In vitro clonal propagation of vulnerable medicinal plant, Mimosa pudica L.

ISSN: 2321-0516 (Online) International Journal of Current Research and Development Available Online at http://www.journalcrd.com Research Article 2013,July, Vol.2 (1): 18-30 Copy Right, 2013 IJCRD In vitro clonal propagation of vulnerable medicinal plant, Mimosa pudica L. Ramesh.S 1, Chandran.C 1,Venkatesan.G 2. 1 Department of Botany, A.V.V.M. Sri Pushpam College (Autonomous), Poondi, Thanjavur. 2 Department of Botany,Mannai Rajagopalasami Govt.Arts College,Mannargudi-614 001. Thiruvarur.India Author 2 Email: gv2032@gmail.com Abstract Mimosa pudica L. is a medicinal herb. The in vitro regeneration protocol of this plant through shoot tip and nodal, Root tip explants. The explants were culured on MS medium supplemented of different concentrations Benzyl amino acid purine (BAP), Kinetin (KN), 2,4-Dichlorophenoxy acetic acid (2,4-D), IAA, NAA, IBA and GA 3 were (0.5 to 2.5 mg/l) used. Elongation of regenerated shoots tip, nodel, shoot tip using different growth regulators. Induction of roots from the regenerated shoots. Hardened regenerant were acclimatized to the soil. Key Words: Mimosa pudica, clonal propagation, Root, Nodal segments, Shoot tip explants, growth regulators. Introduction Use of plants as a source of medicine has been inherited and is an important. There are about 45,000 plants species in India with concentrated hotspot in the region of Eastern Himalayas, Western Ghats, Andaman and Nicobar Islands. The officially documented plants with medicinal potential are more than 6000 plants. India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world (Nayar, 1979). Mimosa pudica L. is a creeping annual or perennial herb often grown for its curiosity value, as the compound leaves fold inward and droop when touched and reopens within minutes. It belongs to the family Mimosaceae. The species epithet Pudica is a Latin equivalent for Bashful or shrinking because of its curious nature and easy procreation. Mimosa pudica has been extensively used in ayurveda, unani and homeopathi medicine and has become cynosame of modern medicine. It is also used in jaundice, asthma, conjunctivitis, cut wound and glandular swelling liver is considered the key organ in the metabolism, detoxification and secretary functions in the body and its disorders 18

Ramesh et al.,ijcrd 2013,Vol.2(1):18-30 are numerous with the no effective remedies to sterile.distillied water under laminar airflow climate pain Mimosa pudica have been chamber to remove all traces of sterilizing popularly known as thoothache plant. The plants agents (George and Sherrington,1984). has been well documented for its use as species Generally, to disinfect the plant tissues various antiseptic, antimicrobial, antimalarial, sterilized agents have been used. Mercuric immunostimulating and diuretic activity and is chloride was found to be a very effective used as remedy for flu, cough, rabies, disease sterilizing agent at 0.1% concentration in and tuberculosis (Mukesh Chandra et al., 2010). Mumosa pudica. The chlorine gas released from The present study aims to develop an efficient, HgCl 2 was very penetrating that it destroyed the rapid regeneration protocol from Mimosa microorganisms present in most tissues of the pudica Linn. explant (Hall et al.,1993). Materials and methods Culture medium Collection and Sterilization of explants Mimosa pudica was collected from the wild population in Mannargudi, Tiruvarur district during the month of September 2012. The explants were then prewashed with Tween 20 emulsifier (2-3 drops in 100 ml sterile distilled water) for one minute followed by rinsing three times in sterile distilled water. Prior to surface sterilization, antibrowning treatment was given to control phenol exudation from the cutend of the tissues. Before disinfection, the explants were washed with 70% ethanol(v/v) for 10-25 seconds and surface sterilized with 0.1% (v/v) mercuric chloride (HgCl 2 ) for 2-6 minutes. For better contact of the sterilent (0.1% HgCl 2 ) with the explants, they were stirred for few minutes while disinfesting. The surface sterilized explants were finally rinsed in Nutritional support must be essential for optimal growth of a tissue by in vitro. The nutritional supplementation in the medium varies with the species. Selection and preparation of particular media is one of the important steps in the in vitro studies. The nutrient media consists of inorganic nutrients, carbon source and organic supplements. In addition, vitamin and growth regulators are also added to this media. In the present study MS (Murashige and Skoog, 1962) medium supplemented of different concentrations Benzyl amino acid purine (BAP), Kinetin (KN), 2,4-Dichlorophenoxy acetic acid (2,4-D), IAA, NAA, IBA and GA 3 were (0.5 to 2.5 mg/l) used. Elongation of regenerated shoots tip, nodel, shoot tip using different growth regulators.medium with B5 vitamin combinations of different growth regulators are used. 19

