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In Vitro Optimization of Phytohormoneson Micropropagation in Butterfly Pea(Clitoria ternatea L.)Seemab Mukhtar a , Mohammad Anis a b & Naseem Ahmad aa Plant Biotechnology Laboratory, Department of Botany , AligarthMuslim University , Aligarh, Indiab Department of Plant Production, College of Food and Agriculture ,King Saud University , Riyadh, Saudi ArabiaPublished online: 16 Sep 2010.

To cite this article: Seemab Mukhtar , Mohammad Anis & Naseem Ahmad (2010) In Vitro Optimizationof Phytohormones on Micropropagation in Butterfly Pea (Clitoria ternatea L.), Journal of Herbs, Spices& Medicinal Plants, 16:2, 98-105, DOI: 10.1080/10496475.2010.499310

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Journal of Herbs, Spices & Medicinal Plants, 16:98–105, 2010Copyright © Taylor & Francis Group, LLC ISSN: 1049-6475 print/1540-3580 onlineDOI: 10.1080/10496475.2010.499310

WHSM1049-64751540-3580Journal of Herbs, Spices & Medicinal Plants, Vol. 16, No. 2, Jul 2010: pp. 0–0Journal of Herbs, Spices & Medicinal Plants

In Vitro Optimization of Phytohormones on Micropropagation in Butterfly Pea

(Clitoria ternatea L.)

In Vitro Optimization of PhytohormonesS. Mukhtar et al.

SEEMAB MUKHTAR,1 MOHAMMAD ANIS,1,2 and NASEEM AHMAD1

1Plant Biotechnology Laboratory, Department of Botany, Aligarth Muslim University, Aligarh, India

2Department of Plant Production, College of Food and Agriculture, King Saud University, Riyadh, Saudi Arabia

An efficient protocol has been developed for plant regenerationfrom cotyledonary nodes of an in vitro grown Clitoria ternateaseedling. Proliferation of shoots was achieved on MS medium sup-plemented with various concentrations of 6-benzyladenine (BA),kinetin (Kin) and 2-isopentenyl adenine (2-iP) either singly or incombination with a-naphthalene acetic acid (NAA). Of all thecytokinins, BA, Kin, and 2-iP tested on the MS medium, BA(2.5 mM) was optimum for inducing the maximum number ofshoots (9.8 ± 0.86). The highest efficacy for shoot proliferation(13.2 ± 0.31) was achieved in a combination of 2.5 mM BA and1.0 mM NAA. The best condition for rooting was observed on half-strength MS medium augmented with 1.5 mM indole-3-butyricacid. After rooting, plantlets transferred to plastic cups filled withsterile soilrite media exhibited normal development, with morethan 90% growing well even after transfer to field conditions.

KEYWORDS legume, regeneration, acclimatization

Received 25 March 2009.The authors appreciate the assistance provided by the Department of Science and

Technology, Government of India, under the DST-FIST Program (2005) to the Department ofBotany, Aligarh Muslim University.

Address correspondence to M. Anis, Plant Biotechnology Laboratory, Department of Bot-any, Aligarh Muslim University, Aligarh 202 002, India. E-mail: anism1@rediffmail.com

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In Vitro Optimization of Phytohormones 99

INTRODUCTION

Clitoria ternatea L., commonly known as the butterfly pea, is an importantmedicinal legume observed in hedges and thickets throughout India (4).This tropical, twining herb that grows wild is valued as both a livestock for-age and as a medicinal plant. The roots and leaves, diuretic, emetic, andantiperiodic, are used in treating a number of ailments, including bodyaches, infections, and urinogenital disorders. The seeds have laxative andcathartic activity but are considered safe for colic, dropsy, and enlargementof the abdominal viscera. An extract of the plant possess anxiolytic, antide-pressant, anticonvulsant, and antistress activities (9).

In nature, propagation via seeds poses problems owing to poor germinationand death of young seedlings (4) and a high degree of heterogeneity (1). Owingto large-scale and unrestricted exploitation of this natural resource, coupled withlimited cultivation and insufficient attempts for its replenishment, the wild stock ofthis species has been markedly depleted. Further, increasing demand for rawmaterials from pharmaceutical industries and loss of their habitat will eventuallyincrease the demand for this medicinal species, leading to depletion of popula-tions and the probable adulteration of plant material in the medicinal plant trade,endangering human health or undermining product efficacy.

