In vitro mass propagation and conservation of ...

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In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered, high trade medicinal plant of Western Ghats Rameshkumar Ramakrishnan, Joe Virgin Largia Muthiah, Satish Lakkakula, Shilpha Jayabalan & Ramesh Manikandan To cite this article: Rameshkumar Ramakrishnan, Joe Virgin Largia Muthiah, Satish Lakkakula, Shilpha Jayabalan & Ramesh Manikandan (2016): In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered, high trade medicinal plant of Western Ghats, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2016.1149120 To link to this article: http://dx.doi.org/10.1080/11263504.2016.1149120

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Date: 03 February 2016, At: 20:34

Publisher: Taylor & Francis & Societa Botanica Italiana Journal: Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology DOI: http://dx.doi.org/10.1080/11263504.2016.1149120

In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal

Downloaded by [Alagappa University] at 20:34 03 February 2016

explants: A globally endangered, high trade medicinal plant of Western Ghats

Ramakrishnan Rameshkumar, Muthiah Joe Virgin Largia, Lakkakula Satish, Jayabalan Shilpha, Manikandan Ramesh

Department of Biotechnology, Science campus, Alagappa University, Karaikudi, Tamil Nadu, India – 630 004. Tel.: + 91 4565 225215. 

Corresponding author e-mail address: [email protected]

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Abstract Nilgirianthus ciliatus is a globally endangered aromatic slender shrub of Western Ghats with extensive applications in Ayurveda. It is endangered due to its indiscriminate collection and overexploitation to meet the requirements of the pharmaceuticals. The present study deals with the preservation of this endangered plant through in vitro nodal culture. Nodal explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 6–


benzylaminopurine (BA) or 6–furfurylaminopurine individually for primary shoot induction. For multiple shoot induction, primary shoots were transferred onto MS medium containing BA individually or in combination with auxins. Clusters of multiple shoots (up to 24.3) occurred with highest frequency (93.2%) on MS medium fortified with BA (3 mg l−1) and indole–3–acetic acid (0.1 mg l−1). In vitro regenerated plantlets were rooted on half-strength MS medium with maximum rooting frequency (82.2%) obtained in the presence of indole-3-butyric acid (1.0 mg l−1). The rooted plantlets were acclimatized to soil with 100% survival rate. Results of the present study allowed us to develop an efficient regeneration system that will permit to carry out restoration programs of N. ciliatus in Western Ghats. In future, this protocol will be an invaluable tool to produce synthetic seeds for cryopreservation and long term conservation.

Keywords: Endangered species, In vitro propagation, Nilgirianthus ciliatus, Nodal explants, Conservation.

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Abbreviations: BA, 6–Benzyladenine; KIN, 6–Furfurylaminopurine; IAA, Indole–3–acetic acid; NAA, α–Naphthaleneacetic acid; IBA, Indole–3–butyric acid.


1. Introduction Nilgirianthus ciliatus (Nees) Bremek (Syn. Strobilanthes ciliatus Nees) belongs to Acanthaceae family and is a slender aromatic shrub with sub-quadrangular white dotted dark green stems and branches. This plant is endemic to the evergreen forests of southwestern Ghats of India (Thomas & Rajeshkumar 2014) and has officially been included in the RED DATA list of South Indian medicinal plants by the IUCN (Ravikumar & Ved 2000; Ved & Goraya 2007). N. ciliatus is commonly known as “Karvi” in Hindi, “Chinnakurunji” in Tamil and “Karimkurunji” in Malayalam (Khare 2007). In India, this plant is considered as medicinal plant species in high trade and sourced from the tropical forests of Western Ghats (Ved & Goraya 2007). In traditional Indian Ayurvedic system, this plant is extensively used for the preparation of “Sahacara” drug which is popularly used to treat neurological disorders, ulcers, glandular swellings, poisonous infections, itching, leprosy and other skin diseases (Singh & Panda 2005; Thomas & Rajeshkumar 2014). It is one of the potential medicinal plants in many of the Ayurvedic preparations such as “Varanathi thylam” for obesity, “Sahacharadi thailam” to cure neurological disorders and “Kurinji kuzhambu” consumed by women after delivery for good health (Reneela 2011).

