Rapid and reproducible Agrobacterium-mediated transformation of sorghum

Abstract

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF(2), designated NTL(4)/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL(4)/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus occurred in 89% of the transgenic T(0) events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T(1) progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels

“…Potential impact of biotechnology for improving composition and plant properties of herbaceous feedstocks is in its infancy [43,53,90,92,93,110], but represents an exciting area of research. The development of genetically engineered plants with improved feedstock properties could fast-track bioenergy exploitation; although widespread acceptance of these engineered plants are likely to face public opposition, especially for native prairie species such as switchgrass.…”

Genetic transformation of Sorghum bicolor

“…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.…”

The impact of over-expression of NPK1 gene on growth and yield of sorghum under drought stress

“…Further screening was conducted through PCR analysis, the transformation frequency was calculated and found to be within the cited range (Emani et al, 2002;Gao et al, 2005b;Zhao et al, 2000;Howe et al, 2006) from 0.2 to 4.5%, even though these reports were on highly regenerable and transformable genotypes, e.g. P898012 and TX430.…”