Therefore, development of a rapid clonal multiplication system forthis medicinally important legume has become imperative to reduce lossof the plant under natural conditions and to ensure a continuous supplyof plant material for pharmaceutical applications. A number of medici-nally important plant species have been propagated successfully usingmicropropagation techniques (2,3,7,16,19), and in vitro plant regenerationof C. ternatea has been reported from root and hypocotyl explants (12)and leaf (13), node (16), and root segments (18) but with only limitedsuccess. The objective of this study was to develop an efficient methodfor rapid in vitro propagation of C. ternatea using cotyledonary nodeexplants that could be used for successful restoration of C. ternatea plantsfor cultivation under field conditions.

MATERIAL AND METHODS

Plant Material

The fresh mature seeds of C. ternatea collected from a plant growing at thebotanical garden of the Aligarh University were washed thoroughly underrunning tap water for 30 min followed by soaking in 5% detergent(Labolene, Qualigens, India) solution for 15 min. After thorough washing,the seeds were surface-sterilized with 0.1% (w/v) mercuric chloride solutionfor 5 min, followed by repeated washing with sterile distilled water. Thesterilized seeds were randomly seeded on sterilized MS basal medium for

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100 S. Mukhtar et al.

germination. At 7 days after seeding, cotyledonary nodes were excised fromthe aseptic grown seedlings and used as explants.

The nutrient medium consisted of MS salts with 3% (w/v) sucrose supple-mented with varying concentrations of different cytokinins [6-benzyladenine(BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP)]. The medium wasgelled with 0.8% agar, adjusted to pH 5.8 using 1N NaOH before autoclavingat 121°C at 1.06 kg cm−2 for 15 min.

Experimental

All the cultures were maintained in a growth room at 24 ± 2°C and 60% to65% relative humidity under a 16-h photoperiod with a photosynthetic pho-ton flux density of 50 μmol m−2 s−1 provided by cool white fluorescentlamps (Philips, India). For shoot bud induction, the cotyledonary nodeexplants were placed on MS medium supplemented with BA, Kin, and 2-iPat concentrations of 0.5 and 7.5 μM either singly or in combination withNAA at concentrations of 0.5, 1.0, and 2.0 μM. The frequency of explantproducing shoots and number of shoots per explant were recorded after 8weeks. MS medium lacking plant growth regulators was used as a control.All the cultures were transferred to fresh medium every 2 weeks.

Individual microshoots (3 to 4 cm long) with two to three pairs of leaveswere excised aseptically and transferred to half-strength MS basal medium sup-plemented with IBA at different concentrations (0.5 and 2.0 μM) for rooting.After 4 weeks, the percentage rooting, the mean number of roots per shoot,and mean root length were recorded, and those plantlets with well-developedroots and shoots were removed from culture medium, washed gently under tapwater to remove remnants of culture medium, and transferred to plastic cupscontaining soilrite (a mixture of horticulture grade expanded perlite, Irish peatmoss, and exfoliated vermiculite in equal amounts; Keltech Energies, Ltd., Ban-galore, India). The plastic pots containing the seedlings were covered withtransparent glass jars to ensure high humidity and placed under diffuse light for16 h each day in a controlled environment room at 24 ± 2°C and watered witha 25% MS basal solution devoid of organic supplements on alternate days.

After 4 weeks’ growth, the glass jars were removed to acclimatize theplants in preparations for transfer to field conditions. After 4 weeks of acclima-tion, the plants were transferred to earthen clay pots containing local gardensoil and maintained in a greenhouse under normal day-length conditions.

Statistical Analysis

A total of 10 explants were used in each tested treatment, and all experi-ments were repeated three times. The results were subjected to a one-wayanalysis of variance, and pair-wise means were compared using Duncan’sMultiple Range Test (p ≤ 0.05).

