Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria.

Present study was aimed at screening and characterizing thermostable amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir. Four thermophilic isolates were characterized on morphological, biochemical, physiological basis and were authenticated by molecular analysis. By 16S rDNA sequencing, isolates were identified as Anoxybacillus mongoliensis (MBT001), Anoxybacillus flavithermus (MBT002), Bacillus (MBT004). Among all identified strains, MBT003 showed maximum homology with both Anoxybacillus mongoliensis and Anoxybacillus flavithermus. Amylase activity was analyzed qualitatively in starch agar and quantitatively by DNS method. The optimal enzyme production was observed and authenticated by Response Surface Methodology at 7 pH, 70 °C, 1.25% substrate concentration, 300 µL of inocula volume after 48 h of incubation. Optimum amylase activity (4.4 U/mL) and stability (3.3 U/mL) was observed with 1.5% soluble starch at 70 °C. Maximum activity (3.7 U/mL) and stability (1.5 U/mL) was found at pH 8. Enzyme activity was increased in the presence of MgSO4 and CaCl2. Amylase was stable with surfactants and commercial detergents for 30 min. Supplementation of the enzyme with commercial detergent improved the washing ability of the detergent. This investigation has revealed that these thermostable bacteria are excellent source of amylase which can be used commercially for generating economic activity on sustainable basis.

Diversity of culturable thermophilic bacteria from Tata Pani hotspring of Kotli Azad Jammu and Kashmir.

Hot water springs are unique areas populated by mesophiles, thermotolerant and hyperthermophiles. They are the source of diversity of thermophiles, mainly belonging to archaea and bacteria domains. The diversity of thermophiles gives an outline of the huge biological potential that can be exploited for industrial applications.To this end, this study was aimed to isolate and characterise the unexplored thermophilic microorganisms from hot water spring in Tatapani, Tehsil & District Kotli AJK, Pakistan. Around 10 bacterial isolates were identified using morphological, biochemical, physiological and molecular attributes. Sequencing of the 16S rDNA gene of the isolates followed by BLAST search revealed that the strain MBT008 has 100% similarity with Anoxybacilluskamchatkensis. MBT012 showed 99.57% similarity with A.mongoliensis, MBT014 was affiliated with A.tengchongensis with 99.43% similarity, MBT009 showed 99.83% homology with A.gonensis and MBT018, 98.70% similarity with A.karvacharensis. The presence of all this microbial diversity in one common source is of immense importance related to envioronmental and industrial aspects in general and extraction of thermostable enzymes from these thermophiles specifically opens new horizons in the field of industrial biotechnology. These thermophiles are revealing new capabilities and are being manipulated by biotechnologists in utilizing them in different unique ways.

Genetic improvement of peanut (Arachis hypogea L.) genotypes by developing short duration hybrids.

Peanut, the only cash crop of rainfed areas of Pakistan, is facing immense challenges due to global warming. Climatic factors particularly the temperature fluctuations and rain pattern shift significantly impact the production and yield of peanut and unavailability of resilient varieties exacerbate this impact. To deal with the cropping pattern change and yield losses, due to climate vagaries, a study was conducted to develop early maturing hybrids using line into tester mating design. The F1 hybrids from the parental lines were produced in the year 2018 using Line × Tester mating design and then grown in the field in the year 2019 for further evaluation. The hybrids were evaluated based on the early maturity and yield-related attributes in comparison with the parental lines. Based on the general combining ability estimate, line V-3 (Golden), was found as best parent with highly significant values for plant height, days to peg formation, days to maturity, number of pegs per plant, number of pods per plants, number of seeds per plant, 100 pod weight 100 seed weight. Similarly, tester V-7 (PI 635006 01 SD) showed highly significant results of GCA for days to germination, day to 50% flowering, plant height, days to peg formation, days to maturity, number of pegs per plant, number of pods per plants, number of seeds per plant, 100 kernel weight, shelling percentage. All the combinations were evaluated for specific combining ability and significant results were observed for V-3 × V-4 (Golden × PI 619175 01 SD) and V-1 × V-6 (BARI-2000 × PI 564846 01 SD) by developing or maturity and yield-related attributes. The hybrid combinations V-3 × V-5 (Golden × PI 635006 01 SD) followed by V-3 × V-6 showed highly significant results for mid parent heterosis and better parent heterosis for days to 50% flowering, plant height, days to peg formation, number of pegs, days to maturity, number of mature seeds per plant, shelling ratio, 100 pod weight and 100 kernel weight. These parents and hybrid combinations with early maturity genes and high yield attributes can further be used for the development of short duration variety.

