Development of a simple, reproducible and efficient in vitro culture ...

Arun Thuruthikkattil Ram *, Mohammed Shareef Koorikkadan and Prasool Kuniyil, Development of a simple, reproducible and efficient in vitro culture protocol for callus proliferation under varying concentrations of 6-benzylaminopurine (BAP) via leaf and noda l segments, Int.J.Curr.Biotechnol., 2014, 2(6):1-4.

International Journal of Current Biotechnology ISSN: 2321 - 8371 Journal Homepage : http://ijcb.mainspringer.com

Development of a simple, reproducible and efficient in vitro culture protocol for callus proliferation under varying concentrations of 6-benzylaminopurine (BAP) via leaf and nodal segments Arun Thuruthikkattil Ram*, Mohammed Shareef Koorikkadan and Prasool Kuniyil. Post Graduate and Research Department of Botany, St. Albert’s College, Ernakulam, Kerala, India.

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Article History: Received 2 June 2014 Received in revised form 10 June 2014 Accepted 15 June 2014 Available online 20 June 2014

Key words: Centella asiatica L., Scoparia dulcis L., Cardiospermum halicacabum L., Portulaca grandiflora L., 6-Benzylamino purine, callus proliferation.

A B S T R A C T The present study investigates the development of a simple, reproducible and efficient in vitro culture protocol for the callus formation successfully developed using Murashige and Skoog (1962) medium under varying concentrations of 6Benzylaminopurine (BAP) via leaf and nodal segments. The result of the experiment provides the standard method for callus proliferation of four important medicinal plants, Centella asiatica L., Scoparia dulcis L., Cardiospermum halicacabum L. and Portulaca grandiflora L. The main aim of this work is to analyse the response of different explants to various plant growth regulator treatments. During the study, Medium B (IAA 1mg/l and BAP 3mg/l) shows maximum response for the callus proliferation of Centella asiatica L., Scoparia dulcis L. and Portulaca grandiflora L. while Medium C (IAA 1mg/l and BAP 3.5 mg/l) refers the maximum response in Cardiospermum halicacabum L. In conclusion, the result of this experiment is the first report describing the protocol for callus proliferation.

Introduction Tissue culture and in vitro plant regeneration system may provide an alternative mean for the conservation and rapid multiplication of many valuable, rare, threatened and endangered medicinal plants using different strategies of micro propagation. Centella asiatica L., Scoparia dulcis L., Cardiospermum halicacabum L. and Portulaca grandiflora L. are the major plants used in the in vitro culture protocol. Centella asiatica L. belongs to the family Umbelliferae, is used in the treatment of various skin diseases, ulceration, chronic rheumatism, leprosy, malaria, fever, epilepsy, asthma, bronchitis, dropsy, kidney troubles, stomachic, diarrhea, jaundice, carminative, headache, vein insufficiency and mental disorders (Kirtikar and Basu, 1975; Mangas et al., 2009). Scoparia dulcis L. belongs to the family Scrophulariaceae, which is having high medicinal values. Leaf of Scoparia dulcis is used as a cure for gastric ulcer and weakness. Infusion of leaves is used for the treatment of fever, cough, bronchitis, diarrhea and dysentery, and as a diuretic and gargle for toothache (Ghani, 1998). Cardiospermum halicacabum L. (Sapindaceae) is an important medicinal herb traditionally used to treat symptoms of malaria, rheumatoid arthritis, *Corresponding author. Email address: [email protected] Phone: 919496827507 1

curing asthma, piles, diarrhoea, earache, fever and various nervous diseases (Jayabharathi, 1998). Portulaca grandiflora Hook. (Portulacaceae) is used to cure sore throat and skin rashes. It is a putative immune stimulant and also used for detoxification (Chavalittumrong et al., 2007). In vitro propagation of medicinal plant species hold tremendous potential for the production of high quality plant based medicines (Murch et al., 2000). Due to higher demand and unavailability of farming practices, the plant population is decreasing. Therefore, the present study describes the first report on micro propagation conducted with an objective to standardize a simple, reproducible and efficient in vitro culture protocol for the propagation of these medicinally important plants. Materials And Methods Plant material and explant preparation The mature plants were selected from the botanical garden of St. Albert’s college, Ernakulam, Kerala. The explant materials were obtained from the following four medicinally important plants, Centella asiatica L., Scoparia dulcis L., Cardiospermum halicacabum L. and Portulaca grandiflora L. The explants were first washed with running tap water to remove all the soil particles. It was then washed with teepol solution. To remove teepol remains, the explants were washed with double distilled

Int.J.Curr.Biotechnol.

