Molecular characterization of banana var Nanjanagudu rasabale and identification of fruit ripening specific products

Banana is a major fruit crop and India is the place of origin and diversity for banana. The cultivar under consideration for the present study is an endangered one namely “Nanjanagudu rasabale” (NR) having genotype AAB. The present study has established genetic relationship of NR, among other 21 commercially important banana cultivars of South India, by using genetic markers. Analyses using 50 Randomly Amplified Polymorphic DNA (RAPD) and 12 Inter Simple Sequence Repeats (ISSR) primers resulted in the amplification of totally 641 bands of 200 – 3100 bp, of which 382 bands were polymorphic. Based on these data, a genetic similarity matrix was established and a dendrogram for each set of primers was developed by UPGMA. The Genetic similarity coefficients in RAPD analysis ranged from 0.3177 to 0.7818 and in ISSR analysis from 0.1800 to 0.8462. The presence of a specific RAPD (OPC 5800) band was observed for an endemic cultivar NR. A group of 8 cultivars was identified having highly diverse relationship from one another and this information would be useful for generating 2X and 4X breeding populations for further application in breeding secondary hybrids. Since edible banana plants are sterile hybrids, obtaining variants through biotechnological methods would greatly assist in the generation of genetically diverse and agronomically improved clones for which in vitro regeneration is a pre-requisite. Thus aiming, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indole-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2-3 per explant, occurred in the explants incubated at the first step in medium with 24.6 μM and 0.198 μM of IBA. Addition of various levels of (10-50 μM) spermine, spermidine, and putrescine to cultures with secondary embryogenesis showed that about 50% of embryogenic calli rapidly produced secondary embryos only in the presence 40 μM spermine but not in other treatments. The shoot cultures were checked for their performance on solid medium (SM) and partial immersion system (PIS). The rate of shoot multiplication was higher in PIS than in SM. A micropropagation protocol was also developed where high levels of Benzylamino purine (BAP) up to 53.28 μM and Kinetin (Kn) up to 55.80 μM showed direct correlation between the two resulting in a highest number of 80 shoot buds per segment in BAP (31.08 μM) treatment. The plantlets were analysed for their genetic stability using 50 RAPD and 12 ISSR genetic markers that resulted in 625 distinct bands showing homogeneous patterns. The absence of any genetic variation indicates that the micropropagation protocol developed in this study as well as for developing stable regenerants of NR for rapid in vitro multiplication is appropriate and applicable for clonal propagation. Fruit ripening and softening involve depolymerization of complex cell wall components. More than one class of enzymes and other proteins are known involved in this process. In the present study the regulation of fruit softening studied at molecular level and demonstrated the simultaneous activities and gene expressions of expansins (the cell wall loosening proteins) and other cell wall hydrolyzing enzymes. Two types of expansin genes, MaEXPA-1 and MaEXPA-2, were found to be banana fruit-ripening-specific and their expressions significantly and differentially altered by ethylene inducers/inhibitors. Activities of pectin methyl esterase (PME), polygalacturonase (PG) and pectate lyase (PEL) in banana cv. NR fruit were measured over a period of 10 days after ripening was initiated with ethylene. Ethylene-stimulated activities of the above three enzymes was differentially suppressed by GA, MH, SA and IAA whereas ABA, ethrel and smoking stimulated the activities of all hydrolases, except polygalacturonase. These treatments appear to play a major role in up/ down-regulation of the activities of various cell wall hydrolases. To gain a better insight on the molecular regulation of banana fruit ripening, the mRNA Differential Display technique coupled with silver-staining was used and specific transcripts differentially expressed during ripening were first identified. Using 71 primer combinations and four populations of mRNA (Pre-climacteric; Climacteric 1; Climacteric II and post climacteric), a total of 120 transcripts were cloned into T/A cloning vector and sequenced. DNA sequence analyses revealed significant homology to transport protein, sucrose phosphate synthase, heat shock protein, transcriptional regulator, senescence protein. These proteins can be associated to biological processes like primary, secondary and RNA metabolism, signal transduction and stress responses or defense. The RNA dot analysis showed the expression of most of the upand down-regulated genes are specific to fruit and ripening. Since banana lacks information about the molecular regulation of ripening, the results of the present finding provide better insight for the characterization of the changes in gene expression that accompanies the ripening process. The enormous data developed through these studies form the basis for developing chemical formulations for controlled ripening of banana fruit, probably with extended shelf life.