The effect of L-glutamine on the genetic transformation of embryogenic cell suspensions of gentian species (Gentiana lutea L., Gentiana cruciata L., and Gentiana kurroo Royle) using Agrobacterium tumefaciens

Abstract

In this study, established embryogenic cell suspensions of three gentian species, Gentiana cruciata L., Gentiana kurroo Royle, and Gentiana lutea L., cultured in the presence of two media CMS1 and CMS2, were used to determine the effect of L-glutamine on the efficiency of Agrobacterium tumefaciens-mediated transformation. The presence of 1 g @ l !1 glutamine in the co-cultivation medium and a 48-hr co-cultivation period were found to be optimal for all the cultures investigated. In order to regenerate plants in the post-transformation culture, approximately 100 mg of cell aggregates was plated as a single layer on RM1 and RM2 media. Timentin was used in posttransformation cultures for preventing bacterial contamination and enhancing cell viability. The transformants were selected in the presence of 50 mg @ l !1 kanamycin. Transformation was later confirmed by histochemical analysis of the activity of reporter enzyme (β-glucuronidase) and by polymerase chain reaction for the detection of uidA and nptII genes. Five lines of embryogenic cell suspension cultures of the studied species were selected and grown in the presence of 50 mg @ l !1 kanamycin. Finally, 23 embryos were regenerated, of which only 11 converted into T0 transformants of G. cruciata. These transformants continued to grow in the presence of kanamycin. A solid, dark blue coloration of their leaves confirmed stable integration and expression of the uidA gene. The molecular analysis of T0 plants revealed the absence of bacterial contamination. Thus, the short list of plant species that can be transformed by A. tumefaciens with the help of an embryogenic cell suspension is extended by the three species investigated in this study.

Agrobacterium-Mediated Genetic Transformation and Cloning of Reference Genes in Suspension Cells of Artemisia Pallens

“…(Wilson et al, 2018), and Gentiana sp. (Rybczynski and Wojcik, 2019). Suspension cells can generate a large number of plants, and it has been used for various medicinal plants.…”

“…The suspension cells can be developed from any explant type; however, most preferably, an embryogenic callus is used. Several successful cell suspension cultures were reported from various plants such as Artemisia annua (Salehi et al 2019), Catharanthus (Saiman et al 2018), Taxus (Wilson et al 2018), Capsicum (Brito-Sanchez et al 2019, Medicago (Santos et al 2019), Arabidopsis (Permyakova et al 2019), Gentiana (Rybczynski andWojcik 2019), andPanicum (Ondzighi-Assoume et al 2019). Agrobacterium-mediated genetic transformation depends on factors like types of explants, bacterial density, co-cultivation duration, the temperature of co-cultivation, concentration of acetosyringone, etc.…”

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application

“…The first, who used embryogenic callus obtained on zygotic embryos of G. dahurica Fisch as a plant material for transformation by A. tumefaciens and apart from shoot organogenesis also reported regeneration of transformants via SE were Sun and Meng (2010). Another example of an exploration of the embryogenic potential for gentian genome modification was the use of embryogenic cell suspensions of G. cruciata and G. kurroo for Agrobacterium -mediated transformation (Rybczyński and Wójcik, 2019) Although both species were successfully transformed, only somatic embryos of G. cruciata were regenerated at length. In order to obtain transformants of G. kurroo , more complicated system, i.e., electroporation of cell suspension-derived protoplasts, had to be engaged (Wójcik and Rybczyński, 2015).…”

Determination of Carbohydrates Content in Gentiana Cruciata L. By Gc/MS Method

“…The genus Gentiana L. is the largest genus of the family Gentianaceae, which includes more than 400 species [5]. The plants of the family Gentianaceae perform a major role in human lives because of their pharmacological (production of rich, specific secondary metabolites) and horticultural values (beauty of the flowers; transformation in shape and size of the leaves) [9,10]. They accumulate flavonoids, xanthones, iridoids, secoiridoids and other metabolites, typical for exceptional species used in medicine [2,11].…”