מחקר זה נתמך על ידי ההולנדי בטכנולוגיית קרן STW (12385), אשר הוא חלק מהארגון הולנד על מחקר מדעי (בתחרות), אשר ממומן בחלקו על ידי משרד כלכלי פרשיות (OTP גרנט 12385 ל MS).  אנו מודים קרול מ המילטון (אוניברסיטת קורנל, ארה ב) על מתן pCH20, עמוד השדרה של הווקטורים BIBAC-GW.

<ol>
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המחברים מצהירים אין מתחרים אינטרסים כלכליים או אחרים ניגודי עניינים.

קריטי כדי לייצר transgenics עם שילובי יחיד, שלם של transgene הוא הבחירה של וקטור בינארי המשמש. וקטורים משפחה BIBAC שימשו להעביר רצפים של תחומי עניין רבים צמח מינים23,24,25,26,27,28. וקטורים BIBAC, BIBAC-ג'י, ?...

בעת יצירת הצמחים הטרנסגניים, בדרך כלל המטרה היא לקיים את transgene(s) משולב stably הביע. זו יכולה להיות מושגת על ידי עותק בודד ללא פגע שילובים של transgene. שילובים מרובות עלולה לגרום ביטוי מוגבר transgene, אלא גם ג'ין להחרשת. וכמובן transgenes סביר יותר אם רצפים שנוספו מסודרים טנדם או הפוך חזרה1,2,3,4. וקטורים בינארי משמשים הסעות ב Agrobacterium-מתווכת טרנספורמציה ניסויים כדי לספק את רצפי עניין לתוך הגנום הצמח. מספר שילובים לתוך הגנום הצמח תלויה עותק מספר בינארי וקטור ב-5, Agrobacterium tumefaciens6. וקטורים בינאריים נפוצים רבים הן וקטורי העתק גבוהה, ולכן התשואה מספר עותק transgene ממוצע גבוה: 3.3 ל 4.9 עותקים תודרנית5.
ניתן להוריד את מספר שילובים T-DNA באמצעות וקטורים בינארי כי יש מספר נמוך-עותק tumefaciens א, כגון BIBAC7, או על-ידי שיגור T-DNA בין כרומוזום tumefaciens א 5. המספר הממוצע של שילובי transgene במקרים כאלה הוא מתחת 25,8,9,10. בשל היותם עותק יחיד ב- tumefaciens א, וגם ב- Escherichia coli, BIBAC-נגזרים יכול לתחזק ולספק בונה גדול כמו 150 kb11.
GW תואמי BIBAC וקטורים10,12 לאפשר הקדמה קלה של הגנים של עניין לתוך הווקטור באמצעות שער שיבוט. השימוש בטכנולוגיה שער מאוד מפשט את הליך השיבוט, אבל גם מתגבר על בעיות נפוצות המשויך וקטורים נמוך-העתקה-מספר גדול13,14, כגון תשואה נמוכה של DNA ומבחר מצומצם של הגבלת ייחודי אתרים זמינים עבור שכפול7,11. נגזרות pBIBAC-GW זמינים גם התנגדות Glufosinate-אמוניום (pBIBAC-בר-GW) או DsRed זריחה במעילים הזרע (pBIBAC-RFP-GW) לבחירת צמחים (איור 1)10,12. עבור שני וקטורים, גן עמידות kanamycin משמש סמן הבחירה של חיידקים.
לשלב הווקטורים pBIBAC-GW: מניפולציה גנטית ב e. coliו שילובים עותק יחיד (2) שלם ב הפיסקו יעילות גבוהה ועיצוב קל (1). PBIBAC-GW וקטורים התשואה על שילובים בממוצע 1.7 ב תודרנית כמחצית הצמחים הטרנסגניים נושא יחיד משולב T-DNA10.
ביטוי יציב של transgenes הוא דרישה עבור רוב transgenics שנוצר. Transgene יציב הביטוי יכולה להיות מושגת על ידי שילובים ללא פגע, עותק יחיד. עבודה עם הצמחים הטרנסגניים נושא שלם, עותק יחיד שילובים היא, עם זאת, חשוב אפילו יותר אם לדוגמה, המטרה היא לחקור את היעילות של תהליכים מבוססי-כרומטין, כגון מוטגנזה מכוונת, רקומבינציה, או לתקן את התלות של אלה תהליכים על המיקום גנומית ומבנה כרומטין במתחם הכניסה. על האינטרסים שלנו, ללמוד את התלות של oligonucleotide ביים מוטגנזה מכוונת (ODM) על ההקשר המקומי גנומית, קבוצה של קווים כתב עם תוספות ללא פגע, עותק יחיד של גנים כתב מוטגנזה מכוונת היה (איור 2) שנוצר10. באמצעות ערכת שורות, זה הוצג כי היעילות ODM משתנה בין לוקוסים הטרנסגניים משולב במקומות שונים גנומית, למרות רמות הביטוי transgene להיות דומה למדי.

