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Transformation of Salt and Drought Tolerant Genes in Sugarcane (2011-12)

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

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

Kuasha Mahmud
Senior Scientific Officer
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna

Asish Kumar Ghose
Scientific Officer
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna

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

Dr. Mohammad Nurul Islam
Professor
Dept. of Botany University of Dhaka

Abstract/ Executive Summary:

This experiment was conducted to develop transgenic sugarcane varieties which will tolerant to salt and drought stress condition at BSRI, BAU and Du Lab during the cropping season 2011-2012. Salt and drought tolerant genes transformation in sugarcane variety Isd 18 was performed. Evaluation of transformation was tested by bioassay using 150mM (21.66ds/M) salt and Polyethyleneglycol (PEG) at the concentration of 7.5% in the media under in vitro conditions. Transformed plants were able to survive on 150mM (21.66ds/M) and 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 survived at concentration 150mM (21.66ds/M) and 7.5% of PEG but also showed vigorous health at this concentration. Survivals of plants regenerated from transformed explants at concentration 150mM (21.66ds/M) and 7.5% of PEG imply the transformation and expression of target genes (drought and salt tolerance) in sugarcane variety Isd 18. Transformed plants are being maintained under contained controlled conditions by regular sub-culture at an interval of 21 days. Results indicate the possibility of the development of transgenic sugarcane against salt and drought tolerance stresses.

Keywords: Sugarcane, Gene transformation, Salt and drought tolerant

Location: BSRI, BAU and DU Lab

Start Date: 01-07-2011

End Date: 30-06-2012

Program Area: Variety and Species

Commodity: Sugarcane

Objectives:

a. To collect Agrobacterium strains with salt and drought tolerant genes. 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:

Luria-Bertani (LB) broth medium contained 1% Bacto peptone, 0.5% Bacto-yeast extracts and 1% Nacl were used for routine Agrobacterium culture.
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.
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.
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.
All the media for co-cultivation and Agrobacterium suspension culture were supplemented with 30 mg/l acetosyringone.
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.
Optimum inhibitory concentration of Kanamycin were determined using 50, 75, 100 mg/l concentrations of Kanamycin in routine media.
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.
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.
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.
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.
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 Report 2012-13 BSRI

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 2,55,200.00

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

Achievement/Findings:

Genetic transformation of foreign genes and subsequent regeneration was performed in the sugarcane variety Isd 18 which indicates the variety able to receive foreign gene. Transformed plants were able to survive at the medium where 150mM (21.66ds/M) and 7.5% of PEG were used.

Knowledge Management: Report/Proceedings