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Impact assessment of the transgenic sugarcane over
expressing antifungal proteins on endophytic and
rhizospheric microorganisms
By
Dr Iqrar Ahmad
5th January 2012, Faisalabad, Pakistan
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Centre of Agricultural Biochemistry and Biotechnology
UNIVERSITY OF AGRICULTURE FAISALABAD
38040-Pakistan
COLLABORATORS: 1. Dr. Abdul Wakeel ISES, UAF 2. Dr. Shinawer Waseem Ali Instt. Agri. PU 3. Dr. Dr Abdul Rehman Plant Pathology, UAF
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CONTENTS
• Introduction
• Research framework
• Results
- Rhizospheric analysis by Non Culture Technique
- Rhizospheric analysis by Culturing microorganisms
- Analysis of the sugarcane endophytes
INTRODUCTION
world trade organization
industrial liberalization
free market economy
Sugarcane ……. Cash crop of Pakistan
Main source of sugar
Superb target for industrial processing.
Our sugar industry and sugar need is exclusively dependent on the fate of this
crop
Need to augment sugarcane productivity and quality.
Heavy losses, caused by a number of diseases to the sugarcane crop
biotic stress
abiotic stress
Yield losses
temperature
water
salt
light
nutrition
nematodes insects
fungi
bacteria
viruses
yield losses due to the biotic and abiotic stresses
Leaf Symptoms Stalk symptoms
Control of red rot disease:
Fungicides Cultural methods Breeding for resistant cultivars
Biocontrol
Genetic engineering for resistance
• Chitinase + chitosanase = weak and osmotically sensitive fungal cell wall
• Fungal cell wall contain chitin and chitosan
Targeting fungal cell wall as a control strategy
RESEARCH FRAMEWORK
Sugarcane Nursery Establishment
Transfer of Nursery Plants to Earthen pots
Soil Sampling from the Rhizosphere
Metagenomic DNA isolation from soil samples
Analyzing rhizosphere with 18s rDNA and 16s rDNA primers
Analyzing bacteria and fungi by culturing them
Culturing of Endophytes Analysis of Endophytes
RESULTS
M 1 2 3 4 5 6 7 8 9 10 c1 c2c3 M Sampling
4th
3rd
2nd
1st
Metagenomic DNA amplifications with 63F plus 1542R primers . 1-10 =transgenic plants, c1 and c2 = Non transgenic control, C3= PCR –ve control
PCR Profile: Initial Denaturation 95 ˚C, 2 Minutes Denaturation 92 ˚C, 30 Seconds Annealing 48 ˚C, 1 minute Final Extension 72, 8 minutes
Rhizosphere analysis
Primer pair Used: 16s +18s rDNA (27F + 1492R)
PCR Profile: Initial Denaturation 95 ˚C, 2 Minutes Denaturation 92 ˚C, 30 Seconds Annealing 58 ˚C, 1 minute Final Extension 72, 8 minutes
M 1 2 3 4 5 6 7 8 9 10 c1 c2 c3 M Sampling
4th
3rd
2nd
1st
Rhizosphere analysis continued
Primer pair Used: 16s +18s rDNA (63F + 1492R)
PCR Profile: Initial Denaturation 95 ˚C, 2 Minutes Denaturation 92 ˚C, 30 Seconds Annealing 58 ˚C, 1 minute Final Extension 72, 8 minutes
Primer pair Used: 16s +18s rDNA (27F + 1542R)
4th
3rd
2nd
1st
Sampling M 1 2 3 4 5 6 7 8 9 10 c1 c2 c3 M
Cloning and Sequencing
Phylogenetic Tree of cloned sequences
Control-1 Control-2 Transgenic
Fungal colonies growing on PDA media under antibiotic selection. C1 and C2 are controls and T is transgenic rhizosphere.
Mature cultures
Fungal Cultures
Major Fungal Species Identified Fusarium Solani
Fusarium Oxysporum
Rhizoctonia Solani
Trichoderma
Control-1 Control-2 Transgenic
Bacterial colonies growing on LB media . C1 and C2 are controls and T is transgenic rhizosphere.
Bacterial Cultures
40.66
66.66
61
47
54.66
65 64
45
64
P1 P2 P3 C1 C2 P4 P5 P6 P7
Colonies/cm2
Colonies/cm2
No of colonies developed per cm2 on LB media when rhizospheric soil was diluted in ultra pure water and spread using dilution plating method. P1-P7 transgenic rhizosphere, C1 and C2, non transgenic controls.
Bacterial Colony Count
M 1 2 3 4 5 6 7 8 9 10 c1 c2 M c3
27F+1492R
63F+1492R
27F+1542R
63F+1542
Endophytes analysed through rDNA primer
Sugarcane Endophytes
ACKNOWLEDGEMENTS: ADNAN HAMEED M USMAN TAHIR NAVEED MUHAMMAD Sarwar Khan IQRAR AHMA KHAN
THANKS
Microscopy
