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Agrobacterium-mediated Gene Transfer in Potato for Abiotic Stress Tolerance

G. C. Biswas
Scientific Officer
Fruit Research Station, BARI, Binodpur, Rajshahi-6206, Bangladesh

K. M. Nasiruddin
Professor
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh; Bangladesh

M. S. Haque
Professor
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh; Bangladesh

M. Hoque
Lecturer
Department of Agronomy and Seed Science, Sylhet Agricultural University, Sylhet; Bangladesh

A. Hoque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh; Bangladesh

Abstract/ Executive Summary:

The experiment was carried out to standardize the callus induction potentiality and transformation ability of leaf and callus explants of potato cv. Granola during the period of January to June 2009 in the Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. Callus induction ability of leaf explants was observed in response to different concentrations and combinations of growth regulators supplemented to MS medium. The highest number of calli per vial (5.67), percentage of callus induction (94.44%) and callus weight (2.66 g) were observed in MS medium containing 3.0 mgL^-1 NAA and 2.5 mgL^-1 BAP. No callus was observed in medium without growth regulators. Leaf discs and callus explants of potato cv. Granola were inoculated with Agrobaterium tumefaciens strain LB4404 carrying CIPK (abiotic stress tolerant) sense gene and a binary vector pBI121, which contains GUS (reporter gene) and nptII (kanamycin resistance) gene. The transformation work was performed with different infection times and co-cultivation periods. The leaf discs and callus of potato cv. Granola were found GUS positive after inoculation and selection. The highest GUS positive response (83.33%) was recorded from the leaf disc when inoculated for 7 minutes and co-cultured for 5 days. The highest GUS positive response (88.89 %) was observed for 3 minutes inoculation time and 5 days co-cultivation periods in case of callus of potato cv. Granola. These results of GUS histochemical assay indicated that, abiotic stress tolerant CIPK sense gene was transferred in potato cv. Granola.

Keywords: Agrobaterium, Gene, Transfer, Stres, Tolerance

Location: Biotechnology Laboratory of Bangladesh Agricultural University, Mymensingh

Start Date: 00-01-2009

End Date: 00-06-2009

Program Area: Variety and Species

Commodity: Potato

Objectives:

To standardize the callus induction potentiality and to develop abiotic stress tolerant potato through Agrobacterium-mediated genetic transformation

Methodology:

