Stable transformation of Sorghum bicolor protoplasts with chimeric neomycin phosphotransferase II and β-glucuronidase genes

Abstract

Parameters influencing the stable transformation of Sorghum bicolor protoplasts with a chimeric neomycin phosphotransferase II (NPT II) gene by electroporation were investigated. The mean number of kanamycin-resistant calli produced increased in direct proportion to the concentration of DNA used for transformation. Linearization of the plasmid doubled the mean number of kanamycin-resistant calli produced, while the addition of carrier DNA had no effect. The copy number (1-4) of integrated genes was low compared with that frequently reported for PEG-mediated transformation. Two strategies for transforming protoplasts with a nonselectable, β-glucuronidase (GUS) gene were compared. One utilized a plasmid containing a CaMV 35S-NPT II gene covalently linked to a CaMV 35S-GUS gene, and the other strategy utilized the two genes on separate plasmids. DNA from all 77 kanamycin-resistant calli analyzed contained restriction fragments hybridizing to the NPT II probe; approximately 70% of the clones from all transformation treatments contained a 1.7-kb EcoRI/HindIII restriction fragment corresponding to the full-length gene. Of the kanamycin-resistant calli, 38-63% (depending on the transformation treatment) contained GUS-hybridizing fragments, and 8-19% contained the full-length gene. The addition of NPT II and GUS genes on a single plasmid or on separate plasmids did not appear to lead to an appreciable difference in the frequency of cointegration of these genes, although an increased proportion of the plasmid bearing the nonselectable (GUS) gene appeared to favor its cointegration.

Genetic transformation of Sorghum bicolor

“…The first report of direct DNA uptake into protoplasts of sorghum by electroporation was carried out by Ou- Lee et al, (1986) and latter by Battraw and Hall (1991). Hagio et al, (1991) reported stable transformation from suspension cell cultures of Sorghum vulgare through microprojectile bombardment.…”

“…They include cat, npt II, hpt, bar and manA. Neomycin phosphotransferase II (npt II) gene isolated from E.coli conferring resistance to the antibiotic Kanamycin is one of the commonly used selection strategy for sorghum (Howe et al, 2006;Tadesse and Jacobs, 2004;Battraw and Hall, 1991). Hagio et al, (1991) reported the use of Hygromycin B phosphotransferase (hpt) as well as Neomycin phosphotransferase II (nptII) genes to confer Hygromycin and Kanamycin resistance, respectively.…”

“…Phosphinothricin inhibits glutamine synthetase (GS) irreversibly, resulting in inhibition of amino acid biosynthesis. Almost all the transformation studies conducted earlier in sorghum used bar gene as selectable marker gene except for Battraw and Hall (1991) who used nptII and Hagio et al (1991) where nptII and hph genes were used. The herbicide phosphinothricin is used as selection agent at a concentration of 5.0 mg L -1 (Zhao et al, 2000) while others used low concentrations of the same i.e.…”

“…Battraw and Hall (1991) electroporated protoplasts isolated from embryogenic suspension cultures to introduce nptII along with uidA reporter gene. They studied, transient reporter genes expression using different factors such as linearization of the plasmid and the effect of electroporating with two different gene constructs.…”

“…The first attempt of successful transgene integration into sorghum is by electroporating its protoplasts by Ou- Lee et al, (1986) and latter by Battraw and Hall, (1991). In the same year the particle bombardment of a non-regenerable cell suspension was reported (Hagio et al, 1991).…”