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N. ciliatus was reported to contain several phytochemicals viz., lupeol, stigmasterol, betulin, stigmasterol glycoside, taraxerol and 4-acetyl-2,7-dihydroxy-1,4,8-triphenyloctane-3,5dione (Reneela 2011; Venkatachalapathi & Subban 2012). The whole plant has been used for the treatment of paraplegia, ulcers, glandular swellings, itching, cough, oedema, toothache, jaundice, rheumatism, urinogenital tract diseases, strangury fever, bronchitis, gum diseases, whooping cough, chest congestion, rheumatalgia, lumbago, sciatica, limping, leucoderma, dropsy, leprosy,


skin diseases, pruritus, inflammations, scrofula, odontalgia and general debility (Warrier et al. 1994; Khare 2007; Sujatha et al. 2011; Srinivasan et al. 2013). In folk medicine, drinking of N. ciliatus leaf decoction or applying its paste over affected area for relieving rheumatic pain has been practiced. In addition, roots of N. ciliatus possess many therapeutic properties viz., thermogenic, emollient, diuretic, febrifuge, diaphoretic, depurative, anti-inflammatory, expectorant and tonic (Warrier et al. 1994; Srinivasan et al. 2013) and similarly seeds are used to treat gonorrhoea and spermatorrhea diseases (Nambiar et al. 1985). Besides various medicinal values, extracts of the aerial parts of N. ciliatus have been reported to possess biological activities like hepatoprotective (Usha et al. 2013), radical scavenging (Srinivasan et al. 2013), antimicrobial (Venkatachalapathi & Ravi 2013; Varghese et al. 2014), and analgesic activities (Jayaraman et al. 2014). The major phytochemicals present in the stem and root extracts of N. ciliatus are betulin, lupeol and stigmasterol (Reneela & Sripathi 2011), and they were reported to possess anti-inflammatory, hypoglycemic, hepatoprotective, antioxidant and anticancer properties (Alakurtti et al. 2006; Kaur et al. 2011). The widespread curative potentials of N. ciliatus resulted in over exploitation, thus threatening the existence of this plant in the near future (Ved & Goraya 2007). At the time of harvesting, more than 70% of medicinal plants are facing risk due to the harmful collection of plant parts which makes them 4

endangered (Thiyagarajan & Venkatachalam 2013). N. ciliatus has been identified as monocarp and flowers appear only once during its life cycle at the end of 6-7 years of vegetative growth. Therefore, conventional propagation through Nilgirianthus seeds is not feasible to produce enough offsprings due to limited or non-availability of seed, slow germination rate, infrequent flowering. Stem cuttings are also unable to balance this ever decreasing wild stock (Chandrashekara & Ramakrishnan 1993). All these factors enforced this plant to be endangered


in the wild. Taking all these difficulties into consideration, our aim was to develop a high-frequency regeneration protocol for the globally endangered shrub N. ciliatus through nodal explants. In vitro micropropagation has been widely used as an ideal alternative for conservation of valuable species. In recent years, many reports demonstrated successful in vitro propagation of rare, threatened and endangered medicinal plants viz., Quercus euboica (Kartsonas & Papafotiou 2007), Leontopodium nivale (Pace et al. 2009), Gymnema sylvestre (Thiyagarajan & Venkatachalam 2013) and Rauwolfia serpentina (Senapati et al. 2014) through multiple shoot induction and conservation. As the requirement of N. ciliatus biomass increased considerably over the last few years, concrete measures are needed to conserve this highly valuable and worldwide endangered medicinal plant. Since the conventional propagation approach is slow for this plant, tissue culture-based propagation could balance the ever declining plant resources. Hence attempts were made in the present study to establish a rapid in vitro propagation system for N. ciliatus through nodal explants as an important conservatory measure. To the best of our knowledge, this is the first detailed report on mass propagation of N. ciliatus.

