CRISPR/CAS9 IN GOSSYPIUM HIRSUTUM (COTTON) COKER 312 FOR CLCUD COTTON LEAF CURL VIRUS DISEASE RESISTANCE MEDIATED BY AGROBACTERIUM
Keywords:CRISPR/Cas9, Cotton Coker 312, 3gRNA, Agrobacterium EHA-105, Multiplex vector, Transformation, Cloning
The cotton crop is the largest contributor in the world-wide economy. On the other hand, Gemini-virus is the biggest enemy of the cotton crop, causing CLCuVs infection in the African and Asian regions. It destroys the GDP rate to an alarming point. As biotechnology is serving mankind by introducing many conventional techniques like; specific RNA editing and plant breeding and advance techniques like; ZFNs, CRISPR/Cas9, and TALENs. To produce disease-resistant Coker 312 Cotton a CRISPR/Cas9 technique was used due to its site-specific targeting and efficiency. The targeted Rep and ?C1gene play role in CLCuVs replication. An expression vector pHSE-401 containing Cas9 and multiple guided RNAs was cloned. The vector was transformed by excision of hypocotyls of cotton (Gossypium hirsutum) type Coker 312 plants and infection delivery was mediated by EHA-105 Strain agrobacterium. The hypocotyls were grown on MSB regeneration media containing specific antibiotics. Then calluses were cultured in culture media and were planted into soil containing pots and kept under continuous observation. At different growth periods, no symptom of CLCuD was observed in the transgenic plants after 30 days of the growth period. The RNA was extracted at different growth periods and confirmed by PCR. The PCR products were visualized by running it on gel electrophoresis 0.8kb single guide RNA (sgRNA) bands confirmed the CRISPR/Cas9 in cotton Coker 312. Plants containing pHSE-401vector showed fewer virus traits and delay in disease development in 30 days growth period. This proposed project exhibits the desired results with a successful gene editing.