Journal of Pharmaceutical Biology www.jpbjournal.com e-issn - 2249-7560 Print ISSN - 2249-7579 THE EFFECT OF GROWTH REGULATORS ON SHOOT INITIATION, MULTIPLICATION AND ROOTING OF LAVENDER (Lavandula angustifolia L.) - AN SIGNIFICANT MEDICINAL PLANT Leelavathi D 1, Govinda Raju MV 2, Yashoda 3 and Narendra Kuppan* 1 Head of Department of Botany, MES Degree College of Science, Bangalore, Karnataka, India. 2 Block Technology Manager (ATMA), Department of Agriculture, Hunsur, Mysore district, Karnataka, India. 3 Global Scientific Research Foundation, Mathekere, Bangalore, Karnataka, India. ABSTRACT Abstract: Lavandula angustifolia-important Medicinal Plant, Nodal segment from micro propagated plants were used to evaluate the effect of growth regulators on the in vitro callusing and shoot proliferation and rooting from apical bud explants supplemented with BAP (8.88 μμ ) and NAA (5.36 μm). Rooting of the plantlets were obtained, when these shoots were inoculated on rooting medium, MSBM fortified with BAP (8.88µΜ) + NAA (2.68µΜ) + IBA (4.92µΜ) after 38 days of culture. However, rooting rate and root growth increased with increased concentration of NAA with half strength of MS medium. The plantlets were successfully transferred to soil and were grown till they reached maturity. MS basal medium supplemented with BAP 4.44 µμ + Kn 4.64 µμ + NAA 2.68 µμ produced highest frequency of shoots regeneration with highest number of long shoots. This established normal growth and development with 75-85% survival frequency. Key words: BAP: 6-Benzyl Amino Purine, MSBM: Murashige and Skoog Basal Medium (1962), NAA:α-Napthalene Acetic Acid, μm: micro molar, kinetin Kn, IBA- Indole 3 acetic acid. INTRODUCTION Lavandula angustifolia L. commonly known as lavender of the family Lamiaceae It is in great demand for the lavender oil it yields which is used in perfumery, cosmetics, flavouring and pharmaceutical industries. Such aromatic plants are gift of nature it should be protected and propagated [1] lavender is not only valued for its aromatic oil but also for the pharmaceutical properties. Lavender oil has a delightful clean refreshing and sweet odour with esters and linalyl acetate has the main constituents and free linalool is a major component of the English lavender oil, which has a peculiar heavy and slightly camphoraceous odour Lavender oil has a delightful clean refreshing and sweet odour with esters and linalyl acetate has the main constituents and free linalool is a major component of the English lavender oil, which has a peculiar heavy and slightly camphoraceous odour Lavender oil is used in most of the popular scents, its delightfully refreshing odour blends well with many other essential oils. It is also used in toilet soaps, talcum powders and as disinfectant formulations. The oil is used as a flavouring agent in medicine and as carminative. The oil of lavandin is used as a substitute for true lavender oil. On account of its microbicidal action it is used in the form of aerosols, for disinfecting houses, classrooms and public halls in Europe. Micro propagation technique for rapid and large scale propagation of medicinal and aromatic plants has been significantly increased [2-4] and gained importance in the recent times. MATERIAL AND METHODS The in vitro apical bud explants of Lavandula angustifolia were inoculated on Murashige and Skoog s basal medium (1992)(MSBM) fortified with auxins and cytokinins to induce callus, the callus was subcultured on the same medium to obtain profuse, compact whitish green morphogenetic callus. compact whitish green callus was subcultured on MSBM fortified with auxins and cytokinins for bud initiation and multiple shoot formation. All the cultures were maintained in an air conditioned room at a temperature 25 ± 2 o C with white fluorescent light at a photon density of 30-50 EM -2 S -1 under a Corresponding Author:- Narendra Kuppan Email:- narendrakuppan@gmail.com 66 P a g e
