Fusarium oxysporum f. sp. cubense ( Foc ), a devastative soil-borne fungal pathogen causing vascular wilt (i.e. Panama disease) which leads to severe crop losses in most of the banana-growing regions of the world. As there is no single source of effective management practices available so far, understand the pathogenicity of the organism may help in designing effective control measures through molecular approaches. The study aims to compare the proteome of the two pathogenic Foc virulent strains, Race 1 ( Foc R1) and tropical race 4 ( Foc TR4) that are capable of infecting the Cavendish group of bananas using 2-dimensional (2-D) gel electrophoresis, MALDI-TOF/MS and MS/MS analysis. The results of the study revealed that the proteins, peroxiredoxins, NAD-aldehyde dehydrogenase (NAD-ALDH), MAPK protein, pH-response regulator protein palA/rim-20 and isotrichodermin C15 hydroxylase have shared homology with the fungal proteins, which regulate the osmotic stress response, signal transduction, root colonization and toxin biosynthesis. These are the important functions for the pathogen survival in an unfavourable environment, and successful establishment and infection of the banana host. The present study also identified several putative pathogenicity related proteins in both Foc R1 and Foc TR4. Specifically, certain Foc TR4 specific putative pathogenicity related proteins, phytotoxins biosynthesis gene, fructose 1,6-bisphosphate aldolase class II, Synembryn-like proteins found to contribute strong virulence. Overexpression or knockout of the elective genes could help in devising better control measures for the devastative pathogens in the future. To the best of our knowledge, this is the first report on the proteomics of Foc R1 and Foc TR4 strains of Indian origin that infect Cavendish bananas. ### Competing Interest Statement The authors have declared no competing interest.

In the present investigation, the insertional polymorphisms of retro-elements were studied in the Musa germplasm available at ICAR-NRCB field gene bank using IRAP markers. The maximum number of polymorphic bands were produced by the primer pair Nikita and LTR 6150 (48) followed by LTR 6149 and 3′LTR (47) and minimum of 35 bands were produced by the primer pair Sukkula and LTR 6150. The bands produced were scored as 0 (absent) and 1 (present) and the resultant binary data was subjected to diversity analysis. The dendrogram consisted of two major clusters with members of Eumusa and Rhodochlamys in one indicating their genetic closeness and members of the genus Ensete in another cluster. Results of principal coordinate analysis were congruent to those obtained in hierarchial cluster analysis. The molecular markers used in this study could reveal intra and inter-group diversity among the Musa germplasm accessions with similarity co-efficient ranging from 0.41 to 0.99. IRAP marker system has performed excellently clustering the accessions based on both genomic and subgroup levels. The entire germplasm was found to be robust with no duplications indicating the diverse group of accessions available at ICAR-NRCB field gene bank. It has also exhibited high polymorphism and hence could be effectively used to detect the genetic relatedness among diverse genome of Musa.

Banana Bunchy Top Disease (BBTD) caused by the Banana Bunchy Top Virus (BBTV), is the most serious virus disease of banana and plantain worldwide. Detection of the BBTV infection in the planting material could help in the effective management of the disease in banana. A polymerase chain reaction (PCR) assay was used to detect the of banana bunchy top virus at early stages of infection in banana suckers before the expression of symptoms. In the present study, primers were designed for amplification of the BBTV coat protein (CP) gene and the gene of 513 bp size was successfully amplified through polymerase chain reaction (PCR). The amplified PCR product was cloned and sequenced. The homology search of the sequence using NCBI blast showed similarity (99%) to ten CP gene sequences and are 98% similar to four CP gene sequences. 40 randomly selected banana suckers were subjected for the detection of BBTV through PCR. Out of 40, size specific amplification of coat protein gene was found, which indicates infection of planting material with BBTV. The PCR result was confirmed and validated through Dot blot hybridization analysis by using sequences of BBTV CP gene as probe.

