博士スティーブン市会議員、デビッド葉、そしてエリンマクリーは感謝して顕微鏡で助けを認められている。この研究はNIFA特殊作物研究イニシアティブ、USDA SCRI-SREPグラント賞2009から02484番からの助成金で賄われました。ブリアナKinash、ケビンキンロック、ミーガンレオンハルトジョセフ·マッカラム、マリアMcSharryとニコールSallee、優れた技術支援と思いやりの議論を提供した。

<ol>
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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

本明細書に記載された手順は、土壌から電位BDMのdegradersを単離するための第一パスの技法を表し、正常7ヶ月間土壌中に埋設BDMSから菌類を分離するために使用された。同じタイプの新鮮なBDM材に再接種時菌類は孤立菌類が本当に植民あったことを示す、とフィルムは真菌の増殖を阻害するではなかったこと、成長した。プラスチック分解菌や細菌の単離は、潜在的にプラスチックが分解する?...

内訳は、製品の寿命及び耐久性を短縮するため、劣化が歴史的に、プラスチックポリマーの望ましくない属性と考えられてきた。最近では、自然環境1,2,3で廃プラスチックによって提示された環境問題への意識は、生分解性プラスチックを従来のプラスチック材料に魅力的な代替手段を行っている。分解は、（構造的変化、断片化、および分子量の低下、整合性、および強度4,5として定義される）の両方の非生物的プロセス（熱応力、光酸化、加水分解、浸食、機械的ストレス）を含む一連の出来事、を介して行われると生物学的分解6。非生物的プロセスは断片サイズやプラスチックの特性を変えることができるが、微生物は水と二酸化炭素（好気条件での）および/またはメタン（嫌気条件下）への最終的な鉱化に必要とされる。 のための実質的なニッチ生分解性プラスチックは、プラスチックmulchesが土壌水分を保持するために、土壌温度7,8を高めるために、雑草の成長を防止するために使用される農業に存在する。米国だけで数千エーカーの何百もの生分解性プラスチックで構成mulches含むプラスチックmulches 9、で覆われている。作物の生育期に続いて、生分解性mulches（BDMS）を処分するためのオプションは、埋立処分、エネルギー回収10焼却、堆肥化を経由して劣化、または耕作11後の土壌中での劣化があります。これらのうち、少なくとも労働集約的な運命が土壌にBDMSを耕しているが、春の耕作と作付け時に効率的な劣化や石灰化することなく、非作物ヶ月（一般的には冬に）中に、プラスチックの断片が残っている可能性があり、農業機器に干渉し、彼らはかなり野生動物、植物、そして微生物1,2,3,10に影響を及ぼす環境中に残留。 <p claSS = &quot;jove_content&quot;&gt;農業用マルチフィルムを含む多くのプラスチック製品は、ラベルの練習で、 &quot;生分解性&quot;や &quot;堆肥&quot;、劣化や石灰を受けるには、土壌の分解のためにあまりにも非効率的な、および/またはあまりに不完全かもしれない負担が、これらの製品の廃棄のための実行可能な代案。例えば、オキソ生分解性ポリエチレンは、堆肥の温度は25℃で12だったときに℃の堆肥、及び鉱化作用の半分以下の量が発生した58に風化の1年3その後の数ヶ月後にのみ12.4％の石灰化を達成しました。冬には、ほとんどの場所で土壌温度は少ない鉱、おそらくさらに低い微生物の活性が生じ、その結果、これらの温度のいずれよりも低くなる。分解速度を遅くすることに加えて、 &quot;生分解性&quot;という用語の誤用は、農業を含め消費者が13,14によってこれらの製品への不信感につながっている。生分解が変換である二酸化炭素（及び/又はメタン）と微生物4天然由来による水14へのポリマー。そのため、生分解を化学測定されなければならない;基板と微生物の物理的会合は、その材料の微生物分解を意味するものではありません。 農業におけるBDMSの持続可能な利用を検討するための努力の一環として、本研究では、植民地化と市販BDMSを低下農業の土壌に自生する微生物の発見に焦点を当てた。標準的な試験方法は、化学的に非生物的および生物学的手段15,16,17による生分解性プラスチックの分解を測定するために公開されている。しかし、これらの方法は、個々の微生物の種によるプラスチックの劣化に対処する、またはそれらの単離のための方法を提供しない。方法論は、ここより密接に真菌の胞子を持つ検体を接種した後、微生物の分解への抵抗のためにプラスチックを評価するために設計された標準的な方法に似ている18,19。 プラスチックの製剤は公知であり、原料のソースが利用される場合、粉末状プラスチックをクリアゾーン13の可視化により決定寒天ベースのメディアおよび分解中に懸濁させることができる。この方法は、ポリウレタン20、ポリ（ブチレンサクシネートアジペート- コ - ）21、及びポリ（乳酸）22のようなポリマーを分解する微生物を同定するために以前に使用されている。同様の方法は、プラスチックが唯一の炭素源20,23である液体媒体中に純粋なプラスチック粉末を懸濁さ含む。これらの方法は定義されたシステムの利点を有するが、それらは不十分BDMS の in situ分解に模倣する。 BDMS、土壌マトリックス全体に小さな粒子に分散させ、むしろ、フィルムとして販売され、使用されないため、まず、表面積が異なって分配される。第二に、BDMSの化学的構成は純粋なポリマーとは異なります。 BDMSは一般的にそのような添加剤を含んでい充填剤、可塑剤、及び着色剤、及びこれらの添加剤は、それにより微生物の増殖や、石灰化の速度に影響を及ぼすことがある。本研究では一定の商業膜の組成は、そのフィールドレディ形で独自、プラスチックフィルムであったため、この理由のため、および真菌や細菌を単離するために用いた。簡単にするために、以下の方法がどこに細菌の単離のための適切な修正が指摘して、真菌に対してのみ記述されている。 