The experiment was conducted during period of January to June, 2009 in the Biotechnology Laboratory of Bangladesh Agricultural University, Mymensingh to observe the regeneration potentiality and to transform the genetic make up of potato cv. Granola through Agrobacterium tumefaciens starin LBA4404 containing pBI121. This vector includes CIPK sense gene encoding calcineurin B-like protein conferring abiotic stress tolerance. Experimental design and treatments: The experiments were laid in Completely Randomized Design (CRD) with four replications. For callus induction, two different factors were included: i) Different concentrations of NAA (0.0, 2.5, 3.0 and 3.5 mg/L) ii) Different concentrations of BAP (0.0, 2.0, 2.5 and 3.0 mg/L) Agrobacterium- mediated genetic transformation of potato cv. Granola consisted of four factors: i) Different concentrations of cefotaxime (100, 200 and 500 mg/L) ii) Different concentrations of kanamycin (20, 40 and 50 mg/L) iii) Inoculation time (3, 5, 7 and 9 minutes) iv) Co-cultivation period (3, 5 and 7 days). Culture media: For pre-culture and the induction of callus, MS (Murashige and Skoog, 1962) medium supplemented with different concentration of BAP and NAA were used. Two different types of culture media, namely YMB (Yeast Extract Mannitol Broth) medium and LB (Luria Broth) medium were used with kanamycin as antibiotic to grow the strain of genetically engineered Agrobacterium tumefaciens. MS medium without growth hormones was used for co-cultivation. Cefotaxime (500 mg L^-1) was used for washing the explants after co-cultivation. MS medium supplemented with 3.0 mg L^-1NAA and 2.5 mg L^-1BAP and 200 mg L^-1cefotaxime was used for post-cultivation and regeneration purpose. For low selection medium, MS medium supplemented with 3.0 mg L^-1NAA, 2.5 mg L^-1BAP, 20 mg L^-1kanamycin and 100 mg L^-1cefotaxime; whereas for high selection medium, MS medium supplemented with 3.0 mg L^-1NAA, 2.5 mg L^-1BAP, 40 mg L^-1kanamycin and 100 mg L^-1cefotaxime were used. MS medium supplemented with 3.0 mg L^-1NAA, 2.5 mg L^-1BAP, 40 mg L^-1kanamycine and 100 mg L^-1cefotaxime were used for regeneration.Culture techniques: Six pieces of leaf discs were separately cultured on MS medium supplemented with different combinations of BAP and NAA. The culture plates were placed under dark in a room with controlled temperature (25± 2°C).Leaf discs were arranged horizontally on each petridish and gently pressed into the surface of the sterilized pre-culture medium. The culture plates were then placed under dark in a room with controlled temperature (25±2°C). All the explants (callus) were maintained in pre-culture media for 5 to 7 days depending on the explants. For maintenance, one single colony from previously maintained Agrobacterium stocks was streaked on to freshly prepared petridish containing YMB medium having kanamycin. The petridish was sealed with parafilm and kept at room temperature for at last 48 hours. This was then kept at 4°C to check over growth. Such culture of Agrobacterium strain was thus ready to use for liquid culture. The cultures were subculture regularly at each week in freshly prepared media to maintain the stock. For infection from this, Agrobacterium stock single streak was taken in an inoculation loop and was inoculated in a conical flask containing liquid LB medium with 50 mg L^-1kanamycin. The culture was allowed to grow at 28°C for three days to get optimum population of Agrobacterium for infection and co-cultivation of explants. Following the determination of density, the pre-culture explants were dipped into bacterial suspension (OD600= 0.60) for 3, 5, 7 or 9 minutes before transferring them to co-cultivation medium. All the explants were maintained in co-cultivation media for 3, 5 and 7 days. Co-cultured vial containing explants were placed under fluorescent illumination (1500 lux) for 16/8 hours light/dark cycle at 25±2°C.After co-cultivation, the infected explants were washed twice with sterile distilled water and once with sterile distilled water containing 500 mg L^-1cefotaxime. Then the explants were transferred onto post-cultivation medium containing 200 mg L^-1cefotaxime. Following post-cultivation, the explants were transferred onto low selective medium. After low selection the calli were transferred onto high selection medium. For this experiment, survived calli were immersed in X-gluc (5-bromo-4-chloro-3-indoly-l- glucuronide) solution and were incubated at 37°C overnight. A characteristic blue color would be the expression of GUS (β-Glucuronidase) gene in the plant tissue. After ten days the survived callus were transferred to regeneration medium. Recording data: Callus initiation was started after eight days of incubation of explants. The number of callus initiated over a number of days was recorded. The number of explants producing callus in each vial was recorded. The percentage of callus induction was calculated by the following formula: Per cent callus induction = No. of explants induced calli ÷ No. of explants incubated × 100. The weight of callus was measured on the basis of the difference between the weight of vial containing leaf explants before and after callusing. The number of leaf and callus giving positive response to GUS histochemical assay is recorded. The percentage of GUS positive explants is calculated on the basis of the number of calluses assayed for GUS and the total number of calli positive for GUS.

Source of Information: Int. J. Sustain. Crop Prod., 5(3):1-7, 2010

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Histochemical GUS assay performed soon after co-cultivation of explants with Agrobacterium tumefaciens strain LBA4404 containing the gene for GUS expression produced characteristic blue colour with X-gluc. Therefore, the presence of this blue colour gave a preliminary indication of GUS gene transfer from bacterial plasmid into the plant cell. The result of GUS assay depended upon many factors like, variety, inoculation time and co-cultivation periods. From the GUS histochemical assay, the result indicated that the potato cv. Granola showed positive responses towards transformation. The leaf discs and callus of potato cv. Granola were found GUS positive after inoculation and selection.
Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 herbouring CIPK sense gene was used for infection of leaf explants. CIPK sense gives defense against a variety of abiotic stresses tolerance including drought, light, cold and wounding. Agrobacterium tumefaciens strain LBA4404 has nptII gene within its T-DNA, which is abiotic stress tolerant. The results of GUS histochemical assay indicated that abiotic stress tolerant CIPK sense gene was transferred in potato cv. Granola. In this way, we can develop abiotic stress tolerant transgenic potato. Thus, using this protocol, agronomically and economically important gene/genes can be transferred to the locally grown potato variety in further program.

Knowledge Management: Journal