Toldi et al Plant Cell Rep (original) (raw)
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Agrobacterium-mediated transformation of the desiccation-tolerant plant Craterostigma plantagineum
Plant Cell Reports, 1994
An efficient procedure for A g r o b a c t e r i u m tumefaciens-m e d i a t e d transformation of the desiccation-tolerant plant Craterostigma plantagineum has been developed. Leaf explants were inoculated with A . tumefaciens strain GV3101 carrying the gene for kanamycin-or hygromycin-resistance and the 0glucuronidase reporter gene. Parameters which affected the transformation efficiency were the age of the explant, the degree of wounding and the presence of an antioxidant in the medium. Under optimal conditions, calli originated in more than 80% of leaf explants. Transformed plants were obtained from more than 50% of the cultured calli during regeneration in the presence of a suitable antibiotic. The stable integration of T-DNA was confirmed by Southern blot analysis and its expression by assays for 0-glucuronidase activity.
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...
Vegetos, 2018
The present study describes an efficient in vitro culture protocol for direct plantlet regeneration and A grobacterium- mediated genetic transformation of Corchorus capsularis L. cultivar JRC 517. In vitro morphogenetic capacity of different explants was evaluated. Nodal explants with immature axillary buds showed maximum in vitro culture response (95%) with plantlets induction when cultured on MS with 0.1 mg l-1 IAA and 0.1 mg l-1 Kin. A. tumefaciens strain LBA4404 harbouring a binary vector pBI121, containing gusA reporter gene under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and NOS terminator was used in addition with neomycin phosphotransferase (npt-II) as plant selection marker gene. Different parameters viz. O.D600. of Agrobacterium cell suspension: 0.3; one day preculture; one min explants dipping, vacuum infiltration for 10 min at 600 mm Hg pressure; 0.001 ml l-1 concentration of non-ionic surfactant (Tween 20) and two days co- cultivation with 100 μM acetosyringone (AS) were found to be optimum treatment to achieve maximum number of stable genetic transformants (~3.6%). The putative transformants were screened on MS medium supplemented with 50 mg l-1 kanamycin (Kan50) and their transient expression was confirmed through GUS histochemical assay of the reporter gene and PCR analysis. The survivor plants were grown under Kan50 selection pressure, and rooted successfully. Regenerated plantlets were acclimatized, hardened and transplanted to glass house. Stable integration of the transgene into the recipient genome was confirmed by PCR using compatible primers of gusA and nptII, and through Southern hybridization. The transgenic plants showed normal morphology and most of them followed 3:1 ratio of Mendelian inheritance for a single dominant transgene In vitro direct shoot regeneration protocol from nodal explants with concurrent transgenic development deemed to be successfully involving economically important gene/s and trait enrichment in jute.
Plant Science, 2005
High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.
Turkish Journal of Agriculture and Forestry Sciences, 2019
Alfalfa is a fodder crop that accounts for one of the best sources of protein and is widely cultivated around the world. In vitro regeneration of alfalfa has been studied earlier; however, most of the studies were almost intervened with callus formation. In this study, 3 explant sources (cotyledonary node, hypocotyl, and root crown) of two Turkish cultivars (Nimet and Savaş) were excised from young seedlings. Explants were subjected to different concentrations of BAP, BAP-IBA, and TDZ to evaluate the direct in vitro regeneration potential of selected plant parts. Moreover, we transformed the alfalfa plant with pBin19 harboring 35s.GUS-INT_35s.nptII construct to investigate the transformation efficiency and regeneration frequency after bacterial inoculation. The transformation was carried out by Agrobacterium tumefaciens strain GV2260. The highest mean number of regenerated shoots per explant was recorded as 8.5, 6.66, and 6.33 shoots per explant after cotyledonary node explants were treated with BAP (0.40 mg/L), BAP-IBA (1.25-0.06 mg/L), and TDZ (0.55 mg/L), respectively. The highest gene transformation frequency was 9.52% and 6.19% based on PCR and GUS assays. The regeneration frequency was decreased by up to 48.1% under kanamycin selection pressure. The effect of cultivar on shoot regeneration frequency, mean number of regenerated shoots, and gene transformation efficiency was significant. This study contributes to in vitro regeneration of alfalfa crop and its genetic transformation which could be utilized in future gene transformation studies.
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.
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.
Physiology and Molecular Biology of Plants, 2015
An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B 5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a βglucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T 0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T 1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.
Journal of Genetic Engineering and Biotechnology, 2020
BackgroundRecalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized.ResultsGerminated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 μM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T1 plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture.ConclusionsIt concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.
In Vitro Agrobacterium ' Mediated Transformation and Regeneration
2012
of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree ofDoctor ofPhilosophy IN VITRO AGROBACTERIUMMEDIATED TRANSFORMATION AND REGENERATION OF WHITE CLOVER (TRIFOLIUMREPENSL.) By Jason J. Goldman December 1998 Chairperson: Dr. David S. Wofford Major Department : Agronomy White clover is a high quality forage that fixes atmospheric nitrogen and can improve animal fertility. Genetic transformation technology has the potential to enhance existing varieties and to be used in the development of novel varieties for future use. A genotype independent tissue culture regeneration system is required to use these genetic technologies and this has proven to be a limiting factor for white clover. A new regeneration protocol based on germinated seedlings was tested on five white clover cultivars adapted to the USA. This system proved to be much less genotype specific than previous reports, with 39 to 5 1% of the indi...