Ramesh et al.,ijcrd 2013,Vol.2(1):18-30 Results callus after 5 weeks (Table 1).Various Plant tissues normally grow in an organized fashion in which specific cell types differentiate from nonspecialised meristematic cells. Plant developmental processes can be modified by culture in vitro in a suitable nutrients medium concentration of BAP were used (0.5 to 2.5 mg/l), IBA at 0.6 mg/l 80% of single shoot growth with mean shoot length was 2.0 cm.out of different concentrations (0.2 to 1.0 mg/l), IBA at 0.4 mg/l has given 65% at growth with and with the application of plant growth mean shoot length of 1.2 cm and induced for regulators. The interactions of the main groth regulators can be complex, but at the simplest level auxins can cause cell enlargement and amount of basal callus after 3 weeks (Table 2).With constant concentration of NAA at (0.4 mg/l) and IBA (0.6 mg/l) along with various division, cytokinins cause cell division, concentration of BAP (0.5 to 2.5 mg/l) were gibberellins cause elongation, and abscisic acid used. The concentration of BAP at 1.5 mg/l inhibits growth (Michal et al., 1988).Explants of combined with IBA and NAA showed field grown Mimosa pudica (shoot tip, Root, node and leaf) was washed with tap water and Teepol solution and sterilized by 0.1 per cent mercuric chloride (HgCl 2 ). After sterilization explants were inoculated in MS basal medium. After 7 days, growth of shoots was observed. The MS medium was supplemented with different concentrations of hormones (IAA, NAA, BAP, KN, 2,4-D, GA 3 ) and combination of BAP + IAA + NAA. It initiates, basal callus and shoot proliferation. The shoot tip obtained here was used as explant source in approaching experiments. Shoot multiplication from shoot tip explants Among the different concentration (0.5 to 2.5 mg/l) used. 1.5 mg/l was given 100% growth of mean shoot length was 4 cm multiple shoots were observed in 2 mg/l and induced basal maximum growth with 2.3 cm mean shoot length (Table 1).With constant concentrations of IBA (0.6 mg/l) and NAA (0.4 mg/l) along with various concentrations of kinetin (0.5 to 2.5 mg/l) were used. At 2.0 mg/l concentration of BAP with IBA and NAA maximum amount of growth with mean shoot length of 4.0 cm was observed (Table 2). Shoot multiplication from nodal explants Different concentration of KN (0.5 to 2.5 mg/l) used, IBA at 1.5 mg/l has given 70% growth of shoot with a mean shoot length of 3.0 cm (Table 3 and Fig. 1).Different concentration of KN (0.5 to 2.5 mg/l) was used. The maximum amount of growth was observed at 2.0 mg/l with 81% of response with a mean shoot length of 4.5 cm (Table 4). Different concentrations of IBA (0.2 to 1.0 mg/l) used. The highest percentage of 20

