1992; Singh et al., 2003). It is prescribed in case of diabetes, fever and skindiseases. It is also used to improve the immune system of the body. The conventional methods of propagation of Giloy such as stem cuttingshave limited potential for large scale production. Because the plant has poor seed producing capacity. Therefore, in the present investigation, asimple and efficient plant regeneration system is developed for T. cordifolia.

2. MATERIALS & METHODS2a. Source of explantGiloy plants were collected from Kurukshetra University campus, Kurukshetra, Haryana. Leaf segments were used as explants for the in vitroculture work.

2b. SterilizationThe explants were washed in running tap water to remove all the dust particles. It was followed by washing with liquid detergent (Teepol) for10 minutes and again washed several times with tap water to remove all the traces of detergent. Then, in laminar air flow chamber surfacesterilization was carried out by treating with 0.1% (w/v) mercuric chloride solution for 2-5 minutes and subsequently washed 3-4 times withsterile double distilled water to remove all the traces of mercuric chloride. Again explants were treated with 70 % (v/v) ethyl alcohol for 1 min.The medium was sterilized by autoclaving at 121 ºC temperature and 15 psi pressure for 20 min.

2c. Culture medium and conditionsMS basal medium (Murashige and Skoog, 1962) and Woody Plant Medium (Lloyd and McCown, 1980) with 3 % (w/v) sucrose and solidified with0.8 % (w/v) agar were used. Moreover 5 g/l activated charcoal was also used in the present investigation. All the cultures were maintained at25+2ºC under a 16 hours photoperiod with 30 mol m–2 s–1 irradiance provided by cool white fluorescent tubes (40 w-Philips, India).

2d. Callus induction and growthLeaf segments were cultured on MS and WPM supplemented with different concentrations (0.5, 1.0, 2.0 and 4.0 mg/l) of growth regulatorsIAA, NAA; 2,4-D, BAP and Kn, individually. Visual observations like percent response of explant, number of days required for callus induction,colour and texture of callus were periodically recorded. The callus was collected after 60 days of their initiation and growth was measured interms of fresh and dry weights. Fresh weight of callus was taken after removing the excess of moisture on the surface using blotting paper. Dryweight was determined by drying the callus in a hot air oven at 60°C for 24 hours.

2f. Shoot regenerationCallus developed from the leaf segments on induction medium was separated and cut into small pieces and transferred to the MS and WPMsupplemented with 0.5, 1.0, 2.0 and 4.0 mg/l of BAP and Kn for shoot initiation. Observations like the percentage of callus cultures formingshoots, number of days required for shoot induction, number of shoots per culture and shoot length were recorded.

2g. Rooting of in vitro shootsAfter in vitro regenerated shoots attained a height of 2-3 cm, they were excised and planted on half strength MS medium supplemented withdifferent concentrations of IAA, IBA, NAA and 2,4-D for rooting.

2h. Hardening and acclimatization of plantlets in SoilThe rooted plantlets were gently pulled out of the medium. They were washed in running tap water to remove the medium sticking to the root.The plantlets with well-developed roots were transferred to sterilized soil and sand mixture (1:1) in small plastic pots. For initial 15 daysmaintained high humidity around the plants. So, they were covered with transparent polythene bags and made small holes in them for aircirculation. Plants were watered with ¼ MS or WP salt solution on alternate days. Then pots were transferred in Polyhouse.

2i. Statistical AnalysisThe data were collected after 60 days of inoculation of leaf explants. Each experiment was repeated three times with 10 explants pertreatment. All the data were analyzed by ANOVA followed by Duncan Multiple Range Test at P = 0.05.

3. RESULTS AND DISCUSSION3a. Callus initiation and growthCallus induction from leaf explants were reported in many plant species such as Hypericum perforatum (Pretto and Santarem, 2000), Coleusforskohlii (Reddy et al., 2001), Echinacea purpurea (Koroch et al., 2002) and Vanilla planifolia (Janarthanam and Seshadri, 2008). Further, young