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In Vitro Optimization of Phytohormones 101

RESULTS AND DISCUSSION

MS medium lacking growth regulators failed to induce shoot bud prolifera-tion (Table 1). In contrast, all tested concentrations of BA, Kin, and 2-iPfacilitated shoot bud differentiation, with BA being more efficient than Kinand 2-iP for initiation and proliferation of shoots. BA treatment at 2.5 μMproduced the highest shoot regeneration frequency (85.2%), number ofshoots (9.8 ± 0.86), and shoot length (11.0 ± 0.45 cm; Figure 1A). Theseresults support the stimulating effect of BA on multiple shoot formationreported earlier for several medicinal and aromatic plant species(6,7,11,16,20). The regeneration frequencies and number of shootsdeclined, with an increase in the concentration of each cytokinin beyondthe optimal level (2.5 μM; see Figure 1B). A reduction in the number ofshoots at a concentration higher than optimal level has also been reportedfor other medicinal plants (7,10,17). Kin was effective at 2.5 μM, but theeffect was weaker than BA supplemented medium.

For multiple shoot regeneration, the addition of NAA to MS media con-taining BA was more effective than media containing only BA (Table 2).Among the tested concentrations of BA and NAA, the highest shoot regener-ation frequency (88%), number of shoots per explant (13.2 ± 0.31), and

TABLE 1 Cytokinins and Shoot Regeneration from C. ternatea in MS Medium

Growth regulator(μM)

Regeneration(%)1

Shoot formation(No. explant−1)1

Shoot length(cm)1

0.0 00.0 ± 0.00h 0.0 ± 0.00h 0.0 ± 0.00i

BA0.5 41.2 ± 5.39ef 5.6 ± 1.63bcdef 5.7 ± 1.24cde

1.0 54.2 ± 4.50d 6.6 ± 1.32bcde 7.8 ± 1.26bc

2.5 85.2 ± 1.58a 9.8 ± 0.86a 11.0 ± 0.45a

5.0 75.0 ± 2.22b 8.2 ± 1.06ab 8.1 ± 1.01b

7.5 64.8 ± 3.01c 7.6 ± 1.12abc 7.3 ± 0.85bcd

Kn0.5 34.6 ± 2.83fg 3.8 ± 1.24efg 4.0 ± 0.14efgh

1.0 49.0 ± 4.30e 6.4 ± 0.92bcde 5.3 ± 1.20def

2.5 78.2 ± 1.83b 8.6 ± 0.40ab 7.9 ± 0.57b

5.0 69.8 ± 2.56c 7.0 ± 0.54abcd 5.7 ± 0.48cde

7.5 63.2 ± 3.43cd 5.0 ± 0.54cdef 4.6 ± 0.26efgh

2-iP0.5 29.0 ± 0.70g 1.8 ± 0.37g 2.3 ± 0.35h

1.0 43.6 ± 2.54ef 4.2 ± 1.15defg 3.0 ± 0.34gh

2.5 58.4 ± 3.86cd 6.4 ± 0.24bcde 5.6 ± 0.22cde

5.0 66.6 ± 2.94c 3.8 ± 0.58efg 3.7 ± 0.24efgh

7.5 55.2 ± 3.92c 2.8 ± 0.37fg 3.1 ± 0.24fgh

BA = benzyladenine; Kn = kinetin; 2-iP = 2-isopentenyl adenine. 1After 4 weeks’ culture, means ± SE,means followed by the same letter within columns are not significantly different (p ≤ 0.05) usingDuncan’s multiple range test.

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102 S. Mukhtar et al.

maximum shoot length (12.5 ± 0.15 cm) were recorded on MS medium sup-plemented with 2.5 μM BA and 1.0 μM NAA (see Figure 1B ). The positivemodification of shoot induction efficacy by an auxin in combination with acytokinin are in accordance with the findings of Faisal et al. (7), Martin (14),and Faisal et al. (5) in Ruta graveolens, wherein the combination of BA andNAA was most effective for shoot regeneration.

FIGURE 1 Development C. ternatea plantlets. A. Induction of shoots from cotyledonarynodes on MS basal media + 2.5 μM BA. B. Multiplication and proliferation of shoots on basalMS media + 2.5 μM BA +1.0 μM NAA. C. Rooted plantlet on 50% basal MS media + 1.0 μMIBA. D. Acclimatized plantlet of C. ternatea in greenhouse.

