עבודה זו נתמכה על ידי BMBF Forschungsinitiative &quot;BioEnergie 2021 - Forschung für למות Nutzung פון Biomasse&quot; והאשכול של אקסלנס &quot;דלקים תפורים מביומסה&quot;, אשר ממומן באמצעות יוזמת המצוינות על ידי הממשלות פדרליות ואת המדינה הגרמנית לקידום מדע ומחקר באוניברסיטאות גרמנית.

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יש המחברים אין לחשוף.

ביטוי רקומביננטי של cellulases הוא תחום עניין רב, בשל הרצון למערכות ייצור דלק ביולוגי יעילים יותר 1. צמחים מציעים אפשרויות רבות לייצור cellulase מוצלח, עם תכונות כגון טכניקות קצירה כלכלית ושיטות autohydrolysis להיות תכונות רצויות מאוד לייצור דלק ביולוגי בקנה מידה גדולה. יתר על ...

<html> השפלה של ביומסה lignocellulosic והמרתו לדלק נוזלי כבר חזתה כשיטת להפחתת תלות בדלקים מאובנים. המשוכה משמעותית אחד בהקמת מערכות לעיבוד ביומסה מבחינה כלכלית היא בשפלה האנזימטית של תאית hemicellulose 1. מערכות ביטוי צמח מראות פוטנציאל גדול לייצור של אנזימים בקנה מידה תעשייתי. מערכות חקלאיות לצמחי יבול מבחינה כלכלית ובנפח כבר הוקמו, כפי שהם היו במשך אלפי שנים. הייצור של cellulases בצמחים רצוי בשל גורמים כמו קלות autohydrolysis 2, ואת הפוטנציאל למקסם את השימוש ברמות אנזים נמוכים יותר על ידי הגדלת הנעכלות של קירות תא צמח 1. לבסוף, מערכות מפעל מאפשרות את המיקוד של חלבונים רקומביננטי לאזורים הספציפיים של תאי צמח והם מסוגלים לשנות posttranslationally אנזימים שבו נדרשו 3. ve_content &quot;&gt; tabacum Nicotiana (טבק) מאוד נפוץ כאורגניזם מודל ללימודי ביטוי חלבון Heterologous בצמחים, בשל תכונותיה צמיחה המהירה וביומסה הצטברות 4. ביטוי חולף של חלבונים רקומביננטיים הוא טכניקה המאפשרת ייצור חלבון בתקופות זמן קצר 5, תוך שמירה על גמישות כמו ללוקליזציה ולכן שינויי posttranslational של החלבונים שנבחרו, כלומר cellulases. זה מאפשר הייצור של cellulase (ים) לניתוח בסיסי, גם בעת הנחת התשתית לאסטרטגיות ביטוי נוספות בצמחים. משתמש באסטרטגית ביטוי חולפת כאלה, endoglucanase ממשפחת glycosyl hydrolase 5, Cel5A, הנגזרת מהמארח פטרייתי Trichoderma reesei (jecorina Hypocrea) 6 מופק (להלן כTrCel5A). TrCel5A הוא חלבון 42 kDa אשר glycosylated באופן מקורי, והוא פעיל מאוד בHYDroylzing רשתות תאית 7. טכניקת הביטוי חולפת המתוארת כאן מבוססת על מערכת יחסית נפוץ, שחדרו לעלי הצמח עם Agrobacteria נושא את הגן של עניין בוקטור ביטוי מתאים. כדי לאפשר ניתוח מהיר של הצלחה בביטוי Planta, TrCel5A יכול גם לבוא לידי ביטוי חלבון היתוך עם mCherry, חלבון פלואורסצנטי monomeric נגזר במקור sp Discosoma. חלבון DsRed 8, עם שאריות נוספות שש היסטידין (שלו תג) התמזגו C-הסופית (TrCel5A-mCherry). ביטוי של חלבון Heterologous ההיתוך, TrCel5A-mCherry, ובכך תוכל להיות במעקב במסגרת גידול הצמחים על ידי שימוש באור ירוק לבחון פיזור mCherry. אם תרצה, סעיפים דקים של החומר הצמחי יכולים להיבחן תחת אור הירוק במיקרוסקופ כדי לקבוע את הלוקליזציה מסוימת של החלבון. לשם כך עבודה, אות וטראןפפטידים לשבת שולבו במבנה, בתרגום יצוא חלבון Heterologous לreticulum endoplasmic 9. כדי לנתח את הפעילות של cellulases הצמח בא לידי ביטוי, ביניהם TrCel5A, מספר בדיקות פעילות cellulase ניתן להפעיל. לאחר המיצוי של החלבונים הצמחיים המסיסים בסך הכל, TrCel5A יכול להיות מטוהר באופן חלקי באמצעות טכניקת דגירה תרמית. גודל חלבון הוקם באמצעות SDS-PAGE ואחריו מערבי סופג. Zymography יכול לשמש גם כדי לנתח את הפעילות נגד מצעים, למשל תאית אשר כבר-מפוגל carboxy (מה שהופך את תאית carboxymethyl: CMC) למסיסות 10. פעילות Cellulase וglucosidase ניתן לנטר באמצעות fluorophore 4-methylumbelliferyl (4-MU) הקשורים β-D-cellobioside (בשילוב: 4-MUC). שיטה נוספת להערכת פעילות endoglucanase כרוכה ניתוח spectrometric של CMC שנקשר עם אזו דאתם (Remazolbrilliant הכחול R) 11. בנוסף, פעילות חלבון של שני endoglucanases ומגוון של cellulases יכולה להיות פיקוח על ידי שימוש במבחן ניתוח סוכר, כגון hydrazide p-הידרוקסי benzoic החומצה (PAHBAH) assay 12. טכניקות אלה יכולים לשמש כדי להבהיר ולכמת את הפעילות של cellulase הביע עניין.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">4-methylumbelliferyl &#946;-D-cellobioside</td>
