A comparative method for protein extraction and proteome analysis by two-dimensional gel electrophoresis from banana fruit

Bananas and plantains are a major staple food and export product in many countries. Banana fruit tissues contain large amounts of secondary compounds, polysaccharides and other plant polymers which will interfere with protein extraction for gel-based proteomic analysis. Due to presence of very small amount of proteins (approximately 1%) in a large matrix of fruit tissues, it classified as ‘recalcitrant’.  In this connection, assessment of different protein extraction protocols and extraction of high quality proteins from banana fruit tissues is crucial for successful gel-based proteome analysis.   In this study, two different protein extraction protocols were validated to isolate proteins from banana (cv.Grand Naine) fruit peel and pulp tissues, and proteins were resolved by using SDS-PAGE.  A comparative study showed that phenol based method is effective in extracting proteins over TCA-acetone method. Isolated proteins were further subjected to two-dimensional gel electrophoresis separation and stained with colloidal coomassie blue to visualize protein spots. On average 380 protein spots could be detected on coomassie stained two-dimensional gel and our study clearly demonstrated the differential protein accumulation during pre-climacteric and climacteric stages of banana fruit.Â

Deciphering the genetic identity and fidelity of banana through inter simple sequence repeats fingerprinting

Bananas and plantains are a major staple food and export product in many countries. The success and pace of progress of crop-improvement program depend to a large extent on the availability of diverse germplasm and information on their characteristics. Somaclonal variation in the tissue culture is a common phenomenon which makes it mandatory to monitor for genetic stability of plants. Present study aimed to assess the genetic identity of 16 different banana varieties and true-to-type conformity of micro-propagated banana. In the present investigation, genetic relationships of 16 bananas, including some exotic varieties, were assessed based on inter simple sequence repeats (ISSR). Out of 26 ISSR primers screened, 18 ISSR primers were produced totally 2168 clear, reproducible and scorable band classes resulting in a total of 1608 polymorphic bands. The number of scorable bands for ISSR primers varied from 1 [(AC)8 YT] to 13 [(GA)8 YC and (AG)8 YT], with an average of 7bands per primer. Cluster analysis classified varieties into clusters, showing similarities for evaluated banana. Further, micro-propagated banana plantlets of Musa acuminata cv. Bantala and cv. Grand Naine that were developed from suckers were screened for genetic variation, if any, using ISSR markers. Similarly, the mother maintained in the field was also subjected for genetic analysis, where the banding patterns for each primer was highly uniform and monomorphic to the field grown mother clone from which the culture had been established. The preset study demonstrated the utility of ISSR markers in assessing both genetic variability among 16 different banana varieties and genetic uniformity of micro-propagated banana. Further, our study confirmed the true-totype  nature of micro-propagated clones

‘OMICS’-approach to regulate ripening and enhance fruit shelf-life in banana: an important fruit crop for food security

Banana belonging to family Musaceae is the fourth most important crop after rice, wheat and corn. Fruit harvested from bananas and plantains are important components of food-security in the tropical world and provide main source of income to the agrarians through local and international trade. India stands as the major producer of banana with an annual production of 28.4 million tons on 796,500 ha, which contribute to 27% of the world production and about 38% of the total fruit crop production in the nation.  In developing countries, post-harvest losses of fruits and vegetables account for almost 50% of the produce. India, the world’s second largest producer of fruits and vegetables, loses 35–40% of the produce due to excessive softening, every year. Therefore, ripening associated softening is the obvious target to extend the fruit shelf-life and to control the post-harvest losses globally. In this study, proteins were phenol extracted from different fruit tissues (peel and pulp) during different developmental (40, 60, 80 and 90-days after flowering) and ripening stages (2, 4, 6, 8, 10 and 12-days after ripening) of banana (cv.Grand naine), and resolved using global proteome approach. 2-D gel images were further submitted to powerful image analysis software (Image Master Platinum, Version.7.1) for qualitative and quantitative analysis. Several protein spots showed fold-change (increase and decrease in abundance), and some protein spots are unique to certain developmental and ripening stages, after image analysis.  Further, spots of interest were subjected to both MALDI-TOF/TOF-MS and LC-MS/MS (Q-TOF) mass spectrophotometry for protein mass fingerprinting and peptide sequencing, after tryptic digestion of the excised protein spots. In parallel, experiments are in progress to subject the samples for transcriptome (RNAseq.) analysis. Based on protein/gene sequence information corresponding genes will be isolated and cloned, and knowledge will be utilized for RNAi constructs preparation to define individual role of identified key proteins/genes in ripening and enhancing fruit shelf-life in banana