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2022 - 20232
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Author: Mandeep Kaur × End date: 2023 × Start date: 2020 × Thesis Type: Ph.D ×
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    Biochemical characterization and gene expression analysis of pulp pigmentation specific genes in pummelo
    (Punjab Agricultural University, Ludhiana, 2023) Mandeep Kaur; Rattanpal, H.S.
    The present study aimed at morphological and biochemical characterization of pummelo genotypes grown under the subtropical condition of North India along with gene expression analysis for pulp colour developmental genes involved in carotenoid biosynthesis. Morphological characters of pummelo genotypes were studied as per International Plant Genetic Resources Institute, Italy descriptors for citrus. A wide range of genetic diversity was observed for fruit length (102.1-158.6 mm), fruit diameter (98.8-146.7 mm), peel thickness (10.9-21.8 mm), fruit weight (427.2-1150.0 g), peel weight (179.7-526.2 g), juice content (76.6-221.3 ml) and seed number per fruit (51.0-132.0) whereas, a relatively narrow range of genetic diversity was observed for flower length (30.0-41.4 mm), number of petals (4-5) and the number of segments (15.0-20.0). The highest fruit size was recorded in NRCC Pummelo5 and PTF-1. Basic fruit quality attributes and components of the antioxidant defense mechanism were estimated colorimetrically. Among biochemical attributes (total soluble sugars, titratable acidity, total sugars, sucrose and reducing sugars), NRCC Pummelo-3 and PTF-4 had the best quality fruits on account of the maximum sugar-acid ratio. Relatively higher content of vitamin C, total phenols, total flavonoids, o-dihydroxy phenols and total antioxidant capacity (DPPH and activity of antioxidant enzymes - superoxide dismutase, catalase, ascorbate peroxidase and peroxidase) was recorded in PTF-4 and NRCC Pummelo3. With respect to the volatile composition of pummelo genotypes as determined by GC-MS analysis, a total of thirty and sixty one volatile compounds were identified in pummelo peel oil and juice, respectively. Limonene was observed as the most predominant compound in pummelo peel oil as well as juice. Additionally, the expression of pulp pigmentation-specific genes in pummelo genotypes including pink-fleshed (NRCC Pummelo-3 and PTF-4) and white-fleshed (Local) was studied. The genes upstream of the lycopene biosynthesis (HDS, HDR, DXS, DXR, PDS, PSY, ZDS, ZISO, CRTISO and PLIS) were up-regulated whereas the genes downstream of the lycopene biosynthesis (ε-LCY, ε-CHX, β-CHX, CCD1, CCD4, AAO3, A1CYP707, A3CYP707 and A4CYP707) were down-regulated. Relative expression of carotenoid biosynthesis genes was the highest in NRCC Pummelo-3 than PTF-4 with respect to Local genotype. The information generated may assist fruit breeders to shortlist the genotypes in hybridization programs for the development of nutritionally-enriched cultivars.
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    Bioprocessing of corn stover for bioethanol production
    (Punjab Agricultural University, Ludhiana, 2022) Mandeep Kaur; Kocher, Gurvinder Singh
    The present study was conducted with the objective of standardization and evaluation of an efficient and ecofriendly, method for the pretreatment of corn stover and its subsequent conversion to bioethanol. The proximate analysis of corn stover (500 µ) revealed a composition of cellulose (57.4%), hemicellulose (17.5%), lignin (14.4) and ash (2.2%) in the raw corn stover. Among the different chemical pretreatment methods, acid (1.0% H2SO4) –autoclave (15 psi for 90 min) followed by alkali (2.0% NaOH) pretreatment resulted in 86.8 % and 79.4% decrease in lignin and hemicellulose, respectively and 66.0% increase in relative proportion of cellulose. Among the green solvent methods of pretreatment, organosolv pretreatment methods, acetic acid (40:60, 40%) resulted in 84.6 % and 53.6% decrease in lignin and hemicellulose, respectively and 53.7% increase in relative proportion of cellulose whereas deep eutectic solvent pretreatment, choline chloride-lactic acid (1:8 molar, ratio) resulted in 82.5 % and 33.0% decrease in lignin and hemicellulose, respectively and 69.3% increase in relative proportion of cellulose. For biological pretreatment of corn stover, a two fungal consortium of strains viz. Pleurotus ostreatus PAU03 and Phanerochaete chrysosporium MTCC787 was screened for ligninolytic enzyme production by plate assay on lignin modifying enzyme (LME)- basal medium (LBM), supplemented with 0.01 % (w/v) Azure B and 0.02 % (w/v) Remazole brilliant blue dye and inoculated with agar discs ( 10 mm) of active mycelia. Decolourization of the respective dyes was observed with ligninolytic index of 1.5 and 1.22 for Azure B and Remazole brilliant blue dyes, respectively. The consortium culture of two fungal strains viz. P. ostreatus and P. chrysosporium was used for ligninolytic enzyme production, by using corn stover as substrate. Maximum enzyme activity (U/ml) was recorded on 10th day as 48.33, 61.85 and 40.8 for LiP, MnP, laccase, respectively. The enzyme production was scaled upto 4000ml and the crude extract was concentrated (6.7 times) using acetone and 600 ml concentrated enzyme was produced having enzyme activities (U/ml) 68.89, 41.13 and 110.08 for Lacc, LiP and MnP enzymes, respectively. The latter was partially purified by Fast Performing Liquid Chromatograpgy (FPLC) technique. The enzyme activity (U/ml) of 131.96, 130.38 and 94.21 was recorded for Lacc, LiP and MnP enzymes, respectively in the partially purified enzyme, which was further concentrated (1.5 times) and the enzyme activities (U/ml) of 140.9, 168.53 and 98.05 for Lacc, LiP and MnP enzymes, respectively. This partially purified ligninozyme was used for nanoligninozyme synthesis. In the nanologninozyme (enzyme: sodium:silicate nanohydrate, 1:1) showed the enzyme activities (U/ml) of 128.93, 187.09 and 116.94 for for Lacc, LiP and MnP enzymes respectively. The pretreatment of corn stover with nanoligninolzyme under shake flask conditions (50 ml reaction volume) using optimized physico-chemical parameters viz. corn stover concentration, 2.5 g; enzyme volume, 8.0 ml; Mn2+ ions (0.5 mM) and incubation temperature, 45°C in 72 h of enzymatic action resulted in 87.2% and 67.4% decrease in lignin and hemicellulose, respectively and 80.3% increase in relative proportion of cellulose. The saccharification of pretreated corn stover at different concentrations of corn stover (1.0-10mg) with Arrowzyme (commercial cellulase) at enzyme loading of 30 FPU, resulted in maximum release of reducing sugars (0.396 g/gds) at 2.5g concentration of corn stover. Under these optimized conditions, saccharification of organosolv (acetic acid, 40:60) and biological (nanoligninozyme) pretreated corn stover resulted in release of 0.395 and 0.439 g/g, reducing sugars, respectively. The fermentation of organosolv as well as biological pretreated, and Arrowzyme saccharified corn stover hydrolysate resulted in 0.112 and 0.132 g/gds ethanol, respectively. The fermentation efficiency of 66.54 % [33.9 % yield (yps)] and 70.91 % [36.1 % yield (yps)] was recorded for organosolv and biological pretreatment, respectively.