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leaf explants of T. cordifolia were more responsive for callus induction than older ones. This could be attributed to high plasticity of cells atyounger age. The younger tissues are physiologically and biochemically more active as well as they have less rigid cell walls. Therefore, theyshow better callusing potential. In Vanilla planifolia, juvenile leaf explants were more responsive for callus induction than nodal explants(Janarthanam and Seshadri, 2008). Leaf segments cultured on basal MS and WPM served as control. In control cultures, no any response ofexplant was observed. This is probably due to the insufficient level of endogenous growth regulators in explants to induce callus and thereforeit required an exogenous supply. Further, calli formations from leaf explants were depended on medium type and auxin added to the medium.The results suggested that WPM was better as a basal medium for callus induction and differentiation in T. cordifolia. The suitability of WPMover MS medium might be due to its low ionic strength which counteracts salt sensitivity of woody species (Lloyd and McCown, 1980). TheWPM has been found suitable for many other species like Quercus floribunda (Purohit et al., 2002), Echinacea purpurea (Mechanda et al.,2003), Prunus avium (Bhagwat and Lane, 2004), Ziziphus jujuba (Feng et al., 2010) and Ficus religiosa (Siwach and Gill, 2011). Further, leafexplants produced callus at all the four concentrations -0.5, 1.0, 2.0 and 4.0 mg/l of IAA, NAA and 2,4-D in T. cordifolia. Among these, 2,4-D wasmost effective for the induction of callus. According to Murashige (1974); 2,4-D is a most potent auxin and it stimulates callus formation.Presence of 2,4-D has been effective in inducing callus in Paspalum scrobiculatum (Arockiaswamy et al., 2001), Calamus tenuis (Sett et al.,2002), Vernonia cinerea (Baig and Shahzad, 2003), Momordica charantia (Agarwal and Kamal, 2004) and Vanilla planifolia (Janarthanam andSeshadri, 2008). Callus formation started at 0.5 mg/l and increased with increase in concentration of auxins from 0.5 to 4 mg/l (Figure 3).Therefore, the concentration of 4 mg/l of IAA, NAA and 2,4-D was most suitable for callus induction as maximum fresh and dry weights wereobserved at this concentration (Table 1). Further, the callus was initiated near the cut ends of leaf segments. The colours and textures ofdifferent calli are summarised in Figure 1 & 2.

3b. Shoot regenerationThe leaf derived calli were divided into small pieces and cultured on WPM supplemented with 0.5, 1.0, 2.0 and 4.0 mg/l concentrations of BAPand Kn individually for shoot formation. The results are summarized in Table 2 & Figure 4. From the table, it is clear that the concentration of 2mg/l BAP as well as Kn was optimum for callus organogenesis. BAP at this level resulted in 63.3 % response with 3.3 ± 0.15 shoots per culturewhereas Kn resulted in 36.7 % response with 1.5 ± 0.07 shoots per culture. Further of the two growth regulator tried, BAP was found to bemore effective than Kn for shoot regeneration. Similar observations were made in several plant species including Capsicum annum (Singh andShukla, 2001), Geoderum purpureum (Mohapatra and Rout, 2005), Solanum melongena (Solanki et al., 2006), Curculigo orchioides (Nagesh,2008), Rubus (Wu et al., 2009), Vanilla planifolia (Tan et al., 2011) and Citrus jambhiri (Savita et al., 2011). In the present investigation in T.cordifolia, addition of 5 g/l AC in the medium prevented browning of medium and explants and increased percent shoot induction from callus(Figure 4). It is due to the fact that AC has a very fine network of pores with large surface area that gives it a unique adsorption capacity (Bakeret al., 1992). Positive effect of AC on plant regeneration has been reported by many workers (Chang et al. 2001; Wang et al. 2005 and Guo et al.2007).

3c. Rooting of in vitro shootsIn the present report, roots failed to appear in in vitro plants on MS basal medium. Roots were induced on half-strength MS mediumsupplemented with IAA, IBA, NAA and 2,4-D. Among these, IBA was most effective in rooting as it induced healthy and elongated roots (Figure5.A). The promotive effect of IBA on rooting has been reported in Pterocarpus marsupium (Chand and Singh, 2004), Bacopa monnieri(Mohapatra and Rath, 2005), Chlorophytum borivilianum (Sharma and Mohan, 2006), Datura metel (Khan et al., 2010) and Cinnamomumcamphora (Sharma and Vashistha, 2010). IBA at 0.5 mg/l concentration was optimum for rooting which resulted in to 46.7 % response with 2.1± 0.11 roots initiated in MS medium (Table 3). At higher concentration of IAA, IBA, NAA and 2,4-D; response decreased and inhibited rootingand callus formation was observed at the base of shoots.

3d. Hardening and acclimatization of plantlets in SoilIn the present investigation, in T. cordifolia, for successful acclimatization to natural conditions and normal growth a careful and gradualtransfer of in vitro regenerated plantlets was necessary. Therefore, after about a month, the plantlets with well-developed roots were shiftedto pots in Polyhouse where they grew normally with 70 % survival rate (Figure 5.B).

