Larry A. Gilbertson

Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants

Abstract. After the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. There are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. We have tested the Cre/lox system from bacteriophage P1 for its ability to precisely excise stably integrated marker genes from chromosomes in transgenic maize plants. Two strategies, crossing and autoexcision, have been tested and demonstrated. In the crossing strategy, plants expressing the Cre recombinase are crossed with plants bearing a transgene construct in which the selectable marker gene is flanked by directly repeated lox sites. Unlike previous reports in which incomplete somatic and germline excision were common, in our experiments complete somatic and germline marker gene excision occurred in the F1 plants from most crosses with multiple independent Cre and lox lines. In the autoexcision strategy, the cre gene, under the control of a heat shock-inducible promoter, is excised along with the nptII marker gene. Our results show that a transient heat shock treatment of primary transgenic callus is sufficient for inducing cre and excising the cre and nptII genes. Genetic segregation and molecular analysis confirmed that marker gene removal is precise, complete and stable. The autoexcision strategy provides a way of removing the selectable marker gene from callus or other tissues such as embryos and kernels.

Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.

A novel real‐time polymerase chain reaction method for high throughput quantification of small regulatory RNAs

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are important players of both transcriptional and post-transcriptional gene silencing networks. In order to investigate the functions of these small regulatory RNAs, a system with high sensitivity and specificity is desperately needed to quantitatively detect their expression levels in cells and tissues. However, their short length of 19-24 nucleotides and strong similarity between related species render most conventional expression analysis methods ineffective. Here we describe a novel primer for small RNA-specific reverse transcription and a new TaqMan technology-based real-time method for quantification of small RNAs. This method is capable of quantifying miRNA and siRNA in the femtomolar range, which is equivalent to ten copies per cell or fewer. The assay has a high dynamic range and provides linear readout of miRNA concentrations that span seven orders of magnitude and allows us to discriminate small RNAs that differ by as little as one nucleotide. Using the new method, we investigated the expression pattern of gma-miRMON1, a novel miRNA identified from soybean leaves. The results were consistent with our results obtained from Northern blot analysis of gma-miRMON1 and Affymetrix microarray analysis of the gma-miRMON1 precursor, suggesting that the new method can be used in transcription profiling.

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.

A test for ectopic exchange catalyzed by Cre recombinase in maize

A maize line expressing Cre recombinase as well as the recipient line without the transgene were assayed for evidence of ectopic recombination within the maize genome. Such a test is valuable for understanding the action of Cre as well as for its use to recombine two target lox sites present in the chromosomes. Pollen examination and seed set tests of material expressing Cre provided no evidence of ectopic recombination, which would be manifested in the production of translocations or inversions and result in pollen abortion and reduced seed set. Root-tip chromosome karyotype analysis was also performed on material with and without Cre expression. Chromosomal aberrations in Cre+ material were not observed above the background level.

Cre/lox Mediated Marker Gene Excision in Transgenic Crop Plants

Selectable marker genes are used in most plant transformation processes. Frequently, however, the selectable marker gene does not confer useful traits on the transgenic plant, and there are a number of advantages to removing the selectable marker gene after regeneration of the plant, including reducing the overall complexity of the transgene array, removal of redundant genetic elements, and improved regulatory and public acceptance, especially when antibiotic resistance marker genes are used as selectable markers. A number of site-specific recombinases of prokaryotic or yeast origin have been shown to function in transgenic plants for marker removal, including Cre/lox from bacteriophage P1 (Hoess and Abremski, 1990). We chose the Cre/lox system to develop technologies for marker removal in commercially important crop species.

RepB C-terminus mutation of an ori pRi vector affects plasmid copy number in Agrobacterium and transgene copy number in plants

A native repABC replication origin, ori pRi, was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299H mutation in Agrobacterium cells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation

Key messagevirGmutant strains of a nopaline type ofAgrobacterium tumefaciensincrease the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from thetzsgene in thevirGmutant strains is responsible for the improvement.AbstractStrains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.