Genome Editing in Brassica juncea Using CRISPR/Cas9 Technology.

Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.

Studying Salt-Induced Shifts in Gene Expression Patterns of Glucosinolate Transporters and Glucosinolate Accumulation in Two Contrasting Brassica Species.

Brassica crops are well known for the accumulation of glucosinolates-secondary metabolites crucial for plants' adaptation to various stresses. Glucosinolates also functioning as defence compounds pose challenges to food quality due to their goitrogenic properties. Their disruption leaves plants susceptible to insect pests and diseases. Hence, a targeted reduction in seed glucosinolate content is of paramount importance to increase food acceptance. GLUCOSINOLATE TRANSPORTERS (GTRs) present a promising avenue for selectively reducing glucosinolate concentrations in seeds while preserving biosynthesis elsewhere. In this study, 54 putative GTR protein sequences found in Brassica were retrieved, employing Arabidopsis GTR1 and GTR2 templates. Comprehensive bioinformatics analyses, encompassing gene structure organization, domain analysis, motif assessments, promoter analysis, and cis-regulatory elements, affirmed the existence of transporter domains and stress-related regulatory elements. Phylogenetic analysis revealed patterns of conservation and divergence across species. Glucosinolates have been shown to increase under stress conditions, indicating a potential role in stress response. To elucidate the role of GTRs in glucosinolate transportation under NaCl stress in two distinct Brassica species, B. juncea and B. napus, plants were subjected to 0, 100, or 200 mM NaCl. Based on the literature, key GTR genes were chosen and their expression across various plant parts was assessed. Both species displayed divergent trends in their biochemical profiles as well as glucosinolate contents under elevated salt stress conditions. Statistical modelling identified significant contributors to glucosinolate variations, guiding the development of targeted breeding strategies for low-glucosinolate varieties. Notably, GTR2A2 exhibited pronounced expressions in stems, contributing approximately 52% to glucosinolate content variance, while GTR2B1/C2 displayed significant expression in flowers. Additionally, GTR2A1 and GTR1A2/B1 demonstrated noteworthy expression in roots. This study enhances our understanding of glucosinolate regulation under stress conditions, offering avenues to improve Brassica crop quality and resilience.