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Establishment of Optimal Conditions for an Agrobacteriummediated Transformation in Four Tomato (lycopersicon Esculentum Mill.) Varieties Grown in Bangladesh

APARNA ISLAM
Biotechnology Programme, Department of Mathematics and Natural Sciences, BRAC University, 66 Mahakhali C/A, Dhaka-1212, Bangladesh.

JEBUNNESA CHOWDHURY
Biotechnology Programme, Department of Mathematics and Natural Sciences, BRAC University, 66 Mahakhali C/A, Dhaka-1212, Bangladesh.

ZEBA I. SERAJ
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh.

Abstract/ Executive Summary:

An efficient transformation protocol for tomato by reducing complexity and increasing transformation efficiency in four varieties namely, Bina tomato-3, Bina tomato-5, Bahar and Pusa Ruby. Transformation of cotyledonary leaf explant was performed with Agrobacterium tumefaciens strain LBA4404, harboring binary vector pBI121 having GUS and nptII marker genes. Frequency of transient GUS expression showed that the transformation competence in tomato was highly influenced by several factors, like optical density of Agrobacterium suspension, incubation period, co-cultivation period etc. In the present study, cotyledonary leaf explant from all four tested varieties found to be efficiently transformed by bacterial suspension having optical density (OD600) of 0.79 with 15 min incubation and 3 days of co-cultivation period. All these conditions along with pre-culture of explants prior to transformation gave better regeneration response following Agrobacterium infection. Moreover, for successful regeneration of transformed shoot 200 mg/l kanamycin was found to be most effective selection pressure. As all four varieties showed similar response the present protocol can be considered as a simple and genotype-independent reproducible protocol.

Keywords: Solanaceae, Transformation parameter, GUS expression, Cotyledonary leaf explant

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Tomato

Objectives:

To establish a simple, genotype independent, efficient and reliable transformation methodology.

Methodology:

Seeds of four varieties of tomato (Lycopersicon esculentum Mill.) namely, Bahar (BR), Bina tomato-3 (B-3), Bina tomato-5 (B-5) and Pusa Ruby (PR) were used in this study. Among these B-3 is a summer variety, while B-5 and Bahar BR are winter varieties of Bangladesh. Seeds of Bina varieties were collected from Bangladesh Institute of Nuclear Agriculture (BINA), Mymansing while seeds of the popular Indian variety, Pusa Ruby was collected from the local market. Seeds of all four varieties were surface sterilized for 1 min with 70% ethanol, followed by 5.25% Clorox treatment with two drops of tween 20 and shaken continuously for 5 - 8 minutes. Seeds were then repeatedly washed with distilled water and kept in a rotator shaker (EDISON; NJ, U.S.A) for 24 - 36 hr in 150 rmp at 28°C while immersed in sterile distilled water. Afterwards seeds were placed on MS(Murashige and Skoog 1962) medium for germination. Subsequent cotyledonary leaves were excised from 8 - 10 days old seedlings and were transversely cut into two to three segments for use as explants. Genetically engineered A. tumefaciens strain LBA4404 containing plasmid pBI121 of 14KDa (binary vector) was used for infection in the transformation experiments. This binary vector contains the udiA gene (Jefferson et al. 1987) encoding GUS (β- glucuronidase) enzyme and the nptII gene (Hoekema et al. 1983) encoding neomycin phosphotransferase II conferring kanamycin resistance, both driven by the NOS promoter and NOS terminator. Overnight grown liquid Agrobacterium culture was used for infection of cotyledonary leaf explants. Prior to infection optical density of the bacterial suspension was determined at 600 nm with the help of a spectrophotometer (Analytikjena Specord 50, Germany). Bacterial suspensions with optical densities of 0.79, 0.64 and 0.42 were used. The explants with or without pre-culture were immersed in the Agrobacterium suspension for infection for different time periods, ranging from 10 to 20 minutes and cocultured for two - three days in dark. For selection of transformed cells to regenerate into shoots various concentrations of kanamycine (50, 100, 150, 200 and 300 mg/l) was evaluated to determine the best selection pressure in regeneration media. Sensitivity of the explant tissue was compared with control having no kanamycin. Each experiment was performed thrice with 15 explants. Following co-cultivation the explants were subjected to transient GUS histochemical assay to score the efficiency of transformation. From each batch of transformation experiment, randomly selected 50 - 60% treated explants were examined for GUS activity by GUS histochemical assay (Jefferson et al. 1987). The explants were submerged in the substrate X-gluc (5-bromo-4-chloro-3-indolyl glucuronide) and incubated at 37ºC for two - three days. They were then washed in 70% alcohol and scored for transient GUS expression. A characteristic blue color as a result of GUS gene expression in the plant tissue was recorded as a positive result. Each experiment was performed in triplicate and proper control was done with explants not infected with Agrobacterium. As a positive control transformed tobacco plant having nptII in the genome was used. Two-way ANOVA was performed considering either optical density or incubation period with different tomato varieties as two factors. All statistical analyses were performed using the GraphPad Prism version 5.00 (www.graphpad.com). p value was considered at the 5% level of significance to deduce inference of the significance of the data.

Source of Information: Journal of Bangladesh Academy of Sciences, Vol. 34, No. 2, 171-179, 2010

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The experiments revealed that transformation frequency in tomato is strongly influenced by various factors. The methodology presented here is simple, repeatable and gives high frequency of transformation. Most importantly this protocol is made considering the highest transformation frequency without compromising regeneration capacity to ensure large amount of trans genic plant development. Several reports on tomato regeneration have already been made (Begum and Mia 1993, Chowdhury 2009, Sarker et al. 2009). In future, using this transformation coupled with the regeneration protocol already established, attempts will be made to develop transgenic tomato to improve our varieties for better yield and quality.

Knowledge Management: Journal