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Callus Induction and Cell Culture of Castor (ricinus Communis L. Cv. Shabje)

M A Rahman
Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

M A Bari
Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Context: Tissue culture is an important tool in plant biotechnology that allows for an increase in biomass or metabolite production by utilizing several techniques in callus or cell cultures. Ricin is a toxic protein that can be extracted from the castor bean as secondary metabolite. The ricin has been used experimentally in medicine to kill cancer cell. We developed callus and cell culture technique for the possible extraction of ricin from the cell suspension culture of Ricinus communis. Objectives: The present investigation aimed to develop the cell culture technique of R. communis L. cv. Shabje and tried to establish a protocol for cell suspension culture of castor for possible extraction of ricin from cell extract. Materials and Methods: The hypocotyl explants of R. communis L. cv. Shabje were used as experimental materials. Cultured on Murashige and Skoog medium supplemented with different concentrations and combinations of BAP, NAA, 2,4-D and IAA for callus induction. For cell culture, the media were used without agar with different concentrations and combinations of these hormones. Results: For callus induction the combination of BAP 2.0 mg/l + 0.5 mg/l NAA showed the best performance but in case of cell culture the combination of BAP 2.0 mg/l and 0.2 mg/l NAA showed the best result. Conclusion: The present investigation clearly established and demonstrated the method of obtaining cell suspension culture and important secondary metabolite ricin could be obtained from cell suspension culture of R. communis L. holding promises to explore cell culture industry for ricin production.

Keywords: Castor, Hypocotyl, Embryogenic callus, Cell culture and Regeneration.

Location: Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Castor

Objectives:

The present investigation aimed to develop the cell culture technique of R. communis L. cv. Shabje and tried to establish a protocol for cell suspension culture of castor for possible extraction of ricin from cell extract.

Methodology:

The hypocotyl explants of R. communis L. cv. Shabje, were used as experimental materials in the present investigation. Seeds were collected from the research field of the Institute of Biological Sciences, Rajshahi University, Rajshahi, Bangladesh. Seeds were washed thoroughly under running tap water and then treated with 1% Savlon and 2-3 drops of Tween-80 for about 10 min. This was followed by successive three washing with distilled water to make free the seeds from Savlon and Tween-80. Surface sterilization was carried out with 0.1% HgCl₂ for 6-7 min followed by gentle shaking. After this treatment, the seeds were rinsed 4-5 times in sterile distilled water to make free the seeds from HgCl₂. Sterilized seeds were partially decorticated and aseptically germinated in glass bottle containing 50 ml of autoclaved (121ºC temperature for 20 minutes at 1.1 Kg/cm² pressure) MS (Murashige and Skoog 1962) medium, fortified with BAP (1.0 mg/l), 30 gm/l sucrose and 0.8 gm/l agar. Callus culture: Hypocotyl segments as explants were taken from 8 days old in vitro grown seedlings of the plant. The explants were cultured in 9 cm petri dish and placed horizontally on the callus induction medium. The MS medium supplemented with 3% sucrose and different concentrations of NAA, 2, 4-D, IAA and BAP in combination were compared for the induction of callus. The data for callus initiation were scored after 4 weeks of culture. Cell culture: Rapidly proliferating friable calli subcultured for 18 days, were aseptically transferred to MS liquid medium supplemented with 2.0 mg/l BAP + 0.05 mg/l NAA, 2.0 mg/l BAP + 0.2 mg/l NAA and 2.0 mg/l BAP + 0.5 mg/l NAA in three lots in 250 ml flasks. The flasks were placed on a rotary shaker (100 rpm). After 4 days, the liquid medium containing cells and micro calli were filtered through a 500 micron mesh sieve. The filtrates containing cell were maintained in the laboratory. To observe the growth efficiency, flasks containing the liquid medium with cell culture were kept in an orbital shaker. Cell growth was measured by weighting the cells in 5 ml liquid medium taken after every two days. On the other hand, to obtain callus, some cells were distributed to petri dishes (4 cm) containing the fresh semi solid medium at 25ºC in dark for 35-42 days of incubation. Micro calli were appeared in the plates initiating induction of callus from cell aggregates.

Source of Information: J. bio-sci. 20: 161-169, 2012, ISSN 1023-8654

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present investigation clearly established and demonstrated the method of obtaining cell suspension culture and important secondary metabolite ricin could be obtained from cell suspension culture of R. communis L. holding promises to explore cell culture industry for ricin production.

Knowledge Management: Journal