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In Vitro Regeneration of Pumpkin (cucurbita Maxima) Through Shoot Apical Meristem

M E Haque
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi 6205, Bangladesh

D Rezwana
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi 6205, Bangladesh

M A Islam
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi 6205, Bangladesh

B Sikdar
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi 6205, Bangladesh

Abstract/ Executive Summary:

Context: Pumpkin (Cucurbita maxima) is very important vegetable crop. It is damaged by several types of diseases, especially viral diseases which reduce the total yield of pumpkin. This is very difficult problem and such type of problem can be overcome by meristem culture. Objectives: To develop suitable protocols for indirect regeneration of pumpkin plant through shoot apical meristem. Materials and Methods: Meristems were isolated from 15-21 days in vivo grown plants by collecting shoot tips and were prepared (0.2-0.5 mm) under 4X zoom stereo-microscope remaining two leaves primordia. Inoculation of explants was made singly per culture vessel in semisolid MS fortified with different concentrations and combinations of cytokinin and auxin. Results: Among different concentrations, 1.0 mg/l BAP showed best response for callus induction. Calli were sub cultured for shoot formation in MS containing BAP singly or combination with GA3 and 1.5 mg/l BAP + 0.1 mg/l GA₃ gave better result. In vitro regenerated shoots were rooted well in ½ strength MS with 0.5 mg/l IBA and micro plants were acclimatized successfully in natural condition. Conclusion: Indirect regeneration of pumpkin plants through shoot apical meristem has been established.

Keywords: Growth regulators, In vitro regeneration, Pumpkin, Cucurbita maxima.

Location: At DNA and Chromosome Research Laboratory, Department of Genetic Engineering and Biotechnology, Rajshahi University

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Pumpkin

Objectives:

To develop suitable protocols for indirect regeneration of pumpkin plant through shoot apical meristem.

Methodology:

The study was carried out at DNA and Chromosome Research Laboratory, Department of Genetic Engineering and Biotechnology, Rajshahi University. Meristems were isolated from 15-21 days in vivo grownplants. Shoot tips were cut carefully with the help of forceps and dissecting blade then tips were taken into a conical flask and thoroughly washed under running tap water for 30 mins to remove loose contaminates attached to explants. Then these explants were washed with distilled water containing 1% savlon (v/v) and 4 drops of Tween-80 for 20 mins to remove gummy substances. That was followed by successive 3 washing with distilled water to make the material free from savlon. Subsequently the materials were transferred to running laminar air flow hood and sterilized by 0.05% HgCl₂ for different periods of (1-10 mins) time to ensure contamination and tissue killing free culture. To remove every trace of the strident, the materials were then washed at least 6 times with sterile distilled water. Meristems were prepared (0.2-0.5 mm) under 4X zoom stereo-microscope. Microscope and other materials were properly sterilized by 70% ethanol before starting isolation. Meristem were prepared by excising leaves remaining two leaves primordia with apical meristem. Inoculation of explant was made singly per culture vessels. During inoculation, special care was taken that the explant must touch the medium equally. Each culture tube containing more or less 15 ml of nutrient medium was supplemented with different concentrations of a single hormone or combination of hormones. In all the cases, explants were inoculated to semi solid MS (Murashige and Skoog 1962) medium for callus, shoot and root induction with different concentrations and combinations of cytokinin and auxin. The inoculated culture vessels were incubated in a growth chamber containing special culture environment. First the cultures were taken in dark chamber for two days then transferred in the growth chamber illuminated by 40 watts white fluorescent tubes fitted at a distance of 30-40 cm from the culture shelves. The cultures were maintained at 25 ± 2°C under the warm fluorescent light intensity varied from 2000-3000 lux. After ten weeks, the well rooted plantlets were taken out very carefully from test tubes and roots were washed gently and transplanted to small pot containing sterilized soil. They were covered with transparent polythene bags to maintain high humidity and kept in the growth chamber for seven days. Within seven days the plantlets began to form new leaves and resumed fresh growth.

Source of Information: J. bio-sci. 18: 104-107, 2010 ISSN 1023-8654

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Hormonal differences and simple rapid in vitro regeneration protocol of L. cylindrica and L. acutangula have been established which will help in conservation and propagation of these two important species.

Knowledge Management: Journal