INTRODUCTION:

Euphorbia hirta L. is a medicinal, rhizomatous herb distributed in southern-western Ghats of India and northern east coast of Tamil Nadu^[1]. It is the largest genus of family Euphorbiaceae with about 1600 species. All species of Euphorbia exudes a milky juice when broken, which is more or less poisonous and used as an ingredient in arrow poisons. E. hirta is distributed throughout the hotter parts of India and Australia, often found in waste places along the roadsides^[2].

It is a small, erect or ascending annual herb reaching up to 50 cm, with hairy stems. The leaves are opposite, elliptical, oblong or oblong-lanceolate, with a fainty toothed margin and darker on the upper surface^[3].

E. hirta possesses antibacterial, anthelmintic, antiasthmatic, sedative, antispasmodic, antifertility, antifungal, and antimalarial properties^[4]. In East and West Africa extracts of the plant are used in treatment of asthma and respiratory tract inflammations. It is also used for coughs, chronic bronchitis and other pulmonary disorders in Malagasy. The plant is also widely used in Angola against diarrhea and dysentery, especially amoebic dysentery. In Nigeria extracts or exudates of the plant are used as ear drops and in the treatment of boils, sore and promoting wound healing^[5]. This plant has been studied by various workers and a number of active constituents have been isolated. Afzelin, quercitrin and myricitrin have been isolated from the methanolic extract of E. hirta^[6].

E. hirta has great ethano-medicinal value but so far very little work has been done for in vitro cloning and characterization of this plant^[7]. Present study demonstrates direct regeneration of shoots from the surface of explants on MS medium.

Materials and Methods

The plant material was collected from the east coast of south India which includes the coastal Puducherry and Tamil Nadu. The survey was conducted to select the superior and healthy plant material from January to December, 2012. The fresh growth of the plant material was collected from different places and brought to the biotechnology lab. Some plants were maintained in our green house to get better and healthy plants. Apical shoot tips and shoot segments were used as explants to initiate the cultures.

2-3 cm long shoots each with 1-2 nodes were used as explants. The explants were cleansed, dressed and washed with soap water. Explants collected were treated with 0.1% Bavistin for 5-10 minutes. These were surface sterilized with 0.1% HgCl₂ for 4-5 minutes and washed 5-6 times with autoclaved distilled water under Laminar Air Flow Cabinet, (Technico Systems, Chennai) and inoculated aseptically onto MS basal medium^[8].

The explants were inoculated vertically on MS medium for culture initiation. Different concentration and combination of cytokinins (BAP and Kn ranging from 1.0 to 5.0 mgl^-1) and IAA ranging from 0.1 to 2.0 mgl^-1 were incorporated in the medium for induction of buds from the explants. These cultures were incubated at 28±2°C in diffused light (22 µ molm^-2 s^-1 Photosynthetic Photon Flux Density, PPFD) for 8-10 days. The light was provided by fluorescent tubes and incandescent bulbs.

The in vitro regenerated shoots were multiplied by repeated transfer of mother explants on fresh medium and subculturing of in vitro produced shoots on fresh medium. For multiplication of cultures, MS medium supplemented with various concentration and combination of cytokinins (BAP and Kn ranging from 0.1 to 2.0 mgl^-1) were used. The cultures were incubated at 28±2°C temperature, 60-70% RH and 30-35 µmol m^-2s^-1 PPFD for 12 h/d.

For the rooting of in vitro produced shoots, the shoots were isolated of appropriate size and these were rooted on half strengths of MS medium containing auxins (IAA and IBA ranging 0.5 mgl^-1 to 3.0 mgl^-1).

The in vitro rooted plantlets were washed with autoclaved distilled water to remove adhered nutrient agar and then transferred to soil mixture (sand, soilrite, organic manure and black soil in 1:1:1:1 ratio) filled in bottles moistened with one-fourth strength of MS basal salts. After 15 days the caps were loosened and finally removed. Plants hardened in bottles were transferred to polybags containing soil mixture. The hardened plantlets were finally transferred to the pots.

OBSERVATION AND DATA ANALYSIS:

The cultures were regularly subcultured on fresh medium after 4-5 weeks interval. The observations were taken after every seven days of inoculation. The experiments were repeated thrice with ten replicates per treatment. The rate of multiplication represents number of shoots produce per explant on a specific medium after number of days of its inoculation as mentioned in the results. The data were subjected to statistical analysis.

