Effect of Different Media and Growth Hormones on Shoot Multiplication of In Vitro Grown Centella asiatica Accessions

Medicinal plants are the traditional source of many pharmaceutically important compounds. In recent times, they are utilized by the pharmaceutical companies for the preparation of several formulations. Centella asiatica is one of the important traditional medicinal plant belonging to family Apiaceae and commonly known as Gotu kola in India. It is an important perennial medicinal herb found in the tropical and subtropical countries like India, Sri Lanka and Bangladesh. C. asiatica contains several triterpene, saponins like asiaticoside, asiatic acid, sapogenins, madecassic acid, vellarin, adecassoside, glycosides and centelloside [1]. Leaves contains high amount of triterpenoids [2]. It possesses several important properties like antileprotic, anti-stress, anti-feedent, anti-tuberculosis activities, wound-healing properties [3,4], antibacterial, antherosclerosis and fungicidal activity [5]. It is used in the treatment of leprosy, wound, cancer, fever, allergies [6], abscesses, asthma, catarrh, convulsions, dysentery, eczema, gonorrhea, hypertension, bronchitis, headache, jaundice, pleuritis, rheumatism, ulcers, spasms, tuberculosis, urethritis, etc. [7]. Leaves of this plant are rich in Vitamin B, C, and minerals such as magnesium, potassium, calcium, phosphorus and aluminium [8,9]. It is also used as brain tonic and blood purifier [10]. C. asiatica contains various flavonoids which include quercetin and kaempferol, rutin and naringin [11]. Roots are rich in amino acids like aspartic, glutamic, serine, alanine, threonine, histidine and lysine [12]. Underground parts contain many polyacetylenic compounds [12]. Due to its medicinal importance, this plant is overexploited and there is a decline in the population of the Centella asiatica. International Union for Conservation of Nature and Natural resources (IUCN) listed it as threatened plant and endangered species [13]. Tissue culture techniques can play an important role in the clonal multiplication of elite clones of this plant as well as conservation of its germplasm. For this study, different accessions of Centella asiatica have been used. An accession refers to the collection of plant material from a single species which is collected at one time from a specific geographical location. Each accession is an attempt to capture the diversity present in a given plant population. Accession number is given a unique identifier, and it is used to maintain associated information in the database. It exhibits significant variations in morphological parameters like growth of leaf, flowering, stomatal frequency, etc. The purpose of using different accession was to choose the best accession for the phytocompounds production. To the best of our knowledge there is no study which describes the effect of different media on the shoot multiplication of this plant and it provides an opportunity to explore the role of media and growth hormone on the enhancement of in vitro culture of different accessions of C. asiatica. In this study, effects of different media like MS, Gamborg’s B5 and Nitsch and the concentrations of plant growth hormones on the growth of three Centella asiatica accessions were observed and reported.


Introduction
Medicinal plants are the traditional source of many pharmaceutically important compounds. In recent times, they are utilized by the pharmaceutical companies for the preparation of several formulations. Centella asiatica is one of the important traditional medicinal plant belonging to family Apiaceae and commonly known as Gotu kola in India. It is an important perennial medicinal herb found in the tropical and subtropical countries like India, Sri Lanka and Bangladesh. C. asiatica contains several triterpene, saponins like asiaticoside, asiatic acid, sapogenins, madecassic acid, vellarin, adecassoside, glycosides and centelloside [1]. Leaves contains high amount of triterpenoids [2]. It possesses several important properties like antileprotic, anti-stress, anti-feedent, anti-tuberculosis activities, wound-healing properties [3,4], antibacterial, antherosclerosis and fungicidal activity [5]. It is used in the treatment of leprosy, wound, cancer, fever, allergies [6], abscesses, asthma, catarrh, convulsions, dysentery, eczema, gonorrhea, hypertension, bronchitis, headache, jaundice, pleuritis, rheumatism, ulcers, spasms, tuberculosis, urethritis, etc. [7]. Leaves of this plant are rich in Vitamin B, C, and minerals such as magnesium, potassium, calcium, phosphorus and aluminium [8,9]. It is also used as brain tonic and blood purifier [10]. C. asiatica contains various flavonoids which include quercetin and kaempferol, rutin and naringin [11]. Roots are rich in amino acids like aspartic, glutamic, serine, alanine, threonine, histidine and lysine [12]. Underground parts contain many polyacetylenic compounds [12]. Due to its medicinal importance, this plant is overexploited and there is a decline in the population of the Centella asiatica. International Union for Conservation of Nature and Natural resources (IUCN) listed it as threatened plant and endangered species [13]. Tissue culture techniques can play an important role in the clonal multiplication of elite clones of this plant as well as conservation of its germplasm. For this study, different accessions of Centella asiatica have been used. An accession refers to the collection of plant material from a single species which is collected at one time from a specific geographical location. Each accession is an attempt to capture the diversity present in a given plant population. Accession number is given a unique identifier, and it is used to maintain associated information in the database. It exhibits significant variations in morphological parameters like growth of leaf, flowering, stomatal frequency, etc. The purpose of using different accession was to choose the best accession for the phytocompounds production. To the best of our knowledge there is no study which describes the effect of different media on the shoot multiplication of this plant and it provides an opportunity to explore the role of media and growth hormone on the enhancement of in vitro culture of different accessions of C. asiatica. In this study, effects of different media like MS, Gamborg's B5 and Nitsch and the concentrations of plant growth hormones on the growth of three Centella asiatica accessions were observed and reported.