photoperiod regime of 16 hours light and 8 hours dark cycles. The in vitro raised plants with well-developed roots were subjected for hardening in a potting mixture, soilrite mixture containing peat: perlite: vermiculite (1:1:1v/v). After 40 days of hardening, the plants were transferred to the soil. The principal component Linalool was analysed by Gas Chromatography in the in vivo and in vitro stem and leaves of lavender. RESULTS The apical bud explants were inoculated on MS Basal Medium (Fig.1) callus was induced at the cut ends of the in vitro apical bud explants after 18 days of culture on MS basal medium fortified with BAP (8.88 µμ) + NAA (5.36 µμ) (Fig. 2a) 35 days old culture (Fig. 2b). After 48.days of subculture on the same medium large amount of profuse whitish green callus was observed (Fig.3). Shoot bud differenciation was observed after transferring it on MS Basal medium fortified with BAP (4.44 µμ) + Kn (4.64 µμ) + NAA (2.68 µμ) after 28 days of culture (Fig 4) the maximum number of shoot bud (3-5) formation was observed (Fig.4) Further, multiplication of shoot buds was noticed after 42 days of subculture (Fig.5). The well developed healthy, elongated multiple shoots (40) were observed after 56 days of subculture (Fig.6) (Table3).These shoots developed roots when inoculated on root inducing medium on MSBM fortified with BAP (8.88µΜ) + NAA (2.68µΜ) + IBA (4.92 µμ). After 38 days of culture multiple shoots with well developed roots were observed (Fig.7) and the plantlets were subjected to hardening (Figs.8&9). Field grown plants Fig.10) Field grown plants with inflorescene.fig.11) Stem and leaves of L.angustifolia in vivo and in vitro plants were subjected to phytochemical analysis for the determination and comparison of the principle component linalool. Table 1. Effect of different concentrations of cytokinin and auxins on in vitro apical bud explant of Lavandula angustifolia for callus induction Basal media BAP(µM) NAA (mg/l) NAA (µμ) Culture duration Response (%) MS 8.88 0.5 2.68 18 days 72 MS 8.88 1.0 5.36 18 days 92 MS 8.88 1.5 8.05 18 days 72 MS 8.88 2.0 10.74 18 days 67 MS 8.88 2.5 13.42 18 days 63 2,4-D (mg/l) 2,4-D (µμ) MS 8.88 0.5 2. 26 18 days 53 MS 8.88 1.0 4.52 18 days 68 MS 8.88 1.5 6.78 18 days 57 MS 8.88 2.0 9.04 18 days 50 MS 8.88 2.5 11.30 18 days 47 Table 2. Effect of different concentrations of cytokinin and auxins for the growth of the callus from in vitro apical bud explant of Lavandula angustifolia Basal BAP NAA NAA Culture Quantity Fresh Weight Dry Weight media (µμ) (mg/l) (µμ) duration of callus X *+ SD X *+ SD MS 8.88 0.5 2.68 36 + 14.58±0.68 0.81± 0.4 MS 8.88 1.0 5.36 36 +++ 18.46±2.23 2.50±0.51 MS 8.88 1.5 8.05 36 ++ 14.56±0.50 1.20±0.38 MS 8.88 2.0 10.74 36 ++ 13.59±0.53 0.80±0.41 MS 8.88 2.5 13.42 36 ++ 12.61±0.73 0.70±0.31 2,4-D (mg/l) 2,4-D (µμ) MS 8.88 0.5 2.26 36 + 10.65±0.66 0.75±0.28 MS 8.88 1.0 4.52 36 ++ 13.63±0.58 1.02±0.31 MS 8.88 1.5 6.78 36 ++ 11.52±0.50 0.69+0.30 MS 8.88 2.0 9.04 36 ++ 10.49±0.44 0.59±0.31 MS 8.88 2.5 11.30 36 + 09.56±0.43 0.48±0.17 Table 3. Anova Table (Fresh Weight ) SV DF SS MSS F cal ratio F tab value** CD Treatment 9 614.11 68.23 36.48 2.00 1.72 Errors 90 168.68 1.87 Total 99 728.79 67 P a g e
Table 4. ANOVA TABLE (Dry Weight ) SV DF SS MSS F cal ratio F tab value** CD Treatment 9 69.99 7.77 64.75 2.00 0.11 Errors 90 11.06 0.12 Total 99 81.50 Note: * : Mean of 10 replication ** : Significance F Value @ 5%level + : Scanty ++ : Moderate +++ : Abundant Table 5. Effect of different concentrations of cytokinins and auxins on regeneration from the callus of in vitro apical bud explant of Lavandula angustifolia Basal media Growth regulators (µm) Apical bud BAP KN NAA Response % No. of shoots /culture MS 2.22 2.32 2.68 40 18 MS 4.44 4.64 2.68 88 40 MS 6.66 6.96 2.68 66 30 MS 8.88 9.28 2.68 55 25 MS 11.11 11.60 2.68 44 20 BAP KN 2,4-D MS 2.22 2.32 2.26 35 16 MS 4.44 4.64 2.26 71 32 MS 6.66 6.96 2.26 55 25 MS 8.88 9.28 2.26 46 21 MS 11.11 11.60 2.26 37 17 Figure 1. Chromatogram of Linalool in vivo leaf of L. angustifolia Figure 2. Chromatogram of Linalool in vitro leaf of L. angustifolia 68 P a g e
