Methods and compositions for increasing harvestable yield via editing ga20 oxidase genes to generate short stature plants

An oxidase and plant technology, applied in the field of trait composition, can solve problems such as the inability to find semi-dwarf high-yield corn varieties

Pending Publication Date: 2021-03-26
MONSANTO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, other cereal crops, such as maize, have not been successfully increased in yield through manipulation of the GA pathway
In fact, some mutations in GA pathway genes have been associated with multiple off-types that are incompatible with yield in maize, which has prevented researchers from finding semi-dwarf high-yielding maize by manipulating the GA pathway Variety

Method used

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  • Methods and compositions for increasing harvestable yield via editing ga20 oxidase genes to generate short stature plants
  • Methods and compositions for increasing harvestable yield via editing ga20 oxidase genes to generate short stature plants
  • Methods and compositions for increasing harvestable yield via editing ga20 oxidase genes to generate short stature plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1. Phenotypic observations of maize plants with edited GA20 oxidase-3 or GA20 oxidase-5 genes.

[0103] Several genome-edited mutations were generated in the endogenous GA20 oxidase_3 and GA20 oxidase_5 genes in maize plants to test the phenotypic effects of knocking out each of these genes. A series of 10 single-stranded guide RNAs (sgRNAs) encoding targeting constructs were generated for each of the GA20 oxidase_3 and GA20 oxidase_5 genes along the genomic sequence target of each gene. to different locations. An additional series of 10 sgRNAs were generated, each targeting both the GA20 oxidase_3 and GA20 oxidase_5 genes at similar or different positions along the genomic sequence of each gene. Targeted genome editing is performed by delivering sgRNA to maize explants together with expression of the Cas9 protein such that a DSB or nick occurs at or near the gRNA's genomic target site, which can then be incompletely replaced. Repair to introduce mutations at ...

Embodiment 2

[0108] Example 2. Identification of maize plants with various combinations of edited GA20 oxidase_3 and GA20 oxidase_5 mutant alleles.

[0109] As described in Example 1, maize plants were edited by a CRISPR / Cas9-based approach using a guide RNA (gRNA) that specifically targets one or both of the GA20 oxidase_3 and GA20 oxidase_5 genes. Both genes were targeted simultaneously (see Table 2). A total of 30 gRNA constructs were transformed into maize. Leaf samples from R0 plants were collected and analyzed for InDel by Fragment Length Analysis (FLA) analysis. Putative mutant alleles identified by FLA were sequenced using gene-specific primers and standard deep sequencing protocols to confirm one or more mutations. Table 3 provides a list of the mutant alleles (ga20ox3-1 to ga20ox3-12) and their sequences in the 12 edited GA20 oxidase_3 genes. Table 4 provides a list of the mutant alleles (ga20ox5-1 to ga20ox5-11) and their sequences in the 11 edited GA20 oxidase_5 genes. R0 p...

Embodiment 3

[0125] Example 3. Editing of both GA20 oxidase_3 and GA20 oxidase_5 reduces active GA levels in plants.

[0126] R2 plants with edited alleles in GA20 oxidase_3, GA20 oxidase_5, or both genes, as well as those with genes targeting GA20 oxidase_3 and GA20 oxidase_5, were tested in the field for Transgenic inbred maize plants (SUP_GA20Ox3 & Ox5 ("SUP_Ox3 & Ox5")) of suppressed artificial microRNA suppression constructs. Various physiological traits were measured, including plant height to ear node, plant height to uppermost ligule, ear height, ear length, ear diameter, kernels / ear, kernels / unit area, single kernel weight, stem diameter at R3 stage and grain yield estimates. Plants containing homozygous mutant alleles of both GA20 oxidase_3 and GA20 oxidase_5 genes (ie, double homozygous ga20ox3 / ga20ox5 mutants) displayed semi-dwarf phenotypes with altered plant architecture. The homozygous single ga20ox3 mutant and the homozygous single ga20ox5 mutant showed slightly higher plan...

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Abstract

The present disclosure provides compositions and methods for the editing or mutating of specific subtypes of GA20 oxidase genes and specific zygosity combinations of those edits or mutations. Modifiedplants, and plant parts and cells thereof, having mutations reducing the expression or activity of GA20 oxidase genes are further provided with improved characteristics, such as reduced plant heightand increased lodging resistance, but without off-types. Methods are further provided for making modified plants, and plant parts and cells thereof, having one or more mutations in specific subtypes of GA20 oxidase genes.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 631,412, filed February 15, 2018, and U.S. Provisional Application No. 62 / 710,302, filed February 16, 2018, both of which are incorporated herein by reference in their entireties. [0003] Incorporation of Sequence Listings [0004] A Sequence Listing entitled "P34605WO_SEQ.txt" with a size of 382 kilobytes (measured in MS-WINDOWS) and created on February 15, 2018 accompanies the submission and is hereby incorporated by reference in its entirety. Background technique [0005] The present disclosure relates to compositions and methods for improving traits in corn, such as lodging resistance and increased yield. [0006] Gibberellins (gibberellic acid or GA) are plant hormones that regulate several major plant growth and developmental processes. During the 20th century, manipulation of GA levels in semi-dwarf wheat, rice and sorghum plant species ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/00C12N15/63A01H5/10
CPCA01H5/10A01H6/4684C12N9/0071C12N15/8213Y02A40/146C12N15/8261
Inventor S·曼朱纳斯L·A·赖默奎斯T·L·斯勒温斯基
Owner MONSANTO TECH LLC
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