Agrobacterium - Mediated Transformation of Secondary Somatic Embryos from Rosa Hybrida L. and Recovery of Transgenic Plants (original) (raw)
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Agrobacterium- mediated transformation of chickpea using mature embryos
Chickpea is an important food legume, valued for its seeds with high protein content. Its production is bedeviled by major biotic and abiotic stresses. Plant transformation is a relatively new tool developed with potentially huge benefits for plant improvement. In this study, a transformation protocol based on the inoculation of decapitated mature embryos with the Agrobacterium tumefaciens strain AgL0 was used. Two chickpea lines, ILC482 and ICC12004 were transformed by the binary vector pCGP1258 which contains the bar and the gusA genes. Regenerated explants were selected on 10 mg/l of phosphinothricin. The putative transgenic explants were subjected to hormone-free media for rooting or were grafted on 5-day old non-transgenic rootstock. Transgene to chickpea was confirmed by the polymerase chain reaction (PCR). The expression of the bar gene encoding for the enzyme phosphinothricin acetyl transferase and confering resistance to the herbicide phosphinothricin (PPT)was detected by surviving plants after painting the leaves with 600 mg/l PPT. The activity of gus gene encoding for β-glucuronidase was confirmed by histochemical which revealed GUS-positive expression in T 0 and T 1 plants.
Journal of Tissue Culture Methods, 1989
Somatic embryos have been successfully used as a target tissue for transformation and regeneration of transgenic walnut plants. Walnut somatic embryos, initiated originally from developing zygotic embryos, proliferate numerous secondary embryos from single cells in the epidermal layer. These single ceils in intact somatic embryos are susceptible to transformation by genetically engineered Agrobacterium tumefaciens and provide a means to regenerate nonchimeric transgenic plants. This gene transfer system has been made more efficient using, a) vector plasmids containing two marker genes encoding f3-glucuronidase {GUS) and aminoglycoside phosphotransferase (APH(3')II) and B) a more virulent strain of Agrobacterium. This system should be applicable to any crop that undergoes repetitive embryogenesis from single Agrobacterium-susceptible cells.
Production of Transgenic Plants via Agrobacterium-Mediated Transformation in Liliaceous Ornamentals
Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol. II, 2006
Studies on the Agrobacterium-mediated production of transgenic plants in several Liliaceous ornamentals, Lilium spp., Agapanthus spp., Muscari armeniacum and Tricyrtis hirta are described. Different strains of A. tumefaciens were used, all of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII) gene, the intron-containing ȕ-glucuronidase (GUS) gene, and the hygromycin phosphotransferase (HTP) gene in the T-DNA region. Utilization of organogenic or embryogenic calluses as a target material for transformation and acetosyringone (AS) treatment during inoculation and/or co-cultivation with Agrobacterium were found to be critical for successful production of transgenic plants in Liliaceous ornamentals. Following transfer of co-cultivated organogenic or embryogenic calluses onto hygromycin-containing media, several hygromycin-resistant (Hyg r) tissues were obtained, and complete plants were subsequently developed from these tissues. Most of the plants were verified to be transgenic plants by GUS histochemical assay and PCR analysis. For Lilium 'Acapulco', A. praecox ssp. orientalis 'Royal Purple Select' and M. armeniacum 'Blue Pearl', Southern blot or inverse PCR analysis revealed the integration of 1-5 copies of the transgene into the genome of transgenic plants, but most of them had 1 or 2 copies. Agrobacterium-mediated transformation systems thus established may be useful as a tool for molecular breeding as well as molecular biological studies.