Figures 3 showing various stages of cultures Graph 1. Effect of different concentrations of auxins on in vitro apical bud explant of Lavandula angustifolia for callus induction Graph 2. Effect of different concentrations of auxins on the growth of the callus from in vitro apical bud DISCUSSION It was found that BAP 8.88 µμ and NAA 5.36 µμ concentration was effective in inducing in vitro apical ud callus of L. angustifolia in comparison to 2,4-D 4.52 µμ. it was found in the present study, in vitro apical bud explants of L. angustifolia exhibited large amount of callus and multiple shoot formation with a fresh weight of 18.46 gm producing multiple shoots 40 in number and apical bud has induced callus after 18 days of culture. In the present study, higher concentration of NAA suppress the rate of shoot regeneration and enhance the proliferation of callus instead of organogenesis. MS basal medium supplemented with BAP 4.44 µμ + Kn 4.64 µμ + NAA 2.68 µμ produced highest frequency of shoot 69 P a g e
regeneration with highest number of long shoots. Frequency of shoot regeneration, number of shoots per callus and number of regenerated shoots were recorded at every subculture. The frequency of shoot regeneration reached its maximum after the fourth subculture (Table1). Published reports were available apical/axillary bud callus of different plants [3-14]. Further, the in vivo stem and leaf of L.angustifolia revealed major compounds such as Linalool was found to be 0.0079 % and in in vitro stem and leaf 0.004 %. REFERENCES 1. Leelavathi D And Narendra Kuan. Protocol For Rapid Clonal Multiplication Using In vitro Apical Bud Of Lavandula angustifolia, IOSR journal of pharmacy and biological sciences, 7(3), 2013, 96-98. 2. Bajaj YPS. Protoplast isolation, culture and somatic hybridization. In, Reinert, j. and Bajaj y.p.s (Eds.) Alied and fundamental aspects of plants cell tissue and organ culture, Springer-verlag, New York, 1977, 449-467. 3. Calvo MC and Segura J. In vitro morphogenesis from explants of Lavandula latifolia and Lavandula stoechas seedlings. Scientia Horticulturae, 36, 1988, 131-137. 4. Panizza, M and Tognoni F. Clonal Propagation, Callus formation and regeneration of Lavandin. Scientia Horticulturae, 37, 1988, 157-163. 5. Pratibha Misra and Chaturvedi HC. Influence of inorganic salts on cytokinin induced caulogenesis in leaf segments of Rosmarinus officinalis L. Plant Science, 79, 1991, 229-235. 6. Neena Kumari and Pardha Saradhi. Regeneration of plants from callus cultures of Origanum vulgare L. Plant Cell Reports, 11, 1992, 476-479. 7. Tawfik AA, Read PE and Cuett SL. Rosmarinus officinalis L. (Rosemary), In vitro culture, regeneration of plants, and the level of essential oil and monoterpenoid constituents. Biotechnology in Agriculture and Forestry, 41, 1998, 350-365. 8. Dronne S, Colson M, Moja S and Faure O. Plant regeneration and transient GUS expression in a range of lavandin (Lavandula x intermedia Emeric ex Loiseleur) cultivars. Plant cell, Tissue and Organ Culture, 55, 1999, 193-198. 9. Tsura M, Koda M, Inoue M. Comparative effect of different types of cytokinin for shoot formation and plant regeneration in leaf-derived callus of lavender (Lavandula vera DC). Scientia Horticulturae, 81, 1999, 331-336. 10. Youssef AA and Rady MR. Effect of salt stress on the essential oil content and composition of Rosmarinus officinalis callus cultures. Egypt. J. Hort, 27(1), 2001, 69-79. 11. Sairam Reddy P, Rosaline Rodrigues and Ram Rajasekharan. Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell, Tissue and Organ Culture, 66, 2001, 183-188. 12. Leelavathi D, Narendra Kupan, Yashoda, An Efficient Protocol for in vitro Multiplication of origanum vulgare L- An Important Medicinal Plant Using Cytokinin and Auxins. Int.J.Biopharm.Nanomed.Sci, 3(1), 2014, 166-173. 13. Leelavathi D and Narendra Kuan. Plant regeneration from in vitro axillary bud of Ocimum basilicum l. - an important medicinal plant. International Journal of Biology and Allied Sciences, 2(11), 2013, 2137-2141. 14. Leelavathi D and Narendra Kuan. In vitro regeneration from apical bud explants of Rosmarinus officinalisl.-an Important Aromatic and Medicinal plant. Banat s Journal of Biotechnology. 4(8), 2013, 1-4. 70 P a g e