The present research work was conducted at the Plant Biotechnology Laboratory, Department of Horticulture, Patuakhali Science and Technology University, during May 2013 to April 2014.Winter variety of BARI tomato-14 was used as the plant material. Stems, leaves and shoot tips of the tomato plant cultured on MS medium were used as the plant materials. The seeds were collected from the Regional Horticulture Research Station (RHRS), Lebukhali, Patuakhali.
Macronutrients
Stock solutions of macronutrients were prepared with 10 times of the BM in 1 litre of dw. Ten times the weight of the salt required for 1 litre of medium were weighed accurately upto four decimal places, using a balance (Model no. # AY 220 Shimadzu corporation, Japan) and dissolved thoroughly in 750 ml of distilled water and final volume was made up to 1 litre by adding dw. The stock solution was poured into a clean glass bottle. The bottle was labeled properly and stored in a refrigerator at 4°C for subsequent use.
Micronutrients
The stock solutions of micronutrients were made up to 100 times of the BM with the 1000 ml of dw. The stock solution was filtered, labeled and stored in a refrigerator at 4°C for later use.
Growth regulators
Besides the nutrients, addition of growth regulators (hormones) such as auxins and cytokinins to the medium to support good growth of tissues and organs were made. The following growth regulators were used in the present investigation:
Auxin: NAA (Naphthalene Acetic Acid) and IAA (Indole Acetic Acid)
Cytokinin: BAP (6-Benzyl Amino Purine)
The above three growth regulators were dissolved in 1 N NaOH solvent. To prepare the stock solutions of above hormones, 10 mg each of the hormones was placed in a clean 100 ml volumetric flask and then dissolved in 1 ml of respective solvent. The volume was then made up to 100 ml with dw. The solution was then stored at 4°C for use.
Axenic culture
Sterilized seeds were placed onto seed germination medium in test tubes. In each test tube, 4 seeds were inoculated. The culture was then incubated in incubation room till the germination of seeds. It was noticed that seeds started growing in dark and later they were transferred to light. Thirty days old seedlings were used as the source of explants.
Explants culture
The seedlings raised in axenic culture were used as the source of different kinds of explants. Leaf disc with 0.5 cm2, stem and shoot tip segments with 0.5 cm in length were used as explants. Attempts were made for the induction of callogenesis and organogenesis using different explants in MS medium supplemented with different growth regulators.
Stem segment culture
Stem segments measuring 0.5 cm were excised from each seedling using sterile scalpels and forceps. Three pieces of stem segments were arranged horizontally on each culture vessel and gently pressed into the surface of the sterilized culture medium with various concentrations and combinations of BAP and NAA.
Leaf disc culture
Leaf discs measuring 0.5 cm2 were cut with the sterile scalpels and forceps and 3 pieces were placed on the sterile culture medium with various concentrations and combinations of BAP and NAA.
Shoot tip culture
For inoculation, the shoot tip segments were cut into 0.5 cm long pieces and three pieces of those were cultured horizontally on MS medium supplemented with 0, 0.25 or 0.5 mg/L NAA + 1.0, 2.0 or 3.0 mg/L BAP.
The culture vessels with inoculated explants were incubated both in dark and light in a temperature controlled growth room (25 ± 1°C) under 16 hours photoperiod with a light intensity of 1500 lux and the relative humidity of 60-70%. Day to day observations was carried out to note the response.
- Transfer of plantlets from culture vessel to soil
Plantlets of the 5-7 cm length with well developed roots were removed from culture vessel with the forceps. Medium attached to roots were gently washed out with tap water. The plantlets were kept on glass plates and the plates were moistened to prevent the plantlets from drying and to reduce the effect of sudden shock. Each plantlet was transferred into a small earthen pot containing garden soil, sand and well rotten cowdung at the ratio of 1:2:1. Transplantation of the plantlets was done in the afternoon. Immediately after transplantation, the plantlets were irrigated with fine spray of water and the plantlets along with pots were covered with transparent polyethylene bags (Ishag et al., 2009) to prevent desiccation. The pots were kept in the laboratory and irrigated regularly at an interval of 2 days. The plantlets established within 5 to 7 days and the polythene bags were removed.