Weed control methods involving aad-1 plants and pre-plant and/or pre-emergence herbicide application
A herbicide, plant technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, herbicides and algicides, etc., which can solve problems such as differences in spatial or temporal expression patterns
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Embodiment 1
[0182] Example 1: Transformation and selection of AAD1 event pDAS1740-278
[0183]AAD1 event pDAS1740-278 was generated by WHISKER-mediated transformation of maize line Hi-II. The transformation method used is described in US Patent Application #20090093366. The FspI fragment of plasmid pDAS1740 (also known as pDAB3812) (Figure 1) was transformed into maize lines. This plasmid construct contains a plant expression cassette containing the RB7MARv3::maize ubiquitin 1 promoter v2 / / AAD1v3 / / maize PER5 3'UTR::RB7MARv4 plant transformation unit (PTU).
[0184] Generate many events. Those events that survived and produced healthy haloxyfop-resistant calli were assigned unique identifiers representing putative transformation events and were serially transferred to fresh selection medium. Plants were regenerated from tissue derived from each unique event and transferred to the greenhouse.
[0185] Leaf tissue was harvested for molecular analysis to confirm the presence of the AAD-1 ...
Embodiment 2
[0186] Example 2: Characterization of pDAS1740-278-9 events via Southern blotting
[0187] Southern blot analysis was used to establish the integration pattern of the inserted DNA fragment and to determine the insert / copy number of the aad-1 gene in event pDAS-1740-278-9 (DAS-40278-9). Data were generated to demonstrate the integration and integrity of the aad-1 transgene inserted into the maize genome.
[0188] Southern blot data suggested that the pDAS1740 / Fsp I fragment insert in maize event DAS-40278-9 existed as a simple integration of a single intact copy of the aad-1 PTU from plasmid pDAS1740. Detailed Southern blot analysis using probes specific to genes, promoters, terminators, and other regulatory elements contained in the plasmid region and descriptive restriction enzymes with cleavage sites located within the plasmid , and generate hybrid fragments within the plasmid or fragments (border fragments) that span the junction of the plasmid and the maize genomic DNA. ...
Embodiment 21
[0189] Example 2.1: Maize Leaf Sample Collection and Genomic DNA (gDNA) Isolation
[0190] gDNA prepared from leaves of individual plants of aad-1 maize event DAS-40278-9. gDNA was extracted from tissue harvested from individual plants carrying aad-1 maize event DAS-40278-9. Transgenic maize seeds from 5 different generations of event DAS-40278-9 were used. Twenty individual maize plants (from 4 plants per generation) were selected for event DAS-40278-9. Additionally, gDNA was isolated from conventional maize plant XHH13 without the aad-1 gene (which contained a genetic background representative of the subject lines).
[0191] Prior to gDNA isolation, leaf punches were collected from each plant to test for aad-1 protein expression using a rapid test strip kit (American Bionostica, Swedesboro, NJ) following the manufacturer's recommendations. Each leaf punch sample was given a score of + or - representing the presence or absence of aad-1, respectively. Only 5 generations ...
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