Genetic diversity of common bean (Phaseolus vulgaris L.) ecotypes from Pakistan using Simple Sequence Repeats.

Common bean (Phaseolus vulgaris L.) is a legume crop grown all over the world and is a very important food of mountain population of Pakistan for protein intake. The Western Himalayan Mountains are rich in biodiversity including unexplored landraces of the common bean crop. Unfortunately, very little attention has been given to this valuable crop in Pakistan, and it is being exported, majorly from Ethiopia, to meet the country's requirements. The exploitation, utilization, preservation and multiplication of existing germplasm within the area are very important for sustainable production of the crop and enhancing the nutrition value for the local community in mountain regions. A research study was conducted for evaluation of biological diversity of common bean landraces from Azad Kashmir and Northern areas of Pakistan using morpho-physiological and molecular markers. Thirty-five common bean ecotypes along with one check variety were collected from different altitudes of Azad Kashmir and Northern Pakistan and screened for biological diversity. Morphological characterization revealed high genetic diversity in parameters including stem anthocyanin, growth type, days to flowering, pods/plant and 100 seeds weight. Genomic characterization using SSR markers, for allelic diversity evaluation among germplasm, also provided diverse profile with 83.3% polymorphism in banding pattern. The bulk of gene pool diversity evaluated within bean landraces may help to initiate breeding program for common bean improvement.

Genetic characterization of flowering and phytochrome genes in peanut (Arachis hypogaea L.) for early maturity.

BACKGROUND: Peanut (Arachis hypogaea L.) production and cropping pattern is highly influenced by the climatic factors including temperature and rain pattern fluctuations. It is one of the most important cash crop in the rain fed areas of Pakistan and its production, under changing climatic conditions, that can be improved by developing short duration varieties. The present study was based on the molecular characterization of the maturity associated gene families in the peanut under two light conditions. METHODS AND RESULTS: Genomic analysis based on the in silico study of important gene families for early maturity associated attributes like flowering time, their pattern, duration and photoperiodism was done for a comprehensive mapping of maturity related genes. Phytochromes genes Phy A, Phy B and Phy E and flowering genes FT2a, Ft5a and COL2 were selected for in silico characterization for protein based analysis including Multiple Sequence Alignment (MSA), and Neighbor Joining (NJ) tree. MSA and NJ trees of the peanut with Arabidopsis thaliana and Glycine max showed a clear picture of the phylogenetic relationship on the basis of selected gene proteins. Expression profile of phytochrome and flowering genes revealed that photoperiod conditions i.e. short and long days, have great influence on the Phy A, Phy B and Phy E, Ft2a, FT5a and COL2 gene expression pattern. In current study, the relative expression of all studied genes was found higher in short day light condition at flower initiation stage of the plants than in the long light day condition with exception of COL2 gene protein. CONCLUSIONS: The molecular characterization based on the in silico study of the particular genes and qPCR based gene expression profiling of the selected genes provided an evidence of the role of these genes and their comparative analysis under two photoperiodic conditions.

Identification of bacterial communities in extreme sites of Pakistan using high throughput barcoded amplicon sequencing.

Microorganisms thrive nearly everywhere including extreme environments where few other forms of life can exist. Geochemistry of extreme sites plays a major role in shaping these microbial communities and microbes thriving in such harsh conditions are untapped sources of novel biomolecules. To understand the structure and composition of such microbial communities, culture-independent bacterial diversity was characterised for two extreme sites in Pakistan, Khewra salt range and Murtazaabad hot spring. Barcoded amplicon sequencing technique was used to study the microbial communities. Physicochemical analysis of these sites was also conducted to study the dynamics of microbial communities under stressed conditions. Metagenomic sequencing of salt range soil samples yielded of 40,433 16S rRNA sequences, while hot spring sediments produced 76,449 16S rRNA sequence reads. Proteobacteria were predominant in saline soil while Firmicutes were most abundant in hot spring sediment. The taxonomic analysis of saline samples revealed 914 operational taxonomic units (OTUs) while that of hot spring sequences were clustered into 726 distinct OTUs. OTUs from genus Alkalibacillus were most abundant in hot spring sediments, whereas Haloarcula were more prevalent in saline soil. Some unidentified sequences were also present at each taxonomic level. Multivariate analysis indicated that electrical conductivity and pH are the major environmental factors involved in modelling microbial communities. This study revealed a poly-extremophilic microbial community in the Murtazaabad hot spring and characterised the unexplored halophilic microbial diversity of saline soil of Pakistan.