이 연구는 네덜란드 기술 재단 STW (12385), 네덜란드 조직에 대 한 과학적 연구 (NWO)의 부분 이다 고는 부분적으로 경제 통상 부 (OTP 부여 12385 ms)에 의해 자금이 지원 됩니다.  우리는 pCH20, BIBAC-GW 벡터의 등뼈를 제공 하기 위한 캐롤 M. 해밀턴 (코넬 대학, 미국) 감사 합니다.

<ol>
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저자 아무 경쟁 금융 관심사 또는 다른 충돌을 선언합니다.

생성에 중요 한 제 닉은 transgene의 단일, 그대로 통합으로 사용 되는 이진 벡터의 선택입니다. BIBAC 가족 벡터 많은 식물 종23,,2425,26,,2728관심사의 순서를 전달 하기 위해 사용 되었습니다. BIBAC 벡터, BIBAC-GW를 포함 하 여 높은 효율으로 단일 복사 통합...

유전자 변형 식물을 생성할 때 일반적으로 목적은 안정적으로 표현 하는 통합된 transgene(s) 것입니다. 이 transgene의 그대로 단일 복사본 통합 하 여 얻을 수 있습니다. 여러 통합은 transgene의 증가 표현 뿐만 아니라 유전자 입을 발생할 수 있습니다. Transgenes의 입을 경우 삽입된 시퀀스 협동 또는 거꾸로 반복1,2,,34에서 배열 된다 확률이 높다. 이진 벡터 Agrobacterium에 셔틀으로 이용 된다-식물 게놈으로 관심의 시퀀스를 제공 하는 변환 실험을 중재. 식물 게놈으로 통합의 수 Agrobacterium tumefaciens5,6. 이진 벡터의 복사본 수에 따라 달라 집니다. 많은 일반적으로 사용 되 이진 벡터는 높은 복사 벡터 및 따라서 높은 평균 transgene 복사본 수를 산출: 애기53.3 4.9 복사본.
BIBAC7또는 T-DNA A. tumefaciens 염색체5에서 시작 하 여 낮은 복사 번호 A. tumefaciens에 있는 이진 벡터를 사용 하 여 T-DNA 통합의 수를 낮출 수 있습니다. 이러한 경우 transgene 통합의 평균 수는 25,8,,910아래입니다. 따른 단일-복사 A. tumefaciens 에 대장균에서 BIBAC 파생 상품 수 유지 및 구문 150 kb11로 제공.
GW 호환 BIBAC 벡터10,12 게이트웨이 복제를 사용 하 여 벡터에 관심사의 유전자의 도입에 쉽게 수 있습니다. 게이트웨이 기술 사용 하 여 복제 프로시저를 단순화 하지만 또한 큰 낮은 복사 번호 벡터13,14, 낮은 DNA 수율 등 독특한 제한의 제한 된 선택과 관련 된 일반적인 문제를 극복 7,11복제에 사용할 수 있는 사이트. PBIBAC-GW 파생 상품 Glufosinate 염화 (pBIBAC-바-GW) 어느 저항 또는 DsRed 형광 씨 외 투 (pBIBAC-RFP-GW) 식물 (그림 1)10,12선택에 사용할 수 있습니다. 두 벡터에 대 한 대 저항 유전자는 박테리아에서 선택 마커로 사용 됩니다.
PBIBAC-GW 벡터 결합: (1) 쉬운 디자인 및 유전자 조작 대장균및 (2) 그대로 단일 복사 통합에서 planta에 높은 효율에. 들고 단일 유전자 변형 식물의 약 절반으로 애기 에 평균 1.7 통합에 pBIBAC GW 벡터 수확량은 통합 T DNA10.
안정적인 표현의 transgenes 대부분 제 닉 생성에 대 한 요구 사항입니다. 안정적인 transgene 식 그대로, 단일 복사 통합 하 여 얻을 수 있습니다. 그러나 유전자 변형 식물 그대로, 단일 복사 통합 운반 작업은,, 훨씬 더 중요 한 경우 예를 들어 목표 mutagenesis, 재결합, 또는 복구, 등 이러한 의존 chromatin 기반 프로세스의 효율성을 연구 하는 것입니다 genomic 위치 및 삽입 사이트에서 chromatin 구조에 처리 합니다. 우리의 관심 지역의 게놈 문맥에 의존 oligonucleotide 감독 mutagenesis (ODM)의 공부 mutagenesis 취재 원 유전자의 손상, 단일 복사 통합 기자 라인의 집합 생성된 (그림 2)10이었다. 이 일련의 줄을 사용 하 여, 보였다 ODM 효율 되 고 오히려 유사한 transgene 식 수준에도 불구 하 고 다른 genomic 위치의 통합 유전자 변형 loci 사이 변화 한다.

