Optimization of micropropagation and Agrobacterium-mediated gene transformation to spinach (Spinacia oleracea L.)

Spinach is one of the dioecious plant which is considered as a model plant in genetic and molecular studies of sex determination because of its special characteristics such as low chromosome number and short life cycle. An efficient protocol for Spinacia oleracea Agrobacterium-mediated gene transformation was developed. The leaf disks, roots, hypocotyls and cotyledons of this plant were inoculated with LBA4404. LBA4404 carrying pCAMBIA3301 binary vector with 35SCaMV gusint and 35SCaMV bar cassettes. Effects of two preparation condition (induction of vir genes and noninduction) were considered. Also effects of different number days of co-cultivation and pre-culture of explants were examined. After co-cultivation, the explants were transferred to regeneration medium containing 250 mg·L-1 Carbeniciline. Transient expression efficiency was calculated based on the number of blue spots per explants one week after inoculation. Based on the results of transient expression, stable transformation was carried out. After formation of callus the histochemical GUS assay was carried out on some parts of them and other parts were leaved for being regenerated.