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Optimization of Agrobacterium-mediated Genetic Transformation in Indica Rice

M.A.N. Nazim-Ud-Dowla
Department of Genetics and Plant Breeding Patuakhali Science and Technology University, Dumki, Patuakhali-8602, Bangladesh

N.U. Ahmed
Department of Genetics and Plant Breeding Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

L. Hassan
Department of Genetics and Plant Breeding Patuakhali Science and Technology University, Dumki, Patuakhali-8602, Bangladesh

Abstract/ Executive Summary:

This experiment was conducted during the period from August 2004 to April 2005 in the Biotechnology & Genetic Engineering Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh. In this experiment, an efficient and reproducible protocol for the production of transgenic rice plants was developed by inoculating mature embryo of three indica rice genotypes with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121, which contains selectable marker gene nptII conferring resistance to kanamycin and the GUS reporter gene. The transformation experiment was performed by optimizing two important parameters: infection time and co-cultivation period. Infection was most effective when explants were inoculated for 15 minutes (72.59% GUS positive) and co-cultivated for four days (70.37% GUS positive) with Agrobacterium. Among the varieties, BRRI dhan-33 showed the highest response to GUS assay (65.33% GUS positive). Transformation percentage (putative) was also highest in BRRI dhan-33 (4%). 10 minutes infection time and co-cultivation period of three days was found effective for regeneration of shoots (2.45% and 4.45%, respectively). Among the varieties, BRRI dhan-33 produced the highest percentage (61.11%) of rooted shoots.

Keywords: Transformation, Indica rice, GUS, Agrobacterium

Location: Biotechnology & Genetic Engineering Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

Start Date: 00-08-2004

End Date: 00-04-2005

Program Area: Variety and Species

Commodity: Rice

Objectives:

To study the transformation potentiality of three indica rice varieties in accordance with the optimization of preculture and co-cultivation time required for Agrobacterium mediated genetic transformation.

Methodology:

Plant Materials - Three varieties of indica rice namely, BRRI dhan-29, BRRI dhan-33 and Binnatoa were used in that experiment. Agrobacterium Strain and Plasmid - Genetically engineered Agrobacterium tumefaciens strain LBA4404 was used for infection in the transformation experiment. This strain contains plasmid pBI121 of 14 KDa (binary vector).This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct: The udiA gene encoding GUS (β-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptII gene encoding neomycin phosphotransferase II (nptII) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 KDa) containing the virulence genes. Media Used - YMB (Yeast extract Mannitol Broth) and LB (Luria Broth) medium were used for bacteria culture and maintenance, MS medium supplemented with 1 mg L^-1 2,4-D and 50 mg L^-1L-Tryptophan for callus induction and co-cultivation. Along with these 100 μM acetosyringone was added to enhance the bacterial activities. MS liquid medium supplemented with 500 mg L^-1 cefotaxime were used for washing of explants after co-cultivation, for Post-cultivation and shoot differentiation MS medium supplemented with 6 mg L^-1kinetin, 0.5 mg L^-1 NAA and 200 mg L^-1 cefotaxime were used. MS medium supplemented with 6 mg L^-1 kinetin and 0.5 mg L^-1 NAA along with increasing rate (20 to 30 mg L^-1) of kanamycin and decreasing rate (100 to 75 mg L^-1) of cefotaxime were used for selection and regeneration and MS medium + 0.5 mg L^-1 IBA + 50 mg L^-1 kanamycin + 50 mg L^-1 cefotaxime were used for rooting Sterilization and Placement of Seed - Mature seeds of three Oryza sativa var. indica were dehusked manually without interrupting the embryo. The dehusked seeds were then washed thoroughly in distilled water followed by dipping in 70% ethyl alcohol for three minutes with vigorous shaking and washing. Surface disinfection’s was done by the use of 30% clorox for fifteen minutes. Sterilized mature seeds were cultured directly as explant in MS medium supplemented with different concentrations of hormones and sucrose required as per treatment. Ten explants were horizontally placed in a petridish containing about 20 mL solid medium. Infection and Incubation - The Agrobacteria grown in liquid LB media were used for infection and incubation maintaining the optical density of OD6₀₀=0.6 to get suitable and sufficient infection of the explants, freshly excised explants were wounded and immersed in a culture of the preinduced bacteria for 5-15 minutes with gentle shaking and then transferred them to cocultivation medium. GUS (β-glucuronidase) Histochemical Assay - From each batch of explants following each transformation experiment, randomly selected cocultured tissues were examined for GUS histochemical assay. For this experiment co-cultured explant tissues were immersed in X-gluc (5-bromo-4- chloro-3-indolyl glucuronide) solution and were incubated at 37^oC overnight. A characteristics blue color would be the expression of GUS (β-glucuronidase) gene in the plant tissue. Proper control for GUS histochemical assay was done with the explants having no Agrobacterium infection. After X-gluc treatment explants were transferred to 70% alcohol for degreening. Following degreening explants were observed under stereomicroscope.

Source of Information: Thai Journal of Agricultural Science 2008, 41(3-4): 127-133

Article Link (Online Journal): www.thaiagj.org

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The transformation experiment was performed by optimizing two important parameters: infection time and co-cultivation period. Infection was most effective when explants were inoculated for 15 minutes (72.59% GUS positive) and co-cultivated for four days (70.37% GUS positive) with Agrobacterium. Among the varieties, BRRI dhan- 33 showed the highest response to GUS assay (65.33% GUS positive). Transformation percentage (putative) was also highest in BRRI dhan-33 (4%). 10 minutes infection time and co-cultivation period of three days was found effective for regeneration of shoots (2.45% and 4.45%, respectively). Among the varieties, BRRI dhan-33 produced the highest percentage (61.11%) of rooted shoots. We have got a simple protocol for rice transformation but to make it more precise and reproducible further research considering many other factors with more laboratory facilities should be carried out.

Knowledge Management: Journal