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European Journal of Medicinal Plants 4(11): 1356-1366, 2014

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Development of Simple, Cost Effective Protocol for Micropropagation of Tylophora indica (Burm f.) Merill., an Important Medicinal Plant Pooja Patel1 and Rajani Nadgauda1* 1

Department of Plant Cell and Molecular Biology, IIAR, Puri Foundation, Gandhinagar, Gujarat, India. Authors’ contributions

This work was carried out in collaboration between all authors. Author RN designed the study and analysed the results. Author PP performed the experiments, noted the results and wrote the first draft of the manuscript. All authors read and approve the final manuscript.

th

Original Research Article

Received 9 April 2014 th Accepted 29 May 2014 th Published 27 July 2014

ABSTRACT Aims: The present studies were initiated to develop a cost effective protocol for micropropagation, as a mean for conservation of medicinal plant- Tylophora indica (Burm f.) Merill. The plant is threatened and needs immediate conservation, therefore, the study was undertaken with following objective:  In vitro multiplication of Tylophora indica using nodal axillary bud proliferation and through organogenesis of callus. Study Design: For all experiments ten replicates were used per treatment and all the experiments were repeated three times. Data have been presented as Mean ± Standard deviation. Place and Duration of Study: Department of Plant Cell and Molecular Biology, Indian Institute of Advanced Research (IIAR), Gandhinagar, Gujarat, India. Methodology: For in vitro plant regeneration, micropropagation and organogenesis techniques were used. For micropropagation, surface sterilized nodal explants were inoculated on different shoot inducing media and further multiplication was obtained. Root containing shoots were transferred to pot containing pre autoclaved mixture of soil and soilrite. For organogenesis, surface sterilized leaf explants were inoculated on different types of callus inducing media. ____________________________________________________________________________________________ *Corresponding author: Email: [email protected];

European Journal of Medicinal Plants, 4(11): 1356-1366, 2014

Results: All the nodal explants were sprouted at a very high frequency, i.e. 98% and sprouted buds elongated up to 8cm on three different media. Dissected explants grew further and average height of shoots reached 9.5cm±0.80cm within 30 days. Interestingly, -1 root formation was observed on the same media; so that the best media was 0.4mg L -1 BA and 0.1mg L Kn for both initiation as well as for multiplication. For organogenesis, the -1 -1 fragile callus was observed on media containing 2mg L 2,4-D and 0.1mg L Kn. Green pigmented calli were transferred to MS media, where it regenerated in to shoots and roots, simultaneously Conclusion: The protocol of micropropagation through axillary bud proliferation described here is very simple, repetitive and cost effective, which can be easily utilized for commercial cultivation. On shoot multiplication media, root formation observed, thereby making the process is one step; which is very easy to follow. Keywords: Asclepiadaceae; micropropagation; Tylophora indica; 6-Benzyl amino purine; 2,4dichlorophynoxy acetic acid; kinetin.

1. INTRODUCTION Tylophora indica (Burm f.) Merill. (Family: Asclepiadaceae) is a perennial climber, native to South and East India. The plant is medicinally important source as several alkaloids including tylophorine, tylophorinine and anti cancerous tylophorinidine. T. indica contains 0.2-0.3 % of alkaloids having phenanthroindalizidine and furoquinoline framework [1,2]. Tylophora has become a more popular treatment for asthma, inflammation [3,4], bronchitis, allergy and dermatitis [5,6]. Tylophora also seems to be a good remedy in traditional medicine as anti-psoriasis. The amount of active compound found is high in root; therefore the roots are used as a drug in Ayurveda. Due to overexploitation for commercial purposes, T. indica has become a threatened plant species. Conventional propagation is difficult in Tylophora due to low seed viability and germination rate [7]. The progeny raised through the seeds shows remarkable genetic variation which is not suitable for commercial cultivation. Stem cuttings failed to produce proper root when treated with different growth regulators [8]. Moreover, in vitro plant regeneration through axillary bud proliferation is an easy and economic way of obtaining a large number of homogeneous plants within a short span of time. Sharma and chandel, reported the micropropagation of T. indica via axillary shoot proliferation [9]. After that Faisal et al. reported callus mediated shoot organogenesis as an alternative method to achieve a higher rate of shoot multiplication [10]. Kalimuthu and Jeyaraman reported, in vitro multiple shoot production from leaf explants [11]. This paper describes the micropropagation protocol through axillary bud proliferation and through organogenesis from callus cultures.