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2. Materials and methods 2.1. Plant material and explant sterilization Nilgirianthus ciliatus plants were collected from MS Swaminathan Research Foundation, Wayanad, Kerala and maintained in shade house, Department of Biotechnology, Alagappa University, Tamil Nadu. About 2.0-cm long nodal explants were collected from field-grown


plants and cleansed with running tap water for four times to remove surface contaminants and rinsed in 0.1% (w/v) Bavistin solution for 20 min. Then the explants were immersed in 0.1% (w/v) mercuric chloride (HgCl2) for 4 min and washed with sterile distilled water thrice. Finally the explants were treated with 70% (v/v) ethanol for 1 min and rinsed four times in sterilized distilled water. After surface disinfestations, processed aseptic nodes were trimmed and used for in vitro propagation.

2.2. Primary shoot induction and culture conditions Surface sterilized nodal explants (approximately 1-1.5 cm) were inoculated on Murashige and Skoog (MS) (Murashige & Skoog 1962) medium amended with various concentrations of 6benzylaminopurine (BA: 1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) or 6–furfurylaminopurine (KIN: 1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) for primary shoot induction. Sucrose 30 g l-1 (w/v) was used as carbon source and medium pH was adjusted to 5.8 by using 1 N NaOH or 1 N HCl prior to autoclaving at 121 oC for 20 min. Agar (0.8%, w/v) was used as solidifying agent. All the cultures were maintained in a growth chamber (Sanyo versatile environmental test chamber,

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Japan, model no: MLR 351 H) with 16–h light/8–h dark at 25 ± 1 °C with 50 μmol photons m−2 s−1 of cool white fluorescent light.

2.3. Shoot multiplication and maturation Young green primary shoots emerged from the nodes were transferred to two different


experimental conditions for shoot multiplication. 2.3.1. Experiment I Effect of two different cytokinins viz., BA or KIN was studied individually. For that, in vitro derived primary shoots were inoculated on MS medium enriched with various concentrations of BA (1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) or KIN (1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) for multiple shoot induction. The explants were subcultured every 15 days on fresh medium to maintain their growth and proliferation; multiple shoots were elongated on the same medium. The shoot induction percentage, number of shoots induced per explant and shoot lengths were analyzed after the third subculture using three replicates.

2.3.2. Experiment II In experiment II, in vitro derived primary shoots were inoculated on MS medium supplemented with 3 mg l−1 BA along with various concentrations of auxins such as Indole–3– acetic acid (IAA: 0.05, 0.1, 0.2, and 0.3 mg l−1), Indole–3–butyric acid (IBA: 0.1, 0.2, 0.3, and 0.4 mg l−1) and α–Naphthaleneacetic acid (NAA: 0.1, 0.2, 0.3, and 0.4 mg l−1) for multiple shoot 7

induction. The explants were subcultured onto identical new medium for every two weeks. After the third subculture, the elongated shoots were subjected to various rooting treatments.

2.4. In vitro rooting of regenerated shoots The elongated in vitro derived green shoots (6 cm in size) were transferred to various


strengths of MS media (1/8, 1/4, 1/2, and 3/4) alone or in combination with various concentrations of auxins like IAA, IBA, and NAA (0.5, 1.0, 1.5, and 2.0 mg l−1) for in vitro rooting. Sucrose 1.5% and agar 0.8% were added and pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for 20 min. For each concentration, 350 explants with three replicates were tested. For each treatment, percentage of root induction, average number of roots per shoot and mean length of induced roots were determined after four weeks of incubation. Then, the plantlets with an abundant root system were taken out from the culture container and washed with sterile distilled water to remove media traces.