REFERENCES1. Agarwal M, Kamal R. In vitro clonal multiplication of Momordica charantia

L. Indian J Biotechnol, 2004, 3, 426-4302. Anonymus. The useful plants of India, CSIR (Publication and Information

Division). New Delhi, 19923. Arockiasamy S, Prakash S, Ignacimuthu S. High regenerative nature of

Paspalum scorbiculatum L., an important millet crop. Curr Sci, 2001, 80,496-498

4. Baig N, Shahzad A. In vitro vegetative multiplication of Vernonia cinereathrough shoot tip culture. Bionotes, 2003, 5, 65

5. Baker FS, Miller CE, Repik AJ, Tolles ED. Activated carbon. Kirk-OthmerEncycl Chem Technol, 1992, 4, 1015-1037

6. Bhagwat B, Lane WD. In vitro shoot regeneration from leaves of sweetcherry (Prunus avium) ‘Lapins’ and ‘Sweetheart’. Plant Cell Tiss Org Cult,2004, 78, 173-181

7. Chand S, Singh AK. In vitro shoot regeneration from cotyledonary nodeexplants of a multipurpose leguminous tree, Pterocarpus marsupiumRoxb. In Vitro Cell Dev Biol-Plant, 2004, 40, 464-466

8. Chang SH, Ho CK, Chen ZZ and Tsay JY. Micropropagation of Taxus maireifrom mature trees. Plant Cell Rep, 2001, 20, 496-502

9. Feng JC, Yu XM, Shang XL, Li JD, Wu YX. Factors influencing efficiency ofshoot regeneration in Ziziphus jujuba Mill. ‘Huizao’. Plant Cell Tiss OrgCult, 2010, 101, 111-117


Page62

10. Guo Y, Bai J, Zhang Z. Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Plant Cell Tiss OrgCult, 2007, 89,151-157

11. Gururaj HB, Giridhar P, Ravishankar GA. Micropropagation of Tinosporacordifolia (Willd.) Miers ex Hook. f. & Thoms- a multipurpose medicinalplant. Curr Sci, 2007, 92, 23-26

12. Janarthanam B, Seshadri S. Plant regeneration from leaf derived callus ofVanilla planifolia Andr. In Vitro Cell Dev Biol-Plant, 2008, 44, 84-89

13. Khan S, Tyagi P, Kachhwaha S, Kothari SL. High frequency plantregeneration and detection of genetic variation among micropropagatedplants of Datura metel L. Phytomorphology, 2010, 60, 1-8

14. Koroch A, Juliani HR, Kapteyn J, Simon JE. In vitro regeneration ofEchinacea purpurea from leaf explants. Plant Cell Tiss Org Cult, 2002, 69,79-83

15. Kumar S, Narula A, Sharma MP, Shrivastava PS. Effect of copper and zincon growth, secondary metabolite content and micropropagation ofTinospora cordifolia: a medicinal plant. Phytomorphology, 2003, 53, 79-91

16. Lloyd GB, McCown BH. Commercially feasible micropropagation ofmountain laurel, Kalmia latifolia by use of shoot-tip culture. Proc Int PlantProp Soc, 1980, 30, 421-427

17. Mechanda SM, Baum BR, Johnson DA, Arnason JT. Direct shootregeneration from leaf segments of mature plants of Echinacea purpurea(L.) Moench. In Vitro Cell Dev Biol-Plant, 2003, 39, 505-509

18. Mohapatra A, Rout GR. In vitro micropropagation of Geoderumpurpureum R. Br. Indian J Biotechnol, 2005, 4, 568-570

19. Mohapatra HP, Rath SP. In vitro studies of Bacopa monniera- animportant medicinal plant with reference to its biochemical variation.Indian J Exp Biol, 2005, 42, 373-376

20. Murashige T. Plant propagation through tissue culture. A Rev PlantPhysiol, 1974, 25, 136-166

21. Murashige T, Skoog F. A revised medium for rapid growth and bioassayswith tobacco tissue cultures. Physiol Plant, 1962, 15, 473-497

22. Nagesh KS. High frequency multiple shoot induction of Curculigoorchioides Gaertn.: shoot tip V/S rhizome disc. Taiwania, 2008, 53, 242-247

23. Pretto FR, Santarem ER. Callus formation and plant regeneration fromHypericum perforatum leaves. Plant Cell Tiss Org Cult, 2000, 62, 107-113

24. Purohit KV, Lok MS, Palani SK, Nandi, Rikhari HC. In vitro regeneration ofQuercus floribunda Lindl. through cotyledonary nodes: an important treeof Central Himalaya. Curr Sci, 2002, 83, 312-315

