Biology subjects

Karn, A.

Publications and source records attributed to Karn, A..

4 recordsLinked to original sources

bioRxiv2026

An indirect organogenesis-based citrus transformation system monitored using visible reporter markers

Genetic transformation of citrus is essential for functional genomics and trait improvement, yet remains limited by prolonged regeneration cycles, genotype-dependent responses, and inefficient screening of transformed tissues. Here, we established an Agrobacterium-mediated indirect organogenesis-based transformation system for Carrizo citrange and evaluated two visible reporter systems, the anthocyanin regulator ROSEA1 and the betalain biosynthetic cassette RUBY, for monitoring transformed tissues throughout regeneration. An optimized regeneration protocol enabled visible callus ini-tiation within 7 days following a 5-day pre-culture treatment and recovery of PCR-confirmed transgenic plantlets within 5 months. Both visible reporters were compatible with callus development and shoot regeneration throughout the indirect organogenesis workflow. During callus proliferation, visible pigmentation closely corresponded with GFP fluorescence, enabling rapid, non-destructive identification of transformed tissues. However, reporter performance diverged during shoot regeneration: RUBY maintained stable pigmentation throughout regeneration, whereas ROSEA1-associated pigmentation progressively declined despite continued GFP fluorescence. Consequently, RUBY exhibited a 4.7-fold higher pigmenta-tion-based detection rate than ROSEA1 at the shoot stage and showed closer agreement with GFP-based detection. Putative transgenic events were confirmed by PCR in independent lines of both constructs, together with GFP fluorescence in leaves and root tips. Together, these findings establish an efficient indirect organogenesis-based transformation platform for citrus and demonstrate that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, whereas RUBY provides more reliable visual identification during shoot regeneration and plant recovery.

plant biology↗

bioRxiv2026

miR319 promotes de novo shoot regeneration by repressing LsTCP4 in lettuce

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

plant biology↗

bioRxiv2024

14-3-3 epsilon inhibits premature centriole disengagement by inhibiting the activity of Plk1 and Separase.

The 14-3-3 protein family regulates several pathways in mammalian cells, including centrosome duplication. However, the precise mechanisms by which 14-3-3 paralogs regulate the centrosome cycle remain unclear. To identify the mechanisms by which 14-3-3{varepsilon} regulates centrosome duplication, we altered two conserved acidic residues in the 14-3-3{varepsilon} phospho-peptide-binding pocket that regulate complex formation and dissociation with the associated ligands, D127 and E134, to Alanine. Altering these residues to Alanine led to opposing effects on centrosome duplication; the D127A mutant inhibited centrosome duplication, while cells expressing the E134A mutant showed the presence of supernumerary centrosomes. We demonstrate that 14-3-3{varepsilon} does not inhibit centriole duplication, as reported for 14-3-3{gamma}, but inhibits centriole disengagement. Using a combination of pharmacological and genetic approaches, we demonstrate that 14-3-3{varepsilon} inhibits the activity of Plk1 and Separase, leading to disengagement defects that ultimately lead to decreased proliferation and cell death. Our work demonstrates that different 14-3-3 paralogs regulate different steps in the centrosome cycle, and disrupting complex formation between 14-3-3{varepsilon} and Plk1 or Separase could be a novel therapeutic strategy in tumor cells.

cell biology↗

bioRxiv2020

Stable QTL for malate levels in ripe fruit and their transferability across Vitis species

Malate is a major contributor to the sourness of grape berries (Vitis spp.) and their products, such as wine. Excessive malate at maturity, commonly observed in wild Vitis grapes, is detrimental to grape and wine quality and complicates the introgression of valuable disease resistance and cold hardy genes through breeding. This study investigated an interspecific Vitis family that exhibited strong and stable variation in malate at ripeness for five years and tested the separate contribution of accumulation, degradation, and dilution to malate concentration in ripe fruit in the last year of study. Genotyping was performed using transferable rhAmpSeq haplotype markers, based on the Vitis collinear core genome. Three significant QTL for ripe fruit malate on chromosomes 1, 7, and 17, accounted for over two-fold and 6.9 g/L differences in ripe fruit malate, and explained 40.6% of the phenotypic variation. QTL on chromosomes 7 and 17 were stable in all and in three out of five years, respectively. Variation in pre-veraison malate was the major contributor to variation in ripe fruit malate (39%) and their associated QTL overlapped on chromosome 7, indicating a common genetic basis. However, use of transferable markers on a closely related Vitis family did not yield a common QTL across families. This suggests that diverse physiological mechanisms regulate the levels of this key metabolite in the Vitis genus, a conclusion supported by a review of over a dozen publications from the past decade, showing malate-associated genetic loci on all 19 chromosomes.

genetics↗