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. Primers were designed for the PCR amplification of BBTV coat protein (CP). In the present study, to confirm the virus infection and to quantify the viral load in the plants, samples from infected and healthy banana were subjected to PCR and southern blot hybridization. There was no distinguishable difference in viral load with respect to age of the plants was observed. The BBTV capsid protein (CP) gene located on component 3 was cloned in an expression vector pET28a (+). Expression of the CP in E. coli BL21 cells was induced by adding isopropyl-3-D-1-thiogalactoside (IPTG) to a final concentration of 1 mm. The expressed CP which migrated as a protein of approximately 19.5 kDa in Sodium Dodecyl Sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was identified by its molecular weight. Hence it is concluded that the CP gene of BBTV was cloned and expressed in E. coli can be used for antibody rising or adding an affinity purification tag will enable us to produce pure monoclonal antibody in future.

Bananas are one among the world’s leading food crops, after rice, wheat and maize. Almost ninety percent of production is consumed in the production areas, especially in the poorest countries in Africa, Latin America and Asia. In certain regions, pureed banana is the first solid food given to infants. Bananas contribute to reducing food insecurity in producer country populations. Their composition, which includes high carbohydrates and minerals, makes them a staple calorie resource for over 500 million inhabitants of tropical countries. Considering the nutrition aspect, it is the world’s leading fruit crop, and in terms of economical value it is ranked as fifth economically important agricultural crop in world trade. In the global production of banana India contributes 29.19% as leading country. Bananas face numerous environmental challenges, particularly with fungal, bacterial as well as the major threatening disease like banana bunchy top virus. The problem is further aggravated by the limited diversity of banana cultivars around the world. Conventional breeding methods have limited success due to low female fertility, sterility, ploidy levels and poor seed set, besides the process is time consuming. These problems point to the necessity of developing alternate strategies for banana improvement through advancement of biotechnology tools like tissue culture and transgenic technology to improve the bananas. In this regard I will be discussing the current status of Banana improvement using biotechnology and future prospects.

Plantlets of the banana cultivar ‘Virupakshi’ (AAB) were regenerated from somatic embryos derived from embryogenic cells of calli from immature male flower explants. Induction of calli from explants was favored by a relatively moderate concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water. A suspension culture of the callus-derived embryogenic cells in Murashige and Skoog (MS) basal medium (pH 5.3) with 2 mg/L 2,4-D, 1 mg/L indole-3-acetic acid (IAA), 1 mg/L NAA, 45 g/L sucrose and 20 g/L maltose produced synchronously proliferating cells with the potency to be induced into somatic embryos on an 8 g/L agarose-solified SH basal medium (pH 5.7) with recommended vitamins, 1 mg/L IAA, 1 mg/L naphthaleneacetic acid (NAA), 0.2 mg/L 2-ip, 40 g/L sucrose, 20 g/L maltose, 10 g/L dextrose. The young somatic embryos differentiated into regenerable mature somatic embryos on an 8 g/L agarose-solidified MS basal medium (pH 5.7) with 1 mg/L NAA, 100 ml/L coconut water, 30 g/L sucrose, 30 g/L maltose and 100 mg/L glutamine. The mature somatic embryos regenerated into plantlets on a 2.5 g/L gelrite-solidified MS-based medium with 2.5 mg/L 6-benzylaminopurine (BAP), 1 mg/L gibberellic acid (GA3), 100 mg/L L-glutamine, 30 g/L sucrose and 2.5 mg thidiazuron (TDZ). The total duration from explant stage, for the development of plantlets of 10 to 15 cm height, which could withstand hardening process, was 16 months. The plantlets were morphologically normal, suggesting normal development without somaclonal variation. The present regeneration protocol for ‘Virupakshi’ has great potential for preserving the endangered germplasm by micropropagation and its improvement by transgenic technology against the deadly Banana bunchy top virus and other economically important diseases. Keywords : Hill banana, in vitro micropropagation, male flower bud explants, somatic embryogenesis, Virupakshi African Journal of Biotechnology Vol. 12(6), pp. 563-569

One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana.

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.