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Genetic Transformation in Tossa Jute Through Agrobacterium Vectors

A.S.M. Yahiya
Senior Scientific Officer
Genetic Resources and Seed Division Bangladesh Jute Research Institute, Manik Mia Avenue, Dhaka-1207

Abstract/ Executive Summary:

The combined effects between the varieties and BAP (6-Benzyl Amino Purine) concentrations showed statistically significant differences. The highest average number of regenerated cotyledons (4.857) was recorded in the var. O-9897 with 3 mg/l BAP which was statistically identical with the var. O-72 (3.597) with 3 mg/l BAP. The lowest average number of regenerated cotyledons (1.867) was counted in the both vars. O-9897 and O-72 with 1 mg/l BAP. The combined effect of varieties and A. tumefaciens also showed a statistically significance in respect of number of cotyledons produced shoot. The highest number of shoot producing cotyledons (3.70) was recorded in the variety O-9897 with A. tumefaciens which was statistically identical (3.60) with the variety O-72. In Gus histochemical assay, it was found that the two varieties showed positive responses towards transformation. Between the two varieties O-9897 showed the highest response (86.67%) and O-72 showed the poorest response (80.00%) to GUS assay. Control explants did not show any response to the assay. A. tumefaciences strain LBA4404 carrying a binary vector pBI121, which contains selectable marker gene nptII conferring resistance to kanamycin and the GUS reporter gene. After co-cultivation and selection, histochemical GUS assay was performed in two varieties (O-9897 and O-72). In the transformed explants, GUS reporter gene was expressed blue colour in the explants tissues. Non-transformed explants did not show any colour.

Keywords: Jute (Corchorus olitorius L.), Genetic transformation, Agrobacterium vector.

Location: Genetic Engineering Laboratory of Bangladesh Jute Research Institute (BJRI), Dhaka.

Start Date: 01-03-2007

End Date: 28-02-2008

Program Area: Variety and Species

Commodity: Jute

Objectives:

To observe the status of the genetic transformation by Agrobacterium vector of locally available jute varieties so that the desired genes can be introduced for the development of disease as well as pest resistant varieties.

Methodology:

The seeds of C. olitorius used in the experiment were collected from Gene Bank of Bangladesh Jute Research Institute (BJRI) and conducted in the Genetic Engineering Laboratory, Genetic Resources and Seed Division, BJRI, Dhaka during March 2007 to February 2008. Sterilized seeds were germinated on the surface of 50 ml aliquots of agar-solidified hormone free MS based medium and were inoculated in three 100 ml capacity of conical flasks and was placed in a growth room at 28⁰C under 1.0 Wm^-2 of daylight fluorescent illumination with 12 hour photoperiod. The cotyledons were excised from 8-9 days old seedlings with attached petioles were used as explants and were cultured in 250 ml conical flasks containing 50 ml of agar-solidified MS medium supplemented with IAA (0.5 mg/l) and BAP (3.0 mg/l). Eight explants were inoculated in each culture flask and were placed in a growth room and maintained at 28⁰C under 1.0 Wm^-2 of daylight fluorescent tubes with a 12-hour photoperiod. Data were recorded after 6 weeks culture. Genetically engineered A. tumefaciences strain LBA449004 was used for infection in the transformation experiment. This strain contains plasmid pBI121 of 14KDa (binary vector).

Source of Information: Int. J. Sustain. Agril. Tech. 5(8): 01-06, November, 2009.

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 50000

Total Budget (Tk.) : 50000

Achievement/Findings:

It may be concluded that, in Gus histochemical assay, it was found that the two varieties showed positive responses towards transformation. Between the two varieties O-9897 showed the highest response (86.67%) and O-72 showed the poorest response (80.00%) to GUS assay. Control explants did not show any response to the assay. After co-cultivation and selection, histochemical GUS assay was performed in two varieties (O-9897 and O-72). In the transformed explants, GUS reporter gene was expressed blue colour in the explants tissues. Non-transformed explants did not show any colour.

Knowledge Management: Journal