A well-developed root system is crucial for the rapid growth, asexual reproduction, and adaptation to the drought environments of the watercress. After analyzing the transcriptome of the watercress root system, we found that a high concentration of auxin is key to its adaptation to dry conditions. For the first time, we obtained DR5::EGFP watercress, which revealed the dynamic distribution of auxin in watercress root development under drought conditions. Via the application of naphthylphthalamic acid (NPA), 4-biphenylboronic acid (BBO), ethylene (ETH), abscisic acid (ABA), and other factors, we confirmed that auxin has a significant impact on the root development of watercress. Finally, we verified the role of auxin in root development using 35S::NoYUC8 watercress and showed that the synthesis of auxin in the root system mainly depends on the tryptophan, phenylalanine, and tyrosine amino acids (TAA) synthesis pathway. After the level of auxin increases, the root system of the watercress develops toward adaptation to dry environments. The formation of root aerenchyma disrupts the concentration gradient of auxin and is a key factor in the differentiation of lateral root primordia and H cells in watercress.

Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the distribution details of auxin in the growth process of watercress. Our findings provide an effective method for gene function research and lay the foundation for innovative utilization of germplasm resources of watercress.