			<td style="width:127px;">SIGMA-ALDRICH</td>
			<td style="width:156px;">M6018</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>ROTH</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringon (concentrated)</td>
			<td>ROTH</td>
			<td>6003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - kanamycin</td>
			<td>ROTH</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - rifampicin</td>
			<td>ROTH</td>
			<td>4163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - carbenicillin</td>
			<td>ROTH</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - rabbit anti His(6) pAb</td>
			<td>ROCKLAND</td>
			<td>600-401-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - goat anti rabbit AP, FC pAb</td>
			<td>DIANOVA</td>
			<td>111-055-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azo-carboxymethyl cellulose</td>
			<td>MEGAZYME</td>
			<td>S-ACMC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-cyclodextrin</td>
			<td>SIGMA-ALDRICH</td>
			<td>C4805</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carboxymethyl cellulose</td>
			<td>ROTH</td>
			<td>3333.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellulase from Trichoderma reesei ATCC 26921</td>
			<td>SIGMA-ALDRICH</td>
			<td>C2730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Congo red</td>
			<td>SIGMA-ALDRICH</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomasie brilliant blue G 250</td>
			<td>ROTH</td>
			<td>9598.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>ROTH</td>
			<td>8337</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>ROTH</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper - Rotilabo blotting paper</td>
			<td>ROTH</td>
			<td>CL67</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NBT/BCIP</td>
			<td>SIGMA-ALDRICH</td>
			<td>72091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES buffer</td>
			<td>ROTH</td>
			<td>4256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>ROTH</td>
			<td>8388</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate</td>
			<td>ROTH</td>
			<td>3580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylsulfate</td>
			<td>SERVA</td>
			<td>20765</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>ROTH</td>
			<td>6771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soil, Einheitserde classic&#160;</td>
			<td>PATZER GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer - Infinite M200</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>SIGMA-ALDRICH</td>
			<td>S-5390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Hydroxy benzhydrazide&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>H9882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ROTH</td>
			<td>1058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV light source - BLAK RAY</td>
			<td>UVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome - VT1000S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

