The present investigation was carried out at the Plant Biotechnology Lab and the ‘’Horticultural Germplasm Centre”, Department of Horticulture, Patuakhali Science and Technology University, Patuakhali during February 2013 to March 2014.The virus tested three potato cvs. Diamant, Cardinal and Granula were obtained from the University of Rajshahi, Rajshahi-6205, Bangladesh. The internodal segments of the plantlets grown in vitro were used for the establishment of culture.The following methods were used for the present investigation, each for specific purposes. Those were:For callus initiation: MS medium supplemented with 2,4-D and Kinetin. For shoot initiation: MS medium supplemented with BAP and GA3. For root initiation: MS medium supplemented with IAA and GA3. Field Evaluation: Performance of varieties under different growth parameters and yield attributes. The study was conducted at the Plant Biotechnology Lab, Department of Horticulture, Patuakhali Science and Technology University, Patuakhali.Growth regulators Besides the nutrients, addition of growth regulators (hormones) such as auxin and cytokinin to the medium to support good growth of tissues and organs were made. The following growth regulators were used in the present investigation:
Auxin: 2,4-D ( Dichlorophenoxy acetic acid) and IAA (Indole Acetic Acid)
Cytokinin: BAP (6-Benzyl Amino Purine), KIN (Kinetin)
Gibberellin: GA3 ( Gibberellic Acid)
The growth regulators (IAA, 2,4-D, kin and BAP) were dissolved separately in 1 N NaOH solvent and GA3 was dissolved in 50% ethanol. To prepare the stock solution of the above hormones, 10 mg 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.
The conical bottles containing the medium were autoclaved (Model no. LAC-5060S; Korea) with 15 psi at 121°C for 20 minutes. After autoclaving, the bottles containing the medium were allowed to cool down under a laminar airflow cabinet.
Preparation of microplants: Under the above aseptic conditions, the virus tested potato plantlets were taken out from the test tube with a pair of sterilized forceps and the plant was cut into small pieces in such a way that each piece contained at least one node. Then the cut pieces were inoculated on hormone free MS medium. The test tubes with inoculated nodal segments were incubated in growth room.
Preparation of explants: Under aseptic conditions, one month old microplants (3-5 cm in height) were taken out from the test tube and placed on a sterilized petridish. The intermodal segments were cut aseptically into small pieces (3 mm) with a sterilized scalpel blade.
Inoculation: Internode segments (3 mm in length) were prepared inside the laminar airflow cabinet aseptically from the in vitro plantlets using a fine sterile forceps and scalpel. The excised explants were then inoculated into each test tube containing 15 ml MS medium supplemented with 2,4-D ( 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L) with the fixed concentration of kinetin (0.25 mg/L) as per treatments. The test tube was covered and sealed with a film of paraflim.
Effect of 2,4-D and KIN on callus proliferation: The present experiment was consisted of two factors:
Factor A: Plant growth regulator (2,4-D and Kinetin) There were seven levels with factor A
MS with 2,4-D 0.0 mg/L + KIN 0.25 mg/L
MS with 2,4-D 0.5 mg/L + KIN 0.25 mg/L
MS with 2,4-D 1.0 mg/L + KIN 0.25 mg/L
MS with 2,4-D 1.5 mg/L + KIN 0.25 mg/L
MS with 2,4-D 2.0 mg/L + KIN 0.25 mg/L
MS with 2,4-D 2.5 mg/L + KIN 0.25 mg/L
MS with 2,4-D 3.0 mg/L + KIN 0.25 mg/L
- Factor B: Variety
- There were three levels with factor B. Diamant, Cardinal and Granula.
So, there were total 21 (3x7) treatment combinations.