<table><tbody><tr><td>Kanamycin sulphate monohydrate</td><td>Duchefa</td><td>K0126</td><td></td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa</td><td>G0124</td><td></td></tr><tr><td>Rifampicin</td><td>Duchefa</td><td>R0146</td><td></td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma</td><td>T-3383</td><td></td></tr><tr><td>DB3.1 competent cells</td><td>Thermo Scientific - Invitrogen</td><td>11782-018</td><td>One Shot &lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant &lt;em&gt;E. coli&lt;/em&gt; strain can be used instead&amp;nbsp;&amp;nbsp;</td></tr><tr><td>DH10B competent cells</td><td>Thermo Scientific - Invitrogen</td><td>18290-015</td><td></td></tr><tr><td>Gateway LR clonase enzyme mix&amp;nbsp;</td><td>Thermo Scientific - Invitrogen</td><td>11791-019</td><td></td></tr><tr><td>tri-Sodium citrate dihydrate</td><td>Merck</td><td>106432</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>EDTA disodium dihydrate</td><td>Duchefa</td><td>E0511</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Scientific&amp;nbsp;</td><td>EO0491</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Honeywell Fluka</td><td>13423</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Merck</td><td>108346</td><td></td></tr><tr><td>Electroporation Cuvettes, 0.1 cm gap</td><td>Biorad</td><td>1652089</td><td></td></tr><tr><td>Electroporator Gene Pulser</td><td>BioRad</td><td></td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Calbiochem</td><td>442613</td><td></td></tr><tr><td>D(+)-Maltose monohydrate 90%</td><td>Acros Organics</td><td>32991</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100</td><td></td></tr><tr><td>Silwet L-77</td><td>Fisher Scientific</td><td>NC0138454</td><td></td></tr><tr><td>Murashige Skoog medium</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Glufosinate-ammonium (Basta)</td><td>Bayer</td><td>79391781</td><td></td></tr><tr><td>Restriction enzymes</td><td>NEB</td><td></td><td></td></tr><tr><td>Ethidium Bromide</td><td>Bio-Rad</td><td>1610433</td><td></td></tr><tr><td>Electrophoresis system</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106498</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100316</td><td></td></tr><tr><td>Blotting nylon membrane Hybond N+</td><td>Sigma Aldrich</td><td>15358</td><td>or GE Healthcare Life Sciences (RPN203B)</td></tr><tr><td>Whatman 3MM Chr blotting paper</td><td>GE Healthcare Life Sciences</td><td>3030-931</td><td></td></tr><tr><td>dNTP</td><td>Thermo Fisher</td><td>R0181</td><td></td></tr><tr><td>Acetylated BSA</td><td>Sigma-Aldrich</td><td>B2518</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Merck</td><td>805740</td><td></td></tr><tr><td>Sephadex G-50 Coarse</td><td>GE Healthcare Life Sciences</td><td>17004401</td><td>or Sephadex G-50 Medium (17004301)</td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate&amp;nbsp;</td><td>US Biological</td><td>S5010</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Sigma-Aldrich</td><td>D7656</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>Merck</td><td>106346</td><td></td></tr><tr><td>Storage Phosphor screen and casette</td><td>GE Healthcare Life Sciences</td><td>28-9564-74</td><td></td></tr><tr><td>Phosphor imager</td><td>GE Healthcare Life Sciences</td><td>Typhoon FLA 7000</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>Stratalinker 1800</td><td></td></tr><tr><td>cling film (Saran wrap)</td><td>Omnilabo</td><td>1090681</td><td></td></tr><tr><td>Agarose</td><td>Thermo Scientific - Invitrogen</td><td>16500</td><td></td></tr><tr><td>Boric acid</td><td>Merck</td><td>100165</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Blauw&amp;rsquo;; DNA ladder.</td><td>MRC Holland</td><td>MCT8070</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Rood&amp;rsquo;; DNA ladder</td><td>MRC Holland</td><td>MCT8080</td><td></td></tr><tr><td>Hexanucleotide Mix</td><td>Roche</td><td>11277081001</td><td></td></tr><tr><td>Large-Construct Kit</td><td>Qiagen</td><td>12462</td><td></td></tr><tr><td>Heat-sealable polyethylene tubing, clear</td><td>various providers</td><td></td><td>the width of the tubing should be wider than that of blotting membrane</td></tr><tr><td>Heat sealer</td><td></td><td></td><td></td></tr><tr><td>Membrane filter disk</td><td>Merck</td><td>VSWP02500</td><td></td></tr><tr><td>Magnesium chloride</td><td>Merck</td><td>105833</td><td></td></tr><tr><td>Hybridization mesh</td><td>GE Healthcare Life Sciences</td><td>RPN2519</td><td></td></tr></tbody></table>

generating transgenic plants with single-copy insertions using bibac-gw binary vector

