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Agrobacterium-mediated Genetic Transformation of Potato for Abiotic Stress Tolerance

S. N. Begam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

K. M. Nasiruddin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. Yasmin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Two potato varieties, Cardinal and Heera, were used to optimize the Agrobacterium-mediated genetic transformation system in potato for abiotic stress tolerance. MS medium supplemented with different concentrations and combinations of NAA and BAP were used to obtain callus induction from leaf explants of potato cv Cardinal and Heera. Maximum percentage of callus induction (95.83%) was observed in MS medium supplemented with 4 mg/L NAA and 2 mg/L BAP. The highest callusing was found in Cardinal and the lowest in Heera. Calluses were inoculated with Agrobacterium tumefaciens strain LBA4404 (pB1121) carrying CIPK sense gene, selectable marker gene nptII gene conferring resistance to kanamycin and the GUS reporter gene. Both the varieties were found GUS positive after inoculation and selection. Cardinal had maximum percentage of survived callus (25%) on selection media. Stable intregration and expression of the transgenes were confirmed by GUS histochemical assay.

Keywords: Potato, Agrobacterium mediated transformation, Abiotic stress

Location: Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Potato

Objectives:

To optimize the Agrobacterium- mediated genetic transformation system for abiotic stress tolerance.

Methodology:

The present investigation was carried out in the Biotechnology Laboratory of the Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. Two varieties of potato (Cardinal and Heera) were used in the experiment. Leaf explants were used for callus induction. The induced calli were then used for Agrobacterium-mediated genetic transformation. Genetically engineered Agrobacterium tumefaciens strain LBA4404 (pB1121) containing the uidA gene encoding GUS (β-glucuronidase), the nptII gene and the CIPK sense gene encoding calcineurin B-like protein conferring abiotic stress tolerance driven by CaMV promoter and NOS terminator within the right border (RB) and left border (LB) region of the construct was used. For the induction and maintenance of callus from leaf, MS (Murashige and Skoog, 1962) medium containing different concentrations and combinations of BAP (0, 2, 4 and 6 mg/L) and NAA (0, 1, 2 and 4 mg/L) was used. YMB medium was used for Agrobacterium culture aintenance and LB medium was used as Agrobacterium working culture medium for transformation. Calli were infected in liquid bacterial strain and co-cultivated for 3 days on the MS medium without growth regulators. After co-cultivation, explants were washed with sterile water with added cefotaxime (500 mg/l) and transferred onto MS medium supplemented with 3 mg/L NAA, 4 mg/L BAP and 200 mg/L cefotaxime. For low selection, MS medium supplemented with, 3 mg/L NAA and 4 mg/L BAP, 20 mg/L kanamycin and 100 mg/L cefotaxime was used. For high selection and regeneration, MS medium supplemented with 3 mg/L NAA and 4 mg/L BAP, 40 mg/L kanamycin and 100 mg/L cefotaxime was used. Transformation ability was assessed through histochemecal assay of GUS reporter gene in callus tissue. The experiments were laid in Completely Randomized Design (CRD) with three replications. The analysis of variances for different parameters were performed and means were compared by the Duncan’s Multiple Range Test (DMRT).

Source of Information: Progress. Agric. 19(2) : 37-43, 2008 ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16919/11879

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

After antibiotic treatment the survived calli were transferred to regeneration medium. The regeneration medium comprised with MS medium containing 3 mg/L NAA and 4 mg/L BAP, 100 mg/L cefotaxime and 40 mg/L kanamycin and root was obtained from three Cardinal and two Heera calli (Plate 1). The system developed here would be used for the introduction of agronomically important trait in potato and regeneration of transgenic plants. However, more studies should be conducted including molecular analysis of transgenic plants. Cardinal had maximum percentage of survived callus (25%) on selection media. Stable intregration and expression of the transgenes were confirmed by GUS histochemical assay.

Knowledge Management: Journal