2. MATERIALS AND METHODS 2.1 Plant Materials and Explant Sterilization for Shoot Regeneration and Callus Induction Nodal axillary buds and young, fresh leaves were collected from the medicinal garden of the IIAR, Gandhinagar, Gujarat, India. Explants were washed under running tap water for 30

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minutes to remove contaminants, followed by treatment with 1.0% detergent, teepol (v/v, Himedia) for 10 minutes. After through washing, under the laminar air flow unit, the explants were surface sterilized with 0.1% mercuric chloride solution (w/v; Merck) for 2-3 minutes and rinsed 4-5 times with double distilled water before inoculation. The explants were inoculated on sterile Murashige and Skoog’s media (MS) [12] with various combinations of Plant Growth Regulators (PGRs).

2.2 Media Preparation and Growth Conditions Seven different media, listed in (Tables 1 and 2) were prepared. These media consisted of MS, 2% sucrose supplemented with various combinations and concentrations of PGRs. The pH of all the media was adjusted to 5.8±0.02 and autoclaved at 121ºC for 15 minutes under 15psi. Cultures were incubated under 8/16h light/ dark, at 25±2ºC.

2.3 In vitro Plant Regeneration through Axillary Bud Proliferation 2.3.1 Initiation of culture The surface sterilized nodal explants (3cm) were inoculated on different shoot induction media (SM) (Table 1). The frequency with which explants produced shoots, the number of sprouts per explant and the shoot length were recorded after 5 weeks of culture. 2.3.2 Shoot proliferation All the sprouted buds were dissected. Sprouted buds were removed from their original tissue and inoculated on SM A, and E media. 2.3.3 Acclimatization of the plants Shoot formation was associated with simultaneous root formation in same media. These rooted plants were removed from culture, rinsed with water the roots were dipped in 2% bavistin solution for 5 seconds and then transferred to pots containing pre autoclaved mixture of soil and soilrite. The potted plants were covered with transparent polythene to ensure high humidity and watered every after 3 days interval for 2 weeks. Polythene was removed after 2 weeks in order to acclimatize plants to field conditions.

2.4 Organogenesis 2.4.1 Initiation of callus Callus was initiated from leaf explants (1cm). These surface sterilized leaf explants were separately inoculated on another set of Callus inducing Media (CM) with different auxins (Table 2). MS media devoid of growth regulators served as control. The cultures were incubated in dark condition for 7 days. Data was recorded as the percent of the leaf explants giving rise to callus induction. 2.4.2 Proliferation of callus For callus proliferation CM B, C and D media were used. The cultures were incubated under 8/16h light/ dark, at 25±2ºC

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2.4.3 Regeneration Regeneration of callus was done on CM C media and CM C media without containing 2,4-D. Cultures were incubated under 8/16h light/ dark, at 25±2ºC. In all experiments ten replicates were used per treatment and all the experiments were repeated three times.

3. RESULTS 3.1 Axillary Bud Proliferation and Plant Formation As recorded in (Table 1), which shows data on initiation all the explants were sprouted with a very high frequency reaching up to 98%. These sprouted buds elongated up to more than 8 cm on SM A, SM B media. These sprouted shoots were dissected and inoculated on the same media and incubated further under light condition. It was observed that dissected explants grew further, reaching to the average height of 9.5cm±0.80cm within 30 days. Interestingly root formation was observed on the same media. The multiplication process was done through the intermodal explants of in vitro grown shoots (Fig. 1). The best media -1 -1 was 0.4mg L BA and 0.1mg L Kn for both initiation as well as for multiplication. The multiplication process was done through the internodal explants of in vitro grown shoots. Thus from the one sprouted shoots 5-6 internodes were produced which in turn gave rise to a complete plant. Therefore the ratio of multiplication was 1:5. The best media was found to -1 -1 be the one containing 0.4mg L BA and 0.1mg L Kn for both initiation as well as for multiplication. Table 1. Effect of different growth regulators on initiation of axillary bud proliferation from nodal explant Media

Control SM A SM B SM C SM D SM E SM F

Plant growth regulators -1 (mg L ) BA Kn Adenosine hemisulphate 0.4 0.1 0.4 0.02 2.0 0.1 2.0 0.02 4.0 0.1 4.0 0.02

Sprouting (%)

