RNA mediated resistance against banana bunchy top virus (BBTV) in banana

Abstract

The study entitled “RNA mediated resistance against Banana bunchy top virus (BBTV) in banana” was conducted at the Department of Plant Pathology, College of Agriculture, Vellanikkara and National Research Centre for Banana, Trichy during the period from 2018 to 2023. The study aimed at developing banana lines that have resistance against Banana bunchy top virus (BBTV) through RNAi, using the ligation independent cloning (LIC) method. The RNAi approach targeted the replicase gene of BBTV. The process began with the amplification of replicase gene fragment that contains the dicer substrate siRNA region. Gene specific primers with adaptor sequences were used to amplify the DNA fragment, creating the sense and antisense fragments of the RNAi construct. The LIC vector which specifically contained four adaptor sequences (LIC1 to LIC4) was linearized with SmaI followed by treatment with T4 DNA polymerase and dTTP. Simultaneously, the amplified gene fragments were treated with T4 DNA polymerase in the presence of dATP, facilitating the development of 5' extending single stranded tails. Subsequently, the T4 DNA polymerase treated vector and gene fragments with sticky ends were mixed and incubated. The splicing of the cohesive ends on the insert fragments and the vector resulted in the circularization of the vector. The prepared vector, housing the desired sense and antisense fragments was transformed into E. coli DH5α. All the transformed colonies, obtained on antibiotic selection medium underwent initial screening via colony PCR, demonstrating cent per cent transformation. Further confirmation involved plasmid isolation, verification of intron, restriction digestion and sequencing. The next phase was transforming Agrobacterium EHA105 strain with the prepared vector by modified freeze-thaw method. The success of transformation was confirmed by verifying the presence of sense and antisense fragments through colony PCR. For plant transformation, embryogenic calli of banana cv. Nendran were developed from the immature male inflorescence of 0.5 cm in size, by culturing on MS medium supplemented with different combinations of hormones viz., 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA) and picloram. The embryogenic calli development was observed after five months, with the highest per cent in MS medium with 2,4-D (2 mg l-1) and IAA (1 mg l-1). In order to minimize the time delay, the embryogenic cell suspension (ECS) obtained from NRCB, Trichy was used for plant transformation. The co-cultivation of Agrobacterium carrying the desired construct and ECS was done in the presence of acetosyringone for three days, in the dark at 24 to 25 ⁰C, followed by washing of excess Agrobacterium with cefotaxime. The co-cultivated embryogenic cells were transferred to a selection medium containing kanamycin (100 mg l-1). The transformed selected embryogenic cells were then transferred to the embryogenesis medium for 35 to 40 days, followed by culturing on embryo germination medium, under light. The germinated embryos were cultured in Petri plates for one and half months, followed by regular subculturing and maintenance in tissue culture bottles, till they attained the three to four leaf stage. The plants were rooted in a rooting medium for 30 to 40 days, and then acclimatized in a containment facility. The PCR assay with the primers for amplification of sense and antisense fragments confirmed the presence of RNAi construct in the acclimatized plants. These plants were then tested for BBTV resistance by aphid (Pentalonia nigronervosa) challenge inoculation. The transformed plants remained healthy, while the typical bunchy top symptoms appeared within 25 days on the non-transgenic control plants. However, it was observed that two out of the forty eight transgenic lines gradually developed yellowing symptoms, indicating varying levels of resistance among the transformation events. Therefore, further evaluation of these transgenic lines is crucial to better understand the resistance levels. The study has successfully demonstrated the efficacy of transgenic lines in conferring BBTV resistance through RNAi approach. By utilizing the ligation independent cloning, the hairpin construct preparation was done in more efficient manner. The transgenic lines developed in the study represent a promising tool, offering the scientific community a proactive defense against unmanageable BBTV epidemics in the absence of resistant lines.