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Chrysanthemums (Chrysanthemum morifolium Ramat.), highly regarded in the ornamental plant industry and second only to roses in the cut flower market worldwide, have traditionally been enhanced through crossbreeding and natural mutations, yielding a diverse array of flower morphologies and colors. Recent elucidations of the molecular mechanisms underpinning their physiology and ecology have paved the way for the development of new cultivars using advanced biotechnological tools. This chapter provides an overview of the in vitro methods used for germplasm conservation and describes advancements in various tissue culture techniques, including somatic embryogenesis, embryo rescue, anther culture, and protoplast culture. It also discusses genetic modifications aimed at improving flower color and floral architecture analysing, made possible by the identification of pigment biosynthetic genes and various homeotic genes. Furthermore, this chapter outlines transformation protocols for genetic engineering, producing viroid-free chrysanthemums, explores the role of artificial intelligence in optimizing in vitro cultural conditions, and discusses future prospects in chrysanthemum biotechnology.