90.00±2.0 98.01±1.2 92.10±1.5 92.00±2.0 93.00±1.5 90.02±1.0 89.00±2.0

Average number of sprouts per explant 1.2±0.1 2.60±0.2 2.0±0.05 2.2±0.3 2.5±0.1 2.2±0.25 2.0±0.1

Average length of shoot per explants (cm) 1.5±0.50 9.5±0.80 9.9±0.70 6.8±0.07 7.0±0.20 6.5±0.20 6.1±0.40

Media: Murashige and Skoog’s. SM: Shoot induction Media. BA- 6-Benzyl amino purine, Kn=Kinetin -2 -1 and Adenosine hemisulphate. Incubated under (80 μMm S ) light condition. Data were presented as Mean±Standard deviation, n=3

3.2 Field Transfer and Acclimatization Rooted shoots were transferred to pots, containing soil and soilrite (1:1). Transferred plantlets showed normal phenotypic appearance. During hardening, shoots elongated and leaves turned green.

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3.3 Organogenesis 3.3.1 Callus induction The leaf explants swelled in the media and initiated callus in dark condition after 7 days of incubation. The callus was initiated from cut edges, midrib and veins portion at the abaxial side of the leaf explant. Frequency of callus formation varied with the concentration of and combination of growth regulators (Table 2). The callus obtained on CM a media gave rise to compact callus with profuse root formation (Fig. 2). Fragile calli were obtained on CM C media (Table 2). Therefore callus obtained on CM C media was used for further experiments. Table 2. Effect of different growth regulators on response of callus Media Control CM A CM B CM C CM D CM E CM F

-1

Plant growth regulators (mg L ) NAA 2,4-D Kn 2.0 2.0 2.0 0.1 3.0 3.0 4.0 -

% of explant forming callus 0.00±0.0 60.00±1.2 70.01±0.2 95.00±1.1 70 .00±0.0 50.00±0.6 55.00±0.3

Days taken for growth of callus 0 30 24-26 25 25 25 25

Media: Murasige and skoog. NAA=α- Naphthalene acetic acid, 2,4-D=2,4-Dichlorophynoxy acetic acid, Data were presented as Mean±Standard deviation, n=3

3.3.2 Callus regeneration The callus proliferated on CM C media in dark condition within 15 days. On subculture the callus showed green pigmentation under light condition within 7 days. Green pigmented calli -1 (Fig. 2). And callus obtained on NAA containing media were transferred to MS+2mgL BA media and MS without any growth hormones; response obtained on MS media was 85±0.50 percent calli. Average number of shoots and roots per calli was 2.0±0.05 and 1.5±0.02 respectively. However the callus obtained on NAA did not show any shoot formation and continue to grow as roots

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-1

-1

Fig. 1. (A) Sprouting on MS+0.4mg L BA+0.1mg L Kn within 7 days. (B) Showing shoot multiplication with well developed root. (C) Acclimatized plantlets 1361

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Fig. 2. Initiation of callus from cut edge of the abaxial surface of the explant on -1 -1 MS+2mg L 2,4-D+0.1mg L Kn within 7 days of incubation. (B) Growth of callus on same medium after subculturing showing green pigmentation. (C) Callus with profuse root on NAA containing media. (D) Shoot and root formation observed on MS media

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4. DISCUSSION 4.1 Axillary Bud Proliferation Plantlet Formation Shoot regeneration from nodal explants was obtained. For shoot proliferation, cytokinins are one of the most important factors affecting the response as reported in Spire abumalda, garlic, Pissardii plum [13-16]. Induction of shoot was observed on BMS medium -1 -1 supplemented with BA (5 mg L ) and adenine hemisulphate (0.5mg L ) from mature leaf -1 explants of T. indica [17]. BAP (2mg L ) supplemented media was used for shoot regeneration, wherein total number of shoots were 35-40 formed per explant. Faisal et al. has reported BA as more efficient growth regulator than others with respect to initiation and subsequent proliferation of shoots [18]. They reported the highest number of shoots (8.6±0.71) with the maximum average shoot length of (5.2±0.31cm) on MS medium -1 supplemented with 2.5μM BA, 0.5μM NAA and 100mg L AA [18]. Kalimuthi reported, shoot -1 regeneration through leaf on MS media containing 2.5mg L and the highest number of shoots was 7 per explant [11]. Both the above reports used half-strength MS-medium -1 supplemented with 0.5mg L IBA for root formation [10,11,18]. Faisal et al. in 2003 reported, the shoot formation through intervention of callus on MS media containing 10μM 2,4,5-T and 5μM Kn [10]. In the present study, the axillary shoot formation took place on MS media -1 -1 containing 0.4mg L BA and 0.1mg L Kn with the highest number of shoots in the initial stage reaching up to (2.60±0.2) with the height (9.5±0.80cm). Further multiplication was obtained through internodal sectioning, thus, achieving multiplication ratio was 1:5. The root formation took place simultaneously during multiplication stage. Therefore, there was no need to transfer microshoots to different rooting media. All the rooted shoots transferred to field with 97% surviving rate after 3 months. -1

