In vitro and in planta Agrobacterium tumefaciens madiated transformation of Arabidopsis thaliana (original) (raw)
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Plant Cell Reports, 1992
The efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana was compared with different organs, Arabidopsis ecotypes, and Agrobacterium strains. Efficiency of shoot regeneration was examined using hypocotyl, cotyledon and root explants prepared from young seedlings. Hypocotyl explants had the highest regeneration efficiency in all of the four Arabidopsis ecotypes tested, when based on a tissue culture system of callus-inducing medium (CIM: Valvekens et al. 1988) and shoot-inducing medium (SIM: Feldmann and Marks 1986). Histochemical analysis using the 13glucuronidase (GUS) reporter gone showed that the gusA gone expression increased as the period of preincubation on CIM was extended, suggesting that dividing cells are susceptible to Agrobacterium infection. In order to obtain transgenic shoots, hypocotyl explants preincubated for 7 or 8 days on CIM were infected with Agrobacterium containing a binary vector which carries two drug-resistant genes as selection markers, and transferred to SIM for selection of transformed shoots. Of four Arabidopsis ecotypes and of three Agrobacterium strains examined, Wassilewskija ecotype and EHA101 strain showed the highest efficiency of regeneration of transformed shoots. By combining the most efficient factors of preincubation period, Arabidopsis ecotype, tissue, and bacterial strain, we obtained a transformation efficiency of about 80-90%. Southern analysis of 124 transgenic plants showed that 44% had one copy of inserted T-DNA while the others had more than one copy.
Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
Nature Protocols, 2006
Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01-0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20-30 plants per pot) within 2-3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.
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.
Facile transformation of Arabidopsis
Plant cell reports, 1991
A protocol is described for the simple, rapid and efficient production of transgenic Arabidopsis plants. The procedure was developed using growth regulator regimes that promote adventitious embryogenesis during or immediately following Agrobacterium mediated transformation. Both the RLD and Columbia genotypes of Arabidopsis were transformed using slightly different growth regulator regimes. For the Columbia genotype two modifications of the protocol were identified which substantially improved regeneration. Cold treatment of the plants used as a source of root explants resulted in a three-fold increase in the number of morphogenic sectors produced. A more important modification was the inclusion of 25 mg/l silver nitrate (an inhibitor of ethylene action) to the medium used for shoot regeneration. This provided a ten-fold increase in the number of shoots produced. These procedures made it possible to obtain over 100 putative transformants of RLD or Columbia from a single 10 cm petri ...
Agrobacterium-Mediated Plant Transformation under in Planta Conditions
This review describes methods of Agrobacterium-mediated T-DNA transfer to plant vegetative and generative cells under in planta, ex planta and " floral dip " conditions, including designing and testing methods that require the in vitro cultivation of transgenic plant cells and tissues. At present, most methods of Agrobacterium-mediated transformation are based on the coincubation of plant vegetative organs and tissues (leaves, roots, stems, or meristems) with bacterial cell suspensions. Adult plants are then regenerated from the cultivated cells or tissues. This approach gives rise to chimeric transformants and bottlenecks with the regeneration from the cultivated cells or tissues of some monocotyledonous plants. Alternatively, T-DNA integrates into the plant genome as a result of treatment of the male and female plant gametophytes with Agrobacterium cells containing activated vir genes by using the " floral dip " method and its variations. Since the transformation frequency is not sufficiently high, especially for monocotyledonous plants, factors affecting the transformation frequency and the Agrobacterium-mediated T-DNA transfer mechanism have been analyzed.
Plant methods, 2006
The Agrobacterium vacuum (Bechtold et al 1993) and floral-dip (Clough and Bent 1998) are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown. To avoid problems associated with large bacterial liquid cultures, we investigated whether bacteria grown on plates are also suitable for plant transformation. We demonstrate here that bacteria grown on plates can be used with si...
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...
Advances in Bioscience and Biotechnology
Arabidopsis thaliana, one of the simple protocol and widely used to produce transgenic Arabidopsis. As the efficiency and ease of getting a transformant is very much time consuming effort and less number of the transformants people get, we have developed a little modified transformation protocol to avoid the disparities. Four types of inoculums (inoculum-1, inoculum-2, inoculum-3 and inoculum-4) were used to check the transformation efficiency out of which Inoculum-3 showed the highest rate of transformation among the four types. 0.07% Twin-20 also acts in same manner as silwet L-77 to increase the rate of transformation efficiency and glucose instead of sucrose can be used in inoculum to transform Arabidopsis. After vacuum infiltration keeping the Agrobacterium infected plants for 7 -8 hrs horizontally in low light at 28 0 C temperature condition, considered best to get an increased number of transformed seeds. Modified protocol produced ~ 12% -14% increase in transformants. Selection pots (kanamycin supplemented soil filled pots) in place of selection plates (Kanamycin supplemented Murashige and Skoog agar plates) proved beneficial as no MS medium and no aseptic condition is required for selection of transformed plants. This increase in transformation efficiency consequently increased the percentage of homozygous and single copied stable transgenic lines. P. Das et al.
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.
In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from 'Valencia' sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l -1 was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l -1 anaphthaleneacetic acid (NAA), 0.5 mg l -1 6-benzyladenine (BA) and 0.5 mg l -1 kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD 600 of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l -1 BA, 0.5 mg l -1 KT, 0.1 mg l -1 NAA, 50 mg l -1 kanamycin and 250 mg l -1 cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.