Mother plant of Evolvulus nummularius 

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... herbs are moving from fringe to mainstream use, as a greater number of people endeavor to opt for herbal formulations over the allopathic compounds, since these are devoid of side effects and cost effective (Dubey et al., 2004). Popular demand and scientific interest in complementary or alternative medicine, particularly medicinal botanicals, has increased considerably in recent years (Borchers et al., 1997). Screening and indexing of many plants, which have been used in traditional Indian and Chinese medicines since time immemorial, resulted in novel therapeutics, useful for the treatment of various ailments of man such as rheumatism, kapha, pitha, blood pressure, cancer etc. (Anonymous, 1948; Anonymous, 1959). The traditional health care systems, including Ayurveda, were transmitted from generation to generation by „Gurukula‟ and practice was dependent on the intimate knowledge which was passed on for many years by the Guru while he was treating each individual case to the disciple (Unnikrishnan, 2004). The earlier examples of western medicine had the influence of plant medicines used in ancient times. Current examples are the use of the cardiac glycosides from the purple foxglove, Digitalis purpurea , morphine and opiates from poppy, Papaver somniferum , reserpine from Rauvolfia serpentina and quinine from cinchona officinalis. Similarly vincristine from catharanthus roseus and taxol from taxus baccata were introduced as drugs for the treatment of cancer (Posey, 1998). Conservation of medicinal plants and capability to utilize them in a sustained manner are essential for the well being and continued survival of man (Ghimire et al., 2004). Medicinal plants are the most important source of life saving drugs fo r the majority of the world‟s population. The biotechnological tools are important to select, multiply and conserve the critical genotypes. In-vitro regeneration holds tremendous potential for the production of high-quality plant-based medicine (Leena and Jaindra, 2003). The continuous use of plant derived products for health care problems has resulted in the establishment of various pharmacological laboratories (Fereidoon, 1999). The growing use of medicinal herbs and their practical utility has created new importance in opening of pharmacological and pharmocognosy laboratories that provide adequate information of diverse chemical substances and their usefulness in treating various ailments (Julia et al., 2006). First significant attempt of isolated plant cells on artificial nutrient medium was done by Haberlandt in 1902. Later, long term calli cultures were established from carrot cambium by Gautheret, 1939 and Nobecourt, 1939. Subsequently various culture media were reported to obtain cultured cells in diverse plant explants of several taxa by the work of Gautheret, 1940, Hildebrandt et al., 1946, Nitsch 1951, Reinert and White, 1956, Murashige and Skoog, 1962, White, 1963, Gamborg et al., 1968, Schenck and Hidebrandit, 1972 etc. Investigations by Minocha, 1980, Bornman, 1983, Villalobos et al., 1984 on culture conditions; White and Gilbey, 1966, Mulder-Krieger et al., 1982 on nutrient requirements; Oka and Ohyama, 1975, Minocha, 1987, Nadel et al., 1991 on hormonal requirements; MacRae and VanStaden, 1990, Pochet et al., 1991 on gelling agents etc. revealed some other important aspects of culture. Evolvulus nummularius is commonly called Kidney weed (Miller 1997) and locally known as Bichhamalia, Krishna ankaranti ; Jungi-ba, Tandi kode baha (Kambaska) Lakshmi krantha ( Fig. 1 ). It is a perennial herb used as anthelmintic and in the treatment of bronchial asthma. Medicinally E.nummularius is highly useful in large quantities and hence in vitro cultivation is necessary in the present studies. Fresh plant material was collected at Visakhapatnam and the explants were sterilized within 4 hours. Glass double distilled water was used for the preparation of culture media. After addition of all constituents of media, the pH was adjusted to 5.8 using 0.1 N KOH or 0.1 N HCl. Gelling-agent (agar-agar) was added as per requirement and the medium was steamed to melt the gelling agent. It was then dispensed into test tubes (20 ml per tube) or Erlenmeyer flasks (100 ml per 250 ml flask) or screw capped bottles (50 ml per bottle) and was autoclaved at 121°C at a pressure of 15 lbs for 20 min. When no gelling agent was added, each tube contained liquid medium with a filter paper bridge of Whatman No. 1 filter paper. Heat labile constituents like hormones were filter-sterilized by passing through a Millipore membrane (0.22 μm pore size) (“Millipore Corporation”, USA) and added aseptically to the autoclaved medium just before gelling. All the plant growth regulators used during the course of the present work were added before autoclaving the medium. As per the requirements, the medium was also poured in sterile glass petridishes (12 ml per 55 mm dish, 20 ml per 85 mm dish) in front of a laminar airflow hood. The composition of MS media used for culturing of explants. Healthy explants such as shoot tips and young flowers of Evolvulus nummularius were selected for tissue culture The explants were washed thoroughly under running tap water, followed thrice (3X) with distilled water and submerged in 70% ethanol for 3 min. The explants were again rinsed 3X in sterile, double distilled water and are then submerged in 5% sodium hypochlorite for 10 minutes. Finally the explants were rinsed in 3X sterile, double distilled water. The explants were then treated with 0.5% mercuric chloride for 5 minutes and rinsed 3 times in sterile double distilled water. After the surface sterilization the explants were cultured on different nutrient media under aseptic conditions. To control the phenolic exudation in the cultures of Evolvulus nummularius activated charcoal (1%) was used in culture media. Periodic subculturing (at weekly intervals) to fresh media with same compositions were also tested to overcome the problem. Shoot regeneration potential via callus phase of different explants (shoot tips and flower) of Evolvulus nummularius were studied by culturing on MS medium fortified with different combinations of auxins and cytokinins. The callus and shoot cultures thus obtained were subcultured at regular intervals of 1-2 weeks. Observations were recorded at every week. For root induction in Evolvulus nummularius , the shoots ( size more than 3cm) were excised from primary cultures and cultured on semi solid MS medium supplemented with NAA (0.1-3mg/l) on IAA (0.1-3mg/l) individually. The plantlets, regenerated through various in vitro techniques, with healthy root and shoot systems were taken out from the culture medium and washed gently with sterile distilled water for removing all traces of medium from the plantlet. The washed plantlets were transferred to small plastic cups containing sterile sand. The pots were then covered with polythene bags to maintain high humidity and kept in plant growth chamber. The plantlets were moistened with water two times a day. After fifteen to twenty days, the polythene bags were removed and transferred to larger pots containing sterile sand and soil (1:1) and kept under shade in the net house for another two weeks before transferring to field. Depending upon the size and the availability of the explants, each experiment consisted of 20 ...