2.5. Acclimatization In vitro rooted plants were transferred to plastic packs containing autoclaved mixture of sand, vermicompost and horticulture soil (1:1:1 ratio). These packs were kept in a growth chamber for 30 days. Further, acclimatized plants were individually transferred to plastic pots and maintained in a shade house. The plant survival percentage was determined 30 days after transfer to the shade house. The regenerated plants were gradually exposed to the field for further conservation. 8

2.6. Data analysis All the experiments were carried out in triplicate with a minimum of 350 explants per experiment. The data such as regeneration frequency, shoot number, shoot length, root number and root length were recorded after the third subculture. The data are presented as means ±


standard deviation (SD) and were analysed by ANOVA using SPSS ver 17.0 software (SPSS, IBM). The difference between the mean values was tested using Duncan’s multiple range test at a significance of P≤0.05.

3. Results and discussion 3.1. Effect of cytokinins on primary shoot induction Sterile nodal explants from N. ciliatus were inoculated on MS medium supplemented with various concentrations of BA and KIN (1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) independently for primary shoot induction (Fig. 1a). Green juvenile shoot buds started to arise from the nodal cuttings after one week of culture at 25 ± 1 °C in light (Fig. 1b). Among different concentrations of BA and KIN tested for initial shoot bud initiation, MS medium added with 3.0 mg l−1 BA showed maximum percentage 86.7 of shoot induction whereas 2.0 mg l−1 KIN showed 73.2% of response after third subculture (Table 1). Maximum number of primary shoots (2.1) per explant was observed on MS medium supplemented with 3.0 mg l−1 BA and the produced shoots were dark green in colour (Fig. 1 b and c). BA or KIN concentrations in the shoot induction medium played a crucial role in primary shoot bud initiation in N. ciliatus (Table 1). Beyond the level of 9

3.0 mg l−1 BA or 2.0 mg l−1 KIN, there was no further improvement in shoot initiation percentage; in fact, a decline in primary shoot induction rate, shoot number and shoot length (Table 1) was noticed. Similar results were previously observed in Tylophora indica (Faisal & Anis 2003) and Stevia rebaudiana (Thiyagarajan & Venkatachalam 2012). The superiority of BA over KIN for shoot bud initiation has been reported in medicinal plants like Andrographis paniculata (Purkayastha et al. 2008), Stevia rebaudiana (Thiyagarajan & Venkatachalam 2012)


and Ceropegia evansii (Chavan et al. 2015). Our findings confirm the higher effectiveness of BA as compared with KIN also in N. ciliatus

3.2. Effect of cytokinins and auxins on regeneration 3.2.1. Experiment I. Effect of various concentrations of cytokinins alone in shoot multiplication and maturation The green primary shoots induced on MS medium supplemented with 3.0 mg l-1 BA were subcultured onto MS media containing various concentrations of BA or KIN (1.0, 2.0, 3.0, 4.0, and 5.0 mg l−1) showed multiple shoot induction after three subcultures (Fig. 1d). MS media having 3.0 mg l−1 BA showed an additional response in multiple shoot induction and illustrated highest percentage of regeneration (88.2) with maximum number of shoots (21.6) while 2.0 mg l−1 KIN showed comparatively lesser percentage of regeneration (79.4%) with 4.9 shoots after five weeks of incubation in light at 25 ± 1 °C (Table 2). Increasing the concentration of BA or KIN positively affected multiple shoot induction after the second subculture. The highest number of shoots was observed in medium containing 3.0 mg l−1 BA and beyond this optimum

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concentration shoot number and multiple shoot induction percentage were reduced significantly (Table 2). The superiority of BA on shoot multiplication has been reported in many medicinal plant species viz., Pterocarpus marsupium (Chand & Singh 2004), Ulmus parvifolia (Thakur & Karnosky 2007), Sarcostemma brevistigma (Thomas & Shankar 2009), Cyclea peltata (Abraham et al. 2010) and Gymnema sylvestre (Thiyagarajan & Venkatachalam 2013). This is because the synthetic cytokinin BA can stimulate the metabolism and synthesis of natural cytokinins (e.g.,


zeatin) in plant tissues (Rai et al. 2009).

