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Genetic Transformation of a Local Tomato (solanum Lycopersicum L.) Variety of Bangladesh

Pronabananda Das
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

Aneesa Ansari
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

Mohammad Nurul Islam*
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

R. H. Sarker
Department of Botany, University of Dhaka, Dhaka‐1000, Bangladesh

Abstract/ Executive Summary:

An in vitro regeneration and Agrobacterium‐mediated genetic transformation protocol was optimized for a local tomato variety, BARI Tomato‐8 using cotyledonary leaf and hypocotyls explants. The explants were treated with various growth regulators in MS at different concentrations and combinations. Highest number of multiple shoot induction was observed from both the explants cultured in MS supplemented with 8.88 μM BAP and 0.57 μM IAA. Half strength of MS supplemented with 1.14 μM IAA was found to be the best for root induction from excised shoots. Agrobacterium mediated genetic transformation was carried using pBI121 plasmid harboring β‐glucuronidase (GUS) reporter and nptII selectable marker genes. Transient GUS assay confirmed that both the explants pre‐cultured for two days showed best transformation efficiency in bacterial suspension having optical density (OD) of 0.8 (at 600 nm) for 15 min and co‐cultivation period of 3 days. The shoots regenerated from transformed cotyledonary leaf explants survived at 200 mg/l kanamycin selection. The presence of expected amplicon corresponding to the GUS gene was confirmed by PCR. This protocol paves a way for developing disease resistant tomato variety using target gene/s.

Keywords: Genetic transformation, Agrobacterium, BARI Tomato‐8

Location: Department of Botany, University of Dhaka, Dhaka

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Tomato

Objectives:

The present study has been undertaken to establish an efficient and reproducible regeneration and Agrobacterium‐mediated genetic transformation protocol for a farmer popular tomato variety in Bangladesh such as; BARI Tomato‐8 variety using selectable and screenable marker genes.

Methodology:

The seeds of the tomato variety BARI Tomato‐8 were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur. To generate explants, the seeds of BARI Tomato‐8 were treated in 0.1% (v/v) mercuric chloride for 10 ‐ 15 min with vigorous shaking, washed three times with autoclaved distilled water and inoculated onto the cotton bed for germination. These seeds were kept in the dark till the germination took place and finally transferred to 16‐hourslight and 8 hours dark condition at 25 ± 2ºC in growth room. The seedlings were used for explants preparation. The cotyledonary leaf and hypocotyls were first separated. The leaves were cut at tip and the base, removing the meristematic tissue, whereas the hypocotyls were segmented into approximately 0.5 cm in size. The explants were placed on a pre‐culture media for 0, 1 and 2 days before infection. MS in combination with different hormones were used for regeneration and transformation experiments. The pH of the medium was adjusted to 5.8. The medium was then autoclaved. Different combinations of hormone and supplements like IAA, Kn, GA3, NAA, BAP and 2,4‐D were added for multiple shoot regeneration (Table 1). In case of rooting, three types of media were used, i.e. MS containing 1.14 μM IAA, half strength of MS containing 0.98 μM IBA and half strength of MS containing 1.14 μM IAA. The antibiotic kanamycin was used to select the transformed plants after infection with Agrobacterium tumefaciens carrying pBI121 plasmid containing nptII gene. Kanamycin was dissolved in distilled water and sterilized by filter. Once the autoclaved media was cooled, the antibiotic was added. The regenerated shoots were subcultured regularly at an interval of 12 ‐ 15 days and the concentration of kanamycin was gradually increased (e.g. 100, 150, 200 mg/l) in the regeneration medium. Agrobacterium tumefaciens strain LBA4404 containing plasmid pBI121 of 14 kb (binary vector) was used for the transformation. This binary vector contains uidA gene (Jefferson et al. 1987) encoding GUS, driven by CaMV 35S promoter and NOS terminator and nptII gene (Herrera‐Estrella et al. 1983) encoding neomycin phosphotransferase II conferring kanamycin resistance within the right border (RB) and left border (LB) region of the T‐DNA. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 kb) containing the virulence genes. The hypocotyls and the cotelydonary leaf explants were prepared and placed in a pre‐culture media for 0, 1 and 2 days before infection. Healthy explants that responded to pre‐culture, as evident by swelling, were incubated in the bacterial suspension at different optical density with different timing of incubation period. The explants were then soaked on sterile tissue paper and co‐cultured on the same pre‐culture medium for 2 days at 28°C. The explants were washed twice with autoclaved distilled water and finally washed with 300 mg/l of ticarcylin. For selection of transformed shoots, kanamycin containing regeneration medium was used. Cultures were subcultured regularly at an interval of 12 ‐ 15 days and the concentration of kanamycin was gradually increased (e.g. 100, 150 and 200 mg/l) in regeneration medium. Plates were cultured under a 16 hrs light/8 hrs dark cycle at 28°C. The regenerated shoots were excised from the callus and transferred to a rooting medium. Histochemical assay was performed to visualize GUS activity. The leaf tissues which survived the highest kanamycin concentration was selected and incubated in GUS histochemical buffer [50 mM sodium phosphate, pH7.0; 50 mM EDTA, pH 8.0; 0.5 mM K3Fe(CN)6; 0.5 mMK4Fe(CN)6; 0.1% triton X‐100; 1.0 mM X‐gluc (5‐bromo‐4‐chloro‐3‐indolyl β‐D‐glucuronide)] at 37°C for up to 24 hrs. Chlorophyll in leaf tissues was subsequently removed by washing with 70% alcohol after every 3 days. For molecular analysis, the genomic DNA was isolated from the plantlets which survived under the highest selection pressure using modified CTAB method (Islam et al. 2011). Using the isolated DNA, PCR was performed with GUSA‐769 (nucleotide sequence: TGG CTG TGA ACG CAC AGT TCA) and GUSA‐10 (nucleotide sequence: CCT GTA GAA ACC CCA ACC CG) primers for molecular confirmation of the genetic transformation. The conditions for PCR was 95°C for 5 min, 94°C for 1 min, 56°C for 1 min, 72°C for 1 min and repeated for 30 cycles. The final extension was maintained 72°C for 10 minutes and paused at 4°C. The amplified band was checked in the agarose gel.

Source of Information: Plant Tissue Cult. & Biotech. 25(1): 87‐97, 2015 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It can be concluded that the regeneration response of the cotelydonary leaf explants was better than hypocotyls and subsequently, transgenic plants obtained from cotyledonary leaf explants following the regeneration protocol. Therefore, this protocol endeavors a new and efficient way for obtaining transgenic plants which can be used to transfer useful candidate genes conferring disease, insect and pest resistance in the BARI Tomato‐8 variety of Bangladesh.

Knowledge Management: Journal