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जीनोम संपादन के साथ खाद्य सुरक्षा
Food Security with Genome Editing
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ICAR-CICR Genome Editing Project
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ICAR-Sugarcane Breeding Institute (SBI)

Coimbatore, Tamil Nadu, India

Collaborating Centre Principal Investigator (CC-PI)

Dr. A. Ramesh Sundar

Dr. A. Ramesh Sundar

HoD (Plant Pathology)

Principal Investigators involved in the project

Dr. A. Ramesh Sundar

Dr. A. Ramesh Sundar

HoD (Plant Pathology)

Dr. R. Manimekalai

Dr. R. Manimekalai

Principal Scientist

Dr. C. Appunu

Dr. C. Appunu

Senior Scientist

Dr. K. Lakshmi

Dr. K. Lakshmi

Senior Scientist

Dr. P. T. Prathima

Dr. P. T. Prathima

Senior Scientist

Dr. G.S. Suresha

Dr. G.S. Suresha

Senior Scientist

Dr. R. Valarmathi

Dr. R. Valarmathi

Scientist

Co-Principal Investigators (Co-PIs)

Dr. C. Viswanathan

Dr. C. Viswanathan

Principal Scientist

Dr. R. Ramesh

Dr. R. Ramesh

Principal Scientist

Dr. A. Jeevalatha

Dr. A. Jeevalatha

Senior Scientist

Dr. H. Mahadevaswamy

Dr. H. Mahadevaswamy

Scientist

Dr. Shweta Kumari

Dr. Shweta Kumari

Scientist

Target Traits in Sugarcane

Crop Variety Trait
Sugarcane (Saccharum officinarum L.) Co 86032 Drought
Co 86032 Sucrose
Co 99004 Tiller
Co 86032 Biomass
Co 13013 Flowering
CoC 671 / Co 0238 Red rot

Research Methods & Expected Outcomes

DST

Work on drought and salt tolerance focused on a regulatory protein involved in stress adaptation. Three guide RNAs representing different target positions were designed and synthesized. These were cloned in the pRGEB32 vector independently and confirmed through sequencing and restriction analysis. A total of 105 putative edits of sugarcane variety Co 86032 were developed through biolistic and Agrobacterium-mediated transformation. Molecular characterization is in progress. To improve editing efficiency, a construct with three guide RNAs was also developed, and calli are currently in the regeneration stage.

SWEETs

SWEETs are sugar transporters essential for sugar accumulation and biomass improvement. Guide RNAs were cloned into the pRGEB31 plasmid vector and transformed into Agrobacterium tumefaciens strain LB4404. Embryogenic calli, after infection with the transformed Agrobacterium, were cultured on hygromycin-containing selective media to isolate putative edited calli, which were subsequently transferred to regeneration media for shooting and rooting.

VAI

A growth-regulating candidate gene was isolated and cloned from sugarcane. In silico analysis identified conserved domains. A construct with three guide RNAs was developed and mobilized into Agrobacterium. Transformation experiments in sugarcane are currently in progress.

HTD2 and TB1

Two candidate genes associated with branching regulation were identified as key targets. Three guide RNAs for each were cloned into the pRGEB32 vector under the U3 promoter. Callus was initiated from the low tillering variety Co 99004 and transformed using particle bombardment. These plasmids are also being used for Agrobacterium-mediated transformation.

F5H

Four knockout CRISPR constructs were designed for a lignin-modifying candidate gene. Cloning in the pRGEB32 vector was confirmed by restriction and sequencing analysis. Putative edited lines from two constructs are currently in the rooting stage.

TFL

A total of 40 and 182 calli are under selection with Construct 1 (monocistronic design) and Construct 2 (polycistronic design), respectively. A construct for RNP-mediated transformation has also been prepared, with 125 calli currently on selection media.

CAX4

Coding sequences from sugarcane cultivars were cloned and sequenced. A qPCR assay validated their expression in response to pathogen infection. Efficient target sequences and PAM sites were screened to minimize off-target effects, and three guide RNAs were cloned in the pRGEB32 vector and mobilized into Agrobacterium. Agrobacterium-mediated transformation of red rot susceptible cultivars CoC 671 and Co 0238 with the construct is currently in progress.

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