Volume 2; Issue 6; June, 2014

water for three minutes. The surface sterilized material was taken into sterilized room and placed in the incubation chamber in a beaker containing double distilled water. Then the explants were washed in 70% ethanol for one minute. After that they were washed with double distilled water for three times. These explants were sterilized with sterile double distilled water for three times. Culture media and conditions Following disinfection, leaf segments were cultured in a hormone free MS medium to grow the explants needed to study the response under varying hormonal concentrations. The MS medium used for the culture was supplemented with 440 mg/l CaCl2, 8 g/l agar and four growth regulator concentrations; Indole 3-Acetic Acid (IAA) is kept constant with varying concentration of BAP. pH was adjusted to 5.7 with 1N NaOH and 1N HCl. The solution was heated in a water bath at 80 0C for 25 minutes with constant stirring. The medium was then poured into the culture tubes not allowing the agar to solidify before transfer. Before transferring into tubes, necessary hormones are added. The four sets of media used are summarized in the Table 1. It was then autoclaved at 15 lbs/121 0C for 20 minutes. The autoclaved tubes along with the medium was kept in a slanting position in a rack and kept to solidify and cool before inoculation. The prepared medium was kept for 24 hours before inoculation. Explants should have the size 5 mm to 10 mm. Sterilized explants of suitable size were placed on the medium by pressing it gently for providing the maximum contact with the medium. Before and after inoculation, the flaming of the rim of culture tubes was done to avoid microbial attack. The culture tubes were plugged tightly. The culture tubes were kept in a dark period for 48 hours. After that the culture tubes were transferred to the incubation room and incubated at 25 0 C to 27 0C. Light (1000 lux) was given for five periods of 8 hours per day. Results and Discussion During the present investigation, studies were conducted to establish cultures from the selected plants, Centella asiatica L., Scoparia dulcis L., Cardiospermum halicacabum L. and Portulaca grandiflora L. The leaf and nodal segments obtained from the mature plants were used in this study. MS media fortified with IAA in combination with BAP were investigated for callus induction. MS media supplemented with fixed concentration of IAA (1 mg/l) in combination with varying concentrations of BAP for callus induction. Bulging is considered as an early stage of cell division in in vitro culture. A balance between IAA and BAP growth regulators is mostly required for the formulation of calli. The explants were enlarged 2-3 times than that of the original size and white, green friable and green compact callus initiation occurred within five days after inoculation. The presence of green colour in the initial stages of callus may be due to the retention of chlorophyll pigment in callus. Higher frequency of callus proliferation was observed on the leaf and nodal explants in 3 to 4 week old cultures. Among varying concentrations used, Medium B shows maximum response for the callus proliferation of Centella asiatica L., Scoparia dulcis L. and Portulaca grandiflora L. while Medium C refers the maximum response in Cardiospermum halicacabum L. (Figure 1). A successful protocol for the regeneration of callus cultures of Centella asiatica was established by Patra et


al. (1998), in which the stem and leaf explants were cultured on MS media supplemented with 2.0 mg/l kinetin and 4.0 mg/l of NAA. Multiple shoot formation was observed in Scoparia dulcis L. (Figure 1-c) which is obtained from Medium B. No other explants show such responses. Rashid et al. (2009) in their studies produced multiple shoots from shoot tips and nodal segments of Scoparia dulcis on MS medium supplemented with different concentration of cytokinin for multiple shoot induction. The maximum number of shoot induction was exhibited in a different concentration of IAA and BAP, in another study (Karthikeyan et al., 2009). The results clearly showed that calli derived from nodal segments of Cardiospermum halicacabum could regenerate on MS medium supplemented with BAP in combination with IAA. The addition of lower concentration of IAA along with optimum concentration of BAP significantly improved the callus proliferation from the explants. Many studies related to the in vitro organogenesis underline the importance of auxin/ cytokinin ratio in the culture medium. Existing reports suggest that auxins at lower concentrations along with cytokinins have a critical role in plant regeneration in several systems like Petasites hybridus (Wldi et al., 1998), Eucalyptus grandis (Luis et al., 1999), Hybanthus enneaspermus (Prakash et al., 1999), Coleus forskohlii (Sairam et al., 2001), Saccharum spp. (Chengalrayan and Gallo-Meagher, 2001), Coffea Arabica (Zoriniants et al., 2003) and Eleusine indica (Yemets et al., 2003). Safdari and Kazemitabar (2009) were found that callus formation from Portulaca grandiflora leaf explants was very effective in most combination of IAA and BAP. In the present study, narrow fleshy leaves were used as explant initiation and callus proliferation was highly observed in the medium B. All the media showed responses in which medium B has shown as high growth rate. The present work also exposes some similarities with the earlier findings on Bacopa monnieri L. (Prasool et al., 2013). Orton (1979) classified callus from his studies on the Hordeum, as three different types based on morphology. Type A represents hard, large, discrete, yellow coloured nodules more or less watery in appearance. Type B represents large physically hard nodes, each one bearing single meristems. Type E represents slightly friable callus with loosely associated single cells and clumps or large amorphous parenchymatous cells almost white in colour. In the present investigation, callus obtained from Scoparia dulcis L. and Cardiospermum halicacabum L. belongs to Type A while from Centella asiatica L. and Portulaca grandiflora L. belongs to Type E of Orton. Conclusion The present study is the first to describe fruitful direct regeneration protocol for the important medicinal plants using leaf and nodal segments as explants. The study would be of great help in the maintenance and conservation of these fast depleting species and would also be adopted for use in genetic manipulation and transformation techniques. Acknowledgements We express sincere thanks to Dr. L. Jose, Post Graduate Research Department of Botany, St. Albert’s college, Ernakulam, Kerala, India, for providing support to us carrying out investigation of in vitro culture studies and also for guidance. And also, we duly acknowledge the help extended by Syama M. Mohanan, School of Letters,


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Table 1: Showing Growth Regulators and Medium Used. Medium

A

B

C

D

Indole 3-Acetic Acid (IAA) mg/l

1

1

1

1

6- Benzylaminopurine (BAP) mg/l

2.5

3

3.5

4

Figure 1: Callus Culture

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