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Optimization of Agrobacterium Tumefaciens Mediated Genetic Transformation Protocol for Aromatic Rice

M. R. Hossain
Department of Genetics and Plant Breeding , Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

L. Hassan
Department of Genetics and Plant Breeding , Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

A. K. Patwary
Department of Genetics and Plant Breeding , Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. J. Ferdous
Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

A rapid, efficient and reproducible genetic transformation protocol was optimized for four aromatic rice varieties by using the established plant regeneration protocol. Mature embryos were inoculated with Agrobacterium tumefaciens strain EHA105 carrying a binary vector pIG121-Hm with GUS (reporter gene) and hpt (hygromycin resistance) gene and the transformation experiment was performed by optimizing two important parameters viz. infection times and co-cultivation periods. The highest response to GUS assay was showed by Kalizira (70% GUS positive) followed by Pusa Basmati 1 (66.67%) when the explants were inoculated for 25 minutes and co-cultivated for three days. Twenty five minutes infection time (9.44%) and three day co- cultivation period (8.06%) were found effective for percentage of transgenic shoot regeneration. The highest percentage of putative transgenic shoots was regenerated by variety Kalizira (33.33%) followed by Pusa Basmati 1 (20.0%) and Radhunipagol (13.33%). Among the varieties, Kalizira produced the highest percentage (60%) of rooted shoots. Kalizira also showed the highest survival rate in growth chamber (75%) and in field condition (60%). The performance of Tulsimala was poor for almost all the cases.

Keywords: Agrobacterium, GUS, hpt gene, Transformation and Aromatic rice

Location: BAU, Mymensingh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

The present piece of research work was framed and materialized to standardize an efficient, high frequency and reproducible genetic transformation protocol for aromatic rice using Agrobacterium tumefaciens as a vector.

Methodology:

Sterilized mature embryos along with endosperm (dehusked grain) of four aromatic rice varieties viz. Kalizira, Radhunipagol, Tulsimala and Pusa Basmati-1 were placed horizontally with gentle press onto the surface of the sterilized callus induction medium.This binary vector contains the udiA gene (Jefferson et al., 1987) encoding GUS (β-glucuronidase) reporter gene, driven by CaMV 35S promoter, the (nptΙΙ) gene (Herrera-Estrella et al., 1983) encoding neomycin phosphotransferase ΙΙ conferring kanamycin resistance driven by NOS promoter and NOS terminator in its right and left border region of the construct. The vector also contains the hpt gene. In this study, one way selection was done using kanamycin. A single streak from previously maintained Agrobacterium stock was inoculated in liquid LB medium + 50 mgL-1 kanamycin. The culture was allowed to grow at 28⁰C to get optimum population of Agrobacterium for infection. Freshly excised calli were immersed into bacterial suspension (OD600=0.6) for 20, 25 and 30 minutes with gentle shaking to get suitable and sufficient infection. The infected calli were blotted dry to remove excess bacterial suspension and were co-cultivated for 2, 3 and 4 days on co-cultivation media and incubated under fluorescent illumination with 16/8 hours light/dark cycle at 25±20C for 2-4 days. The intensity of light was maintained at 1500 lux. Transient GUS assay was done at the end of co-cultivation with randomly selected fifteen inoculated calli according to the method described by Jefferson (1987). The infected explants were washed three times with sterile distilled water and once with liquid MS media supplemented with 200 mg l-1 of Cefotaxime and then were transferred onto post-cultivation medium for 15 days. For shoot regeneration, 10 calli were transferred to selection and regeneration medium. The aseptically separated shoots were again cultured on conical vials for rooting on rooting medium. When the plantlets become 5-8 cm in length with sufficient root system, they were taken out from the vials and placed in hardening chamber and the survived plants were transferred to pot to acclimatize in field condition. Daily observation was carried out at every step to note the responses and the contaminated vials were discarded immediately to avoid contamination. The analyses of variances were done and means were compared by the Duncan's Multiple Range Test (DMRT) using MSTATC program.

Source of Information: J. Bangladesh Agril. Univ. 7(2): 235–240, 2009 ISSN 1810-3030

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present study thus found Kalizira to be best variety in terms of Agrobacterium mediated genetic transformation. It can be recommended that Kalizira will be a potential variety if any gene(s) of interest viz. salinity tolerant &/or disease resistant is aimed to transfer to aromatic rice variety.

Knowledge Management: Journal