RESULTS AND DISCUSSION:

E. hirta is a high valued medicinal plant used in the treatment of gastrointestinal disorders, bronchial and respiratory diseases, and in conjunctivitis from centuries. Extracts of E. hirta have been found to show anticancer activity and strongly reduced the release of prostaglandins I_2,E_2, and, D₂^[9]. The aqueous extract also inhibits aflatoxin contamination in rice, wheat, maize, and mustard crops^[10].

Micropropagation protocols based on the tendency of excised stem tip, nodes or lateral buds to form single or multiple axillary shoots have been utilized to clone a number of plant species^{[11, 12]}. Nodal shoot explants of E. hirta collected in the months of March and November were found to be most suitable for culture initiation in present investigation. Shoot buds appeared within 7-15 days from the surface of the explants. Maximum numbers of shoots were appeared from the lower part of explants, which is in direct contact with the medium as compared to the upper part of the explants (Fig. 1F and 2A). Explants collected from immature plants respond earlier (within 7 days) than the explants collected from old plants. Mature leaves were also utilized as explants for the initiation of callus in cultures in E. hirta by Baburaj et al. ^[7]. But direct shoot regeneration was not observed by them. Nodal segments induced multiple shoots in many plants like Ceropegia candelabrum ^[13], Echinopus spinossimum ^[14] etc. Castellanos et al. ^[15]induced multiple shoots from the nodal shoot segments of Poinsettia (E. pulcherrima).

Percentage of response varied from 37% to 100% on MS medium. Nodal segments in E. hirta showed higher rate of multiple shoot induction when BAP alone was used with MS medium. When cytokinin enriched media were supported with auxins for nodal cultures caulogenesis was recorded, with a reduced number of shoots. Similar types of results were observed by Shekhawat et al. ^[16]. Small amount of callus was also observed by Baburaj et al. ^[7] when NAA was used with BAP in case of E. hirta leaf culture. No multiple shoot induction was observed in nodal explants when cultured in media without cytokinin. MS medium supplemented with BAP was found to be more effective than Kn. 2.0 mgl^-1 of BAP proved to be the most effective for induction of multiple shoots. On this medium 6-7 shoots were induced from the lower part of the explants (Fig. 1A and 1B).

The rate of shoots multiplication was significantly different according to the various concentrations and combinations of cytokinins supplemented. Cytokinin level produced a significant response upon the number of shoots formed per explant. Amongst various cytokinins tested for shoot multiplication, MS medium containing 0.5 mgl^-1 each of BAP and Kn proved to be the best for shoot multiplication (Fig. 1C). The effect of different cytokinins and auxins, their concentrations and combination on shoot multiplication and rooting is shown in Fig. 2. About 44-56 shoots were regenerated in a single culture flask on this media combination (Fig. 2B, 2C and 2D). The present results were better than the findings of ^[7]. They regenerated 15-22 shoots from the callus when the callus was transferred to MS medium supplemented with BAP and NAA. Sub-culturing was found to be essential after a period of 20-25 days; otherwise necrosis and tip burning of the shoots were observed.

Fig. 1A and 1B: Effect of BAP and Kinetin concentrations on % response and induction of number of shoots from the explants of E. hirta.

Fig. 1C: Effect of BAP and Kinetin concentrations on shoot multiplicaiton in E. hirta.

Fig. 1D and 1E: Effect of auxins (IBA and IAA) concentrations on root initiation from the shoots in vitro.

Fig. 1F: Induction of multiple shoots from the surface of explants.

Fig. 2A. Induction of multiple shoots from the base of explant.

Fig. 2B and 2C. Elongation of multiple shoots.

Fig. 2D. Multiplication of shoots on MS medium supplimented with 0.5 mgl^-1 BAP and Kn.

Fig. 2E and 2F. Induction of roots from the isolated shoots in vitro on half strength MS medium with IBA.

Fig. 2G and 2H. Acclimatization of plantlets in culture room and in green house.

The individual shoots produced in culture were excised and transferred on root induction medium. Half strength MS medium containing 2.0 mgl^-1 IBA was found to be most suitable medium for induction of viable roots from shoots within 2 weeks (Fig. 1D, 1E, 2E and 2F). On this combination 89% shoots were rooted. Auxins have been widely used for in vitro and ex vitro rooting of plants. ^{[15 and 17 ]}reported that auxins are essential for root growth and development. These results were contrary to the findings of Baburaj et al. ^[7], they produced roots from the shoots on half strength MS medium augmented with 2.0 mgl^-1IAA in E. hirta. In most of the plant systems, IBA was used for efficient rooting ^{[18, 19, 20]}. In vitro rooted plantlets were transferred to soil mixture filled in glass bottles moistened with one-fourth strength of MS basal salts (Fig. 12G). These were kept in the green house. After 30 days these plantlets were transferred to the pots (Fig. 2H).