유전자 변형 식물을 생성할 때 일반적으로 목표는 transgene의 안정적인 식 것입니다. 이 transgene의 단일, 그대로 통합 다중 복사 통합 유전자 입을 대상이 종종 필요 합니다. 다른 pBIBAC 파생 상품 같은 세균성 인공적인 염색체 (pBIBAC-GW)에 따라 게이트웨이 호환 이진 벡터 고효율 단일 카피 transgenes의 삽입을 수 있습니다. 원래 pBIBAC에 개선으로 게이트웨이 카세트는 되었습니다 복제 pBIBAC GW에 관심의 시퀀스 지금 통합 될 수 있다 쉽게 벡터 전송 DNA (T-DNA)에 게이트웨이 복제 하 여 그렇게. 일반적으로, pBIBAC GW와 변환 결과 0.2-0.5 효율 %, 그것에 의하여는 제 닉의 T-DNA의 본래 단일 복사 통합 수행. PBIBAC-GW 벡터 Glufosinate 염화에 저항 또는 선택 식물, 씨 외 투에서 DsRed 형광와 저항 대에 박테리아에서 선택 항목으로 사용할 수 있습니다. 여기, 프로토콜의 시리즈 pBIBAC GW를 사용 하 여 유전자 변형 식물 생성의 과정을 통해 독자를 안내 하는 표시 됩니다: 진상을 공장 로 변환 하는 선택의 pBIBAC GW 벡터에 관심의 시퀀스에서 시작 Agrobacterium, 제 닉, 그리고 테스트 DNA 럽을 사용 하 여 삽입의 intactness 및 복사 번호에 대 한 식물의 선택. 주의는 DNA 더 럽 히 전략을 인식 하는 단일 및 다중 loci에서 단일 및 다중 복사 통합 주어 집니다.

BIBAC-GW 시스템을 사용 하 여, 식물에서 ODM을 공부 기자 구문이 생성된10했다. 구문을은 게이트웨이 항목 벡터 pENTR gm12 설계 되었고에 pBIBAC-바-GW (그림 1) 게이트웨이 LR 재결합 반응을 사용 하 여 삽입.
애기는 pDM19, BIBAC-바-GW 플라스 변환 정지 codon 위치 120 eYFP 독서 프레임...

Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

PBIBAC-GW 이진 벡터를 사용 하 여 생성 유전자 변형 식물을 그대로 단일 복사본 삽입, 쉬운 과정을 만든다. 여기, 프로토콜의 시리즈는 유전자 변형 애기 식물을 생성 하 고 intactness에 대 한 식물을 테스트 과정을 통해 독자를 안내 하 고 삽입의 수를 복사 하 여.

BIBAC-GW 이진 벡터를 사용 하 여 단일 복사본 삽입을 가진 유전자 변형 식물 생성

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

generating-transgenic-plants-with-single-copy-insertions-using-bibac

Research

JoVE Journal

Genetics

12.4K 조회수.  University of Amsterdam. PBIBAC-GW 이진 벡터를 사용 하 여 생성 유전자 변형 식물을 그대로 단일 복사본 삽입, 쉬운 과정을 만든다. 여기, 프로토콜의 시리즈는 유전자 변형 애기 식물을 생성 하 고 intactness에 대 한 식물을 테스트 과정을 통해 독자를 안내 하 고 삽입의 수를 복사 하 여.

비디오: Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

깁슨 조립 및 유전자 총을 사용하여 담배 일시적인 유전자 발현

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

연구

환경과학

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

식물 단백질 곤충 세포에서 분 비 생산 잠재 식 벡터를 수정

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

생화학

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

유도할 수 있는, 애기 thaliana 통해 LhGR 중재 트랜스에 셀 형식 관련 식-활성화

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

유전학

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

모듈형 골든 게이트 복제를 사용하여 다중 유전자 구조물의 신속한 조립

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

생체공학

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

생물학

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

감자와 니코티아나 벤타미안의 농아침투 및 PVX 농병감염

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

<meta charset="utf8"></head><body>쌀 육종에서 저항성 전분을 위한 CRISPR 기반의 효율적인 게놈 편집

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

<meta charset="utf8"></head><body>저자 스포트라이트: 최적의 표현형 및 농업 형질을 얻기 위해 CRISPR/Cas를 사용한 벼 육종 간소화

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

게놈 편집 기술을 이용한 기능성 벼를 위한 설계에 의한 육종

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

아마의 꽃 - 딥 변환 (<em&gt; LINUM usitatissimum</em&gt;) 높은 변환 속도와 형질 전환 자손을 생성합니다

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

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