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Efficient Plant Regeneration from Embryogenic Cell Suspension Culture of Two Deepwater Rice (oryza Sativa L.) Varieties

L. KHALEDA
Department of Genetic Engineering and Biotechnology, University of Chittagong-4331, Bangladesh.

M. AL-FORKAN
Department of Genetic Engineering and Biotechnology, University of Chittagong-4331, Bangladesh.

Abstract/ Executive Summary:

Cell suspension cultures were initiated from 28-30 d old scutellar-derived embryogenic calli of two deepwater rice varieties HAJA-1 and HAJA-8 and maintained on R2 liquid medium containing 2 mg l-1 2,4-D. The formation of fine embryogenic cell suspensions and subsequent plant regeneration were dependent on plant genotype and culture media. Variety HAJA-8 was found less viable than the other variety HAJA-1. In general, it was observed that variety HAJA-1 showed better performance compared to variety HAJA-8 to R2 medium for the initiation of embryogenic cell lines and the rate of the cell growth was higher in this variety. Plant regeneration was not obtained from cell suspensions of both varieties when cells were cultured on regeneration media semi-solidified with 0.4% (w/v) agarose. Plant regeneration from cell suspensions of the both varieties were obtained when the agrose concentration of the regeneration media was increased from 0.4 to 1% (w/v). The highest plant regeneration frequencies were obtained from cell suspensions of the varieties HAJA-1 and HAJA-8 (48%) and (42%), respectively, pre-treated with water for 5 h and cultured on MS medium supplemented with 2 mg l-1 BAP+1 mg l-1 Proline. Washing cell suspensions with water for a prolonged period of time (15 h and more) inhibited plant regeneration.

Keywords: Deepwater rice, Cell suspension, Plant regeneration, Embryogenesis.

Location: Department of Genetic Engineering and Biotechnology

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

The present study was undertaken with a view to developing an efficient plant regeneration protocol from cell suspension cultures of deepwater rice.

Methodology:

Plant materials and media: Seeds of two deepwater rice varieties HAJA-1 and HAJA-8 collected from the Regional Station, Habiganj of Bangladesh Rice Research Institute, were used to initiate callus cultures on MS medium (Murashige and Skoog 1962) and cell suspension culture on R2 (Ohira et al. 1973) liquid medium. Initiation and maintenance of cell suspension culture: Cell suspension cultures were initiated from 28-30d scutellar-derived embryogenic calli. At first, embryogenic calli portions were transferred to 25 ml flasks each containing 7 ml of liquid R2 medium. Four cell suspension lines of discrete calli were initiated for each variety. Then cultures were incubated on shaker at 60 rpm at 26+2°C in the dark. During the first stage of initiation, 6 ml culture medium was replaced at the day 3 and every 3-day. After 4 weeks culture were transferred by pouring into 100 ml flask and replacing all the (spent medium) culture with 22 ml of fresh R2 medium. After 2 weeks, suspensions were sub-cultured every 7 d by the transfer of 1 ml PCV with 7 ml spent medium to 22 ml fresh R2 medium. After 60 d, 2 ml PCV of suspension culture with 10 ml of spent medium were transferred to 250 ml flasks containing 40 ml R2 medium. The cell suspensions were routinely sub-cultured on every 7 d. Estimation of growth rate of cell suspension culture: Established cell suspension culture (2 ml PCV) with 10 ml of spent medium and 40 ml fresh medium were transferred to 250 ml flasks. These flasks were incubated under growth conditions as described earlier but without subculturing from day 0 and daily, 10 ml of cell suspension was taken out by pipette and left for settling down and measured Packed Cell Volume (PCV) over an assessment period of 0-16 d. Data were collected from 3 flasks per variety with five reading taken per flasks. Plant regeneration from cell suspension culture: Cell suspensions of variety HAJA-1 and HAJA-8 initiated in R2 medium, were evaluated for plant regeneration potential on several regeneration media whereby plant growth regulators (PGRs), choice of plant growth regulators, agarose concentration and pre-treatment with water on plant regeneration were investigated. Three-month-old cell suspensions of varieties HAJA-1 and HAJA-8 were used for these experiments. After 4 d sub-cultured, approximately 1 g. fresh weight of cells was subjected to the following different experiments.+

Source of Information: The Chittagong Univ. J. B. Sci.,Vol. 5(1 &2):91-103, 2010.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Washing the cell suspensions with water for a prolonged period of time (15 h and more), inhibited plant regeneration. Therefore, increased plant regeneration frequencies cannot be attributed to any osmoticum effects. This system could be employed to produce large scale of plant from small quantity of mature embryos seeds as starting materials. Embryogenic suspension culture can be established by continuously culturing the calli in a liquid medium for continuous source of experimental material. It seems that the present study shows a simple method to enhance regeneration frequency from suspension cells, which can be used for genetic transformation studies.

Knowledge Management: Journal