2.1 Experimental site: The experiment was carried out at the Plant Tissue Culture Laboratory of Agrotechnology Discipline, Khulna University, Khulna-9208, Bangladesh, during April 2010 to January 2011.
2.2 Type of explants: In vitro grown potato explants were used as source materials for microtuberization and minituber production. 2.3 Constituents of media for microtuberization of potato: For microtuber formation, MS medium supplemented with BAP-5.0 mg l-1 , Chloro Choline Chloride (CCC)-500 mg l-1 and different concentrations of sucrose-5%, 6%, 7%, 8%, 9%, 10% is used.
2.4 Substrates used in pots for minituber formation: Following substrates were employed for minituber production: 1. Soil 100% 2. Sand 100% 3. Coconut dust 100% 4. Soil : Sand 50% : 50% 5. Soil : Coconut dust 25% : 75% 6. Soil : Coconut dust 50% : 50% 7. Soil : Coconut dust 75% :25% 8. Directly into the field
2.5 Experimental design All the experiments were laid out in a Completely Randomized Design (CRD) with five replications for each treatment. In that case, a plastic pot containing different substrates with a single healthy plant was considered as a replication.
2.6 Procedure for microtuberization After taking all the precautions to ensure aseptic condition, in vitro grown healthy plantlets were selected for further inoculation. All inoculation and aseptic manipulations were carried out under a laminar airflow cabinet. planlets were removed carefully and cut the shoot tips measuring 2 cm in length. Then they were placed onto nutrient media containing test tubes for microtuberization. After inoculation of the explants, the microtuber induction cultures were incubated in the dark at 24°C and relative humidity 65%, maintaining a temperature of 18o C. Microtuber was first observed at 35 days after inoculation. The experiment was laid out in a Completely Randomized Design (CRD) with ten replications. A test tube (15 cm length) containing 15 ml medium with a single shoot tip of potato was considered as a replication.
2.7 Procedure for minituber production For minituber production of potato, in vitro cultured plants were transplanted into plastic pots (4 inch size) with different substrates (soil, sand, coconut dust and their different combination) and some were directly into the field. Soil, sand and coconut dust were autoclaved at 121o C for 15 minutes before transplantation. After proper mixing of different substrates according to each treatment, they were kept in plastic pots. Few minutes later after watering, plants were transferred into pots. Transferred plants were placed into growth chamber with 85% humidity for 24 hours. After that, they were kept in net house.
2.8 Analysis of data Recorded data for all of the experiments were analyzed for ANOVA (Analysis of Variance) with the help of computer using MSTAT-C program and the means were compared according to Duncan’s Multiple Range Test (DMRT) and Least significance Differences (LSD).