25. Raghu AV, Geetha SP, Martin G, Balachandran I, Ravindran PN. In vitroclonal propagation through mature nodes of Tinospora cordifolia (Willd.)Hook. f. & Thoms.: an important ayurvedic medicinal plant. In Vitro CellDev Biol-Plant, 2006, 42, 581-588

26. Reddy SP, Rodrigues R, Rajasekharan R. Shoot organogenesis and masspropagation of Coleus forskohlii from leaf derived callus. Plant Cell TissOrg Cult, 2001, 63, 183-188

27. Savita, Singh B, Virk GS, Nagpal AK. An efficient plant regenerationprotocol from callus cultures of Citrus jambhiri Lush. Physiol. Mol. Biol.Plants, 2011, 17, 161-169

28. Sett R, Kundu M, Sharma P. Regeneration of plantlets throughorganogenesis in Calamus tenuis. India J Plant Physiol, 2002, 7, 358-361

29. Sharma H, Vashistha BD. In vitro propagation of Cinnamomum camphora(L.) Nees & Eberm using shoot tip explants. Ann Biol, 2010, 26, 109-114

30. Sharma U, Mohan JSS. In vitro clonal propagation of Chlorophytumborivilianum Sant. et Fernand., a rare medicinal herb from mature floralbuds along with inflorescence axis. Indian J Exp Biol, 2006, 44, 77-82

31. Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC.Chemistry and medicinal properties of Tinospora cordifolia (Guduchi).Indian j Pharmacol, 2003, 35, 83-91

32. Singh RR, Shukla R. In vitro shoot differentiation in Capsicum annum L.Var. ascuminata. J Indian Bot Soc, 2001, 80, 301-303

33. Siwach P, Gill AR. Enhanced shoot multiplication in Ficus religiosa L. in thepresence of adenine sulphate, glutamine and phloroglucinol. Physiol MolBiol Plants, 2011, 17, 271-280

34. Solanki JJ, Powar PK, Maheshwari VL. Efficient plant regeneration inSolanum melongena L. Physiol Mol Biol Plants, 2006, 4, 307-311

35. Tan BC, Chin CF, Alderson P. Optimisation of plantlet regeneration fromleaf and nodal derived callus of Vanilla planifolia Andrews. Plant Cell TissOrg Cult, 2011, 105, 457-463

36. Wang Q, Jaana Laamanen J, Uosukainen M, Valkonen JPT.Cryopreservation of in vitro grown shoot tips of raspberry (Rubus idaeusL.) by encapsulation-vitrification and encapsulation-dehydration, PlantCell Rep. 2005, 24, 280-288

37. Wu JH, Millar SA, Hall HK, Mooney PA. Factors affecting the efficiency ofmicropropagation from lateral buds and shoot tips of Rubus. Plant CellTiss Org Cult, 2009, 99, 17-25.


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Table 1Fresh and dry weights of callus produced by leaf explants of T. cordifolia cultured on MS & WPM supplemented with IAA, NAA and 2,4-D

Medium Concentrationsof auxins

(mg/l)

IAA NAA 2,4-D

FW (g) DW (g) FW (g) DW (g) FW (g) DW (g)

MS

Control 0.0 ± 0.00h 0.0 ± 0.00f 0.0 ± 0.00i 0.0 ± 0.00f 0.0 ± 0.00g 0.0 ± 0.00g

0.5 0.45 ± 0.05g 0.08 ± 0.01e 0.84 ± 0.05h 0.15 ± 0.02e 1.37 ± 0.07f 0.24 ± 0.02f

1.0 0.70 ± 0.05f 0.14 ± 0.02d 1.36 ± 0.06f 0.22 ± 0.03d 1.86 ± 0.06e 0.33 ± 0.02e

2.0 1.08 ± 0.06d 0.20 ± 0.02c 1.80 ± 0.07d 0.30 ± 0.04c 2.12 ± 0.07d 0.38 ± 0.03de

4.0 1.32 ± 0.07b 0.26 ± 0.04b 2.01 ± 0.07c 0.35 ± 0.04bc 2.46 ± 0.08c 0.43 ± 0.04cd

WPM0.5 0.73 ± 0.05f 0.14 ± 0.01d 1.12 ± 0.06g 0.22 ± 0.02d 2.07 ± 0.07d 0.40 ± 0.03d

1.0 0.91 ± 0.05e 0.18 ± 0.02c 1.68 ± 0.06e 0.34 ± 0.03c 2.51 ± 0.08c 0.48 ± 0.03c

