Integrated biotechnological and molecular strategies for conservation of endangered wild <i>Brassica</i> species toward climate-resilient agriculture

Agricultural sustainability is increasingly challenged by climate variability, habitat loss, and erosion of genetic diversity by narrowing the genetic base of cultivated crops. Wild relatives of crop species harbor valuable adaptive traits for climate resilience, yet many remain underexplored and endangered. The present study aimed to develop an integrated conservation framework combining biotechnological propagation, molecular characterization, and ecological reintroduction of endangered wild Brassica species. Field surveys were conducted in arid and semi-arid regions of northwestern India to collect ten wild and allied species. In vitro micropropagation protocols were optimized using Murashige and Skoog medium with different hormonal combinations, followed by acclimatization and field reintroduction. Molecular characterization was performed using DNA barcoding (matK, rbcL, and cox2) along with ISSR and SCoT markers to assess genetic identity and diversity. Brassica tournefortii exhibited a maximum shoot regeneration rate of 81.2% on medium supplemented with 2.0 mg L⁻¹ BAP (6-benzylaminopurine) and 0.5 mg L⁻¹ NAA (α-naphthaleneacetic acid), while B. insularis showed the highest callus induction (77.9%) at 2.0 mg L⁻¹ 2,4-D. Reintroduced plantlets demonstrated a field survival rate of 72%. Molecular analyses revealed 89% interspecific polymorphism and clear ecological clustering among species. This study demonstrates the effectiveness of integrating micropropagation with molecular diagnostics and ecological restoration, highlighting endangered wild Brassica species as valuable genetic resources for climate-resilient agriculture.