Micropropagation Studies of Senna Alata (L.) Roxb. an Anti Allergenic Plant

Senna alata (L.) Roxb. , an important medicinal plant, belongs to the family Ceasalpiniaceae. It is used in several ways for different ailments like skin infections against fungi constipation and intestinal worms etc. The present investigation outlines the in vitro propagation of S. alata, to develop a standard protocol to initiate multiple shoot induction which provides a good source of pharmacologically active plant constituents like chrysophanic acid, sennosides and anthraquinones which were used in an industrial level for preparation of drugs. An in vitro propagation has been investigated by MS medium containing 6-Benzyl adenine (8.8μM and 6.6μM), Kinetin (6.9μM and 9.2μM), Gibberellic acid (1.4μM) and 3% sucrose which induced maximum number of shoots and beneficial shoot lengths by nodal and hypocotyl explants. The rooting was initiated at half strength MS medium consist indolebuteric acid (7.3μM), which promoted well rooted plantlets and showed highest percentage (81%) survival in the field conditions without any morphological variation. Introduction Senna alata L. is a large shrub with very thick finely downy branches. It is named for its flower buds which grow in a column and looks like fat yellow candles each complete with a flames. It is found commonly in Somalia, Saudi Arabia, some parts of Pakistan and Kutch area of Gujarat. It is largely cultivated in Madurai, Ramanathapuram, Salem and Tiruneleveli, districts of Tamilnadu for its medicinal values. It occurs in diverse habitats in the tropics and containing some chemical substances like, chrysophanic acid, rhein, emodin, sistosterol and sennoside A, B, C and b-physcione. It is used in several ways for different ailments like skin infections against fungi, constipation and intestinal worms etc. The leaves are pounded and rubbed on the skin to cure eczema, ringworm and fungal infections (Pieme et al. 2006; Palanichamy and Nagarajan, 1990). The ethanolic extract of S. alata leaves showed efficient results against antifungal (Sule et al. 2010), bacterial activity (Ibrahim and Osman, 1995; Khan et al. 2001; Ogunjobi and Abiala, 2013), analgesic activity (Villasenor et al. 2002) and used in treatment of pityriasis vesicular, anti inflammatory activity. Stem bark could be potential source of active antimicrobial agents, and a detailed assessment of its in vivo potencies and toxicological profile is therefore advocated. The present investigation report delivered the micropropagation of S. alata through nodal and hypocotyl explants and subsequent establishment of plantlets in the field. Material and Methods Matured pods were collected in the months of December 2010 from Mulugu Division of Warangal District, Andhra Pradesh, India. Seeds were extracted manually and air dried at normal temperature. Complete dry seeds were considered for testing of germination. The morphological traits of pod (pod length, width and number of seeds per pod) and seed (seed length, width and thickness) were measured. The experiment was laid out in complete randomized designed with five replications each, 100 seeds were taken for replication under both in vitro and in vivo conditions. Seeds were germinated in natural soil and in vitro on MS basal medium. But in both the cases showed effective seed germination of around 85-86% was seen, when explants taken from such plant lets. Hormones obtained from Himedia laboratories and analytical grade chemicals were used for preparing the stock solutions and subsequent media preparation. The basal medium consisted of MS salts (Murashige and Skoog 1962) supplemented with B5 vitamins (0.5mg/l nicotinic acid, 0.5mg/l pyridoxine HCl, 1mg/l Thiamine HCl and 100mg/l Myoinsosital (Gamborg et al. 1968). 30g/l sucrose and 0.8% agar for gel rite. The pH of the medium was adjusted to 5.8 prior to autoclaving, at molten medium was dispensed in to test tube (Borosil) at 20ml per tube and plugged with non absorbent cotton. Culture tubes containing the media were autoclaved at 121oc for 20min. Surface sterilized explants were inoculated vertically and horizontally on to the culture medium one or two explants per each test tube. All the cultures were maintained at 25 ± 2oc under 16h photoperiod at photosynthetic flux of 35-50 μmol m251 provided by 100/daylight florescent lamps. Hypocotyl and nodal explants were cultured on MS medium supplemented with 3% sucrose with 0.8% agar and supplemented with different combinations and concentrations of plant growth regulators including 2.2-13. 3μM BA, 2.4-11. 5μM Kinetin and addition of 0. 5μM Gibberellic Acid. Elongated shoots were excised from each culture passage and transferred to half strength MS medium containing 3% sucrose and 0.8% agar. The medium was further supplemented with different concentrations of Indole Acetic Acid (IAA), IBA and NAA individually. Plantlets with well developed roots were removed from the culture medium and washed the roots gently for removal of agar traces, and then plantlets were transferred to plastic cups containing autoclaved garden soil, farm yard manure and sand (1:1:1). Each was irrigated with one-eighth strength MS basal salt solution devoid of sucrose and inositol every 4th day for 2 weeks. The potted plantlets were covered with porous poly ethylene sheets for maintain high humidity and maintained inside the culture room conditions. The relative humidity was reduced gradually after 40 days the plantlets were transferred to a Botanical garden and kept under shading in a net house for further growth and development. The morphological characteristics, growth characteristics and floral features were examined. Total plants developed in vitro (81%) were transferred on to the field. The developed protocol will be useful for large scale production at industrial level. Statistical analysis Ten to 15 explants were used per treatment in each replication. Ten replications were considered and repeated thrice. The observations were recorded on the frequency (number of cultures responding for axillary shoot proliferation and root development) and the number of shoots per explant,