3.2.2. Experiment II. Effect of BA in combination with auxins for shoot multiplication and regeneration MS medium amended with 3.0 mg l-1 BA was supplemented with three different types of auxins viz., IAA (0.05, 0.1, 0.2, and 0.3 mg l−1) or IBA or NAA at various concentrations (0.1, 0.2, 0.3, and 0.4 mg l−1) to evaluate shoot multiplication and regeneration of N. ciliatus (Table 3). Among the different formulations tested, BA (3.0 mg l-1) in combination with IAA (0.1 mg l−1) improved the multiple shoot induction percentage (93.2) with a maximum number of shoots (24.3) and subsequent maximum shoot elongation of 6.8 cm after three weeks of incubation in light (Table 3; Fig. 1d). The next best response was found with BA (3.0 mg l-1) and NAA (0.2 mg l−1) followed by BA (3.0 mg l-1) and IBA (0.2 mg l−1) combinations (Table 3). The superiority of IAA compared to other auxins for shoot multiplication was also proved in Cucumis melo (Tabei et al. 1991) and Ceropegia evansii (Chavan et al. 2015) plants. The combination of cytokinin and auxin was found to play an important role in regulating the cell cycle, hormone signaling, transport, and organogenesis and shoot branching in plant development (Su et al. 11

2011; Muller & Leyser 2011). In our study, all three type of auxins showed various percentages of multiple shoot induction at 0.1–0.4 mg l-1 concentrations, while all four auxins beyond 0.2 mg l-1 resulted in reduced multiple shoot induction, mean length of shoots and number in N. ciliatus (Table 3). Our results are in agreement with the recent study of Shilpha et al. (2014) in Solanum trilobatum and Chavan et al. (2015) in Ceropegia evansii, wherein the stimulatory response of


BA and IAA gave the higher regeneration frequency with multiple shoots.

3.3. Effect of medium formulations and auxin concentration on in vitro rooting For efficient in vitro rooting in N. ciliatus, we have initially tested various strengths (1/8, 1/4, 1/2, and 3/4) of MS medium. Among the different strengths of MS media tested, 1/2 strength MS basal basal medium proved to be the best one and induced highest percentage of roots (59.1%), root number per explant (4.5) and root length (3.7 cm) (Fig. 2). Further 1/2 strength MS basal medium supplemented with various concentrations of auxins enhanced rooting frequency (Table 4). Among them 1/2 strength MS medium prepared with 1.5% sucrose and 1.0 mg l−1 IBA provided the highest number of roots (14.2) with an average length of 8.9 cm after four weeks incubation in light (Table 4; Fig. 3a, b). Among the three auxins tested, IBA at 1.0 mg l-1 yielded the highest rooting percentage (82.2%) whereas NAA at 1.5 mg l−1 and IAA at 1.5 mg l−1 gave 42.9% and 35.1% of root induction, respectively after four weeks incubation in light (Table 4). Formerly, 1/2 strength MS medium supplemented with IBA has been proved to be efficient for in vitro rooting of Gymnema sylvestre (Komalavalli & Rao 2000), Clitoria ternatea (Singh & Tiwari 2012), Centaurea cineraria (Valletta et al. 2015) and Ceropegia evansii (Chavan et al. 2015) species. The better root-inducing capacity of IBA over other auxins was also shown in 12

other medicinal plants such as Cyclea peltata (Abraham et al. 2010), Lawsonia inermis (Ram & Shekhawat 2011), Gymnema sylvestre (Thiyagarajan & Venkatachalam 2013), Solanum trilobatum (Shilpha et al. 2014) and Rauwolfia serpentina (Senapati et al. 2014). In agreement with previous findings, 1/2 strength MS supplemented with IBA was the best combination for in


vitro rooting of N. ciliatus.

3.4. Acclimatization of plantlets In vitro derived plantlets transferred to plastic packs and initially maintained in a growth chamber for 30 days showed the best response for acclimatization of N. ciliatus plants (Fig. 3c). Acclimated plants were subsequently transferred to a shade house. Finally, after new leaves were formed, elongated plants were fruitfully established in the shade house at the Department of Biotechnology, Alagappa University, Karaikudi for conservation with 100% survival rate (Fig. 3d).

