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Transformation of Abiotic Stress Tolerance Gene in Sugarcane

Dr. Md. Amzad Hossain
Chief Scientific Officer
Head,Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna

Nadira Islam
Scientific Officer
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna

Dr. Md. Abdus Samad Miah
Director General
BSRI, Ishurdi, Pabna

Khondoker Md. Nasir Uddin
Professor & Head
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh- 2200

Abstract/ Executive Summary:

This experiment was conducted to develop transgenic sugarcane varieties which will tolerant to salt and drought stress condition at BSRI and BAU Lab during the cropping season 2009-2010. The evaluation of drought tolerance genes transformation in sugarcane variety Isd 34 was performed using medium stressed with osmoticum polyethyleneglycol (PEG) at the concentration of 2.5, 5.0 and 7.5% was used. Transformed plants were able to survive up to 7.5% of PEG supplemented medium while the non-transformed plants were completely died out within 21 days after culture. The transformed plants were not only survive at concentration 7.5% but also showed vigorous health at this concentration. Survivals of plants regenerated from transformed explants over 7.5% PEG imply the transformation and expression of target genes (drought tolerance) in sugarcane variety Isd 34. Transformed plants are being maintained under contained controlled conditions by regular subculture at an interval of 21 days. Results indicate the possibility of the development of transgenic sugarcane against drought tolerance stresses.

Keywords: Sugarcane, Gene, Transformation

Location: BSRI and BAU Lab

Start Date: 01-07-2008

End Date:

Program Area: Variety and Species

Commodity: Sugarcane

Objectives:

a. To collect Agrobacterium strains. b. To culture and maintain Agrobacterium strains. c. To develop co-culture protocol with Agrobacterium and sugarcane explants. d. To optimize the Agrobacterium-mediated genetic transformation system in sugarcane. e. To develop transgenic sugarcane varieties

Methodology:

a. Luria-Bertani (LB) broth medium contained 1% Bacto peptone, 0.5% Bacto-yeast extracts and 1% Nacl were used for routine Agrobacterium culture. b. Commercial sugarcane varieties were used as experimental materials. Excised basal innermost area of spindle leaf as explant were cultured on MS-M1 medium (MS medium containing 100 mg/l myo-inositol, 500 mg/l casein hydrolysate, 2 mg/l 2,4-D, 3% sucrose and 0.6% agar) and MS-M2 (MS medium containing 7.5 mg/l NAA, 3% sucrose and 0.6% agar) for callus initiation, sub-culture and shoot development respectively. c. For transformation, Agrobacterium strains (LBA4404 containing plasmid with PsCBL, PsCIPK and Gly1 genes for salt and drought tolerance) were cultured in LB medium supplemented with 50 mg/l Kanamycin, according to the strains at 100rpm under 37 ?C for 24-48 hours. Then the bacterial cells were suspended in MS-M1 liquid medium and were used for infection of callus produced on MS-M1 medium and leaf segments. d. Callus (21-28 days after initiation on MS-M1 medium in the dark at 25 ?C) were sub-cultured on fresh MS-M1 medium for 7-14 days. Then the callus (3-5 mm in diameter) and separated leaf segments were immersed in the Agrobacterium suspension for 60, 90 and 120 minutes for infection. Infected callus were cultured on solid MS-M1 medium and leaf segments on MS-M2 for co-cultivation. Co-cultivation were made in the dark at 28-30 ?C for 3-14 days. e. All the media for co-cultivation and Agrobacterium suspension culture were supplemented with 30 mg/l acetosyringone. f. The co-cultivated materials were washed two times with liquid MS-M1 and MS-M2 media supplemented with 500 mg/l Cefotaxime according to Agrobacterium strains. Then washed materials were transferred to MS-M1 and MS-M2 solid media containing 50, 75,100 mg/l Kanamycin according to bacterial strains and maintained 14-21 days for selection. After selection, the cultures were sub-cultured regularly on regeneration media (MS-M2 + Kanamycin 50 mg/l for leaf segments and MS-M3 containing MS + BAP 2mg/l + Kn 1mg/l + Kanamycin 50 mg/l for callus) at an interval of 2-3 weeks. Kanamycin concentration were escalated to 75 mg/l and then 100 mg/l. g. Optimum inhibitory concentration of Kanamycin were determined using 50, 75, 100 mg/l concentrations of Kanamycin in routine media. h. After co-cultivation and selection, the expression of GUS were analyzed histochemically in callus tissues and leaf segments as well as shoots from resistance callus and leaf segments. i. On the other hand shoots from resistant callus and leaf segments were cultured on the regeneration medium under fluorescent light with 16 h photo period. j. Regenerated shoots were cultured on rooting medium MS-M4 (MS basal medium supplemented with 5.0 mg/l NAA or IBA) containing 50 mg/l Kanamycin. k. To identify and confirm the stable incorporation of introduced genes and its expression in the transformed plants two different assays viz. GUS assay (to identify putative transformed shoots) and genetic analysis using PCR were carried out. l. After PCR confirmation transferred plants were tested against salt and drought stresses using in vitro methods and were maintained under in vitro contained conditions.

Source of Information: Annual Research Programme 2009-2010 & Annual Report 2010-2011

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 2,55,200.00

Total Budget (Tk.) : 2,55,200.00

Achievement/Findings:

In vitro evaluation of genetic transformation of drought tolerance gene was performed in the sugarcane variety Isd 34 which indicates that, this variety able to survive at the medium where 7.5% PEG were used.

Knowledge Management: Report/Proceedings