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Development of Transgenic Rice (oryza Sativa L.) Plant Using Cadmium Tolerance Gene (ycfi) Through Agrobacterium Mediated Transformation for Phytoremediation

Md. Mahmudul Islam
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi, 6205, Bangladesh

Md. Khalekuzzaman
Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi, 6205, Bangladesh

Abstract/ Executive Summary:

Cadmium has adverse effects on plant development and ultimately reduces production. The investigation was undertaken for successful gene transfer and phytoremediation through an efficient Agrobacterium-mediated genetic transformation method using cadmium tolerance gene (YCF1). Embryogenic calli induced after 20 days of highly regenerating rice cultivar BRRI dhan29 and Agrobacterium strain GV 3101 was transformed with binary vector pCAMBIA 1303-YCF1 which contained the hygromycin phosphotransferase (HPT) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene, were used for genetic transformation in the experiment. The transformed colonies were selected on 15 mg L^-1 hygromycin and 50 mg L^-1rifampicin to select hygromycin resistant shoots. Hygromycin-resistant shoots were subsequently rooted on root induction medium. Rooted plantlets were transferred to pot-soil, hardened and grown in a greenhouse until maturity and stable integration, expression of YCF1 gene was also confirmed by using PCR analysis. The maximum transformation efficiency of 22% was obtained using 500 mg L^-1 cefotaxime as a bacteriostatic agent to inhibit growth of Agrobacterium and 100 mM acetosyringone in co-cultivation medium. Southern blot analysis was performed to confirm that transgenes (HPT and YCF1) were stably integrated into the plant genome. All transgenic plants showed single-copy of transgene integration in the host genome. This transgenic rice plant will uptake cadmium from soil and will protect rice grain from cadmium and store into cell vacuoles of rice plants. As a result soil will be free from cadmium through phytoremediation process.

Keywords: Agrobacterium, Cadmium, Oryza sativa, Phytoremediation, Transformation

Location: Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Sciences, University of Rajshahi, Rajshahi

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To develop cadmium tolerance transgenic rice plant using the yeast cadmium factor 1 (YCF1) gene through Agrobacterium mediated genetic transformation and to study phytoremediation.

Methodology:

Seed sterilization: Seeds of rice cultivars BRRI dhan29 were collected from the Regional Research Station of Bangladesh Rice Research Institute (BRRI), Rajshahi, Bangladesh. The seeds were dehusked manually to preserve the embryos from mechanical damage. The dehusked seeds were surface sterilized in 70% ethanol for 1 min and then shaken for 30 min on a gyratory shaker at 200 rpm in 2.6% w/v sodium hypochlorite (50% Clorox) containing 3 drops of Tween 20 per 100 mL Clorox solution. The seeds were rinsed in sterile distilled water and cultured on callus induction media. Culture media and culture conditions: The Basic Medium (BM) was composed of MS salts and organic compounds, 30 g L^-1 sucrose and 8 g L^-1 agar. The pH was adjusted at 5.7 before adding the gelling agent and the media were autoclaved for 20 min at 121°C and 1.07 kg cm^-2. Petri dishes with 25 mL of medium and sealed with Parafilm were used. Callus induction: Ten mature embryos from isolated sterilized seeds were placed individually in each Petri dish containing 25 mL of modified MS with various concentrations of 2, 4-D singly and 2, 4-D with NAA. The seeds were incubated in the dark at 25±2°C. Only embryogenic calli were used for genetic transformation. The percentage (%) of Callus Induction Frequency (CIF) for each group was calculated using the following formula: CIF (%) = (Total No. of mature embryo that produced callus/Total No. of mature embryo plated) x 100. Selection of embryogenic callus: Embryogenic callus of indica rice (Oryza sativa L.) cultivars namely BRRI dhan29 can be described as yellowish and granular callus, compact, greenish-yellow, granular with smaller cells and very dense cytoplasm callus. These types of embryogenic callus were selected for genetic transformation. Bacterial strain and plasmid vector construction: The recombinant plasmid vector pCAMBIA 1303-YCF1 containing hygromycin phosphotransferase (HPT) and yeast cadmium factor 1 (YCF1) genes was introduced into Agrobacterium tumefaciens strain GV 3101 by Agrobacterium mediated transformation by heat-shock method. The HPT gene confers resistance to the antibiotic hygromycin as plant selection marker and the YCF1 gene was introduced into the vector as a target gene with the aim of enhancing heavy metal tolerance and accumulation. Agrobacterium strain culture and infection: Agrobacterium strain GV 3101 was cultured on liquid YEP medium containing kanamycin (50 mg L^-1), rifampicin (50 mg L^-1) and agar (8 g L^-1) for 3 days at 27°C in the dark. The bacteria were collected and suspended in medium containing acetosyringone (100, 200, 400, 600 and 1000 ppm). For Agrobacterium infection, the density of the bacteria was adjusted (OD₆₀₀ = 1.2, 1.1, 0.9, 0.8 and 0.6) and the rice calli were immersed in a bacterial suspension for 25 min. Excess bacteria were removed by blotting the calli on filter paper. The calli were transferred to Petri dish containing MS medium. The plates were sealed with parafilm to prevent evaporation of the medium and submitted to 3 days of co-cultivation at 25±2°C in the dark. Calli were then washed twice in sterile water to remove Agrobacterium. The co-cultured calli were blotted dry on filter paper and plated on MS medium supplemented with rifampicin (50 mg LG1) and hygromycin (0 to 30 mg L^-1). The plates were sealed with surgical tape and incubated at 25±2°C using a 16 h light. Proliferating hygromycin resistant calli were transferred to the same fresh medium. After shoots formation from hygromycin resistant calli and transferred on selection medium. Explants were sub cultured every three weeks followed by harvest of shoots which appeared. Suitable concentration of selective agent (hygromycin) for transformant selection: To determine the effect of hygromycin concentrations on shoot regeneration, 20 days old were placed on shoot induction medium (MS medium supplemented with 2.0 mg LG1 BAP, 1.0 mg LG1 NAA and 1.5 mg KING1) with hygromycin (0, 5, 10, 15, 20 and 30 mg LG1) in Petri dishes and the cultures were maintained at previously described conditions. The regeneration response was evaluated under the selection conditions after 3 weeks of culture in vitro. Regeneration: Proliferating Hygromycin resistant calli were transferred to the same fresh medium. Additionally, non- infected by Agrobacterium embryogenic calli were included as controls. The number of shoots and in vitro plants per embryogenic calli were determined after ten weeks of culture on regeneration medium that consisted of MS mineral salts supplemented 2.0 mg LG1 6-benzylaminopurine (BAP), 1.5 mg LG1 Naphthalene Acetic Acid (NAA) and 1 mg L^-1 kinetin, 30 g L^-1 sucrose and 6 g L^-1 agar. The explants were cultured in the dark at 26±1°C. The percentage of calli with shoots and regeneration rate were calculated using the following formula: Embryogenic calli with shoots (%) = (No. of calli with shoots/Total of embryogenic calli) x 100, and Regeneration (%) = (No. of in vitro plants/Total of embryogenic calli) x 100. PCR analysis of transformation: Genomic DNA was isolated from transformed shoots. The PCR analyses were carried out by using YCF1 gene two primers namely, forward 5’TAC CGA GGA ACT TTA GTA GTG3’ and reverse 5’ TGG CAT CAT AAT AAC TAG TAT 3’ for amplification of YCF1 gene transformants and 5’ CAT GTG TAT CACTGG CAA ACT GT 3’ (forward) and 5’ GTA CTT CTA CACAGC CAT CGG TC 3’ (reverse) for the HPT gene. The reaction mixture (20 μL) of PCR composed of 1.0 μL DNA template, 2.0 μL 10x buffer, 1.0 μL (2.5 mM) dNTPs, 2.0 μL (25 mM) MgCl₂, 1.0 μL of each primer (F/R), 0.4 μL TaqDNA polymerase and ddH₂O 13 μL. Reaction procedures were carried out at 94°C for 4 min followed by 25 cycles at 94°C for 1 min, 56°C for 45 sec and 72°C for 1 min. After the final cycle, the reactions were maintained at 72°C for 5 min before completion. Finally, PCR products were analyzed on 1% agarose gel with 0.5x TBE buffer. Southern blot analysis of the regenerated plants: Leaves of the fully-grown putative transgenic rice plants were used to extract total genomic DNA by the methods described by McCouch et al. (1988). Southern blot analysis was performed to confirm the stable integration of YCF1 genes in to the transgenic rice. DNA samples (5 μg) were digested with restriction endonuclease EcoRV and then fractioned on 0.8% agarose gels and transferred to a Hybond-N membrane according to manufactures instructions. The 500 bp PCR amplified HPT gene and 700 bp PCR amplified YCF1 gene were labeled with a-32P dCTP using the Rediprime II random prime abeling system and used as hybridization probes. The probes were labelled with α-32P dCTP using rediprime labelling kit. Membranes were washed twice at room temperature in 2x SSPE/1% Sodium Dodecyle Sulphate (SDS) for 10 min and at 65°C in 1x and 0.1 x SSPE/0.1% SDS for 15 min each time and then autoradiograph.

Source of Information: Asian Journal of Agricultural Research 9 (4): 139-154, 2015; ISSN 1819-1894

Article Link (Online Journal): DOI: 10.3923/ajar.2015.139.154

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

By using Agrobacterium tumefaciens strain GV 3101 carrying plasmid pCAMBIA 1303 harbouring YCF1 gene, obtained several transgenic rice lines of elite rice cultivar BRRI dhan29. They were highly resistant to heavy metals especially cadmium. The presence and expression of the YCF1 genes were confirmed by qualitative and molecular analyses. The obtained transgenic lines could be useful for phytoremediation and rice breeding in the world.

Knowledge Management: Journal