Rapid and reproducible Agrobacterium-mediated transformation of sorghum

• Published in: Plant cell reports Vol. 25; no. 8; pp. 784 - 791
• Main Authors: Howe, A, Sato, S, Dweikat, I, Fromm, M, Clemente, T
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.08.2006; Springer Nature B.V
• Subjects: Agrobacterium radiobacter; Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; beta-glucuronidase; Biological and medical sciences; Biotechnology; Blotting, Southern; Chromosome Segregation; Chromosome Segregation - genetics; Chromosomes, Plant; Chromosomes, Plant - genetics; embryo (plant); embryology; Embryonic Development; Experiments; forage crops; Fundamental and applied biological sciences. Psychology; gene expression regulation; Genetic engineering; Genetic technics; genetic transformation; genetics; Genotype; Genotypes; grain sorghum; Methods. Procedures. Technologies; microbiology; micropropagation; neomycin phosphotransferase II; plant regeneration; Plant Tumor-Inducing Plasmids; Plant Tumor-Inducing Plasmids - genetics; Plants, Genetically Modified; reporter genes; Reproducibility of Results; Sorghum; Sorghum - embryology; Sorghum - genetics; Sorghum - microbiology; Sorghum bicolor; Time Factors; Transformation, Genetic; Transgenic animals and transgenic plants; Transgenic plants; Monocotyledones; Embryo; GUSPlus; Genetic transformation; Immature embryo; Sorghum; nptII; Agrobacterium; Gramineae; Angiospermae; Bacteria; Spermatophyta; Genetic engineering; Transgenic plant; Rhizobiaceae
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-005-0081-6

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 pTiKPSF2, designated NTL4/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL4/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus TM 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 T0 events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T1 progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.