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Factors Enhancing Virulence Response of Agrobacteria in Cucumber (cucumis Sativus L.)

A. K. M. Mohiuddin
Dept of Biotechnology. University Putra Malaysia, 43400 Serdang, Selangor DE, Malaysia, Department of Biotechnology and Genetic Engineering. Mawlana Bliasliani Science and Technology University, Santosh, Tangail- 1902, Bangladesh .

Zaliha C. Abdullah
CRAUN Research Sdn. Bhd., Lot 3147. Block 14, J Sultan Tengah, 93055, Kuching, Sarawak, Malaysia.

M. K. U. Chowdhury
Department of Biology, Claflin University, 400 Magnolia Street, Orangeburg, SC 29115, USA.

K. Harikrishna
Sime Darby Technology Centre, Biotechnology Div., 2 Jln. Tandang 46050, P J, Selangor D.E., Malaysia.

Suhaimi Napis
Department of Biotechnology and Genetic Engineering. Mawlana Bliasliani Science and Technology University, Santosh, Tangail- 1902, Bangladesh

Abstract/ Executive Summary:

The virulence response of five wild type Agrobacterium tumefaciens (tumorigenic) strains and five wild type A. rhizogenes (oncogenic) strains was studied in cucumber (Cucumis sativus L.), cv. Spring Swallow. Several physical and chemical factors e.g. Agrobacterium strains, tissue type (stem and petiole tissues), light intensity, dark incubation, concentrations of acetosyringone (a phenolic compound) were tested. The virulent response of Agrobacteria was found to be depend on plant tissue type, Agrobacterium strain, tissue-Agrobacterium interaction and both chemical-physical factors. Specific light intensity, 2500-lux, as well as the acetosyringone concentration, 20-µM, played important role in virulence response. Among the different strains, LBA 4404 and 15834 were highly virulent to cucumber compared to the other strains tested. A. tumefaciens strains A348 and A281 were failed to initiate crown galls in both stem and petiole tissues, however, in the presence of acetosyringone they produced crown galls. Similarly, A. rhizogenes strain A4 initiated hairy roots in the presence of acetosyringone. All developed crown galls and hairy roots showed an autonomous growth on a hormone free medium containing cefatoxime. Thus, enhancement of virulence response was not only dependent on Agrobacterium strains, but also on particular physical and chemical factors also play vital role.

Keywords: Tumourigenic and oncogenic Agrobacterium, Virulence response, Cucumber, Physical- chemical factors

Location:

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Cucumber

Objectives:

To study virulence response of Agrobacterium in Cucumber (Cucumis sativus L.).

Methodology:

Seeds of the cucumber cultivar Spring Swallow (SS) used for this study were soaked in distilled water for approximately 10-20 minutes prior to manual removal of the testa. The peeled seeds (with endotesta) were treated with 70% denatured ethanol for one minute, and then surface sterilised by a solution of 20% (v/v) Clorox, a commercial bleach containing 5.25% sodium hypochlorite, with a dose of 6 drops (per liter) of Tween 20 for 15 minutes. Seeds were then washed extensively, 3-4 times, with sterile water. Healthy and unwrinkled seeds were used for germination (one seed per box) aseptically in Magenta boxes (GA 7, 110-mm height) on hormone free MS full strength basal medium supplemented with 2% phytagel. The medium was adjusted to pH 5.7 before autoclaving at 121°C for 15 min at 1.05 kg/cm^-2 pressure (15-20 psi). Magenta boxes were sealed with semi-transparent and moisture-resistant Parafilm^R The seeds were then incubated in an incubation chamber at 24±1ºC under 16h photoperiod of 2500-lux light intensity provided by cool-white fluorescent and incandescent lights. LB (Luria-Bertani) medium (broth) containing 1% Bacto-peptone, 0.5% Bacto-yeast extract and 1% NaCI (w/v) was used for A. tumefaciens culture. Similarly, for A. rhizogenes YMB medium (Yeast Mannitol Broth) containing K₂HPO₄, MgSO₄.7H₂O, NaCI, yeast extract and mannitol were used. Both the media were adjusted to pH 7.2 before autoclaving at 121°C for 15 min at 1.05 kg/cm^-2 pressure (15-20 psi). For YMB medium- mixture (i) K₂HP0₄, NaCI, yeast extract and mannitol and mixture (ii) MgSO₄. 7H₂O were autoclaved separately. Five strains of both tumorigenic A. turnefaciens and oncogenic A. rhizogenes were cultured with the LB and YMB media, respectively and agitated at 150-175 rpm overnight at 27ºC. A completely randomised design (CRD) was used for all the experiments. Descriptive statistics such as mean and standard error were used for percentage of crown gall and hairy root induction as well as the average number of hairy roots produced from petiole and stem ex plants of cucumber species.

Source of Information: The Agriculturists 7(1 & 2): 72-81 (2009), ISSN-1729-5211

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Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present study confirmed that physical and chemical factors particularly specific light intensity, 2500-lux, as well as the acetosyringone concentration,20-µM, played important role enhancement of virulence response In agrobacteria. Application of these factors in future gene transformation study will facilitate the production of transformed plants with novel genes.

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