Biology subjects

Pathak, B.

Publications and source records attributed to Pathak, B..

4 recordsLinked to original sources

bioRxiv2026

Efficient In Vitro Regeneration from Cotyledon Nodes and In Planta Genetic Transformation in Elite Peanut Cultivars

Peanut (Arachis hypogaea L.), a vital oilseed and food legume, is cultivated across the globe. Genetic improvement via conventional breeding faces limitations from narrow diversity and reproductive barriers, underscoring the need for tissue culture-based regeneration and transformation platforms. This study optimizes an efficient, reproducible in vitro regeneration protocol using cotyledonary node explants from three Indian elite cultivars: GG-20, GJG-9, and TAG-37A. Explants from aseptically germinated seedlings were cultured on Murashige and Skoog (MS) medium with varying cytokinins (e.g., BAP 0-4 mg/L) and auxins (e.g., NAA 0.1-0.9mg/L), yielding direct multiple shoot induction without callus, minimizing somaclonal variation. Optimal shoot proliferation occurred on full-strength MS + 2 mg/L BAP for GG-20/GJG-9 (88.9% efficiency) and 4 mg/L BAP for TAG-37A ([~]89-100% efficiency); rooting peaked on half-MS + NAA (up to 88.9% in GG-20). Regenerated plants acclimatized successfully in greenhouse conditions. Additionally, a robust in planta Agrobacterium tumefaciens (EHA105, pGFPGUSPlus) transformation via plumular meristem pricking in GG-20 achieved 7.69% efficiency. Transgene integration was confirmed by GUS assay and PCR (GUS/hptII), with [~]64% soil establishment.

plant biology↗

bioRxiv2022

Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a

Overexpression of Arabidopsis Dehydration Response Element Binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. While the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be colocalized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding when compared to non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a overexpressing lines, suggesting antagonism between heat and dehydration stress pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and rapid response towards stress tolerance as well as attenuation of the signal to prevent deleterious effects of the runoff response.

plant biology↗

bioRxiv2020

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus

Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co-integration and co-expression of the genes could present a bottleneck. Recombinase-mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site-specific integration locus is needed. Here, the recombinase technology based on Cre-lox recombination was evaluated for developing multigenic lines harboring constitutively-expressed and inducible genes. Targeted integration of a 5 genes cassette in the rice genome generated a precise full-length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance-stability of the multigene locus. Co-localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each others expression pattern. In summary, high rate of co-integration and co-expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co-segregating traits. Significance StatementRecombinase-mediated site-specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

plant biology↗

bioRxiv2018

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock inducible CRISPR/Cas9 system for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using a soybean heat-shock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS) inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted by transforming rice with HS-CRISPR/Cas9 vectors, and the presence of targeted mutations was determined before and after the HS treatment. We found only a low rate of targeted mutagenesis before HS, but an increased rate of mutagenesis after HS treatment among the transgenic lines. Specifically, only [~]11% of transformants showed characteristic insertions-deletions at the ambient room temperature, but a higher percentage ([~]45%) of callus lines developed mutations after a few days of HS treatment. Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after several weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutant lines that independently segregated from Cas9. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing.\n\nSignificance StatementA method for the temporal control on gene editing based on the use of heat-shock induced expression of CRISPR/Cas9 is described, which was efficient in producing heritable mutations in the rice genome. We assume this method will be useful for targeting essential genes and improving the precision of CRISPR/Cas9.

plant biology↗