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Shelake, R. M.

Publications and source records attributed to Shelake, R. M..

4 recordsLinked to original sources

Generation of Parthenocarpy tomato on multiple commercial cultivars utilizing CRIPR/Cas9 system

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials.

plant biology↗

Characterization of yield and fruit quality parameters of Vietnamese elite tomato lines generated through phenotypic selection and conventional breeding methods

Tomato (Solanum lycopersicum L.) is the second most important vegetable crop after potatoes, and global demands have been steadily increasing in recent years. Conventional breeding has been applied to breed and domesticate tomato varieties to meet the need for higher yield or superior agronomical traits that allow to sustain under different climatic conditions. In the current study, we applied bulk population breeding by crossing eight tomato accessions procured from the Asian Vegetable Research and Development Center (AVRDC) with three heat-resistant tomato inbred lines from Vietnam and generated ten elite tomato (ET) lines in the F8 generation. The individual F8 lines exhibited robust vigor and adaptability to Vietnamese climate conditions. Among the ten lines, ET1 and ET3 displayed indeterminate growth. ET2 showed semi-determinate, while all the other lines had determinate growth. The different ET lines showed distinctive superior agronomical traits, including early maturing (ET4, ET7, and ET10), highly efficient fruit set (ET1), higher yield (ET1, ET8, ET10), jointless pedicels (ET2), and partial parthenocarpy (ET9). Molecular analysis revealed that the ET3 line consisted of Ty-1 and Ty-3 loci that positively contribute to Tomato yellow leaf curl virus (TYCLV) resistance in tomato plants. The elite tomato lines developed in this study would contribute significantly to the Vietnamese and Asian gene pool for improved tomato production and may be a valuable resource for various breeding goals.

plant biology↗

IRI-CCE: A Platform for Evaluating CRISPR-based Genome Editing Tools and Its Components

Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs.

bioengineering↗

A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed \"gel shifting\" appears to be common for histidine-rich proteins but not yet studied in detail. We investigated \"gel shifting\" in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining \"gel shifting\" of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed \"metal gel-shift\" demonstrating an intimate link between Ni2+ binding and \"gel shifting\". To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66{+/-}0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact \"metal-gel shift\" band on SDS-PAGE. In view of presented data metal-binding and \"metal-gel shift\" models are discussed.

biochemistry↗