VECTORS CONSTRUCTION FOR THE DEVELOPMENT OF COTTON LEAF CURL DISEASE AND INSECT RESISTANCE IN COTTON

Abstract

COil on is a high value agri cultuml product. ;;iduptcd mainly for textile fib"r and .:'Ilso for production of wgetablc oil. Collon leaf curl Jisease is c<Ju::.ed by whltclly· transmitted beg.olllovirlis. which require addi tional sa tellite DNA components like DNA p to induce ::.ymploms. ACI gene (replication associated protein) is absolutely required for repii<.:ution of viral DNA In addition to virus. cotton plant is allacked by a llumber or insect pests; the major ones are the cotton bolh:vo nns and collon aphids that cause t!xtensive damage. The insects are mainly cOJ1l rol\ed by chemical sprays, so the main o~jeclivc of th is study was to engineer collon against leaf curl disease and insect pests (bollworm) complex by transformi ng with both AC I (vi rus) and cry/ Ab (bacteria) genes. Ini tia ll y, primers were designed to amplify the truncated rep (ACI) gene from DNA A of colton leaf curl virus (CLCuV). Amplified rep gene (757 bp) was first cloned in plant ex pression vector pN6 in sense and antisense orientation. ACI based RNAi cassette was then lifted from pN6 along \\lith CaMV35S promoter and poly A tail and subcloned into the plant transformation vector pBS389. For insect (bollworm) rcsistHnCl.'. ClylAb (2.l kb) 0(" Bacilllls r/lIIrillgiensis was cloned in plant expression vector pJJ1 60 Imdcr constitutive CaMV35S promoter and then subclonrd into plant tran sformation vector pGA482. Both ACI based RNAi and B1 (cfylAb) constructs \vere transformed into AgrobaclerillfJI 11III1efilciens strain (LBA 4404). AgrobacleriulJI (using equal concentration of Agrobacleril.lfl1 cultures) mediated transformation of hypocotyls or 7·8 days old plants of Cokcr·312 was carried out in four batches. Overall callusing and embryogenic efficiency of cotton calli were 53% and 20% respectively. Molecular analysis of the 5 randomdly se lected embryogenic calli was performed. Total genomic DNA \Vas iso latcd and peR was performed on DNA isolated from five calli, which indicated the presence of both specific (truncated ACI and C/y lAb) and I'lpf/J gcnes. These plants will be t:ha llengcd to virus/insect in containment fac ility. It is expected that transgenic plants produced in this study will resist against CLeuD and insect attack.