Factors influencing Agrobacterium-mediated transient expression of gusA in rice

Abstract

Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

Purine synthesis and increasedAgrobacterium tumefacienstransformation of yeast and plants

“…The most striking results were observed with inhibitors that block both purine and pyrimidine synthesis (azaserine and acivicin). The magnitude of this effect is many times greater than similar effects reported in previous studies (25,(30)(31)(32)(33). A less-pronounced effect was observed for the purine synthesis inhibitor mizoribine, whereas the pyrimidine synthesis inhibitor 5-fluorouracil had no effect.…”

Agrobacterium-mediated genetic transformation of date palm (Phoenix dactylifera L.) cultivar "Khalasah" via somatic embryogenesis

“…Agrobacterium-mediated genetic transformation based gene transformation technology for the development of particular trait, improvement of yield and to develop disease free transgenic has been almost exclusively focused on crops of high economic importance (Birch, 1997;Gheysen et al, 1998;Li et al, 1992;Cervera et al, 1998;Saini and Jaiwal, 2007;Shrawat and Good, 2011;Abalaka and Mohammed, 2011;Ramamoorthy and Kumar, 2012;Li et al, 2015). Unfortunately, other species native to developing countries has not attracted the interest of the well-known multinational seed and biotechnology companies due to their low socioeconomic status and minimal contribution to the national treasury.…”

“…In the present study, 100 mg/l kanamycin was effective for the selection of transgenic somatic embryos. It has been reported that high concentrations of selection agent were effective for the selection of transformed callus and organs in other plant system (Li et al, 1992;Cervera et al, 1998;Saini and Jaiwal, 2007;Shrawat andGood, 2011, Junaid et al, 2012a;Junaid et al, 2012b;Liu et al, 2014;Palla and Pijut, 2015).…”

The Salt-Stress Signal Transduction Pathway That Activates the gpx1 Promoter Is Mediated by Intracellular H2O2, Different from the Pathway Induced by Extracellular H2O2

“…pBI63-3 had a promoter fragment of 1,653 bp upstream of the uidA ATG, which we consider as a fulllength promoter, whereas pBI63-2 and pBI63-1 had fragments of 1,003 and 515 bp, respectively. Plasmids pBI63-3, pBI63-2, and pBI63-1 were introduced into Agrobacterium tumefaciens strain EHA105, which is a EHA101 derivate, Agropine-type, Ti-plasmid, pTiBo542 rif r (Li et al, 1992), and single colonies harboring the relevant plasmids were grown and used for transformation.…”