Explant Source: Shoots from 2 years old plants of A. racemosus grown in the medicinal plant nursery, Department of Botany, <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />University of Rajasthan, were taken as explants.<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />
Culture Medium : For all the experiments MS medium was used. The constituents given in Table 2a, were dissolved in double distilled water and the different stock solutions were made. The volume of stock solution was maintained as per requirements of the experiments. The stock solutions were stored in the refrigerator and used with in a month of their preparation. Stocks of growth regulators were made according to Table 2b, and were added to the medium before autoclaving as per need of the experiments. All auxins were dissolved in few drops of absolute alcohol and cytokinin in few drops of 1 N NaOH and made into required volume. 3% sucrose was added in each combination as carbon source and 1.0% agar was used for solidification of the culture medium. pH of the medium was adjusted to 5.8 by adding 1 N HCl or 1 N NaOH before autoclaving the medium. The medium is then transferred to Erlenmeyer conical flasks (100 ml) and culture tubes (250x15 mm) of Borosil. Approximately 40 ml and 20 ml medium were dispensed in each flask and tube respectively, and ten replicates were taken for each set of experiment. The medium was sterilized in an autoclave at 15 psi for 20 min.
Surface sterilization of explants : The explants collected from nursery of Botany Department, were washed thoroughly with tap water and were kept under running tap water for 30 min, then surface sterilized with 70% alcohol for 3 min followed by 0.1% HgCl2 with a few drops of liquid detergent for 2-5 min depending upon the plant material. These were subsequently washed 4-5 times with sterilized double distilled water to remove the traces of sterilant. At last, suitable explants were selected and were transferred to in sterilized petriplate for inoculation in culture vessels.
Inoculation and Incubation : Explants were inoculated horizontally on to medium in culture vessels with each culture vessel containing single explant. All cultures were incubated at 25±2 oC at 50-60% RH under cool white fluorescent light at 30 mE.m-2.s-1 with a 16 h photoperiod. Temperature, RH and light conditions were manipulated according to experimental conditions to get optimal results.
Regeneration was carried out in following stages
Induction of multiple shoot buds and callus : For induction of shoot buds and callus, desired explants were inoculated on the medium containing various levels of growth regulators or their growth adjuvants. Initially cultures were examined every day upto a week of inoculation, later weekly and finally morphogenic responses were recorded after 30-40 days.
Proliferation of multiple shoot buds and callus : Multiple shoot buds were sub cultured after 40 days on fresh media once and then on medium containing various concentrations of auxins and cytokinis. Callus (with or without shoot bud initials) obtained during primary culture was transferred to fresh medium after 4-5 weeks of culture in order to evaluate whether, callus showed any morphogenic response or continued to proliferate as such. Renewable callus was stocked by
subculturing the same on the callus induction medium for further experimentation.
The regenerable callus was also further subjected to shoot induction medium. Once the shoot buds were initiated, the same was further sub cultured in order to have shoot formation. Thus, large number of proliferated shoots was obtained.
Rooting of elongated shoots : Elongated shoots were subjected to root induction medium for which number of experiments were conducted. Thereby, developing a regenerable protocol for a complete plant.
Hardening of plantlets and pot transfer : All the plantlets thus obtained in vitro from axillary buds, shoot tips or from callus were carefully removed from culture vessels. They were thoroughly washed with water to remove the traces of media and agar, hardened and then transfer to soil.
A record of growth and development was maintained in terms of mean number of multiple shoots formed and their mean length at an internal of 7 days upto 45 days. Further, change in number and length of roots were recorded at 7 days internal upto 60th day.