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 Transformation and Constitutive Expression of Pgnhx1 from Pennisetum Glaucum L. in Oryza Sativa L. Cv. Binnatoa

S.M. Touhidul Islam
Department of Biochemistry and Molecular Biology, University of Dhaka

R.S. Tammi
Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar

Sneh L
Department of Biochemistry and Molecular Biology, University of Dhaka

Singla Pareek
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Z.I. Seraj
Department of Biochemistry and Molecular Biology, University of Dhaka

Abstract/ Executive Summary:

In an effort to improve salinity tolerance in rice, the Pennisetum glaucum vacuolar Na+/H+ antiporter gene (PgNHX1) was transformed and expressed in Bangladeshi rice Binnatoa under the constitutive promoter CaMV35S. Transgenic status of the plant was confirmed by PCR and Southern blot hybridization. RT‐PCR and Western dot‐blot analyses were performed to validate the expression of the transgene at RNA and protein levels, respectively. At 160 mM (ECe of 16 dS/m) NaCl stress the transgenic seedlings showed enhanced tolerance compared to controls

Keywords: Salinity tolerance, PgNHX1, CaMV35S, Binnatoa

Location:

Start Date:

End Date:

Program Area: Knowledge Management

Commodity: Rice

Objectives:

The authors have performed the Agrobacterium‐mediated transformation with the same construct having the gene PgNHX1 under the constitutive promoter CaMV35S in a Bangladeshi rice cultivar Binnatoa, which has been found to be very tissue culture responsive with greater than 4% transformant recovery and confirmed its expression for conferring enhanced salt tolerance.

Methodology:

Agrobacterium‐mediated transformation through electroporation: Agrobacterium tumefaciens strain LBA4404 was purchased from Netherlands Culture Collection of Bacteria (NCCB) (www.bacterio.cict.fr/collections.html). The pCAMBIA 1301/CaMV35S‐PgNHX1 construct was obtained as plasmid DNA from ICGEB (International Centre for Genetic Engineering and Biotechnology), New Delhi, India under an MTA between ICGEB and the University of Dhaka. The plasmid was transformed into E. coli strain DH5α, isolated and transformed into A. tumefaciens LBA4404 by electroporation. Positive clones were selected by lysate PCR (Sambrook et al. 1989) using forward primer (5ʹ‐ATG GCT GTG TTC AGC AGG AC‐3ʹ) and reverse primer (5ʹ‐ TCA CCA AAA ACA TGT CTT CAT CTC‐ 3ʹ). Agrobacterium culture and pre‐induction: One confirmed clone of LBA4404 containing pCAMBIA 1301/CaMV35S‐ PgNHX1 construct was streaked on AB medium (Chilton et al. 1974) supplemented with 50 mg/l kanamycin and incubated at 28°C for 48 ‐ 72 h, until colonies appeared. A single colony was transferred to 5 ml AB liquid medium containing the same selective antibiotic and the culture was allowed to grow overnight with shaking at 200 rpm. The overnight culture was transferred to 50 ml Bacterial Preinduction Medium (AB salts, 10 g/l glucose, 75 mM MES Buffer pH 5.6, 2 mM sodium phosphate buffer, acetosyringone (AS) 250 μM, pH 5.6) containing the same selective antibiotic according to modified protocol of Khanna and Raina (1999) (Rasul 2005) and grown overnight under the same conditions as described above. Bacteria were collected by centrifugation in a 50 ml tube at 4000 rpm for 10 minutes. The supernatant was removed and the bacteria washed in 10 ml of bacterial resuspension medium (AB medium with 36 g/l glucose, 68.5 g/l sucrose, AS 250 μM, 50 mg/l kanamycin, pH 5.2), pelleted and resuspended again in 10 ml ofbacterial resuspension medium. The culture was adjusted to 0.005 OD with the same medium and used for infection and cocultivation.

Source of Information: Plant Tissue Cult. & Biotech. 19(1): 25-33, 2009 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Expression of the gene under the constitutive promoter CaMV35S shows slightly better tolerance compared to the inducible promoter ABRC These workers reported seed set at 100 mM NaCl without providing any details of reproductive stage tolerance compared to tolerant or sensitive controls. Detailed studies of the tolerance of the transgenic plants at reproductive stage under the constitutive promoter CaMV35S will be reported elsewhere. The transgenic lines have been advanced to T₃ generation by selfing and will be advanced further with selection to achieve homozygosity of the transgene. These stable plants will be crossed with popular varieties to obtain the transgene in the background of a modern, agronomical superior variety so that high‐yielding salt tolerant improved varieties can be provided to farmers of Bangladesh

Knowledge Management: Journal