Ramesh et al.,ijcrd 2013,Vol.2(1):18-30 growth response with 60% and mean shoot concentration (Table 6).The rooted plantlets length of 4.5 cm observed at 0.4 mg/l (Table 3 were hardened in unsterilized soil substrate and Fig. 1). Various concentrations of IBA (0.5 showed 51.8% of plant growth and it is to 2.5 mg/l) were used with concentrations of incubated in 20 2 C (Table 7 and Fig KN (0.4 mg/l) and NAA (0.6 mg/l). Out of these 5).Whereas the rooted plantlets were hardened 1.5 mg/l IBA showed maximum amount in unsterilized soil substrate showed 65.5% of response (80%) with mean shoot length of 2.0 plant growth which was incubated in shade. cm (Table 4). Repeated subcultures of shoot tip Discussion and nodal explants further enhanced multiple Medicinal plants are becoming very rare and shoot initiation response. Even BAP at the level extinct and some plants were endangered in of 1.5 mg/l maximum number of shoots were nature. These plants can be regenerated (or) observed.with constant concentration of NAA grown by the micropropagation technique. The at 0.5 mg/l and BAP with various concentrations in vitro technique can profitably be utilized for (0.5 to 4.0 mg/l) were used. The concentration the regeneration of plants. Micropropagation has of BAP at 2.5 mg/l combined with NAA been made successful through the uses of showed maximum growth of multiple shoot various explants such as leaf, node, inter node, initiation growth response (Table 4 and Fig.2). shoot tip and also by various parts of the Shoot elongation plants.in the present study, different explants The dwarf shoots observed on the shoot tip like shoot tip and node of Mimosa pudica L. explants which was inoculated on the MS were cultured on different concentrations of medium supplemented with BAP at 1.5 mg/l, hormones (BAP, KN, IAA, NAA, IBA and GA 3 (1.0 mg/l) was found to be the suitable GA 3 ) of hormones (BAP, KN, IAA, NAA, IBA concentration for high frequency of shoot and GA 3 ) and different combination of (BAP + elongation. The entire plantlets was grown upto IAA + NAA, KN + IAA + NAA; BAP + IAA). 35 cm (Table 5 and Fig.2). It showed that the nodal explants, shoot tip Rooting and Hardening explants after one week of inoculation response The elongated shoots were excised from the like shoot initiation. culture tube and subcultured subsequently Explants from in in vitro raised shoots in BAP rooted on MS media containing IBA. The supplemented medium were found with more highest rooting was obtained at IBA of 2.0 mg/l response than those of wild plants. Saritha and Naidu (2007) achieved induction of more 21

Ramesh et al.,ijcrd 2013,Vol.2(1):18-30 number of auxillary bud developments in Mimosa pudica L. from shoot tip explants on medium containing KN is proved to be superior to BAP for inducing from shoots. Arachis stenosperma and A. villosa is contrast in the present experiment BAP induced more number of multiple shoots than the KN, and the BAP was found to be less efficient in shoot multiplication (Fig.2). In Mimosa pudica shoot tip and nodal explants cultured on BAP containing medium showed maximum number of multiple shoots (Haw and Keng, 2003).In accordance with earlier report the present result deals that KN was found less efficient in shoot multiplication maximum number of shoot healthy normal and elongated shoots for the induction was flowering and also for rooting and survival. It was done by transferring the shoot clumps to the elongation medium having lower level of BAP, NAA and GA 3. These results confirm made by Waseem et al. (2009).In the present experiment, healthy shoots with maximum growth have been observed on BAP (2.0 mg/l) and GA 3 (1.0 mg/l) is suggested that the lower level of BAP and higher level of GA 3 influences the growth of shoots on the shoot elongation medium (Fig.2). But in contrast BAP alone at low concentration had a promoting effect on shoot regeneration and also shoot elongation (Saritha et al., 2002). multiplication was observed in IBA Rooting and Hardening concentration (Table 3 and Fig. 1)). In the present experiment, the shoot tip produced more number of shoots than nodal. Among the two cytokinins (BAP and KN) tested with or without auxins BAP was found to the ideal hormone for the induction of multiple shoot. BAP has been reported to overcome the apical dominance, and later it releases lateral buds from dormancy and promote shoot formation. Whereas the KN also produced the maximum number of shoots and sometimes the numbers of shoots from the medium supplemented with KN was low and the shoots formed were thin combined effect of BAP and KN also produced multiple shoots (Fig.4).The importance of this step was to get The previous and earliest observation (Saritha and Naidu, 2007; Singh et al., 2009) best rooting was achieved in the medium with reduced basal nutrients and IBA. This step is very important for the plant survival and critical step in the production of complete entire plantlets. In the present experiment from full to half strength in the basal medium was sufficient for the rooting of shoots.in many plant species IBA is considered as an important and effective growth regulator for the induction of roots (Table 7and Fig 5). In the present experiment 2 mg/l IBA produced maximum number of healthy roots similar observation are already made in Mimosa pudica (Saritha and Naidu, 22

