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Agrobacterium Mediated Genetic Transformation in Potato (solanum Tuberosum L.)

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

M. A. Hossain
Ministry of Establishment, Bangladesh Secretariat, Dhaka-1000, Bangladesh

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

M. G. Rabbani
Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

R. Sultana
Department of Agricultural Chemistry, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Agrobacterium mediated genetic transformation was conducted in two potato varieties (Diamant and Heera) to observe their transformation ability and develop a suitable and reproducible transformation protocol for those potato varieties. Agrobacterium strain LBA 4404 having a binary vector pBI121, containing selectable marker gene nptll (neomycin phosphotransferase) conferring resistance to kanamycin and the gus (ß-g/ucuronidase) reporter gene were used. Leaf and internodal segments were used as explants. Both the varieties were found effective for producing best GUS response (86.66% GUS positive). Regeneration of plantlets (putative transformed plantlets) in kanamycin containing medium showed the highest regeneration in Diamant leaf (5.71%) followed by Heera internode (2.8%).

Keywords: Agrobacterium tumefaciens, Transformation, Potato, Solanum tuberosum L., ß-gtucuronidase (gus), Neomycin phosphotransferase (npt/l)

Location: Biotechnology Laboratories of the Departments of Horticulture and Biotechnology, Bangladesh Agricultural University, Mymensingh

Start Date: 00-01-2004

End Date: 00-05-2005

Program Area: Variety and Species

Commodity: Potato

Objectives:

To develop a reproducible and genotype independent genetic transformation protocol of two potato varieties.

Methodology:

The present investigation was carried out in the Biotechnology Laboratories of the Departments of Horticulture and Biotechnology, Bangladesh Agricultural University, Mymensingh-2202 from January 2004 to May, 2005. Two potato verities viz. Diamant and Heera were used for this experiment. Genetically engineered Agrobacterium tumefaciens strain LBA4404 was used for infection in the experiment. This strain contains plasmid pBI121 of 14 KDa (binary vector). For Agrobacterium culture and incubatuion, two different types of culture media, namely, YMB (Yeast extract Mannitol Broth) and LB (Luria Broth) were used with kanamycin as antibiotic to grow the strain of genetically engineered Agrobacterium tumefaciens. YMB medium was used for Agrobacterium maintenance and LB medium was used as Agrobacterium working culture medium for transformation. For Co-cultivation Murashige and Skoog, 1962 (MS) medium without hormones was used. For washing the co cultivated materials cefotaxime @500mg/l was used. MS medium with 3.5 mg/l NAA (a naphthalene acetic acid), 2.5 mg/l BAP (-benzyl amino purine) and 200 mg/l cefotaxime was used for callus induction. For the selection and regeneration of explants, MS medium with 3.5 mg/l NAA, 2.5 mg/l BAP, 100 mg/l Cefotaxime and 20 mg/l Kanamycin (in low selectiori medium) and 40 mg/l Kanamycin (in high selection medium) were used.

Source of Information: Bangladesh J. Agril. Sci. 32(1): 135-139, January, 2005

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Transformation ability of two kinds of explants, namely, leaf and internode were examined. Following co culture with Agrobacterium, the explants were subjected to histochemical assay to find out the expression of GUS reporter gene within the explant tissue. A very good number of both explant tissues were found positive in transient GUS assay. Between the explants, leaf explant tissues showed higher GUS positive explant (86.66%) than internode (66.66%). Sultana et al. (2004) also reported that potato leaf explants were more effective for Agrobacterium infection. When leaf explants of Diamant cultured in different concentrations of selection media, it was found that two leaflet explants survived and regenerated in both selection media. No internodal explants of Diamant and leaf explants of Heera survived in selection media. Three internodal explants of Heera also survived and regenerated in presence of kanamycin at the concentration 20 mg/l. But two regenerated shoot became albino in selection medium containing kanamycin at the concentration of 40 mg/l.

Knowledge Management: Journal