Advanced Techniques in Biology & Medicine

Advanced Techniques in Biology & Medicine
Open Access

ISSN: 2379-1764

+44 1223 790975

Research Article - (2016) Volume 4, Issue 2

Effect of different media and growth hormones on shoot multiplication of in vitro grown Centella asiatica accessions

Arpita Roy, Koyel Kundu, Gaurav Saxena, Lakhan Kumar and Navneeta Bharadvaja*
Plant Biotechnology Laboratory, Department of Biotechnology, Delhi Technological University, New Delhi-110042, India, E-mail: global@sharda.ac.in
*Corresponding Author: Navneeta Bharadvaja, Plant Biotechnology Laboratory, Department of Biotechnology, Delhi Technological University, New Delhi-110042, India Email:

Abstract

Centella asiatica also known as Gotu Kola is an important medicinal plant which contains several bioactive compounds such as triterpenoid saponins including asiaticoside, madecassoside, centelloside, asiatic acid, etc. In addition, Centella sp. contains other components including flavonoids, phytosterols, tannins, amino acids, sugars, etc. Due to its medicinal importance, this plant is being overexploited and it is essential to conserve this plant. In the present investigation, comparative study of different accessions of Centella asiatica for shoot multiplication in different media and growth hormones was performed. Shoot multiplication and phytocompounds production from different accessions is required to be assessed to choose an accession giving optimum production of phytocompounds. For this, different media containing different combinations of macro and micro nutrients have to be tested that influence the growth of the plant. Assessment of best culture media and concentrations of plant hormone for shoot culture are some of the critical culture conditions to achieve. So there is a need to optimise such conditions that will enhance the growth of the plant. For shoot culture, explant of different accession of Centella asiatica were inoculated in different media i.e. Murashige and Skoog (MS), Gamborg’s B5 and Nitsch medium which were supplemented with standard concentrations of plant growth hormones. The cultures were incubated at 25 ± 2°C with photoperiod of 16 hours. After six week of incubation period, highest growth was found in MS media in all accessions. Further MS medium was supplemented with different combinations of growth hormones. After six week of incubation, MS medium supplemented with 1mg/l BAP showed the highest growth of the plant followed by 2mg/l BAP+0.5 NAA.

Keywords: Centella asiatica, Murashige and Skoog (MS), Gamborg’s B5, Nitsch, 6-Benzylaminopurine (BAP), Naphthaleneacetic acid (NAA)

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.

Materials and Method

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.

MS Medium [14] B5 Medium [15] Nitsch Medium [16]
Components mg/L Components mg/L Components mg/L
Macronutrients Macronutrients Macronutrients
Ammonium nitrate 1,650.00 Potassium  nitrate 2500 Potassium  nitrate 950
Potassium nitrate 1,900.00 Ammonium  Sulphate 134 Ammonium  nitrate 720
Calcium chloride (anhydrous) 332.2 Calcium chloride.2H2O 150 Magnesium sulphate anhydrous 90.34
Magnesium sulfate (anhydrous) 180.7 Magnesium sulphate 122.1 Potassium phosphate monobasic 68
Potassium phosphate monobasic 170 Sodium phosphate monobasic 130.4 Micronutrients
Micronutrients Micronutrients Manganese sulphate. H2O 18.94
Manganese sulfate monohydrate 16.9 Manganese sulphate. H2O 10 Boric acid 10
Ferrous sulfate heptahydrate 27.8 Boric acid 3 Molybdic acid (sodium salt).2H2O 0.25
Zinc sulfate heptahydrate 8.6 Potassium iodide 0.75 Zinc sulphate.7H2O 10
Boric acid 6.2 Molybdic acid (sodium salt).2H2O 0.25 Copper sulphate.5H2O 0.025
Potassium  iodide 0.83 Zinc sulphate.7H2O 2 Ferrous sulphate.7H2O 27.85
Sodium molybdate dehydrate 0.25 Copper sulphate.5H2O 0.025 EDTA disodium salt.2H2O 37.25
Cobalt chloride hexahydrate 0.025 Cobalt chloride.6H2O 0.025 Vitamins
Cupric sulfate pentahydrate 0.025 Ferrous sulphate.7H2O 27.8 Myo-Inositol 100
Disodium EDTA dehydrate 37.26 EDTA disodium salt.2H2O 37.3 Thiamine hydrochloride 0.5
Vitamins Vitamins Pyridoxine hydrochloride 0.5
myo-Inositol 100 Myo-Inositol 100 Nicotinic acid 5
Nicotinic acid 0.5 Thiamine hydrochloride 10 Folic acid 0.5
Pyridoxine hydrochloride 0.5 Pyridoxine hydrochloride 1 Biotin 0.05
Thiamine hydrochloride 0.1 Nicotinic acid 1 Glycine 2
Sugar Sugar Sugar
Sucrose 30000 Sucrose 20000 Sucrose 20000

Table 1: Composition of MS, B5 and Nitsch medium investigated in this study.