-1

The media containing 0.4mg L BA and 0.1mg L Kn was found to be the best media for all the systems viz. initiation, multiplication and root formation. Thus the process is one step, with utilization of minimal media. The plants were successfully transferred to pots for hardening and acclimatization. About 96% of the total plants survived after 8-10 weeks of their transfer to the soil. Starting from 10 sprouting buds, it is possible to achieve more than 5000 plants within 6-7 months with 98% survival rate (Flow chart). In situation to conserve an important medicinal plant such kind of fool proof, good responsive and cost effective protocol is required. In present study root and shoot formation was observed on the same media.

4.2 Organogenesis The induction of callus was mainly influenced by the auxin. In present study callus was raised in callus inducing media (CM A-F, Table 2). Maximum callus induction was observed on CM C. On NAA containing media compact and root containing and compact callus was -1 observed. Rao et al. reported the compact callus on MS media containing 5.5mg L NAA -1 -1 and 0.1mg L Kn [19]. Induction of callus from leaf explants was obtained on 2,4-D (2mgL ) -1 and Kn (1mg L ) in Tylophora indica. The capacity of 2,4-D to induce embryogenic callus has been reported previously in T. indica and in several other crops [20-23]. Harmanjit et al. reported, different types of calli produced on BA and TDZ supplemented Murashige and Skoog (MS) basal medium were selected for shoot induction and somatic embryogenesis studies [24]. Thomas et al. reported, the callus formulation from immature leaf pieces on MS -1 -1 media supplemented with 1.6mg L 2,4-D and 0.34mg L BA [7]. Faisal et al. reported

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adventitious shoots formation regenerated (85%) from the surface of the callus on MS -1 medium supplemented with 1mg L Kinetin [10]. Beena et al. reported differentiation of -1 shoots on MS media supplemented with BA (2mg L ) within 4-5 shoots [25]. Flow chart Number of plants within 6 months

In present study, 95.00±1.1 percent explants produced callus on MS media containing 2mg -1 -1 -1 -1 L BA and 0.1mg L Kn. The callus on 2mg L 2,4-D and 0.1mg L Kn containing media started showing green pigmentation within 7 days. After 10 days the green calli was transferred to MS medium without any hormones. The organogenesis protocol described here shows the regeneration obtained on MS media devoid of any hormone with 85±0.50%, the organogenesis steps occur on minimal media. Induction of both shoots and roots was observed within a week. In present study plants regenerated thorough organogenesis were acclimatized.

5. CONCLUSION Tylophora indica is a medicinally important plant. Due to the demand of tylophorine as an active compound of several medicines in ayurveda, the plant is extensively exploited therefore, in order to conserve plant, several strategies are being used and the protocol of micropropagation through axillary bud proliferation described here is very simple, repetitive and cost effective. It can be easily utilized for commercial cultivation. Further root formation took place on multiplication media therefore entire process is “one step”. This is important to minimize the occurrence of somaclonal variation due to media stress.

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As mentioned earlier the roots are the major source of tylophorine. The callus obtained on MS containing NAA media shows profuse root formation. This system can be exploited for the production of in vitro roots. This protocol is therefore, be very important for plant production and its utilization in genetic engineering studies.

CONSENT Not applicable.

ETHICAL APPROVAL Not applicable.

ACKNOWLEDGEMENTS Financial support for this work was received from the program support project (DBT New Delhi) and Puri Foundation (Koba, Gandhinagar, Gujarat). Authors also acknowledge Dr. Neeraj Jain for her valuable suggestions while preparation of the manuscript.

COMPETING INTERESTS Authors have declared that no competing interests exist.

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Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=608&id=13&aid=5518

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