Plant Science, 2001
A system for producing transgenic plants was developed for the Liliaceous ornamental Agapanthus praecox ssp. orientalis (Leighton) Leighton via Agrobacterium-mediated genetic transformation. Leaf-derived embryogenic calli were inoculated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which harbored the binary vector carrying the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing b-glucuronidase (GUS-intron) genes in the T-DNA region. Following co-cultivation, the calli were transferred to a medium containing 1 mg l − 1 picloram (PIC), 50 mg l − 1 hygromycin and 500 mg l − 1 cefotaxime, on which several hygromycin-resistant (Hyg r) cell clusters were obtained 5-6 weeks after transfer. Agrobacterium strain, co-cultivation period and acetosyringone (AS) treatment during co-cultivation affected the number of Hyg r callus lines produced: the best result was obtained when embryogenic calli were co-cultivated with LBA4404/pTOK233 for 7 days in the presence of 20 mg l − 1 AS. Hyg r calli were transferred to the same medium, but lacking PIC, for inducing somatic embryos. Somatic embryos thus obtained developed into complete plantlets following their transfer to a medium without PIC and antibiotics. All of them were verified to be stable transformants by GUS histochemical assay, PCR and Southern blot analyses.
African Journal of Biotechnology, 2008
In this study, genetic transformation of embryogenic suspension cultures of sweet potato (Ipomoea batatas) cultivar Xu55-2 was conducted utilizing the Agrobacterium tumefaciens strain EHA105 that contains the binary vector pBIN19/SBD2 with SBD2 (starch binding domain 2) gene and neomycin phosphotransferase (NPT II) gene. The presence of the SBD2 gene in the genomic DNA of transgenic plants was verified by PCR amplification and confirmed by Southern blot analysis. Results suggested that cefotaxime (Cefo), at the concentration of 200 mg/L, was able to effectively suppress the growth of Agrobacterium after co-cultivation. The optimal concentration for kanamycin (Kan) was 10 mg/L for selecting resistance calli, somatic embryo formation and plant regeneration. The highest frequency of shoot induction (30.9%) was obtained on the MS medium containing 10 mg/L Kan, 200 mg/L Cefo, 1.0 mg/L abscisic acid (ABA) and 1.0 mg/L gibberellic acid (GA 3).
Plant cell reports, 2005
A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both β-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringonecontaining medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l −1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived Communicated by K. K. Kamo
Agrobacterium – mediated transformation of chickpea (Cicer arietinum L.) mature embryos
Chickpea is an important food legume, valued for its seeds with high protein content. Its production is bedeviled by major biotic and abiotic stresses. Plant transformation is a relatively new tool developed with potentially huge benefits for plant improvement. In this study, a transformation protocol based on the inoculation of decapitated mature embryos with the Agrobacterium tumefaciens strain AgL0 was used. Two chickpea lines, ILC482 and ICC12004 were transformed by the binary vector pCGP1258 which contains the bar and the gusA genes. Regenerated explants were selected on 10 mg/l of phosphinothricin. The putative transgenic explants were subjected to hormone-free media for rooting or were grafted on 5-day old non-transgenic rootstock. Transgene to chickpea was confirmed by the polymerase chain reaction (PCR). The expression of the bar gene encoding for the enzyme phosphinothricin acetyl transferase and confering resistance to the herbicide phosphinothricin (PPT)was detected by surviving plants after painting the leaves with 600 mg/l PPT. The activity of gus gene encoding for β-glucuronidase was confirmed by histochemical which revealed GUS-positive expression in T 0 and T 1 plants.
Tissue culture-based Agrobacterium-mediated and in planta transformation methods
Czech Journal of Genetics and Plant Breeding, 2017
Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the mai...
Production of transgenic Phalaenopsis plants using Agrobacterium tumefaciens
1999
Transgenic Phalaenopsis plants over-expressing glutathione S-transferase (GST) gene were produced by Agrobacterium-mediated transformation method. Protocorms at an early stage after germination were inoculated with A. tumefaciens strain EHA101 (pEKH35S163P) that harbors GST, hygromycin phosphotransferase and neomycin phosphotransferase II genes. A total of 68 independent transgenic plants derived from 6325 mature seeds were obtained 6 months after infection with Agrobacterium. Regenerated plants were grown in greenhouse and all plants bloomed within 2 years. Five months after pollination, 13 seed pods were obtained from 6 plants showing suitable commercial characters. Germinated Phalaenopsis T1 seedlings with GST gene were confirmed by PCR analysis. Northern blot analysis confirmed the expression of GST in hygromycin-resistant plantlets.
Plant Cell, Tissue and Organ Culture, 2004
A protocol for Agrobacterium tumefaciensmediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9-12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l −1 kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.