CONCLUSION:

Direct shoot regeneration from cultured explants is important to minimize somaclonal variation in regenerated plants. The protocol developed for cloning of E. hirta is highly efficient and reproducible. It can be used for large-scale amplification of selected elite clones.

REFERENCES:

1. Rahuman AA, Geetha G, Venkatensan P and Kannapan G. Larcicidal activity of some Euphorbiaceae plant extracts against Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae). Parasitology Research. 2007: 839-846.

2. Sood SK, Bhardwaj R and Lakhanpal TN. Ethnic Indian plants in cure of diabetes. Scientific publishers, India. 2005.

3. Patil SB, Nilofar SN and Magdum CS. Review on phytochemistry and pharmacological aspects of Euphorbia hirta L. J. Pharmac. Rese. Health Care. 1(1); 2009: 113-133.

4. Williamson EM. Major Herbs of Ayurveda. Churchill Livingstone, China. 2002.

5. Ogueke CC, Jude N, Ifeanyi C, Okoli and Beatrice NA. Antibacterial activities and toxicological potentials of crude ethnolic extracts of Euphorbia hirta. Journal of American Science. 3(3); 2007: 11-16.

6. Murakami LYN, Ji H, Abreu P and Zhang S. Antimalarial flavonol glycosides from Euphorbia hirta. Pharm. Biol. 45; 2007: 278–81.

7. Baburaj S, Dhamotharan R and Santhaguru K. Regeneration in leaf callus cultures of Euphorbia hirta Linn. Current science. 56; 1987: 194.

8. Murashige T and Skoog F. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15; 1962: 473–479.

9. Anonymous. The Wealth of India (Raw material), Vol. 3. Council of Industrial and Scientific Research. New Delhi, 2005.

10. Singh P and Sinha KK. Inhibition of aflatoxin production on some agricultural commodities through aqueous plant extracts. J Indian Bot Soc. 65; 1986: 30–2.

11. Al-Bahrany AM and Al-Khayari JM. Micropropagation of grey mangrove Avicennia marina. Plant Cell Tissue and Organ Culture. 2: 2003:87-93.

12. Shekhawat MS and Shekhawat NS. Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and suspension culture. Acta Physiologiae Plantarum. 33; 2011: 1445-1450.

13. Beena MR, Martin KP, Kirti PB and Hariharan M. Rapid in vitro propagation of medicinally important Ceropegia candelabrum. Plant Cell Tissue and Organ Culture. 72; 2003: 285-289.

14. Murch SJ, Wierenga EJ, El-Demerdash MA and Saxena PK.. In vitro propagation of the Egyptian medicinal plant Echinops spinosissimus Turra. Plant Cell Tissue and Organ Culture. 74; 2003: 81-86.

15. Castellanos M, Power BJ and Davey MR. Micropropagation of Poinsettia by organogenesis. Methods in Molecular Biology. 589; 2010; pp 67-75.

16. Shekhawat MS, Shekhawat NS, Harish, Kheta Ram, Phulwaria M and Gupta AK. High frequency plantlet regeneration from nodal segment culture of female Momordica dioica (Roxb.) J. Crop Sci. Biotech, 14(2); 2011: 133-137.

17. Shekhawat MS, Manokari N, Kannan N and Pragasam A. In vitro clonal propagation of Cardiospermum helicacabum L. through nodal segment culture. The Pharma Innovation. 1(7); 2012: 1-7.

18. Loreti F, Morini C and Hu I. Effect of potassium and nitrogen concentration on growth of peach shoots cultured in vitro. Act. Hort. 227; 1998: 311-317.

19. Roy PK, Singh B, Mehta SL, Barat GK, Gupta N, Kirti PB and Chopra VL. Plant regeneration from leaf disc of Lathyrus sativus. Indian Journal of Experimetnal Biology. 29; 1991: 327-330.

20. Shekhawat MS, Kannan N, Manokari M, Revathi J. Induction of Shoots from In vitro Cultured Roots of Oldenlandia umbellata L. – A Dye-yielding Plant. Research Journal of Pharmacognosy and Phytochemistry. 5(1); 2013: 22-25.