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High Efficiency Transformation of U.S. Rice Lines from Mature Seed-Derived Calli and Segregation of Glufosinate Resistance under Field Conditions

^a Agronomy Dep., Louisiana Agric. Exp. Stn., Louisiana State Univ. Agric. Center, Baton Rouge, LA 70803 USA
^b Rice Res. Stn., Louisiana Agric. Exp. Stn., Louisiana State Univ. Agric. Center, Crowley, LA 70527 USA

View larger version (28K): [in a new window]   Fig. 1 Schematic representation of plasmid constructs used in this study. Ubi, maize ubiquitin promoter; nos, terminator of nopaline synthetase; 35S or P-35S, 35S promoter of cauliflower mosaic virus; T-35S, 35S terminator of cauliflower mosaic virus; tml, transcription terminator of a tumor morphology large gene. H, HindIII; P, PstI; E, EcoRI; B, BamHI; S, SmaI

View larger version (94K): [in a new window]   Fig. 2 Production of transgenic rice plants from embryogenic calli of mature seeds of Cocodrie via particle bombardment. (a) Somatic embryogenic calli induced from mature seeds in CI medium. (b) Proliferation of embryogenic calli in subculture CI medium. x 5. (c) Regeneration of embryogenic calli. x 10. (d) GUS gene expression in embryogenic calli 2 d after bombardment with plasmid pAHC25. The calli were stained with 5-bromo-4-chloro-3-indyl-ß-D-glucuronide (X-Gluc). (e) Growth of bombarded calli in the absence (left) vs. presence (right) of 4 mg L-1 bialaphos. (f) Plantlet regeneration (right plate) of transformed resistant calli compared to necrosis of the untransformed calli (left plate) in the PR-2 medium containing 4 mg L-1 bialaphos

View larger version (158K): [in a new window]   Fig. 3 Greenhouse and field test of herbicide glufosinate (Liberty) resistance in R0 and R1 transgenic rice lines. (a) Plantlets regenerated from transformed and untransformed cells were transferred and grown in the greenhouse. (b) Transgenic rice (R0) plants (background) survived while untransformed rice were killed (foreground) 7 d after foliar application of Liberty herbicide at 2000 mg L-1. (c) Transgenic R0 rice plants of Cocodrie at maturity in the greenhouse. (d) Segregation of glufosinate resistance in R1 progeny from selfed R0 seeds at the Rice Research Station, Crowley, LA, in 1998. (e) Stable, elite transgenic rice lines were recovered and grown to maturity in the field, Crowley, 1998. (f) A transgenic glufosinate resistant line showing purple panicles

View larger version (73K): [in a new window]   Fig. 4 Southern blot analysis of pat gene in R0 and R1 transgenic plants of Cocodrie. Lanes 1–3: plasmid pPAT63 digested with EcoRI, equivalent to 1, 5, and 10 copies of the pat gene per rice haploid genome. Lanes 4–14, genomic DNAs digested with EcoRI; Lane 4: untransformed Cocodrie; Lanes 5–10: six R1 progeny plants 16-1, 16-1, 16-3, 16-4, 16-5, and 16-6 derived from transgenic line 1016 (R0, Lane11); Lanes 11–14: four independent R0 transgenic lines, 1016, 1001, 1017, and 1026; Lane 15: undigested genomic DNA; Lanes 16–19, undigested genomic DNAs from four independent R0 transgenic lines corresponding to Lanes 11–14. DNAs were electrophoresed in 1.0% agarose gel, transferred onto Hybond-NX membranes (Amersham), and hybridized with 32 P-labeled 0.45-kb SmaI fragment of PAT63