Ramesh et al.,ijcrd 2013,Vol.2(1):18-30 2007). In the present experiment continuous Michael G.K.Johes, Neil Fish, keith subcultures in shoot multiplication medium Lindsey.1988.plant tissue culture, Methods in improved rooting significantly two incubation molecular Biology.4:499-517. conditions treated for hardening. Mukesh Chandra Sharma and Smita Sharma, 2010. Phytochemical and pharmacological screening of Conclusion combined Mimosa pudica Linn. Mimosa pudica L. is a medicinal herb. The in vitro regeneration protocol is an efficient means Murashige.T., and Skoog.F.1962. a revised medium of ex-situ conservation of plant diversity and for repid growth and bioassays with tobacco tissue vital this medicinal plant maintennance of the culture. Physiologia plantarum.15:473-497. present day speedily dwindling germplasm on Nayar,M.P.1987. in situ conservation of wild flora long term basis, especially for the medicinal resources. Bull.Bot. Surv. India.29: 319-333. plants. Rooted plantlets incubated under shade Saritha, K.V., and Naidu, C.V., 2007. High conditions for hardening gave maximum frequency plant regeneration and in vitro flowering success. of regenerated plantlets of Spilanthes acmella Murr. References An important threatened bioinsecticide medicinal George,E.F. and Sherrington,P.D.1984. Plant plant. Acta. Hort., 756: 183-198. qpropagation by Tissue culture.exogenetical Saritha, K.V., Prakash, E., Ramamurthy, N., and Limited, england. Naidu, C.V., 2002. Micropropagation of Spilanthes acmella Murr. Biol. Pl., 45: 581-584. Hamill,S.D., Sharrock,S.L and Smith, Singh, S.K., Rai, M.K., Asthana, P., Pandey, S., M.K.1993. comparison of decontamination Jaiswal, V.S., and Jaiswal, V., 2009. Plant methods used in initiation of banana tissue regeneration from alginate-encapsulated shoot tips cultures from field collected sukers, Plant of Spilanthes acmella (L.) Murr., a medicinally cell tisusue organ cult.33: 343-346. important and herbal pesticidal plant species. Acta Haw,A.B.,andKeng,C.L.,2003 Physiol. Pl., 31: 649-653. Micropropagation of Spilanthes acmella L., Wasseem, K., Jililani, M.S., and Khan, M.S., 2009. a bioinsecticide plant through proliferation of Rapid plant regeneration of Crysanthemum multiple shoots. J. Appl. Hort., 5(2): 65-68. (Crysanthemum moratorium L.). African J. Biotechnol., 8 (9): 1871-1877. 23

Table 1. Effect of BAP, NAA, IBA in shoot multiplication on MS media from shoot tip explants after 4 weeks of culture Growth regulators (mg/l) BAP NAA IAA Culture showing response (%) Mean shoot / explant Mean shoot length (cm) Basal callus 0.5 - - 52 1 5.1 + 1.0 - - 60 3 4.2 + 1.5 - - 90 4 3.0-2.0 - - 75 2 4.0 + 2.5 - - 60 1 5.2 + - 0.2-40 1 3.2 + - 0.4-60 2 3.4 + - 0.6-80 3 2.0 + - 0.8-60 2 4.0 ++ - 1.0-50 1 5.3 +++ - - 0.2 70 2 1.5 + - - 0.4 90 3 2.0 + - - 0.6 80 4 3.0 + - - 0.8 70 3 1.9 ++ - - 1.0 60 2 1.0 +++ 0.5 0.6 0.4 80 7 1.3 + 1.0 0.6 0.4 80 8 2.0 + 1.5 0.6 0.4 90 8 2.5-2.0 0.6 0.4 70 9 3.0 ++ 2.5 0.6 0.4 65 8 2.7 ++ Shoot induction have been given in different grades viz., - No response, + poor, ++ moderate, +++ high 24

Table 2. Effect of various concentration of BAP, NAA, IBA in shoot multiplication on MS media from nodal explants after 4 weeks of culture. Growth regulators (mg/l) BAP IAA NAA Culture showing response (%) Mean shoot / explant Mean shoot length (cm) Basal callus 0.5 - - 40 2 5.0 + 1.0 - - 60 3 4.8 ++ 1.5 - - 80 4 3.0 +++ 2.0 - - 65 3 4.2 + 2.5 - - 50 2 5.1 + - 0.2-50 1 1.5 - - 0.4-70 3 1.5 - - 0.6-90 2 2.0 - - 0.8-75 2 2.0 - - 1.0-60 1 2.2 - - - 0.2 55 2 1.3 ++ - - 0.4 70 2 1.9 +++ - - 0.6 90 4 1.2 + - - 0.8 50 3 1.6 + - - 1.0 40 1 2.0 + 0.5 0.4 0.6 70 6 1.9-1.0 0.4 0.6 70 8 2.5-1.5 0.4 0.6 90 8 3.0-2.0 0.4 0.6 80 9 3.5-2.5 0.4 0.6 60 8 4.1 - Shoot induction have been given in different grades viz., - No response, + poor, ++ moderate, +++ high 25

