The full sequence from an isolated protoplast to a normal plant was achieved by the establishment of embryogenic calli and regenerated plant from protoplast into plant flowering in vitro of several species in citrus relatives. This efficient system payes the way for extensive usage of genes of citrus relatives in citrus breeding by cell engineering methods. Characteristics of the in vitro flowering were found in Murraya paniculata and Fortunella hindsii among citrus relatives. The flowering was affected by the manipulation of plant growth regulators, nutrients, and environmental conditions. The traits of precocious flowering under in vitro condition of these species can provide the information on the shortening of juvenile period, which is one of the obstacles in citrus breeding, and these can be used as an alternative procedure to breeding techniques pass through biotechnology to agriculture improvement.