2.8.4. Transgenic Breeding for Enriched Glucosinolate/Sulforaphane Content

In recent years, improving the anticancer metabolite glucosinolate/sulforaphane content in broccoli by the genetic engineering of biosynthesis-/regulation-related genes has increased [146–149,152]. Zuluaga et al. reported that the overexpression of *BoMYB29* in DH line AG1012 resulted in the upregulation of the aliphatic glucosinolate pathway and

higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin [146]. Li et al. isolated two tryptophan synthase beta subunit (TSB) genes from broccoli and generated overexpression lines of *BoTSB1* or *BoTSB2* in Arabidopsis, which showed accumulation of tryptophan, indole-3-acetic acid (IAA) and indole glucosinolates; this study provides a target for improving glucosinolates, but no broccoli transgenic plants were generated [147]. Studies on *BroMYB28* revealed its possible role in the biosynthesis of glucoraphanin [152], but its function was not proven in broccoli until 2019 [148]. *Agrobacterium*-mediated transient overexpression of *BroMYB28* in broccoli results in the accumulation of glucoraphanin [148]. Cao et al. generated transgenic broccoli by overexpressing *MAM1*, *FMOGS–OX2* and *Myrosinase* independently or in triple [149]. Compared with wild-type plants, independent transgenes of *MAM1 FMOGS–OX2* and *Myrosinase* enhanced sulforaphane content by 1.7–3.4-, 1.6–2.7- and 3.7-fold, while transgenic plants with the triple gene enhanced sulforaphane content by 1.86–5.5-fold [149].
