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Compositions and methods for transferring biomolecules to wounded cells

A technology of biomolecules and plant cells, applied in the direction of biochemical equipment and methods, botanical equipment and methods, and other methods for inserting foreign genetic materials

Pending Publication Date: 2021-04-16
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many plant species and varieties are difficult to transform, culture and / or regenerate from explants or plant material

Method used

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  • Compositions and methods for transferring biomolecules to wounded cells
  • Compositions and methods for transferring biomolecules to wounded cells
  • Compositions and methods for transferring biomolecules to wounded cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Delivery of TAT-Cre protein into wounded maize callus cells.

[0078] Transgenic maize line A was generated with a recombinant DNA construct in the nuclear genome that included the enhanced CaMV 35S promoter in the 5' to 3' direction and the HSP70 intron in the 5' untranslated region , an nptII selectable marker gene cassette flanked by two lox sites, and then the green fluorescent protein (GFP) encoding gene (see, e.g., Zhang et al., Theor.Appl.Gen.107(7):1157-1168; 2003). In this arrangement, GFP cannot be functionally expressed due to the insertion of the nptII gene between the 35S promoter and the GFP coding sequence. However, in the presence of Cre recombinase, the nptII gene is excised due to the flanking lox sites, which results in the high level of GFP expression that can be observed in most tissues by placing the 35S promoter and GFP coding sequence together ( figure 1 ). Thus, the GFP construct inserted into the genome of maize line A can serve ...

Embodiment 2

[0086] Example 2: Delivery of Cre protein into wounded maize callus cells.

[0087] Embryogenic callus cells were generated from immature embryos of transgenic maize line A as described in Example 1 above. 3-4 grams of callus were blended at high speed for 9-10 seconds in a medium containing 50% concentration of medium 4278 (Table 3) and 0.3M mannitol to obtain callus pieces with a size of 1-2mm fine suspension. After blending, the callus suspension was poured into a sieve, washed with the medium used for blending, and then pipetted onto filter paper.

[0088] Table 3. Composition of Medium 4278

[0089] Element ingredient description quantity MP00927 FN · Iite bulk storage solution (10X) 100.000mL MS_MICRON UTRIENT MS Micronutrients 100.000mL GAMBORGS_B5_500X Gamborgs B5 500X 2.000mL SUCROSE sucrose 30.000g ASPARAGINE_MONOHYD Asparagine monohydrate 1.000g TC_WATER_TO_VOLUME Dilute to the volume with TC water ...

Embodiment 3

[0098] Example 3: Delivery of RNP into wounded maize callus cells.

[0099] The ability to deliver Cre recombinase to injured callus cells suggests that other proteins, ribonucleoproteins and nucleases, can also be added to cells by this method. Embryogenic callus cells were generated from maize immature embryos as described in Example 1 above, and wounded callus suspensions were generated as described in Example 2 above. The blended callus suspension was washed and dried as described above. When the RNP complex solution described below is mixed with injured callus cells, the PEG solution can be added as described above.

[0100] To generate guide RNA-Cas9 ribonucleoprotein (RNP) complex, 20.6 μg Cas9 protein and 8.6 μg RNA were mixed in 1×NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM DTT, pH 7.9, at 25°C) to obtain a 1:2 molar ratio of Cas9 to gRNA, add 1 µl RNase inhibitor (RiboLock; ThermoFisher Scientific) to a total volume of 30 µl, and incubate at room...

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Abstract

The invention provides novel methods and compositions for introduction, transfer or delivery of one or more biomolecules into wounded recipient plant cell(s). Methods for production of a wounded recipient cell culture and the creation of one or more mutations, edits, transgenic insertions, or other genetic changes in the recipient cell(s) are also provided. Product cells produced by such methods, and resulting cells and regenerated plants, plant parts, and progeny plants are further provided. Molecular and genetic analyses, analysis of phenotypes and traits, and use of screenable and selection markers, are also provided to confirm transfer of the biomolecule in to the recipient cell(s) and generation of the mutation, edit, transgenic insertion, or other genetic change in the recipient cell(s), and / or progeny thereof, and in plants or plant parts developed or regenerated from the foregoing.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to US Provisional Application Serial No. 62 / 740,144, filed October 2, 2018, the disclosure of which is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates generally to the fields of agriculture, plant biotechnology and molecular biology. More specifically, the present invention relates to compositions and methods for mutating, editing or genetically modifying plant cells. Background technique [0004] The ability to generate plants with novel combinations of genetic traits is useful for improving crop yields and resistance to pest and disease pressure. In addition to crossing or breeding plants, novel combinations of traits can also be introduced through transgenesis or through various mutagenesis techniques. However, many plant species and varieties are difficult to transform, grow and / or regenerate from explants or ...

Claims

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Application Information

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IPC IPC(8): A01H1/02A01H5/00A01H5/10C12N9/22C12N9/88C12N15/82C12N15/87
CPCC12N15/87C12N15/8214C12N15/8213C12N15/8206C12N15/8207C12N2310/20C12N9/22C12N15/11C12N2800/80
Inventor L·A·吉尔伯特森A·Y·库拉诺夫V·A·西多罗夫
Owner MONSANTO TECH LLC
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