Papers by Larry Gilbertson
Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants
Plant Cell Reports
Selectable marker genes are often required for efficient generation of transgenic plants in tissu... more Selectable marker genes are often required for efficient generation of transgenic plants in tissue culture transformation systems but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from us...
Site‐directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease
The Plant Journal
SUMMARYHigh efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a... more SUMMARYHigh efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation...
Methods and compositions for obtaining transgenic plants of marker-free
PROBLEM TO BE SOLVED: To provide methods and compositions for identifying transgenic seeds that c... more PROBLEM TO BE SOLVED: To provide methods and compositions for identifying transgenic seeds that contain a transgene of interest, but lack a marker gene.SOLUTION: Use of an identification sequence that results in a detectable phenotype increases the efficiency of screening for seeds and plants in which transgene sequences not linked to a gene of interest have segregated from the sequence encoding a gene of interest.SELECTED DRAWING: None
Genetic Transformation a ND H Ybridization
Efficient production of seedling-derived Type I callus was demonstrated for several corn genotype... more Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes in- cluding commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l −1 picloram and 3m g l −1 6-benzylaminopurine, which induced the devel- opment of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l −1 picloram and 0.5 mg l −1 2,4- dichlorophenoxyacetic acid. For lines L4 and L9 the fre- quency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induc- tion from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived cal- lus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluo- rescence protein gene and genes for either neomycin phos- photransferase II or glyphosat...
Compositions and procedures for controlling insect infestations in plants
A transgenic plant that has more than one transgene to reduce or eliminate insect pest infestatio... more A transgenic plant that has more than one transgene to reduce or eliminate insect pest infestation in a plant, or a plant cell thereof, in which said plant or said plant cell comprises an RNAb for the suppression of an essential gene in a plague of target insects and a gene encoding an insecticidal protein of Bacillus thuringiensis that exhibits biological activity against said plague of target insects.
Molecules d'acide nucleique codant une synthase phytoene
HortScience, 2008
The Flavr Savr tomato was introduced as the first genetically engineered whole food in 1994. The ... more The Flavr Savr tomato was introduced as the first genetically engineered whole food in 1994. The commercial event, resulting from transformation with an antisense expression cassette of the endogenous polygalacturonase gene, was sequenced and found to contain two contiguous, linked, transfer DNA insertions. We found polygalacturonase suppression correlates with accumulation of ≈21-nt small interfering RNAs, the hallmark of an RNA interference-mediated suppression mechanism.
Proceedings of the National Academy of Sciences, 1994
In yeast meiosis, crossing-over between homologues is dependent upon double-strand breaks. We dem... more In yeast meiosis, crossing-over between homologues is dependent upon double-strand breaks. We demonstrate that the occurrence of these breaks is independent of pairing between homologues by showing that they occur with normal frequency, timing, and position in the absence of a homologue. This observation supports models that view double-strand breaks as initiating events and crossing-over as a consequence of repair of these breaks.
INCORPORATION OF SEQUENCE LISTING 0002 The sequence listing that is contained in the file named "... more INCORPORATION OF SEQUENCE LISTING 0002 The sequence listing that is contained in the file named "MONS237US seq.txt, which is 286,948 bytes (measured in MS-WINDOWS) and created on Jan. 20, 2011, is filed herewith by electronic submission and incorporated herein by reference BACKGROUND OF THE INVENTION
Alterations du transgene a mediation par recombinaison homologue dans des plantes
L'invention concerne un procede de preparation d'une plante Zea mays transgenique recombi... more L'invention concerne un procede de preparation d'une plante Zea mays transgenique recombinee a partir d'une plante Zea mays transgenique, le transgene dans la plante recombinante possedant une structure genetique alteree par rapport a la structure genetique du transgene dans la plante transgenique, en raison de deletion, d'amplification ou de rearrangement du transgene a mediation par recombinaison homologue.
Methods and compositions for obtaining marker-free transgenic plants
DNA constructs and methods to enhance the production of commercially viable transgenic plants
comprising a promoter with maize endosperm-specific expression operably linked to heterologous DNA; transgenic maize plant cell; improved stress tolerance, yield, process and storage quality
Use of Multiple Transformation Enhancer Sequences to Improve Plant Transformation Efficiency
Methods and DNA Constructs for Autoregulating Transgene Silencing
Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation
Plant cell reports, Jan 9, 2015
KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the tr... more KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains 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 ...
Homologous recombination-mediated transgene alterations in plants
Methods for enhancing segregation of transgenes in plants and compositions thereof
Homologous recombination-mediated transgene deletion in plant cells
Uploads
Papers by Larry Gilbertson