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.

Results and Discussion

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, 5.5 ± 0.22 in accession no.- 342109, 5.0 ± 0.22 in accession no.-347492 and 4.5 ± 0.22 in accession no.-331514. In case of Nitsch media highest shoot multiplication was observed as follow 4.3 ± 0.37 in accession no.-342109, 4 ± 0.33 in accession no.-331514 and 2 ± 0.31 in accession no.-347492. Whereas Gamborg’s B5 media showed lowest shoot multiplication in all the three accessions. Similar results were also reported where MS media was supplemented with 4.0 mg/l BAP+0.1mg/l NAA [17]. The details of each accession are mentioned in Table 2 (Figure 1).

Different media with standard hormones Accessions
342109 347492 331514
Number of Shoots
(M ± SE)
Length of Shoots
(in cm) (M ± SE)
Number of Shoots (M ± SE) Length of Shoots
(in cm) (M ± SE)
Number of Shoots (M ± SE) Length of Shoots
(in cm) (M ± SE)
 
MS + 4mg/l BAP + 0.4mg/l NAA 5.5 ± 0.22 2.6 ± 0.11 5.0 ± 0.22 2.9 ± 0.31 4.5 ± 0.22 2.1 ± 0.22
B5 + 4mg/l BAP + 0.4mg/l NAA 1.0 ± 0.38 1.4 ± 0.22 1.0 ± 0.33 0.6 ± 0.22 2.0 ± 0.38 0.35 ± 0.24
Nitsch + 4mg/l BAP + 0.4mg/l NAA 4.3 ± 0.37 1.25 ± 0.31 2.0 ± 0.31 0.8 ± 0.33 4.0 ± 0.33 0.48 ± 0.21

Table 2: Effect of different media on the number and length of shoots of three different accessions of Centella asiatica after six week of inoculation. Values are expressed as mean ± Standard Error (M ± SE). MS: Murashige and Skoog medium; B5: Gamborg’s B5 medium; BAP: 6-Benzyl amino purine; NAA: Naphthaleneacetic acid.

advanced-techniqe-Centella-asiatica

Figure 1: In vitro culture of Centella asiatica after six weeks of inoculation. I) MS, II) B5 and III) Nitsch Medium.

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 shoots was observed in MS medium with hormonal concentration of 1mg/l BAP followed by 2 mg/l BAP+0.5 mg/l NAA. Multiplication of shoots was observed after 14 days. After six weeks of incubation MS medium supplemented with 1mg/l BAP showed shoot multiplication as follow, 12.5 ± 0.22 in accession no.- 342109, 7 ± 0.22 in accession no.- 347492 and 11.5 ± 0.22 in accession no.- 331514. While MS medium supplemented with 2 mg/l BAP+0.5 mg/l NAA showed shoot multiplication of 5.3 ± 0.38 in accession no.- 342109, 6.2 ± 0.22 in accession no.- 347492 and 8.2 ± 0.33 in accession no.- 331514. Similar results were reported where BAP alone showed the good shoot 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).

MS Media with different hormonal concentrations 342109 347492 331514
Number of Shoots
(M ± SE)
Length of Shoots
(in cm)
(M ± SE)
Number of Shoots
(M ± SE)
Length of Shoots
(in cm)
(M ± SE)
Number of Shoots
(M ± SE)
Length of Shoots
(in cm)
(M ± SE)
 
MS+1mg/l BAP 12.5 ± 0.22 3.5 ± 0.32 7.0 ± 0.22 2.7 ± 0.31 11.5 ± 0.22 2.9 ± 0.24
MS+2mg/l BAP 3.0 ± 0.22 2.5 ± 0.22 3.0 ± 0.37 1.6 ± 0.24 4.0 ± 0.4 1.7 ± 0.31
MS+1mg/l BAP+0.5 NAA 2.7 ± 0.37 1.0 ± 0.31 2.0 ± 0.22 1.3 ± 0.32 3.0 ± 0.38 1.2 ± 0.21
MS+2mg/l BAP+0.5 NAA 5.3 ± 0.38 2.0 ± 0.24 6.2 ± 0.22 2.7 ± 0.24 8.2 ± 0.33 1.5 ± 0.22

Table 3: Effect of different hormone concentrations on number and length of shoots of three accessions of Centella asiatica after six week of inoculation. Values are expressed as mean ± Standard Error (M ± SE). MS: Murashige and Skoog medium; BAP: 6-Benzyl amino purine; NAA: Naphthaleneacetic acid.

advanced-techniqe-growth-accession

Figure 2: Figure showing effect of MS+1mg/l BAP on the growth in accession no-342109 (A), accession no-347492 (B) accession no-331514 (C) and also effect of MS+2mg/l BAP+0.5NAA on the growth in accession no-342109 (D), accession no-347492 (E) accession no-331514 (F).

