The experiments were conducted during the period of July 2005 to May 2006 in the Genetic Engineering Laboratory of Cytognetics Department, Genetic resources and seed Division, Bangladesh Jute Research Institute, Dhaka.
Experimental materials The following genetic materials of Jute (Corchorus capsularis and Corchorus olitorius) were used in the present investigation: a.Corchorus capsularis L. var CVE-3 b. Corchrus olitorius L. var O-72
Methods The following culture media were used in the present investigation depending on specific purposes as mentioned below: For seed germination MS (Murashige and Skoog, 1962) medium. For callus induction and shoot differentiation MS (Murashige and Skoog, 1962) medium supplemented with hormone, pH, vitamin, surfactant, NaCl, FeSO4.
For root induction Half strength MS (Murashige and Skoog, 1962) medium Preparation of culture media For induction of callus and shoot regeneration in jute a number of culture media have been advocated by different scientists of which MS (Murashige and Skoog, 1962) medium was used for investigating the present piece of work. A nutrient medium consists of organic and inorganic salts, irons, a carbon source, some vitamins and growth regulators were used. Based on the types of explants different media along with different concentration were used. Compositions of MS medium formulated by Murashige and Skoog, 1962.
Stock solution (Vitamins) Each of the desired ingredients except myo-inositol were taken at 100 folds (100x) of their final strength in a measuring cylinder and dissolved in 750 ML of distilled water. The final volume was made up to 1000 ml by further addition of distilled water. The solution was dispensed into 10 ml aliquots and stored at – 20o C. Myo-inositol was used directly at the time of media preparation. Steps followed for the preparation of culture media In present investigation, the following steps were followed for preparation of different culture media.
Preparation of MS medium To prepare one liter (1000 ml) of MS medium, the following steps were followed: i) 100 mL of macro-nutrients, 10 mL of micro-nutrients, 100 mL of iron and 10 ml of vitamins were taken from each of these stock solutions into a 2 liter Erlenmeyer flask on a magnetic stirred stirrer. ii) Distilled water was added in the flask to dissolve all ingredients and made the total volume 400 mL. iii) 100 mg of myo-inositol was added directly to the solution and dissolved well. iv) Thirty grams of sucrose was added to this solution and agitated gently to dissolve completely. v) Different concentrations of hormone supplements were added to the solution either in single or in combinations as required and mixed well. vi) The whole mixture was then made up to 500 ML with further addition of distilled water. vii) pH of the medium was adjusted to 5.8 with a digital pH meter with help of 0.1 N NaOH or 0.1 N HCl, whichever was necessary. viii) Seven gram agar was added in 500 mL of water. The mixture was then heated gently with continuous stirring till complete dissolution of agar. Hot agar (500 mL) was then mixed with 500 mL of medium. The mixture was thoroughly mixed by shaking. ix) Required volume of hot medium was dispensed into culture vessels or conical flasks. After dispensing the medium the culture vessels were plugged with cork and/or non-absorbent cotton and marked with different codes with the help of a glass marker to indicate specific hormonal combinations.
Sterilization To ensure aseptic condition in vitro, all instruments, glassware and culture media were sterilized properly by autoclaving. Sterilization of culture media The conical flasks containing prepared media were autoclaved at 1.16 kg cm-2 pressure and 121o C temperature for 20 minutes. For bacteria culture, YMB medium was then poured into sterile Petri dishes in a laminar air flow cabinet and were allowed to cool before use. Sterilization of glasswares and instruments Beakers, test tubes, conical flasks, pipettes, instruments like forceps, scalpels, were wrapped with brown paper packets. Empty flasks were capped with cotton plug and then sterilized in an autoclave at a temperature of 121o C for 20 minutes at 1.16 kg cm-2 pressure. Sterilization of culture room The culture room was initially cleaned by gently washing all floors and walls with a detergent followed by wiping with 70% ethyl alcohol. The process of sterilization was repeated at regular intervals. Generally, laminar airflow cabinet was sterilized by wiping the working surface area with 70% ethyl alcohol. Precautions to ensure aseptic condition Aseptic manipulations were carried out in a laminar airflow cabinet. The cabinet was switched on for at least half an hour before use and cleaned with 70% ethyl alcohol to overcome the surface contaminants. During the entire period of inoculation the autoclaved scalpels, forceps were kept immersed into 70% alcohol contained in a glass jar inside the cabinet. At the time of transfer these were again sterilized by flaming method inside the cabinet. Both the hands were rinsed with 70% alcohol. All measures were taken to obtain maximum contamination free condition during the surgical operation of the explants. The following culture techniques were employed in the present study a) Axenic culture Seeds of test varieties (CVE-3 and O-72) were surface sterilized by immersing in 0.1% (w/v) mercuric chloride (MgCl2) for 20 min, followed by 6 washes with sterile deionized water. Seeds were placed on the surface of 50 ml aliquots of hormone free agar-solidified (0.8%, w/v) MS basal medium (Murashige and Skoog, 1962) contained in a 100 ML conical flasks. In another set of experiment, surgical cotton (1 g approx, in each flask) was used instead of agar in association with MS basal liquid medium to obtain optimum seedling production. Surgical cotton was placed at the bottom of 100 ml flasks. Each flask contained 20 ML of hormone free MS liquid medium. Cultures were placed in a growth room with 28ºC under 1.0 Wm-2 of daylight fluorescent tubes with 12 h photoperiod. Twenty seeds were inoculated in each flask. Seven days old seedlings were used for further research work and data collection.
b) Explants culture The seedlings raised in axenic culture were used as the source of explants. The cotyledons with attached petiole were used as explants. Cotyledon with attached petioles of Corchorus spp. was taken from in vitro grown seedlings for this study. In this case, seedlings were allowed to develop for 7 days to make sure that the undeveloped apical shoot buds were not attached with the petioles. Therefore, the optimum explants source, cotyledons with their attached petioles were excised from 7 days old seedlings and were cultured in 250 ML conical flasks containing 50 ML of agar-solidified MS medium supplemented by IAA (0.5 mg/L) and BAP (2.0 mg/L). Ten explants were placed in each culture flask. The experiments were repeated three times with three replications. Treatments comprised of five different vitamin concentration [0 (control), × 1times (1.5ml/L), × 2 times (3.0 ml/L), × 3 times (4.5ml/L), × 4 times (6.0ml/L)] in modified solid MS medium. The culture flasks were placed under fluorescent light in a growth room with controlled temperature (280 C). The flasks were checked daily to note the appearance of callus and shoot regeneration. The flasks were checked daily to note the development of contamination. Data were recorded 6 weeks after culture.