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Agrobacterium-mediated Transformation of Lentil (lens Culinaris Medik.)

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

Ashapurno Biswas
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Barkat Murtaja Mustafa
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Shirin Mahbub
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M. I. Hoque
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Agrobacterium-mediated transformation system was developed for two microsperma varieties of lentil (Lens culinaris Medik.) namely, Barimasur-2 and Barimasur-4. Transformation ability of different explants like cotyledonary node, decapitated embryo, immature embryo and epicotyl were tested with Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Highest percentage of GUS positive regions were found in epicotyl explants followed by decapitated embryo as detected by transient assays. Decapitated embryo was found to be more effective in formation of multiple shoots on MS medium supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn, 0.1 mg/l GA3 and 5.5 mg/l tyrosine following Agrobacterium infection and selection. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l. Stable expression of the GUS gene was observed in various parts of the transformed shoots. Genomic DNA was isolated from these shoots and stable integration of GUS and nptII genes was confirmed by polymerase chain reaction (PCR) analysis.

Keywords: Lentil, Transformation, GUS expression, Kanamycin resistance

Location: Bangladesh Agriculture Research Institute (BARI), Joydebpur, Gazipur

Start Date:

End Date:

Program Area: Pest Management

Commodity: Lentil

Objectives:

To indentify the Agrobacterium-mediated Transformation of Lentil (Lens culinaris Medik.)

Methodology:

Seeds of microsperma type of two lentil (Lens culinaris Medik.) varieties, namely Barimasur-2 (BM-2) and Barimasur-4 (BM-4) collected from Bangladesh Agriculture Research Institute (BARI), Joydebpur, Gazipur were used in the present investigation. Experiments were conducted on MS medium supplemented with different concentration and combinations of auxins, cytokinins, GA₃ and additives for regeneration of shoots. The cultures were maintained under fluorescent illumination on a 16 h photoperiod at 25 ± 2⁰C. For the preparation of explants the seeds were first washed three times in distilled water and surface sterilized in the laminar flow with 0.1% HgCl₂ for 15 minutes. The seeds were then placed in the dark on 3% sugar - agar medium for germination. The explants were prepared with a scalpel while submerged in the Agrobacterium suspension. The cut explants were kept incubated in the Agrobacterium suspension in a small Petri dish for an additional 30 minutes. They were then blotted dry on a sterilized Whatman filter paper and co-cultured in Petri plates on MS with 0.5 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/l GA3 + 5.5 mg/l tyrosine (regeneration medium) for three days in the dark. Following coculture the explants were washed several times in liquid MS with gentle shaking until no opaque suspension was seen. For the detection of the nptII coding sequence, DNA was subjected to PCR using the following primers and conditions: forward 5’-TGA TTG AAC AAG ATG GAT TG-3´ and reverse 5´-CAT TTT CCA CCA TGA TAT TC-3´. For the GUS gene the primers were : forward 5´-CCT GTA GAA ACC CCA ACC CG-3´ and reverse 5´-TGG CTG TGA CGC ACA GTT CA-3´ (MGW-Biotech, AG, Germany). All primers were used at a concentration of 100 pmol/μl. The plasmid pBI121 isolated from Agrobacterium tumefaciens was used as the positive control. PCR reaction mix of 25 μl contained 2.5 μl of 10 ∇ PCR buffer with 15 mM MgCl₂ (Gene Craft, Germany), 1 μl of 5 mM of the dNTP mix, 1 μl of Red Taq polymerase (Natutech, Germany), 1 μl of each of the respective primers, and 1 μl (50 - 80 ng/μl) of the sample DNA and 17.5 μl ultra pure water. For PCR amplification of the GUS gene, DNA was denatured at 94⁰C for 3 min and then amplified in 30 cycles using 94⁰C for 1 min, 64⁰C for 1 min (annealing) and 72⁰C for 1 min followed by 5 min at 72⁰C. For nptII gene the cycling conditions were 3 min at 94⁰C denaturtion and 30 amplification cycles using 94⁰C for 1 min, 55⁰C for 1 min (annealing) and 72⁰C for 1 min followed by 5 min at 72⁰C. The amplified DNA was run on 1.0% agarose gel and stained with ethidium bromide (0.05 μg/ml).

Source of Information: Plant Tissue Cult. 13(1) : 1-12, 2003 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The protocol reported here is reproducible for the Agrobacterium-mediated transformation of microsperma types of lentil. For this method dry seeds are used as the starting material and regeneration is obtained without an intervening callus phase. Therefore, the transformation method established during this study could be applied to transfer useful candidate genes conferring disease, insect and pest resistance. Decapitated embryo was found to be more effective in formation of multiple shoots on MS medium supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn, 0.1 mg/l GA3 and 5.5 mg/l tyrosine following Agrobacterium infection and selection. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 200 mg/l. Stable expression of the GUS gene was observed in various parts of the transformed shoots. Genomic DNA was isolated from these shoots and stable integration of GUS and nptII genes was confirmed by polymerase chain reaction (PCR) analysis.

Knowledge Management: Journal