Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation |
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Authors: | Yaxin Ge Xiaofei Cheng Andrew Hopkins Zeng-Yu Wang |
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Institution: | (1) Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA |
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Abstract: | Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses,
because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic
calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken
up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was
used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks
of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic
nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and
GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable
tool for functionality tests of candidate genes in forage and turf grasses. |
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Keywords: | Agrobacterium Darnel ryegrass Forage and turf grass Genetic transformation Lolium temulentum Transgenic plant |
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