The experiment was carried out in the Tissue culture Laboratory of Agrotechnology Discipline, Khulna University, Khulna, Bangladesh, during the period from September 2004 to May 2005. The plant materials used in this study were the shoot apices and axillary buds (4-6 cm), which were collected from the selected mature trees of Jackfruit (Artocarpus heterophyllusn Lam.) from Khulna University campus. The shoot tips were collected at the same day of explantation during morning hours. Explants were cultured on agarified MS (Murashige and Skoog, 1962) medium, supplemented with 0.5 mgl-1, NAA (ß- Naphthalene Acetic Acid) fortified with different levels of BA (6-Benzyladenine) viz. 1.0, 1.5, and 2.0 mg l-1. Seven g l-1 agar-agar, 30 g l -1 sucrose, different ratio of auxin and cytokinin for shoot proliferation and multiplication of proliferated shoots.
Preparation of explants: In jackfruit, the apical buds remain covered with a cap like structure called stipule. Unopened buds with one or two leaves along with shoot tip were collected from the selected plant and placed in water and then brought into the laboratory and used as plant materials. After removing the expanded leaves, the materials were washed thoroughly under running tap water. The materials were then suspended in 0.7% PVP solution, an antiseptic plus detergent containing 2% sucrose and were agitated by a magnetic stirrer for 15 minutes. They were then washed thoroughly to remove PVP. After that the plant materials were transferred to the laminar air-flow cabinet. Then the plant materials were dipped in 70% ethanol for 30 seconds and immediately washed with autoclaved sterilized distilled water. This was followed by surface sterilization with 0.2% mercuric chloride (HgCl2) for 10 minutes followed by washing with sterile double distilled water giving 3-5 changes. The surface water of treated material were then dried with the autoclaved tissue paper and kept for few minutes in laminar air-flow on an open Petridish for final drying (Dhar, 1998).
Inoculation of explants for shoot proliferation The explants were prepared by removing the outer covering of the green stipules and excised the inner buds enclosed within the creamy white stipules. The excised explants were then placed vertically on the surface of the medium. The excised explants were cultured with the help of sterilized forceps into the conical flasks (100 ml) containing 30 ml of nutrient medium per conical flask. In each conical flask a single explant was inoculated during first and subsequent sub-cultures. For each treatment 15 explants were cultured for 1st culture, and for subsequentsub-cultures.
Incubation of explants for shoot proliferation: After inoculation of the explants, culture vessels were kept in a growth chamber with 16 hours photoperiod at about 2000 lux and at a temperature of 25±1 ºC. Proliferation of the shoot started within 7-10 days after inoculation. The explants were sub cultured twice on the same medium at 30 days interval. Then finally nodal segments were isolated aseptically from the proliferated shoots and cultured on the same medium.
Experimental design, data collection and statistical analysis: The study was conducted following Completely Randomized Design (CRD). Data on number of shoot proliferated, average number of explants forming callus, average number of nodes per plant, average shoot length, explant proliferation percentage, callus production percentage, relative amount of callus, average number of shoot buds per explants and survivability of cultured shoots were recorded. The means of all the treatments were calculated and the analysis of variances (ANOVA) for all the characters were performed by F-test. The means of treatments were compared by Duncans Multiple Range Test (Gomez and Gomez, 1984).