The aim of this reprint is to present the latest advances in plant genetic engineering and biotechnology published in Plants. In this Special Issue, several important economic and cereal crops, Brassica napus, Brassica juncea, Coffea arabica, Nasturtium officinale, Nicotiana tabacum, Oryza sativa, Passiflora edulis, Solanum betaceum, Solanum tuberosum, and Solanum lycopersicum, are included. Several important gene families, including the cytochrome P450 superfamily, the R2R3-MYB subfamily, the MYB superfamily in Brassica, and the NAC in Passiflora edulis, are extensively analyzed, and the function and potential application of several genes, such as BnCYP704B1a and BnCYP704B1b for genic male sterility, PeNAC-19 for low temperature, and the SUPERMAN-like gene for fruit size control, is determined. A set of new methods is presented for gene transformation and plant propagation. Efficient markers for CMS breeding in oilseed rape and novel reference genes in rice are also reported. In addition, advancements in carotenoid metabolism and in the flavonoid regulation of Brassica, as well as guidelines of genome editing for gene validation and trait improvement, are investigated. In this reprint, leading experts in the field share their insights, research findings, and visions for the future. Overall, this is significant in the sustainable development of crops.