Regeneration and Agrobacterium-mediated transformation studies in carnation (Dianthus caryophyllus L. cv. Turbo) (original) (raw)
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Proceedings of the National Academy of Sciences of the United States of America, 1988
Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F1 seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different Arabidopsis ecotypes. In addition to the neo gene, a chimeric bar gene conferring resistance to the herbicide Basta was introduced into Arabidopsis. The expression of the bar gene was shown by enzymatic assay.
Plant Cell, Tissue and Organ Culture (PCTOC), 2010
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.
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...
Genetic transformation of plants
Proceedings / Indian Academy of Sciences, 1986
Current status of the molecular approaches for integrative genetic transformation of plants is reviewed. Agrobacterium-mediated and direct DNA transformation of protoplasts are considered. Elucidation of the molecular events in natural genetic transformation of plant cells in crown gall disease caused by Agrobacterium tumefaciens, has led to the development of T-DNA based vectors for introducing exogenous DNA into plant cells. Various strategies used for this are discussed. So far, Agrobacteriummediated transformation has been most successful and widely used in dicotyledonous plants. Direct DNA transformation involves either DNA uptake by protoplasts or its mechanical delivery into the cell nucleus. Transformation of both monocotyledonous and dicotyledonous plant cells has been shown by this method. The genes transferred into plants from bacteria, other plant and animal species and various promoters used in chimeric gene construcl~s for the expression of such genes are listed. Organ specific expression and Mendelian inheritance of transferred genes have been demonstrated. Prospects of the transformation technique for the improvement of crop plants are examined. Gene transfers reported so far indicate that the immediate benefits will be in the development of cultivars carrying herbicide resistance or biocide gene(s). Identification, isolation and cloning of DNA sequences governing resistance to plant diseases or pests, and their introduction into improved cultivars could become possible in the future, lsolation, in vitro modification and re-introduction of the modified gene(s) back into the plant genome is yet another possibility for the future.
Development of in Planta Transformation Methods using Agrobacterium tumefaciens
Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol. II, 2006
This chapter describes simple and efficient in planta transformation methods using Agrobacterium tumefaciens for buckwheat (Fagopyrum esculentum M.), mulberry (Morus alba L.), kenaf (Hibiscus cannabinus L.) and rice (Oryza sativa L.). In the methods, meristems of either apical (buckwheat, kenaf and rice) or axillary bud (mulberry and kenaf) of young plants (buckwheat, kenaf, and mulberry) or soaked seeds (rice) were inoculated by A. tumefaciens after being pricked with a needle. The inoculated plants were grown to maturation in pots under nonsterile conditions. Transformation was demonstrated by several lines of evidence obtained by using mostly the progenies of the T1 generation; phenotypic inheritance from T0 plants to the plants of the following generation, detection of transgene and its transmission to the following generation, rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA, detection of ȕ-glucuronidase activity in the transformants and resistance of seed germination of transformants to antibiotics (geneticin or hygromycin).
Optimization of Agrobacterium-mediated genetic transformation in gherkin (Cucumis anguria L.)
Plant Omics, 2013
The present work was aimed to study various factors influencing Agrobacterium tumefaciens mediated genetic transformation of gherkin (Cucumis anguria L). Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene βglucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII) was used for transformation. Factors affecting transformation efficiency, such as Agrobacterium concentration, effect of acetosyringone, pre-cultivation, infection and cocultivation time of Agrobacterium were studied. After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB 5 ) containing 1.5 μM benzylaminopurine (BAP) with 0.5 μM naphthalene acetic acid (NAA), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB 5 medium containing 3.0 μM BAP, 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. Transgenic shoots were elongated in MSB 5 medium fortified with 2.0 μM gibberellic acid (GA 3 ), 100 mg L -1 kanamycin and 300 mg L -1 carbenicillin. The transgenic elongated shoots were rooted in MSB 5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L -1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and direct regeneration of C. anguria.
Plant Tissue Culture and …, 2010
An efficient Agrobacterium-mediated transformation compatible in vitro regeneration protocol was developed for two important varieties of mungbean (Vigna radiata (L.) Wilczek) cultivated in Bangladesh, namely Binamoog-5 and BARI Mung-6. Two different zygotic embryo derived explants, such as cotyledonary node (CN) and cotyledon attached decapitated embryo (CADE) were used for direct organogenesis of shoot. MS supplemented with 4.0 μM BAP was found to be the best for the development of highest number of multiple shoots from CADE in both the varieties of mungbean. While in case CN the best shoot formation was achieved on MS containing 4.0 μM BAP and 0.5 μM NAA in both varieties. Half strength of MS with 2.0 μM IBA was found to be most effective for producing healthy root from regenerated shoots. Following root induction, the in vitro raised plantlets were successfully transplanted to soil for their establishment. Considering overall responses, genetic transformation efficiency was found to be better with CADE explant using Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 conferring GUS and nptII genes. Different factors influencing transformation was optimized during this study. Selection of transformed shoots was carried out by gradually increasing the concentration of kanamycin and such transformed shoots were eventually selected using 200 mg/l kanamycin. Stable expression of the GUS gene was detected in various parts of regenerated transformed plantlets. Transformed shoots were rooted on half strength MS containing 2.0 μM IBA and 100 mg/l ticarcillin. Rooted transformed plantlets were successfully transferred to soil. Stable integration of GUS and nptII genes in the putative transformed shoots was confirmed through PCR analysis.
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.
We established for the first time an Agrobacterium-mediated genetic transformation system for the Himalayan blue pine. Embryogenic tissue derived from vegetative shoot apices of mature trees of Pinus wallichiana were inoculated with A. tumefaciens strain EHA105 fused with a binary vector pBI121. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the-glucuronidase (uidA) reporter gene, was used in the transformation studies. GUS activity was used to monitor transient expression of the uidA gene and to further test lines selected on kanamycin-containing medium. The integration of the transgene (nptII) was confirmed by PCR followed by southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system could be established in P. wallichiana. This provides an opportunity to transfer economically important genes into Himalayan blue pines.
Analysis of Molecular and Morphological Characteristics of Plants Transformed
2015
A defensin gene isolated from chickpea (Cicer arietinum L.), Ca-AFP in the background of pCAMBIA1301 was transferred into tobacco (Nicotiana tabaccum L. var. petit havana) genome following transformation using Agrobacterium tumefaciens strain LBA4404. Although all the explants showed GUS activity after co-cultivation, transgenic tobacco shoots were regenerated only from those explants cultured in presence of bacteriostatic antibiotic carbenicillin under hygromycin selection. Presence of the antifungal gene in the regenerated plants was confirmed by PCR, while integration of the whole T-DNA was demonstrated by southern blot hybridization. Furthermore, southern blot hybridization revealed that most of the transgenics contained single copy of the T-DNA while few were found to have multiple copies. Expression of the GUS and Ca-AFP genes in the transgenic plants was observed. Morphological analysis demonstrated that presence of the transgenes produced no morphological abnormality or yiel...