Development of RNAi construct and optimization of in vitro regeneration and transformation protocol against Banana bunchy top virus

Abstract

Banana bunchy top disease (BBTD) is one of the most destructive viral diseases newlineresponsible for huge destruction in the crop yield, caused by the banana bunchy top newlinevirus (BBTV). Currently, there are no strategies available to completely protect bananas newlineagainst the BBTV. Therefore, in this study, an attempt was made to develop an RNAi newlineconstruct for bunchy top disease management in bananas. Here, Nuclear shuttle protein newline(NSP) gene encoded by BBTV DNA-6 was targeted. The Coat protein gene (CP) newlineencoded by BBTV DNA-3 and Nuclear shuttle protein(NSP) gene encoded by BBTV newlineDNA-6 were initially cloned in pGEMT-easy vector. The clones were confirmed by newlinecolony PCR, restriction digestion, and sequencing. Sequence results were 96.69% and newline94.18% similar to BBTV accessions KX592198 and KX592200 for BBTV-DNA-3 and newlineBBTV-DNA-6 sequences, respectively. Also, NSP gene fragment (500bp) was newlinesuccessfully cloned in sense orientation in an RNAi vector pHANNIBAL. For the newlineprocess of callus induction, 1-10 weeks old immature male flower buds (IMB) were newlineselected. The immature flowers from position 8th to 16th were isolated and cultured on newlineMurashige and Skoog medium (MS) (HIMEDIA) with CaCl2, Vitamins, Sucrose and newlineAgar (REF PT100-10x1L). The medium was supplemented with NAA, IAA, 2,4-D and newlineMalt extract each in the concentration of 1mg/lit. The efficiency of embryogenic callus newlineformation from Grand Naine male flower bud was 6% ± 0.021. Two suppressor genes, newlineDNA3_Jorhat and DNA4_Jorhat showed 98.66% (KX249744) and 97.54% newline(KX249745) similarity with Assam isolates. Transmission study using 15 to 30 aphids newlinewas confirmed by the LAMP detection technique in 10-fold dilutions of DNA newlineconcentrations at 100 ng to 0.0001 ng. Phylogenetic relationships shared by the Jorhat newlineisolates with the different isolates from India and the world allowed a deeper newlineunderstanding of the evolution and spread of viruses. Leaf samples confirmed through newlinetransmission studies were only used for biochemical analysis. These findings indicate newlinethat there was a significant increase...