Plant material
Three different accessions of in vitro grown plantlets of Centella asiatica were collected from the NBPGR (National Board of Plant Genome Research), New Delhi. Nodes of C. asiatica were utilized as explant for this study.

Culture medium and conditions
Effect of different media on shoot multiplication and shoot length: Three different plant tissue culture medium i.e. Murashige and Skoog (MS) (1962) [14] with 3% (w/v) sucrose and 0.8% (w/v) agar, Gamborg's B5 (HiMedia Laboratories Pvt. Ltd., India) [15] and Nitsch (HiMedia Laboratories Pvt. Ltd., India) [16] media were used. The composition of each medium is mentioned in Table 1. These media were supplemented with standard concentration of plant growth hormone i.e. 4.0 mg/L BAP+0.4 mg/L NAA. The pH of media was adjusted to 5.8 with 1N NaOH or 1N HCl and media were autoclaved at 121°C for 20 minutes at 15 psi. The explants were then inoculated in the medium under aseptic conditions and incubated at 25 ± 2°C with photoperiod of 16 hours under cool-white fluorescent tubes for six weeks.

Effect of growth hormones on shoot multiplication and shoot length:
MS medium was augmented with different concentrations of plant growth hormones i.e. 1.0 mg/l BAP, 2.0 mg/l BAP, 1.0 mg/l BAP+0.5 NAA and 2.0 mg/l BAP+0.5 mg/l NAA. Explants were inoculated and cultures were incubated at 25 ± 2°C with photoperiod of 16 hours under cool-white fluorescent tubes for six weeks. Regenerated shoots were subcultured every three weeks in the same media (Table 1).

Data analysis
Visual observations were recorded in terms of number of shoots per explant and the length of each shoot. Experiments were done in triplicates and means of each experiment was carried out to detect the significant differences.

Effect of different media on shoot multiplication and shoot length
This study was an attempt to correlate the effect of different media and plant hormone concentration on the shoot multiplication of Centella asiatica accessions. To initiate the study, nodal explant were taken from in vitro grown plants. Shoot multiplication of Centella asiatica nodal explants cultured on MS, Gamborg's B5 and Nitsch media supplemented with 4.0 mg/L BAP+0.4 mg/L NAA. After two weeks of incubation explants showed the growth response in different media. After six weeks of incubation period it was found that MS media showed highest shoot multiplication as compared to Gamborg's B5 and Nitsch media in all the three accessions. In case of MS media, the highest shoot multiplication was observed as follow, 5.

Effect of growth hormones on shoot multiplication and shoot length
Nodal explants were cultured in MS medium supplemented with different concentrations and combination of auxin (NAA) and cytokinine (BAP) to assess their effect on shoot multiplication of C. asiatica accessions. The highest number of shoots as well as length of III II I    induction. In general, BAP is the most efficient growth hormone for the shoot proliferation [18]. It mimics as an inhibitor agent and function against apical dominance of shoot induction and shoot bud formation [19]. Several studies reported that media supplemented with BAP and NAA have also useful for the shoot multiplication. One article reported that MS media supplemented with 22.2 µM BA+2.68 µM NAA showed highest growth [20] where as another article reported that maximum shoot multiplication was observed at 2 mg/l BAP [21]. Results of this study indicate that large scale propagation of this plant is feasible and several plantlets can be regenerated from a single nodal explant. Details of this experiment are mentioned in Table 3 ( Figure 2).

Conclusion
In this investigation, it was concluded that the MS medium with concentration of BAP 1 mg/l supports the maximum shoot multiplication and length of shoots for all the three accessions of Centella asiatica. It was also observed that the maximum number of shoots and length of shoots obtained for Accession-342109 in comparison to the other two accessions. Further analysis of phytocompounds will be done with all these cultures. Findings in this investigation have proven an efficient media and plant growth hormone concentration for the mass propagation of this plant. These findings would be useful in conservation and micropropagation of this plant. Future efforts are in progress to evaluate the phytocompound present in this plant.