Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.
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This paper presented the results on induction and multiplication of embryogenic calli in liquid medium; on induction of somatic embryo, shoot/root morphogenesis of somatic embryo through culture and on multiplication of somatic embryo tissue in liquid medium.The aim of this study is to lay out the basis for large scale multiplication of these two kinds of tissues with high capable of secondary metabolite production due to their more or less morphological differentiation status.Leaf disks (about 0.5 × 0.5 cm) were cultured on the MS medium with 2 mg/l 2,4-D for callus induction. Callus was subcultured on the MS medium with 1 mg/l 2,4-D + 1 mg/l NAA + 0.2 mg/l kinetin + 10% coconut water for induction of somatic embryo tissue.The embryogenic callus was proliferated in the MS½ liquid medium with 0.5 mg/l 2,4-D + 0.5 mg/l NAA. Depending on the initial medium and the subsequent media, the somatic embryo tissue was cultured for development, via two directions, into population of shoots or roots. The mentioned above tissues are being cultured on shaker in big flask/bioreactor for biomass propagation.
Borer is one of the major pests of sugarcane that can cause a loss of crop yield. Spraying pesticides for the control of borer faces obstacles because the pest lives inside sugarcane stem and sugarcanes with sharp leaves are planted at high density in fields. Transformation of two sugarcane varieties, VN 84 4137 and Suphabury 7, with synthetic Bt genes, cry1Ab and hybrid cry1B-cry1Ab, aims to provide effective resistance against pests, mainly borer. Two strains of Agrobacterium tumefaciens were transformed with plasmids containing cry1Ab gene or cry1B-cry1Ab gene for plant transformation. Sugarcanes were investigated in vitro conditions of cultivation, which indicated that the highest calli formation from the young leaf rolls was obtained on the medium with 3 mg/l 2,4-D for VN84 4137 at 93.33% and 2 mg/l 2,4-D for Suphanbury 7 at 96.67%. The highest number of calli forming shoots was achieved on the medium with the combination of 2 mg/l BAP and 1 mg/l NAA at 100% for two varieties. Preliminary Agrobacterium-mediated transformation of sugarcane was obtained with calli expressing GUS and/or resistant to phosphinothricin at 3 mg/l, which was the lethal threshold for wild-type callus and in vitro young plants.
Four transgenic lines of the Dendrobium cv. Burana White containing the target DsRed gene were obtained following the microprojectile bombardment. Protocorm-like bodies (PLBs, etiolated), cut into 3-5 mm in size, were used as target material for bombardment. Gold particles (0.9 µm) coated by the mixed plasmids (pITB-DsRED with DsRed gene along with the plasmid pITB-BAR with bar gene) (Molar ratio 3:1, respectively) were bombarded into PLBs by the BIO-RAD PDS-1000/He. After 2-day co-bombardment, PLBs were cultured on the Knudson C (1946) selection medium (1 mg/l of BA) with 1-3 mg/l of phosphinothricin (PPT) for several selection cycles. The transient expression of the DsRed gene (2 days after bombardment) was confirmed with a positive result. The PCR analysis of the DsRed, bar genes showed the expected DNA bands-350 bp, 500 bp, respectively. For the detection of the DsRED fluorescence, the transgenic PLBs (etiolated and green) were illuminated with a hand-held LED spotlight (for yellow-green light-560 nm wavelength) and photographed by the CANON digital camera in combination with a red filter. Furthermore, the expression of the DsRED protein in PLB was also readily identifiable under day light/white fluorescent lamp. These results indicate that DsRed is a suitable reporter for genetic transformation of Dendrobium and this system could be used in fundamental research, especially the change of flower color due to the biochemical product of the transgene. This is the first result on genetic transformation of Dendrobium with the DsRed gene for generating transgenic PLBs which can be excited for emitting strong red fluorescence.
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