<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

בעת יצירת הצמחים הטרנסגניים, בדרך כלל המטרה היא שיהיה יציב לביטוי transgene. פעולה זו דורשת שילוב יחיד, שלם של transgene, גם מרובת עותקים שילובים נחשפים לעיתים קרובות ג'ין להחרשת. הווקטור שער תואמת בינארית המבוסס על חיידקי מלאכותי כרומוזומים (pBIBAC-GW), כמו אחרים נגזרים pBIBAC, מאפשר החדרת transgenes עותק יחיד עם יעילות גבוהה. כמו שיפור pBIBAC המקורי, קלטת שער נשלטה לתוך pBIBAC-ג'י, כך הרצפים של עניין ניתן עכשיו בקלות לשלב ההעברה וקטור DNA (T-DNA) על-ידי שיבוט שער. בדרך כלל, השינוי עם pBIBAC-GW תוצאות יעילות של 0.2-0.5%, לפיה מחצית transgenics נושא שילוב יחיד-עותק שלם ב- T-dna. PBIBAC-GW הווקטורים זמינים עם עמידות Glufosinate-אמוניום או DsRed זריחה במעילים seed עבור בחירה בצמחים, ועם התנגדות kanamycin כבחירה של חיידקים. . הנה, סדרה של פרוטוקולים מוצג שמנחים את הקורא לאורך התהליך של יצירת הצמחים הטרנסגניים באמצעות pBIBAC-GW: החל משילוב מחדש את רצפי עניין לתוך וקטור pBIBAC-GW של בחירה, לשתול טרנספורמציה עם Agrobacterium, מבחר של transgenics, ובדיקות את הצמחים על מספר intactness, ולהעתיק הכיסויים באמצעות DNA סופג. תשומת לב ניתנת לתכנון אסטרטגיה blotting DNA לזהות copy יחיד מרובי שילובים-לוקוסים מרובות.

באמצעות מערכת BIBAC-ג'י, כתב בונה לימוד ODM בצמחים היו שנוצר10. המבנים היו בעיצוב שער כניסה וקטור pENTR-gm12 , מוכנס לתוך pBIBAC-בר-GW (איור 1) באמצעות התגובה רקומבינציה שער LR.
תודרנית נהפכו עם pDM19, BIBAC-בר-GW פלסמיד ע...

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

באמצעות pBIBAC-GW וקטור בינארי הופך ייצור הטרנסגניים צמחים עם הוספות עותק יחיד ללא פגע, תהליך קל ופשוט. . הנה, סדרה של פרוטוקולים מוצג מדריך את הקורא לאורך התהליך הטרנסגניים תודרנית מפעלים לייצור, ובדיקות את הצמחים על intactness, להעתיק מספר התוספות.

ייצור הטרנסגניים צמחים עם הוספות עותק יחיד באמצעות וקטור בינארי BIBAC-ג'י

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 Views.  University of Amsterdam. באמצעות pBIBAC-GW וקטור בינארי הופך ייצור הטרנסגניים צמחים עם הוספות עותק יחיד ללא פגע, תהליך קל ופשוט. . הנה, סדרה של פרוטוקולים מוצג מדריך את הקורא לאורך התהליך הטרנסגניים תודרנית מפעלים לייצור, ובדיקות את הצמחים על intactness, להעתיק מספר התוספות.

Video: 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.

ביטוי גנים חולף בטבק באמצעות גיבסון הרכבה והאקדח הגן

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

Environment

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.

שינוי Baculovirus וקטורים ביטוי לייצר מופרש צמח חלבונים בתאי חרקים

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

Biochemistry

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.

Inducible, תאים ייחודיים לסוג ביטוי תודרנית לבנה בתיווך דרך LhGR טרנס-הפעלה

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

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.

הרכבה מהירה של מבנים מרובי גנים באמצעות שיבוט שער הזהב מודולרי

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

Bioengineering

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.

פלטפורמת הפקת MultiBac החלבון המורכבת בEMBL

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

Biology

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.

אגרוינפינציה ו- PVX אגרוינבינציה בתפוח אדמה וניקוטיאנה בנתאמיאנה

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>עריכת גנום יעילה מבוססת קריספר לעמילן עמיד בגידול אורז

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>מחבר זרקור: ייעול גידול אורז עם CRISPR/Cas להשגת תכונות פנוטיפיות ואגרונומיות אופטימליות

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

גידול לפי תכנון לאורז פונקציונלי עם טכנולוגיות עריכת גנום

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.

טרנספורמציה הפרחוני-Dip של פשתן (<em&gt; Linum usitatissimum</em&gt;) כדי ליצור progenies טרנסגניים עם שיעור שינוי גבוה

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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טרנספורמציה הפרחוני-Dip של פשתן (