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Rapid and reproducible Agrobacterium-mediated transformation of sorghum
Authors:Arlene Howe  Shirley Sato  Ismail Dweikat  Mike Fromm  Tom Clemente
Institution:(1) Center for Biotechnology, University of Nebraska, N300 Beadle Center, Lincoln, NE, 68588, USA;(2) Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE, 68583, USA;(3) Plant Science Initiative, University of Nebraska, N300 Beadle Center, Lincoln, NE, 68588, USA
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 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 TM 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.
Keywords:Genetic engineering  Immature embryo  GUSPlus                                    TM" target="_blank">TM                                              nptII
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