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Plant Regeneration Through Nodal Explant Derived Callus in Wood Apple (aegle Marmelos L.)

Ranjoy Das
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.

M. Faruk Hasan
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh.

Harunar Rashid
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh.

Motiur Rahman
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh.

Abstract/ Executive Summary:

This study reports on an improved protocol for callus induction and subsequent regeneration from nodal segment of wood apple (Aegle marmelos L.) Creamish friable competent callus was achieved from nodal segments on MS medium augmented with 4.0 mg1-1 2,4-D within two weeks of inoculation. The callus produced large number of shoots when cultured on MS medium fortified with 2.0 mgl-1 BAP+0.1 mgl-1 NAA within ten days of culture. In vitro raised shoots were rooted on half strength MS medium enriched with 1.0 mgl-1 IBA within fifteen days of culture. The rooted plantlets were successfully established with 80% survival.

Keywords: Plant regeneration, Callus induction, Nodal explant, Aegle marmelos.

Location: Rajshahi University campus, Bangladesh

Start Date:

End Date:

Program Area: Seed Technology

Commodity: Wood apple

Objectives:

The present investigation demonstrates an effective high frequency regeneration method for producing a large number of plants from nodal explants of Aegle marmelos.

Methodology:

Seeds from mature fruits of Aegle marmelos were collected from Rajshahi University campus, Bangladesh. The seeds were washed thoroughly in running tap water for 30 minutes and then soaked in Tween-80 and Savlon for 10 minutes. Then the seeds were washed in distilled water for several times. Finally, the seeds were rinsed in 0.1% HgCl₂ for 10 minute and washed with autoclaved distilled water for several times to remove the traces of sterilant. Then, the sterilized seeds were inoculated to sterile seed germinating medium (MS) in culture bottle. Nodal segments were excised from two week old in vitro grown seedlings and inoculated onto MS medium (Murashige and Skoog 1962) fortified with different concentrations and combinations of auxin and cytokinin. Throughout the experiments, both full strength MS medium and half strength MS medium with 3% (W/V) sucrose and gelled with 0.8% (W/V) agar was used. In all cases, the pH of the medium was adjusted to 5.7 before autoclaving and adding agar. About 10 ml of the medium were dispensed in each culture tube and sealed with nonabsorbent cotton plugs prior to autoclaving at 121^OC for 21 minutes. The cultures were incubated in a culture room at 25+2^OC with a photoperiod of 16 hour at 3000 lux light intensity provided by cool white fluorescent tubes. In this investigation, the basal medium was supplemented with different concentrations of auxin for callus induction. Once the callus developed, they were further cultured for regeneration and elongation in the medium having different concentrations and combinations of auxin and cytokinin. Elongated shoots were rooted on half strength MS medium supplemented with different concentrations of auxin (IBA) singly. After 35 days, well rooted plantlets were obtained. Subsequently, the plantlets were removed from the culture vessels, washed gently under running tap water and planted in pots containing sterile sand, soil and humus in the ratio of 1:2:2. The potted plantlets were covered by polythene sheet to maintain suitable humidity. After sufficient acclimatization, the plantlets were transplanted in the field condition, where 80% plants were survived.

Source of Information: Bangladesh J. Sci. Ind. Res. 44(4), 415-420, 2009

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

After 30 days, well rooted plantlets were achieved. Subsequently, the plantlets were removed from agar medium and planted in small pots containing sterile sand, soil and humus in the ration of 1:2:2. The potted plantlets were covered by transparent polythene sheet to maintain high humidity and within 15-20 days new leaves were emerged from the plantlets that resumed new growth. After 50-55 days, the plants were transplanted in the field condition, where 80% plants were survived and grown satisfactory.

Knowledge Management: Journal