Agrobacterium-mediated Maize Transformation Research Articles

Rapid and highly efficient morphogenic gene-mediated hexaploid wheat transformation.

The successful employment of morphogenic regulator genes, Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2), for Agrobacterium-mediated transformation of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) has been reported to improve transformation by inducing rapid somatic embryo formation. Here, we report two morphogenic gene-mediated wheat transformation methods, either with or without morphogenic and marker gene excision. These methods yield independent-transformation efficiency up to 58% and 75%, respectively. In both cases, the tissue culture duration for generating transgenic plants was significantly reduced from 80 to nearly 50 days. In addition, the transformation process was significantly simplified to make the procedure less labor-intensive, higher-throughput, and more cost-effective by eliminating the requirement for embryonic axis excision, bypassing the necessity for prolonged dual-selection steps for callus formation, and obviating the prerequisite of cytokinin for shoot regeneration. Furthermore, we have demonstrated the flexibility of the methods and generated high-quality transgenic events across multiple genotypes using herbicide (phosphinothricin, ethametsulfuron)- and antibiotic (G418)-based selections.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Advances in Agrobacterium-mediated Maize Transformation.

One of the major limitations of maize transformation is the isolation of a large number of immature embryos using the time-consuming manual extraction method. In this article, we describe a novel bulk embryo extraction method for fast isolation of a large number of embryos suitable for both biolistic- and Agrobacterium-mediated transformation. Optimal gene delivery and tissue culture conditions are also described for achieving high efficiency in Agrobacterium-mediated maize transformation using phosphomannose isomerase (PMI) as a selectable marker.

Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar.

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar. Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03. Agrobacterium strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

Maize transformation technology development for commercial event generation.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

The development of herbicide-resistant maize: stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for 4 × 14 days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4 mg l−1 phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R1 generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Agrobacterium-mediated transformation of maize

Agrobacterium-mediated transformation of maize

Agrobacterium-mediated transformation of maize

Maize may be transformed very efficiently using Agrobacterium tumefaciens-mediated methods. The most critical factor in the transformation protocol is the co-cultivation of healthy immature embryos of the correct developmental stage with A. tumefaciens; the embryos should be collected only from vigorous plants grown in well-conditioned glasshouses. With the protocol described here, approximately 50% of immature embryos from the inbred line A188 and 15% from inbred lines A634, H99 and W117 will produce transformants. About half of the transformed plants are expected to carry one or two copies of the transgenes, which are inherited by the progeny in a mendelian fashion. More than 90% of transformants are expected to be normal in morphology. The protocol takes about 3 months from the start of co-cultivation to the planting of transformants into pots.

The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation.

A new selectable marker system has been adapted for use in Agrobacterium-mediated transformation of maize. This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells are capable of utilizing mannose as a carbon source. Agrobacterium-mediated transformation of immature embryos followed by a pre-selection of 10-14 days prior to selection at a level of 1% mannose and 0.5% sucrose led to the recovery of trangenic lines of a frequency of as high as 30% in about 12 weeks. Molecular and genetic analysis showed that selected plants contained the pmi gene and that the gene was transmitted to the progeny in a Mendelian fashion.

Glyphosate selection of maize transgenic callus lines among genotypes of Ukrainian plant breeding

Aim. Glyphosate selection has a number of advantages over other commonly used selectable markers for maize. There is some natural variability within maize germplasm for degree of sensitivity to glyphosate. We investigated the selective effect of glyphosate for production transgenic maize callus after Agrobacterium-mediated transformation among geno-types of Ukrainian plant breeding. Methods. Agrobacterium-mediated transformation, glyphosate selection in vitro, and PCR analysis were used to obtain transgenic maize callus and to confirm its status. Results. An efficient selectable marker system for production transgenic maize callus lines tolerant to herbicide glyphosate was proposed. Calluses of four maze genotypes of Ukrainian plant breeding and pCB135 vector containing CP4epsps gene were used in Agrobacterium-mediated transformation experiments. Three callus maize lines of DK267×PLS61 genotype containing CP4epsps gene were obtained. Conclusions. The use of glyphosate as a selective agent after Agrobacterium-mediated transformation proved to be effective for transgenic maize callus lines production containing the gene CP4epsps. The success of Agrobacterium-mediated transformation of maize callus strongly depended on the genotype of source ma-terial.
 Keywords: Agrobacterium-mediated maize transformation, CP4epsps gene, glyphosate selection, PCR analysis.