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Transient Gus Expression in Jute (corchorus Capsularis L. Var. D-154) Explants After Infection

A. B. M. Mamun Hossain
Division of Genetics, Institute of Food and Radiation Biology, AERE, Savar, Dhaka, Bangladesh

Golam Ahmed
Division of Genetics, Institute of Food and Radiation Biology, AERE, Savar, Dhaka, Bangladesh

R. I. Khan
Department of Agronomy and Plant Genetics, University of Minnesota, MN 55108, USA.

Serajul Islam
Division of Genetics, Institute of Food and Radiation Biology, AERE, Savar, Dhaka, Bangladesh

Abstract/ Executive Summary:

Two gene constructs, BSF 16 (carrying GUS gene and Basta resistant gene) and Tab7 (carrying GUS gene and kanamycin resistant gene) were used for insertion of useful genes into jute tissues through Agrobacterium tumejaciens infection. Hypocotyl, plumules and cotyledonary bases with petiole from 24 and 48 h old germinating seedlings were used as explants. Successful delivery of genes took place in all types of explants as was evident by GUS assay. The rate of delivery of genes was highest in 48 h old plumule explants followed by cotyledonary base explants. The gene was delivered into the cut surface of the meristematic zone of both plumules and cotyledonary bases from where multiple shoot regeneration took place. However, no transgenic plants grew to maturity.

Keywords: GUS expression, Jute, Agrobacterium tumefaciens

Location: Division of Genetics, Institute of Food and Radiation Biology, AERE, Savar,Dhaka, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Jute

Objectives:

To develop Agrobacterium mediated genetic transformation system in jute. Here, we report the successful delivery of GUS gene into jute tissues by Agrobacterium tumejaciens, confirmed by histochemical GUS assay.

Methodology:

Three types of explants, namely (a) hypocotyl, (b) plumules and (c) cotyledonary bases with petiole were excised from 24 and 48 h old germinating seedlings of Corchorus capsularis L. var. D-154 and were used in all transformation work. Each experimental treatment consisted of four replicate Petri dishes, each with 30 explants. Agrobacterium tumefaciens (strain AGL1), carrying each of the following two gene constructs were obtained from Dr. T. J. Higgins of the CSIRO, Division of Plant Industry, ACT 2601, Australia. Agrobacterium strains were cultured for 18 - 20 h on an orbital shaker (100 rpm) at 30°C in liquid Luria broth medium to obtain OD ~ 600 for bacterial concentration before being used for inoculation. Explants were placed in this suspension for 1 h at room temperature. The explants were then blotted before plating on regeneration media made of MS salts, different growth regulators, sucrose (30 g/l) and agar (6.0 g/l). Cocultivation was carried out for 3 - 5 days in the growth chamber at 28°C with 16 h light period. Some explants from this stage were used for GUS staining and observed under microscope to determine the frequency of infection. The remaining explants were then transferred to selection media. Antibiotics and Basta were dissolved in water and filter-sterilized before being added to the selection media. Explants were maintained at 28° C with 16 h light period and subcultured every four weeks. Explants inoculated with bacteria and placed on media lacking Basta/kanamycin were treated as control. Regeneration media (for cocultivation) were used as (i) MS + 0.5 mg/l BAP+ 50 mg/l tyrosine + 6.0 g/l agar (Sigma, USA), (ii) MS + 0.5 mg/l BAP + 0.07 mg/l NAA + 6.0 g/l agar, (iii) MS + 0.02 mg/l TDZ + 0.07 mg/l NAA + 6.0 g/l agar and (iv) MS + 1.0 mg/l BAP + 0.5 mg/l IAA + 6.0 g/l agar. Selection media were same as the regeneration media but with 300 mg/1 cefotaxime/timetin + 5 - 20 mg/l Basta (for BSF 16)/20 - 50 mg/l kanamycin. Media lacking Basta/kanamycin were treated as control.

Source of Information: Plant Tissue Culture, Vol: 8, (1), pp: 11-18, (June) 1998

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Our results, indicate that Agrobacterium infection of cotyledons with base may lead to more efficient transformation compared to hypocotyl and plumule explants. In case of explants b (plumule), the rate of infection was higher, but the chance of getting transgenic plants was less due to its low regeneration capacity.

Knowledge Management: Journal