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In Vitro Optimization of Phytohormones 103

Superior root development occurred on growth regulator–free, half-strength MS media as compared with root development on full-strength MSmedia (see Figure 1C). Rhizogenesis was facilitated by the presence of alow IBA concentration in half-strength MS medium (Table 3). The maximumfrequency of root formation (88.4%), number of roots per shoot (5.2 ± 0.37),and root length (7.9 ± 0.59) was achieved in half-strength MS medium con-taining 1.5 μM IBA (see Figure 1C). Optimum rooting response using IBAhas been reported for several plants, including Mucuna pruriens (7),Holostemma ada-kodien (14), and Eclipta alba (8).

Plantlets with fully expanded leaflets and well-developed roots weresuccessfully transferred to soilrite and hardened in a growth room for

TABLE 2 Optimal Concentration of Cytokinins with NAA on Shoot Proliferation

Growth regulators(μM)

Regeneration (%)1

Shoot formation (No. explant−1)1

Shoot length (cm)1

0.0 0.0 00.0 ± 0.00f 0.0 ± 0.00h 0.0 ± 0.00i

BA NAA2.5 0.5 80.6 ± 1.20b 8.6 ± 0.24c 8.0 ± 0.15c

1.0 88.0 ± 2.23a 13.2 ± 0.31a 12.5 ± 0.15a

2.0 82.0 ± 0.89bc 10.8 ± 0.37b 9.2 ± 0.08b

Kn NAA2.5 0.5 78.2 ± 0.66cd 6.2 ± 0.37ef 6.2 ± 0.07e

1.0 82.8 ± 1.39bc 9.4 ± 0.24b 8.0 ± 0.07c

2.0 79.2 ± 0.50c 7.2 ± 0.37de 6.6 ± 0.20d

2-iP NAA2.5 0.5 75.4 ± 0.50de 4.6 ± 0.24g 5.7 ± 0.05f

1.0 78.6 ± 0.50cd 8.2 ± 0.37cd 4.9 ± 0.23c

2.0 75.0 ± 0.70de 5.6 ± 0.50fg 4.3 ± 0.05g

BA = benzyladenine; Kn = kinetin; 2-iP = 2-isopentenyl adenine; NAA = naphthalene acetic acid.1After 8 weeks culture, means ± SE, means followed by the same letter within columns are not significantlydifferent (p ≤ 0.05) using Duncan’s multiple range test.

TABLE 3 Effect of MS Strength and Auxin Concentration on Root Induction of C. ternateaMicroshoots

MS medium(% strength)

IBAμM)

Rooting(%)1

Roots(No. shoot−1)1

Root length(cm)1

100 0.0 37.6 ± 1.7f 2.4 ± 0.2efg 2.9 ± 0.3defg

50 0.0 54.8 ± 2.2e 4.2 ± 0.4abc 5.7 ± 0.2bc

50 0.5 60.2 ± 4.0e 2.2 ± 0.4de 4.6 ± 0.9b

50 1.0 88.4 ± 1.2a 5.2 ± 0.4a 7.9 ± 0.6a

50 1.5 81.2 ± 1.9abc 4.2 ± 0.4ab 7.0 ± 0.4a

50 2.0 70.6 ± 3.7d 2.0 ± 0.4de 3.0 ± 0.9bc

MS = Murashige and Skoog basal medium (15); IBA = indole-3-butyric acid.1After 4 weeks’ culture, means ± SE, means followed by the same letter within columns are not signifi-cantly different (p ≤ 0.05) using Duncan’s multiple range test.

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104 S. Mukhtar et al.

4 weeks, and the micropropagated plants could be successfully planted inearthen pots containing garden soil and maintained in a greenhouse (seeFigure 1D). About 90% of the plants transferred to garden soil survived andshowed no detectable variation in morphology or growth characteristics ascompared with their respective donor plants.

In conclusion, the present study describes an efficient method for invitro regeneration of C. ternatea, a valuable medicinal legume. The protocoldescribed was successfully used for mass propagation and could be helpfulfor the improvement of the medicinal content of this pharmaceuticallyimportant plant species.

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