5. Conclusion We have successfully established a rapid protocol for mass propagation of N. ciliatus using nodal explants. Thus the protocol can be used as a potential alternative to conventional propagation as a possible conservatory measure to protect this ever declining globally endangered medicinal plant. In future, this protocol could serve as the first step in the production of hairy roots, secondary metabolites, synthetic seeds and transgenic plants.

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Acknowledgements The corresponding author M. Ramesh and the first author R. Rameshkumar, Project Fellow sincerely acknowledge SERB, Department of Science and Technology, Govt of India for financial support to carry out this work (F.No. SERB/SR/SO/PS/045/2011; dated 31.05.2013).


Also the authors gratefully acknowledge the Bioinformatics Infrastructure Facility of Department of Biotechnology, Alagappa University (funded by Department of Biotechnology, Government of India: Grant No. BT/BI/25/001/2006) for providing the computational facility to do statistical analysis of the study.

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Table 1: Effect of various concentrations of BA and KIN on primary shoot induction from nodes of N. ciliatus. Plant growth

Shooting frequency

Number of shoot buds

regulators (mg l−1)

(%) (mean ± SD)

(cm) (mean ± SD)

1.0

69.6 ± 0.78b

1.2 ± 0.31a

2.0

76.2 ± 0.25c

1.5 ± 0.15ab

3.0

86.7 ± 0.55d

2.1 ± 0.10b

4.0

68.8 ± 0.46b

1.0 ± 0.44a

5.0

63.1 ± 0.40a

1.0 ± 0.52a

1.0

63.7 ± 0.56d

1.4 ± 0.15a

2.0

73.2 ± 0.45e

1.6 ± 0.27a

3.0

59.5 ± 0.49c

1.2 ± 0.16a

4.0

47.9 ± 0.89b

1.0 ± 0.61a

5.0

34.8 ± 0.52a

1.0 ± 0.44a


BA

KIN

The data are presented as means (± SD) of three independent experiments. A minimum of 50 explants were tried for each experiment. Mean

values in each column followed by the same letters are not significantly different by Duncan’s multiple range test at P≤0.05.

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Table 2: Effect of various concentrations of BA and KIN on shoot multiplication from in vitro produced primary shoots of N. ciliatus.

Concentration of

Regeneration frequency

Shoot number

Shoot length (cm)

PGR (mg l−1)

(%) (mean ± SD)

(mean ± SD)

(mean ± SD)

1.0

77.1 ± 0.46c

8.8 ± 0.42c

3.5 ± 0.25c

2.0

84.2 ± 0.72d

17.3 ± 0.55d

4.3 ± 0.15d

3.0

88.2 ± 0.40e

21.6 ± 0.25e

6.4 ± 0.17e

4.0

71.6 ± 0.42b

6.5 ± 0.21b

2.9 ± 0.15b

5.0

65.4 ± 0.26a

5.1 ± 0.30a

2.5 ± 0.20a

1.0

74.1 ± 0.47d

3.6 ± 0.44c

3.1 ± 0.42c

2.0

79.4 ± 0.76e

4.9 ± 0.25d

3.4 ± 0.21c

3.0

64.3 ± 0.59c

2.7 ± 0.26b

2.3 ± 0.20b

4.0

52.1 ± 0.78b

2.4 ± 0.42ab

2.2 ± 0.17b

5.0

44.9 ± 0.26a

1.9 ± 0.25a

1.6 ± 0.42a


BA

KIN



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Table 3 : Effect of 3 mg l−1 BA in combination with various concentrations of IAA, NAA, and IBA on shoot multiplication from in vitro produced primary shoots of N. ciliatus. Plant growth

Regeneration frequency

Shoot number

Shoot length (cm)


(%) (mean ± SD)

(mean ± SD)

(mean ± SD)

