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

Abstract

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 gus-int 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.

In Vitro Plant Regeneration from Petioles of Spinach (Spinacia oleracea L.)

“…Yet, only a few adventitious buds were obtained that could not be grown into whole plant due to lack of root induction. Since then, spinach is supposed to be a recalcitrant plant. , Later, numerous research articles describing protocols of spinach in vitro regeneration were published, and almost all articles suggested a genotype-dependent response of spinach, which has appeared as a major bottleneck affecting its regeneration capacity. ,,,,− The purpose of this study was to establish an efficient and reproducible in vitro regeneration system for the cultivar “Local Palak”. The explants taken from in vitro germinated plantlets were cultured onto various regeneration media containing a variety of plant hormones.…”

“…Only the petioles were found to be totipotent for organogenesis. Although in previous studies, leaf disks, hypocotyls, hypocotyl-based thin layers of cells, root segments, and leaf-based protoplasts have also been utilized in organogenesis. ,,,, According to this study, petioles of our cultivar “Local Palak” exhibited regeneration ability appropriate for plant transformation experiments to produce better germplasm.…”

“…NAA has a significant positive effect on in vitro regeneration. However, it was found that gibberellic acid (GA 3 ), in combination with auxins like NAA, IAA, and 2,4-D was very effective in inducing embryogenic calli spinach. , The effect of GA 3 has been emphasized in many studies for somatic embryogenesis as well as organogenesis. ,,, GA 3 is famous for encouraging the development of shoot primordium in plants, but its effects depend directly on the cultivar used, ,, the explant selected, concentration of GA 3 added to media, amount and types of other plant growth regulators, and the photoperiod and light intensity. The present study revealed the decisive role of GA 3 concentration on direct regeneration of spinach from petiole explants as described in Section .…”