Rapid and reproducible Agrobacterium-mediated transformation of sorghum.
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.
Howe
Arlene
A
Center for Biotechnology, University of Nebraska, N300 Beadle Center, Lincoln, NE, 68588, USA.
Sato
Shirley
S
Dweikat
Ismail
I
Fromm
Mike
M
Clemente
Tom
T
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015139:Blotting, Southern; D020090:Chromosome Segregation / Q000235:genetics; D032461:Chromosomes, Plant / Q000235:genetics; D047108:Embryonic Development; D005838:Genotype; D036741:Plant Tumor-Inducing Plasmids / Q000235:genetics; D030821:Plants, Genetically Modified; D015203:Reproducibility of Results; D045868:Sorghum / Q000196:embryology / Q000235:genetics* / Q000382:microbiology; D013997:Time Factors; D014170:Transformation, Genetic