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Agrobacterium-mediated Genetic Transformation of Two Varieties of Brassica: Optimization of Protocol

M. M. A. KHAN
Department of Biotechnology, BAU

A. B. M. A. H. K. ROBIN
Department of Genetics and Plant Breeding, BAU, Mymensingh-2202, Bangladesh.

M . A. N. NAZIM-UD-DOWLA
Department of Genetics and Plant Breeding, BAU, Mymensingh-2202, Bangladesh.

S. K. TALUKDER
Department of Genetics and Plant Breeding, BAU, Mymensingh-2202, Bangladesh.

L. HASSAN
Department of Genetics and Plant Breeding, BAU, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Two rapeseed varieties, namely Tori-7 and BARI Sarisha-8, respectively, from Brassica rapa and Brassica napus were selected to observe the transformation ability. Petioles were inoculated in Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pBI21 with GUS (reporter) and npt/l (kanamycin resistant) gene. The transformation experiment was performed by optimizing two important factors: preculture time and co-cultivation time and also selected out the best variety. Infection was most effective when explants were pre cultured for 72 hours (80% GUS positive). and co-cultivated for 72 hours (72% GUS positive). The variety Tori-7 showed the best response to GUS assay (65% GUS positive). Callus induction was the highest in Tori-7, which were 6% with 72 hours of preculture period and 9% in 48 hours of co-cultivation. Number of putative transformed plantlets were highest in Tori-7 (7 plants) followed by BARI Sarisha-8 (3 plants).

Keywords: Transformation, Brassica, GUS, Agrobacterium.

Location: Tissue Culture Laboratory and Genetic Engineering & Biotechnology Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

Start Date: 01-07-2004

End Date: 30-05-2005

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To develop a reproducible any efficient protocol for the insertion of molecular genes into Brassica through Agrobacterium tumefaciens vectors and
To standardize the periods of preculture and co-cultivation required for transformation and
To analyze the putative transgenic plants using histochemical GUS assay and PCR technique.

Methodology:

The experiment was conducted in the Tissue Culture Laboratory and Genetic Engineering & Biotechnology Laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh during the period from July 2004 to April 2005. Two cultivars one from Brassica rapa and the other from Brassica napus were taken to standardize different parameters of plant transformation. The varieties were Tori-7 and BARI Sarisha-8, respectively. The germinated seedlings raised in axenic culture were used as the source of explants. Petiole segments were used as explants. After four days, petioles were excised into 3-4 mm pieces from the seedlings. This was carried out by gently holding the base of the petioles with forceps, and slicing through the stem just below the shoot tip using sterilized surgical blades. Ten pieces of petiole segments were arranged horizontally on each petridish and gently pressed into the surface of the sterilized pre-culture medium. The culture plates containing explants were placed under fluorescent light in a room with controlled temperature (25±2°C) using 16 hours photoperiod. All the explants were kept in preculture media for 1-3 days. The petridishes were checked daily to note the response and the development of contamination. To investigate the effect of different treatments of the experiment, data were collected on the following parameters: percent GUS positive, days to callus initiation, percent callus induction, days to shoot initiation, total number of shoots per petridish, number of transgenic plantlets, and number of transgenic shoots with roots. The data for the parameters under present experiments were statistically analyzed wherever applicable. The analyses of variances for different parameters were performed and means were compared by the Duncan's Multiple Range Test (DMRT).

Source of Information: Bangladesh J. Agril. Res. 34(2): 287-301, June 2009 ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

We have got a simple protocol for Brassica transformation hut to make it more precise and reproducible, further research considering many other factors with more laboratory facilities should be carried out.

Knowledge Management: Journal