Thumbnail Image

Thesis

Browse

2016 - 201912020 - 20241
Reset filters
Subject: Agricultural Biotechnology × Author: Sumedha × Type: Thesis ×
Reset filters

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    ThesisItemEmbargo
    CRISPR/Cas9-mediated pectate lyase gene editing for enhanced shelf life of tomato (Solanum lycopersicum L.)
    (Punjab Agricultural University, 2024) Sumedha; Dr Prashant Mohanpuria
    Tomato is one of the most consumed vegetable crops worldwide. Tomato fruits are having an inherently short shelf life which is directly related to fruit softening, caused by cell wall modifying enzymes. Among which pectate lyase (PL) controls fruit softening and thus PL gene was targeted using CRISPR/Cas9 to enhance shelf life of tomato variety Punjab Ratta. In silico study of PL genes from Solanaceous crops including tomato was attempted and three sgRNAs were designed from exon-2 of PL gene. The CRISPR/Cas9 constructs containing sgRNA-1 and 2 were used for transformation of tomato. A new in planta transformation protocol was developed with 10.49 % overall transformation efficiency. Floral buds of 3-8 days of developmental stages were used for dipping in Agro suspension which resulted in setting of 150 fruits. The extracted T0 seeds were selected on kanamycin. A total of 3240 T1 generation plants were regenerated from transgenic tomato fruits (T0), out of which 1620 were found kanamycin resistant which were used for DNA isolation and PCR confirmation of T-DNA integration. The 425 bp PL gene target region from 170 transgenic tomato T1 plants was PCR amplified. Upon sequencing eight T1 tomato transgenic plants were found with PL gene edits containing T insertion, T insertions as well as T substitutions, GC substitution by CG as well as TT insertion downstream to PAM site. PL gene expression and pectin content were decreased in fruits of mutated tomato plants but morphological and biochemical characteristics were more or less similar.
  • Thumbnail Image
    ThesisItemOpen Access
    Cloning and expression of delayed bitterness gene encoding limonoid glucosyltransferase from Kinnow mandarin (Citrus reticulata Blanco)
    (Punjab Agricultural University, Ludhiana, 2016) Sumedha; Mohanpuria, Prashant
    Kinnow mandarin (Citrus reticulata Blanco) is the major citrus fruit crop of north western India and it occupies maximum fruit area in Punjab. But citrus juice processing industry has been suffering from delayed bitterness problem which is mainly because of limonoid aglycones such as limonin content in citrus juices. In order to study citrus limonoid metabolism, limonoid glucosyltransferase (LGT) gene which encodes a natural debittering enzyme was isolated from the fruit tissues of Kinnow mandarin. After confirmation and characterization, this full length gene sequence (1533 bp) was submitted on NCBI. BLASTn and BLASTp analysis of this isolated LGT showed 98% nucleotide sequence identity and 94-99% identity with the amino acids of LGT from different Citrus spp. respectively. The transcript expression of LGT was evaluated in different tissues such as young leaf (YL), flavedo (F), albedo (A), sac covering (Sc) and seed (S) of Kinnow mandarin during early (90-120 days after flowering (DAF)), mid (150-210DAF) and late (240-270DAF) fruit developmental stages using semi-quantitative method. Its expression was highest in flavedo among other tissues. The LGT expression level increases with each fruit developmental stage but in F only, which signifies that delayed bitterness level which is inversely related to LGT transcript level, decreases till the late fruit developmental stage. Further, a prokaryotic overexpression construct was generated by cloning LGT gene into pET-28a vector. This pET28a-LGT construct plasmid was transformed into DH5alpha, and BL21 (DE3) Escherichia coli strain for its protein expression study in future.