Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Agrobacterium-mediated Genetic Transformation of Lentil (lens Culinaris Medik.) with Chitinase Gene Followed by in Vitro Flower and Pod Formation

Subroto K. Das
Department of Botany, University of Barishal, Barishal-8200, Bangladesh

Kishwar Jahan Shethi
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

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

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

Abstract/ Executive Summary:

To investigate the integration of chitinase gene in lentil (Lens culinaris Medik.) namely, BARI masur-4 (BM-4), BARI masur-5 (BM-5) and BARI masur-6 (BM-6) through Agrobacterium-mediated genetic transformation was performed using Agrobacterium strain EHA 105 harboring bar (resistant to phosphinotrycin) and chitinase (gene of interest) gene. Selection of transformed shoots was carried out by gradually increasing the concentration of phosphinotrycin (PPT) up to 2.0 mg/l. Transgenic lentil shoots were produced with an overall frequency of 0.36 in case of BM-4 and BM-6 and 0.34 in case of BM-5, respectively. Most of the selected shoots developed in vitro flowers and pods following their sub-culture on half strength of MS supplemented with 20 mg/l IBA, 0.5 mg/l NAA with 50 mg/l ticarcillin. Seedlings germinated from the seeds were successfully transferred to soil for the development of further progeny. Stable integration of target gene was confirmed through PCR analysis.

Keywords: Lentil, Transformation, Chitinase, In vitro flower

Location: University of Dhaka

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Lentil

Objectives:

To integrate chitinase gene in microsperma variety of lentil through Agrobacterium-mediated genetic transformation.

Methodology:

Cotyledon attached decapitated embryo (CADE) explants of three microsperma varieties of lentil (BM -4, BM-5 and BM -6) cultivated in Bangladesh were used as the plant materials for this investigation. Seeds of these three varieties of lentil were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur and maintained in the Plant Breeding and Biotechnology Laboratory of the Department of Botany, University of Dhaka. Seeds of lentil were surface sterilized by rinsing them with 70% alcohol for 1 min and then kept in 2% sodium hypo chloride supplemented with one drop of tween 20 for 10 min, which was followed by thorough washing with sterile distilled water for 3 - 4 times. The seeds were then soaked overnight in sterile distilled water. For the preparation of CADE explants, overnight soaked seeds were taken in a sterilized petri dish, the seed coats are removed, seeds were split open and removing the root and shoot tips from embryo. Then one part of cotyledon containing decapitated embryo was used as explants. Explants were then placed on MS medium supplemented with 0.5 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/l GA₃ + 5.5 mg/l tyrosin for regeneration of shoots. The cultures were maintained under fluorescent illumination on a 16 h photoperiod at 25 ± 2°C. The hypervirulent Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993) harboring the pGreen II 0229 derivative binary plasmid containing a bar gene and the pSoup helper plasmid, (pGreen website: HYPERLIK “ http://www.pgreen.ac.uk/ ” http://www.pgreen.ac.uk ), (Hellens at.el 2000) was used for transformation. This binary vector contains ‘ bar’ gene (encoding phosphinothricin -acetyl transferase driven by NOS promoter and NOS terminator which conferring phosphinothricin resistance is present as selectable marker) within the right border (RB) and ‘ chitinase’ gene [isolated from Streptomyces olivaceoviridis (ATCC 11238). Chimeric constructs were developed by fusing the Arabidopsis thaliana leader peptide (NCBI, AY081519) to the chitinase gene behind the stilbene synthase promote r (pGIIvstN -Chitin) from grape (Wiese et al. 1994)] within left border (LB) region of the construct. For infection of explants, overnight grown Agrobacterium culture was centrifuged for 10 min at 5000 rpm and the pellet was re -suspended in liquid MS (pH 5.8) to make the Agrobacterium suspension. This Agrobacterium suspension was used for infection and incubation. Prior to this Optical Density (OD) of the bacterial suspension was determined at 600 nm with the help of a spectrophotometer (Shimadzu, Japan). Following the infection and incubation, the explants were soaked in filter papers for a short period of time to remove excess bacterial suspension. All the explants were maintained in co-culture medium in dark condition. After 2 - 4 days the co-cultured explants were washed with distilled water for three-four times until no opaque suspension was seen, then washed for 15 min with distilled water containing 300 mg/l ticarcillin. The explants were then dried with a sterile Whatman filter paper and transferred to regeneration medium with 100 mg/l ticarcillin. After 7 - 10 days, the regenerated shoots were sub-cultured in selection medium containing 0.5 mg/l ppt and 100 mg/l ticarcillin. Cultures were sub-cultured regularly at an interval of 12 - 15 days and the concentration of PPT was gradually increased up to 2.0 mg/l. Shoots survived on selection medium were sub-cultured on half strength of MS containing 20 mg/l IBA and 0.5 mg/l NAA subjected to in vitro flowering and seed formation. The presence of the bar and chitinase genes in the lentil genomic DNA was analyzed by PCR. DNA was isolated from putatively transformants and non-transformed plant using the CTAB method (Doyle and Doyle 1990). For the detection of the bar coding sequence, DNA was subjected to PCR using the following primers and conditions: forward 5’- GAT TTC GGT GAC GGG CAG GA -3´ and reverse 5´- TGC GGC TCG GTA CGG AAG TT -3´. For the chitinase gene the primers were: forward 5´- GGT GAC ATC GTC CGC TAC AC -3´ and reverse 5´- GGT GTT CCA GTA CCA CAG CG -3´ (MGWBiotech, 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 bar and chitinase gene, DNA was denatured at 94°C for 5 min and then amplified in 35 cycles using 94°C for 1 min, 59°C for 1 min (annealing) and 72°C for 1 min followed by 5 min at 72°C. 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. & Biotech. 29(1): 99-109, 2019 (June)

Article Link (Online Journal): DOI: https://doi.org/10.3329/ptcb.v29i1.41982

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It may be concluded that using this protocol it has been possible to develop transgenic plantlets using antifungal genes in other plants with various explants types. Available literature indicated that this may be the pioneering report on the successful development of transgenic microsperma group of lentil plants. However, the frequency of trans formation was rather low which needs to be addressed in the future work of lentil genetic transformation.

Knowledge Management: Journal