2.0 1.18 ± 0.06c 0.24 ± 0.02b 2.14 ± 0.07b 0.40 ± 0.03b 2.93 ± 0.07b 0.56 ± 0.04b

4.0 1.70 ± 0.08a 0.32 ± 0.03a 2.86 ± 0.08a 0.50 ± 0.04a 3.75 ± 0.10a 0.70 ± 0.05a

Values are means ± S.E. of three independent experiments, each consisted of 10 replicates per treatment. Data from 60 days old culture. Means followed by the same letter within columns are not significantly different at P = 0.05 according to Duncan’s Multiple Range Test. FW-Fresh weight, DW-Dry weight.

Table 2Effect of different concentrations of BAP and Kn on shoot formation on calli induced from leaf explants of T. cordifolia cultured on WPM

GrowthRegulators

Concentrations(mg/l)

% response

Time takenfor shootformation

(Days)

Average number ofshoots per culture

Average shoot length(cm)

0.0 - - 0.0 ± 0.00d 0.0 ± 0.00c

BAP 0.5 - - 0.0 ± 0.00d 0.0 ± 0.00c

1.0 53.3 26 2.5 ± 0.12b 0.8 ± 0.03b

2.0 63.3 22 3.3 ± 0.15a 1.3 ± 0.05a

4.0 - - 0.0 ± 0.00d 0.0 ± 0.00c


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Values are means ± S.E. of three independent experiments, each consisted of 10 replicates per treatment. Data from 60 days old culture. Means followed by the same letter within columns are not significantly different at P = 0.05 according to Duncan’s Multiple Range Test. (-) No response

Table 3Effect of different concentrations of growth regulators on rooting of in vitro regenerated shoots of T. cordifolia cultured on MS medium

Values are means ± S.E. of three independent experiments, each consisted of 10 replicates per treatment. Means followed by the same letter within columns are not significantly different at P = 0.05 according to Duncan’s Multiple Range Test. (-)

No response.

Kn 0.5 - - 0.0 ± 0.00d 0.0 ± 0.00c

1.0 - - 0.0 ± 0.00d 0.0 ± 0.00c

2.0 36.7 30 1.5 ± 0.07c 1.3 ± 0.05a

4.0 - - 0.0 ± 0.00d 0.0 ± 0.00c

Growthregulator

Concentrations(mg/l)

% rootinduction

Time taken for rootinduction (Days)

Average number ofroots

Average rootlength (cm)

Control 0.0 - - 0.0 ± 0.00d 0.0 ± 0.00e

IAA 2.0 23.3 35 1.0 ± 0.05c 1.2 ± 0.05c

IBA 0.5 46.7 25 2.1 ± 0.11a 2.3 ± 0.12a

2.0 36.7 30 1.3 ± 0.06b 1.5 ± 0.07b

NAA 0.5 20.0 35 1.0 ± 0.00c 0.6 ± 0.05d

2,4-D 0.5 20.0 35 1.0 ± 0.00c 0.5 ± 0.03d


Page65Figure 1Effect of different concentrations of growth regulators on leaf explants of T. cordifolia cultured on MS medium- (A) Percent callus induction (B)Time taken for callus induction


Page66Figure 2Effect of different concentrations of growth regulators on leaf explants of T. cordifolia cultured on WPM- (A) Percent callus induction (B) Timetaken for callus induction


Page67Figure 3Effect of different growth regulators on leaf explants of T. cordifolia: (A) Callus initiation on WPM + 2 mg/l IAA; (B) Callus on WPM + 4 mg/l IAA;(C) Callus initiation on WPM + 2 mg/l NAA; (D) Callus on WPM + 4 mg/l NAA; (E) Callus initiation on WPM + 2 mg/l 2,4-D; (F) Callus on WPM + 4mg/l 2,4-D.


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Figure 4(A) Callus from leaf segments on WPM + 4 mg/l 2,4-D + 5 mg/l AC; (B)-(C) Shoot differentiation from leaf derived callus of T. cordifolia on: (B)WPM + 2 mg/l KIN + 5 g/l AC, (C) WPM + 2 mg/l BAP + 5 g/l AC

Figure 5(A) Rooting of in vitro regenerated shoot of T. cordifolia on MS + 0.5 mg/l IBA; (B) In vitro regenerated plant in plastic container

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Indian Journal of Science RESEARCH · supplemented with 2.0 mg/l BAP and 5 g/l AC. Rooted plants were grown in the Polyhouse and acclimatized successfully with 70% survival rate