3.0 + 0.05

71.8 ± 0.62a

8.5 ± 0.49a

3.3 ± 0.32a

3.0 + 0.1

93.2 ± 0.40d

24.3 ± 0.25d

6.8 ± 0.21c

3.0 + 0.2

86.4 ± 0.23c

19.4 ± 0.44c

4.9 ± 0.15b

3.0 + 0.3

79.8 ± 0.67b

12.9 ± 0.26b

3.6 ± 0.26a

3.0 + 0.1

71.6 ± 0.44c

14.5 ± 0.26c

3.9 ± 0.21ab

3.0 + 0.2

80.3 ± 0.25d

16.1 ± 0.36d

4.1 ± 0.28b

3.0 + 0.3

62.8 ± 0.21b

9.5 ± 0.31b

3.2 ± 0.10a

3.0 + 0.4

41.4 ± 0.67a

5.2 ± 0.40a

3.1 ± 0.84a

3.0 + 0.1

31.7 ± 0.42c

3.7 ± 0.52b

2.4 ± 0.15ab

3.0 + 0.2

46.2 ± 0.25d

5.1 ± 0.26c

2.9 ± 0.20b

3.0 + 0.3

21.6 ± 0.76b

3.5 ± 0.20b

1.8 ± 0.53a

3.0 + 0.4

18.5 ± 0.81a

2.1 ± 0.23a

1.7 ± 0.53a

MS alone

0.0

0.0

0.0


BA + IAA

BA + NAA

BA + IBA



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Table 4: Effect of different concentrations of IAA, NAA and IBA on in vitro rooting of N. ciliatus.

Plant growth

Rooting frequency (%)

Root number (cm)

Root length (cm)


(mean ± SD)

(mean ± SD)

(mean ± SD)

0.5

15.3 ± 0.40a

1.9 ± 0.17a

1.5 ± 0.42a

1.0

23.2 ± 0.51c

2.7 ± 0.40b

2.0 ± 0.15ab

1.5

35.1 ± 0.56d

3.9 ± 0.21c

3.3 ± 0.15c

2.0

17.1 ± 0.45b

2.0 ± 0.61a

2.1 ± 0.29b

0.5

70.8 ± 0.72c

8.4 ± 0.15b

6.3 ± 0.35b

1.0

82.2 ± 0.51d

14.2 ± 0.36c

8.9 ± 0.49c

1.5

66.3 ± 0.31b

6.9 ± 0.30a

5.1 ± 0.20a

2.0

57.4 ± 0.71a

6.4 ± 0.56a

4.8 ± 0.31a

0.5

18.2 ± 0.68a

2.2 ± 0.10a

1.8 ± 0.17a

1.0

31.8 ± 0.25c

3.1 ± 0.23b

2.8 ± 0.21b

1.5

42.9 ± 0.83d

4.1 ± 0.21c

3.3 ± 0.44b

2.0

28.6 ± 0.60b

2.2 ± 0.18a

1.9 ± 0.25a


1/2 MS + IAA

1/2 MS + IBA

1/2 MS + NAA

The data are presented as means (± SD) of three independent experiments. A minimum of 50 explants were tried for each experiment. Mean values in each column followed by the same letters are not significantly different by Duncan’s multiple range test at P≤0.05.

24

Figure 1: Various stages of in vitro multiple m shoot induction of N. ciliatus using nodaal explants.


(a) Nodal explants excised from wild N. ciliatus plannts used for primary shoot induction; (b) Primary shoots induced on M MS medium supplemented with plant growth regulators BAP (3.00 mg/l); (c) In vitro derived primary shoots after second subculture onn MS medium containing BAP (3.0 mg/l); (d) Multiple shoots induced on MS medium supplemented with BAP (3.0 mg/l) in combination of IAA (00.1 mg /l) after second subculture.

Figure 2: Effect of various streng gths of MS medium on in vitro rooting of N. ciliatus.

25


Figure 3: In vitro rooting and acclimatization of N. ciliatus plantlets.

(a, b) In vitro rooting of N. ciliatus on half-strength MS medium supplemented with IBA 1.0 mg/l (c) Root induced plants were hardened in soil and maintained in the plant growth chamber; (d) In vitro regenerated plantlets growing in shade house.

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