אנזימים משפילים תאית, cellulases, הם מטרות של שניהם אינטרסים תעשייתיים מחקר ו. העליונות של אנזימים אלה באורגניזמים קשים לתרבות, כגון פטריות העובש בנייה וחיידקים אנאירוביים, שהחישה את השימוש בטכנולוגיות רקומביננטי בתחום זה. שיטות ביטוי צמח הן מערכת רצויה לייצור בקנה מידה גדולה של אנזימים וחלבונים באופן תעשייתי שימושיים אחרים. בזאת, שיטות לביטוי החולף של endoglucanase פטרייתי, Trichoderma reesei Cel5A, בNicotiana tabacum הם הפגינו. ביטוי חלבון מוצלח מוצג, פיקוח על ידי הקרינה באמצעות חלבון היתוך mCherry-אנזים. בנוסף, סדרה של בדיקות בסיסיות המשמשות לבחון את הפעילות של ט &#39;הביע transiently reesei Cel5A, כולל-SDS, מערבי סופג, zymography, כמו גם הקרינה ומבחני השפלה מצע מבוסס צבען. המערכת המתוארת כאן יכולה לשמש לייצור תא פעילulase בתקופת זמן קצר, על מנת להעריך את הפוטנציאל לייצור נוסף במפעלים באמצעות מערכות ביטוי מכוננות או מושרה.

TrCel5A וTrCel5A-mCherry באו לידי ביטוי בהצלחה בצמחי טבק באמצעות מערכת חולפת הביטוי (איורים 1 א ו 1 ב &#39;). בחינה של דפוס הביטוי של חלבון היתוך TrCel5A-mCherry חשפה עלים בריאים (איור 1 ג), אשר הראה ביטוי חלבון נרחב תחת אור הירוק (1D איור). <p class="jove_content" styl...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

צמחי טבק ששימשו להפקת cellulase פטרייתי, TrCel5A, באמצעות מערכת ביטוי חולפת. הביטוי יכול להיות במעקב באמצעות חלבון היתוך ניאון, ופעילות החלבון התאפיינה הודעה ביטוי.

ביטוי של רקומביננטי Cellulase Cel5A מ<em&gt; Reesei Trichoderma</em&gt; בצמחים טבק

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

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Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

12.7K Views.  RWTH Aachen University.  צמחי טבק ששימשו להפקת cellulase פטרייתי, TrCel5A, באמצעות מערכת ביטוי חולפת. הביטוי יכול להיות במעקב באמצעות חלבון היתוך ניאון, ופעילות החלבון התאפיינה הודעה ביטוי. 

Video: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

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 ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

תמליל והמטבוליט Profiling להערכת של טבק ועץ צפצפה כחומר גלם לתעשייה המבוססת על ביו

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

מדידת שטפים של חומרים מזינים ומינרליים רעלים בצמחים עם חומרים רדיואקטיביים

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

השימוש ברזולוציה גבוהה אינפרא אדום תרמוגרפיה (HRIT) לחקר הקרח נוקלאציה וקרח רביה בצמחים

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

בקרה בתיווך RNAi של aflatoxins בבוטנים: שיטה לניתוח ייצור mycotoxin וביטוי transgene בבוטנים /<em&gt; אספרגילוס</em&gt; Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

שיפור של שיעור הצמיחה הראשוני של צמחים חקלאיים באמצעות שדות מגנטיים סטטיים

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

הערכה של מתאן והנתיבים תחמוצת החנקן משדה אורז באמצעות חשמל סטטי סגור צ'יימברס שמירה על הצמחים בתוך קראוון

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

פרוטוקול להפקת וידאו תלת ממדי אינפרא-אדום להקפאת בצמחים

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

בתיווך לולאה Assay איזותרמי הגברה (מנורה), עבור זיהוי מהיר של כנימת עש הטבק

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

מנותקת עלה assays לפשט גנים ביטוי לימודי תפוחי אדמה במהלך התפשטות על ידי לעיסת חרק manduca סקסטה

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...