Table 3. Effect of various concentrations of KN, NAA, IBA in shoot multiplication on MS media from shoot tip explant after 4 weeks of culture. Growth regulators (mg/l) KN NAA IBA Culture showing response (%) Mean shoot / explant Mean shoot length (cm) Basal callus 0.5 - - 70 2 2.2 + 1.0 - - 80 3 3.5 + 1.5 - - 90 3 4.0 + 2.0 - - 80 4 4.0 + 2.5 - - 70 4 4.7 + - 0.2-50 2 1.5 + - 0.4-60 3 2.2 ++ - 0.6-80 4 3.0 ++ - 0.8-70 4 4.0 +++ - 1.0-70 3 4.1 +++ - - 0.2 60 2 2.0 + - - 0.4 65 2 3.2 + - - 0.6 80 3 3.8 + - - 0.8 85 4 4.0 ++ - - 1.0 70 14 2.2 +++ 0.5 0.4 0.6 70 3 4.2 + 1.0 0.4 0.6 70 3 3.8 + 1.5 0.4 0.6 80 5 3.5 ++ 2.0 0.4 0.6 90 2 4.5 + 2.5 0.4 0.6 80 2 3.2 ++ Shoot induction have been given in different grades viz., - No response, + poor, ++ moderate, +++ high 26

Table 4. Effect of various concentrations of KN, NAA, IBA in shoot multiplication on MS media from nodal explant after 4 weeks of culture Growth regulators (mg/l) KN NAA IBA Culture showing response (%) Mean shoot / explant Mean shoot length (cm) 0.5 - - 40 2 2.5-1.0 - - 60 2 3.1-1.5 - - 60 4 3.5-2.0 - - 80 4 4.0-2.5 - - 85 3 4.1 - - 0.2-55 2 1.9 ++ - 0.4-60 3 2.7 ++ Basal callus - 0.6-80 3 3.0 +++ - 0.8-80 4 3.2 +++ - 1.0-90 5 4.0 +++ - - 0.2 40 3 1.6 ++ - - 0.4 60 3 1.7 ++ - - 0.6 70 4 2.5 +++ - - 0.8 80 4 3.0 +++ - - 1.0 80 3 3.9 ++ 0.5 0.6 0.4 60 2 2.7-1.0 0.6 0.4 60 2 3.2-1.5 0.6 0.4 80 4 3.9-2.0 0.6 0.4 80 4 4.5-2.5 0.6 0.4 90 5 4.5 - Shoot induction have been given in different grades viz., - No response, + poor, ++ moderate, +++ high 27

Table 5. Dwarf shoots were transferred to shoot elongation media containing BAP and GA 3 in different concentrations Concentrations (mg/l) BAP GA 3 Culture showing response (%) Average shoot length (cm) 1.5 0.2 60 2.2 1.5 0.4 62 3.5 1.5 0.6 70 3.7 1.5 0.8 75 4.0 1.5 1.0 80 4.5 1.5 1.5 87 5.2 1.5 2.0 90 6.2 1.5 2.5 70 4.2 1.5 3.0 60 3.0 Table 6. Effect of various concentration of IBA for rooting on MS media after 4 weeks of culture Growth regulators (mg/l) IBA Culture showing response (%) Average number of roots / explants Average root length (cm) 0.5 60 4 1.0 1.0 70 6 1.5 1.5 70 8 1.9 2.0 80 8 1.9 2.5 80 5 2.0 3.0 60 3 1.9 28

Table 7. Effect of different incubation conditions on the hardening of in vitro raised plantlets Pot condition which plants raised Sterilized soil substrate with MS ½ nutrient solution and incubated in A/C room Unsterilized soil substrate with ordinary water and incubated in shade. Percentage of plants recovered 47.2 51.8 65.5 59.3 Fig.1 Rooted plantlets on MS-B5 medium with IBA (2.%mg/l) after 4 weeks. Fig.2 Shoot elongation on MS-B5 medium with BAP and GA3(1.0mg/l) after 4 weeks. Fig.3 Nodal culture on MS-B5 medium with BAP (1.0 mg/l) after 7 days. Fig.4 Shoot tip explants culture on MS-B5 medium containing KN (2.0mg/l) after 2 weeks. Fig.1 Fig.2 29

Fig.3 Fig.4 Fig.5. Hardend plantlets were transferred to the larger pots containing unsterilized soil substrate. 30