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Methods for improving genome engineering and regeneration in plant ii

A technology for recombining genes and plant cells, applied in botany equipment and methods, biochemical equipment and methods, plant regeneration, etc., can solve problems that hinder the routine implementation of transient gene editing

Pending Publication Date: 2021-03-30
KWS SAAT SE & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These challenges hinder the routine implementation of transient gene editing as a breeding tool for improved plants

Method used

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  • Methods for improving genome engineering and regeneration in plant ii
  • Methods for improving genome engineering and regeneration in plant ii
  • Methods for improving genome engineering and regeneration in plant ii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0388] Example 1. Transient co-expression of a booster gene and a gene of interest (GOI) by co-bombardment.

[0389] Gene cloning and construct preparation

[0390] Maize WUS2 (ZmWUS2) and PLT7 (ZmPLT7) genes were cloned by RT-PCR using total RNA isolated from maize immature embryos of genotype A188. Wheat RKD4 and RKD2 and KWS-RBP1 genes were maize codon-optimized from their protein sequences and synthesized by Integrated DNA Technologies (IDT, San Diego, CA, USA). The enhanced gene fragment was cloned into the expression vector pABM-BdEF1 at the cloning sites of BamHI and HindIII ( figure 1 ) and expressed under the control of the BdEF1 promoter (pBdFE1) and the nos terminator (nos-T). pBdFE1 is a strong constitutive promoter from Brachypodium. Maps of the sequence-confirmed constructs are shown in Figures 2 to 5 and 8 in.

[0391] Maize immature embryos prepared for bombardment

[0392] 9-12 days after pollination, ears of corn (ie A188 or Hi II) with immature em...

Embodiment 2

[0400] Example 2. Transient coexpression of a combination of ZmWUS2 and ZmPLT7 in maize A188 immature embryos promotes early embryogenesis and regeneration.

[0401] Transient co-delivery, embryo preparation and culture are described in Example 1 above. For each bombardment, four premixed DNA plasmids were coated on 100 μg of gold particles with a size of 0.4 μm and co-introduced into the embryonic cotyledon cells of A188 immature embryos at a burst pressure of 650 psi. For one bombardment, the four plasmids were premixed as follows:

[0402] -100ng enhances ZmPLT5 or ZmPLT7 ( figure 2 with image 3 )

[0403] -200ng KWS-RBP1( Figure 4 )

[0404] -100ng pGEP359( Image 6 )

[0405] -150ng pGEP324( Figure 7 )

[0406] Embryos with dense fluorescent signals under a fluorescence microscope were selected ( Figure 9 ), and transferred it from N6OSM to N6-5Ag to induce embryogenic callus. Selected embryos were cultured on N6-5Ag plates with the embryos cotyledons faci...

Embodiment 3

[0411] Example 3. Transient Expression of ZmPLT7 in Maize Hi Immature Embryos Improves Stable Transformation of Co-delivered Reporter Genes

[0412] Examples 1 and 2 describe the preparation of maize embryos, transient bombardment and induction of embryogenic callus. Embryos were cultured in N6-5Ag medium at 27°C in the dark for 14 days. tDT fluorescence was used to monitor the induction and stable transformation of embryogenic callus by observation under a fluorescence microscope. Specifically, boosting was measured by the ability of the absence of selection to increase the transformation frequency (TF) of the tDT reporter gene 12 days after bombardment.

[0413] from Figure 14 The strong and uniform tDT fluorescence signal of the newly generated embryogenic structures indicates the integration and stable transformation of the tDT gene. Stable transformation frequency was defined as the number of embryos induced with at least one stable tDT fluorescent construct from the ...

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Abstract

This document relates to methods and materials for genome engineering in eukaryotic cells, and particularly to methods for increasing genome engineering (i.e. transformation or genome editing) efficiency via co-delivery of combinations of one or more booster polypeptides, and boost genes, with genome engineering components.

Description

technical field [0001] Described herein are novel combinations of regenerative booster (booster) genes and polypeptides, as well as methods and materials for genome engineering in eukaryotic cells, and, inter alia, by co-delivery of booster polypeptides and booster gene and genome engineering components to enhance Methods for genome engineering (ie, transformation or genome editing) efficiency. [0002] Background of the invention [0003] Traditional breeding has provided domesticated plants and animals, while modern biotechnology, especially genome engineering, is expanding breeding capabilities and achieving improvements not achievable with traditional crossing of close species alone. Using biotechnology, multiple traits such as high yield, herbicide tolerance, and insect resistance have been introduced into crops, leading to tremendous advances in global agriculture and food security. However, the presence of foreign DNA in such biotech products raises biosafety and envi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N5/10C12N15/82A01H4/00A01H5/00A01H6/46
CPCC07K14/415A01H4/008C12N15/8201C12N15/821
Inventor 孔吉祥孟玲
Owner KWS SAAT SE & CO KGAA
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