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.

Acknowledgement

Sincere thanks to the Department of Biotechnology, Delhi Technological University for making necessary facilities during this study and National Bureau of Plant Genetic Resources for providing the plant materials.

References

  1. Glasby JS (1991) Dictionary of Plants Containing Secondary Metabolites. Taylor and Francis, London.
  2. Zainol NA, Voo SC, Sarmidi HR, Aziz RA (2008) Profiling of Centella asiatica (L.) Urbaqn Extract. The Malaysian Journal of Analytical Sciences 12:322-327.
  3. Chakraborty T, SinhaBabu SP and Sukul NC (1996) Preliminary evidence of antifilarial effect of Centella asiaticaon canine dirofilariasis. Fitoterapia 67: 110-112.
  4. Srivastava R, Shukla YN, Kumar S (1997) Chemistry and pharmacology of Centella asiatica: a review. J MediArom Plant Sci 19: 1049-1056.
  5. Oyedeji OA,Afolayan AJ (2005) Chemical composition and antibacterial activity of the essensial oil of Centella asiatica growing in South Africa (J). Pharmaceutical Biology 43:249-252.
  6. Inamdar PK, Yeole RD, Ghogare AB, De Souza NJ (1996) Determination of biologically active constituents in Centella asiatica. J. Chromatography742: 127-130
  7. Hausen BM (1993) Centella asiatica (Indian pennywort), an effective therapeutic but a weak sensitizer. Contact Dermatitis 29: 175-179.
  8. Herbert D, Paramasivan CN, Prabhakar R, Swaminathan G (1994) In vitro experiments with Centella asiatica: investigation to elucidate the effect of an indigenously prepared powder of this plant on the acid-fastness and viability of M. tuberculosis. Indian J Lepr 66: 65-68.
  9. Brinkhaus B, Lindner M, Schuppan D, Hahn EG (2000) Chemical, pharmacological and clinical profile of the East Asian medical plant Centella asiatica. Phytomedicine 7: 427-448.
  10. Jorge OA, Jorge AD (2005) Hepatotoxicity associated with the ingestion of Centella asiatica. Rev EspEnferm Dig 97: 115-124.
  11. Zainol MK, Abd-Hamid A, Yusuf S (2003) Antioxidative activity and total phenolic compounds of leaf, root and petiole of four accessions of Centella asiatica (L) Urban (J). Food Chemistry 81:575-581.
  12. Arora D, Kumar M,Dubey SD (2002) Centella asiatica-a review of its medicinal uses and pharmacological effects. Journal of Natural Remedies 2:143-149.
  13. Singh S, Gautam A, Sharma A,Batra A (2010) Centella asiatica L. a plant with immense potential but threatened. Internation Journal of Pharm. Sci Review and Research 4:9-17.
  14. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plant 15: 473-497.
  15. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151-158.
  16. Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163: 85-87.
  17. Das R, Hasan MF, Hossain MS,Rahman M (2008) Micropropagation of Centella asiatica L. an important medicinal herb. Progress. Agri 19: 51-56.
  18. George S, Remashree AB, Sebastian D,Hariharan M (2004)Micropropagation of Centella asiatica L. through axillary bud multiplication. Phytomorphology 54: 31-34.
  19. Wang PJ,Charle A (1991) Micropropagation through meristem culture in biotechnology in agriculture and forestry.Springer Verlag17: 41-44.
  20. Tiwari KN, Sharma NC, Tiwari V, Singh BD (2000) Micropropagation of Centella asiatica (L.), a valuable medicinal herb. Plant cell, Tissue and Organ Culture 63: 179-185.
  21. Karthikeyan K,Chandran C,Klothungan S (2009) Rapid clonal multiplication through in vitro axillary shoot proliferation of Centella asiatica L. Indian Journal of Biotechnology 8: 232-235.
Citation: Roy A, Kundu K, Saxena G, Kumar L, Bharadvaja N (2016) Effect of Different Media and Growth Hormones on Shoot Multiplication of In Vitro Grown Centella asiatica Accessions. Adv Tech Biol Med 4:172.

Copyright: © 2016 Roy A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Top