土壌内の1つの冬のために最近の研究24では、3市販BDMS一実験映画は、一生育期のために、米国の三つの異なる地域での農業のサイトで使用され、その後メッシュ（250ミクロン）の袋に入れて、埋められた同サイトで。 250ミクロンメッシュ開口部は根とほとんどの土壌動物相を除き、土壌侵食25,26を最小限に抑えながら、菌糸が浸透することができます。ナイロン材料は、土壌中の袋の劣化を防止する。掘削、Fに続いてungal分離株はBDM片から回収し、寒天とプレ消毒された新しい、未使用のBDMフィルム5cmの×5センチ、表面消毒広場を除く炭素源なしで最少培地上での増殖のために評価した。フィルムとして使用されるほとんどのプラスチックは、整合性を損なうことなくオートクレーブすることができませんので、UV光は、プラスチック上に存在する任意の微生物細胞を殺すために使用されていました。 ISO 846 19は、70％エタノールとその後の乾燥で表面disinfestingお勧めしますが、この方法を使用する場合は、1は、フィルムのない成分又は添加物に悪影響をエタノールの影響を受けていないことを確認する必要があります。 BDMSはおそらく日光に耐えるように製造されるので、UVは、除染方法として選択した。 一人で最少培地上でより良いBDM作品に育った株は、さらなる研究のために選択した。それは、典型的には農園医学的にないことにより、代謝的に利用されないため、寒天、海藻によって生成された多糖は、微生物のメディアを固化するために使用されるできる微生物はいますが、寒天加水分解酵素は、土壌28から単離された海洋細菌27と寒天分解細菌から単離されている。 BDMポリマーおよび寒天は、潜在的な栄養源として、これらのポリマーが含まれている環境で進化していない土壌菌によって分泌酵素の珍しい基質であることが期待が、両方の基質は（ ステップ7）明細書に記載のプレートバイオアッセイに存在している両方。炭素源としてBDMSではなく、寒天を使用する真菌BDM膜（実験））寒天培地（陰性対照）、 二除いi）を添加しない炭素源を含む寒天固化媒体上に成長を比較することによってのみ、寒天を使用する真菌から区別することができる及びiii）グルコース（陽性対照）。すべての分離株の成長が最少培地に加えてグルコースに期待されている菌類に起因しないグルコース含有プレートは、実​​験に使用される特定の最少培地上で成長することができないかもしれません。ポテンショアルBDM degradersは寒天固化最少培地+、彼らは一人で寒天固化最小培地で生育よりも良いBDMフィルム上に成長する必要があります。最小培地プレート上で生育菌類は寒天degradersまたはoligotrophsであり、またバイオアッセイプレートではなく、フィルム自身（彼らは偶然にもBDMポリマーを分解する場合を除きます）にBDMフィルムに関連付け寒天上に成長すると予想されます。 BDMSの寒天としないの利用に起因する微生物の成長を見ての可能性を排除するために、我々は、定義されたブロス培地（ ステップ9）バイオアッセイと寒天プレート上BDM植民地化のために我々の最初のアッセイを追った。 BDM片バイオアッセイチューブにのみ知られている炭素源を表した。 最初のスクリーニングの後、および分離株のグリセロールストックを復活時には、いくつかの既知の炭素源を含まない液体培地中で定義されて乏しいが、目に見える菌糸を形成した。これらの結果は、取得された単離物のいくつかはoligotであることが示唆されたrophs -炭素、窒素、水環境や大気29,30,31における揮発として存在するのいずれかで溶解し、他の栄養素の非常に少量を捕捉することによって成長する生物。 18SリボソームDNA分析を通じて種の同定には、以前に栄養不良32を示すことが報告された真菌属にマッチした分離株の多くは、このビューをサポートしていました。一般的に腐生ですOligotrophsは、環境30の範囲内の基質利用のための代謝機能の広い範囲を必要とする。したがって、BDMS（おそらく珍しい酵素の機能を必要とする）から、我々は孤立した同菌は貧栄養の能力を実証し、そのような空気中の指紋、ほこり、または微量揮発から皮脂などの微量汚染物質で成長することができたことは驚くべきことではない。 oligotrophsの分離のために、我々は一人でBDM表面にその成長がBDMの内訳を推測するために使用することができなかったと結論付けた。本明細書に記載の方法は、SCへの取り組みを反映している説明善意の BDMの故障のための農業の土壌からREENネイティブBDMの植民。

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potato Dextrose Agar</td>
 <td>Becton Dickinson</td>
 <td>8X05491</td>
 <td></td>
 </tr>
 <tr>
 <td >Agar</td>
 <td>Fisher</td>
 <td>BP 1423-2</td>
 <td></td>
 </tr>
 <tr>
 <td >Chloramphenicol</td>
 <td>Acros Organics</td>
 <td>200-287-4</td>
 <td></td>
 </tr>
 <tr>
 <td >Glutaraldehyde</td>
 <td>Electon Microscopy Sciences</td>
 <td>16216-10</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td >Molecular sieve</td>
 <td>Fisher </td>
 <td>M-8892</td>
 <td></td>
 </tr>
 <tr>
 <td >Ethanol</td>
 <td>Pharmco-Aaper</td>
 <td>E200</td>
 <td></td>
 </tr>
 <tr>
 <td >Contrex</td>
 <td>Decon Labs, Inc.</td>
 <td>5204</td>
 <td></td>
 </tr>
 <tr>
 <td >Parafilm M</td>
 <td>Pechiney Plastic Packaging</td>
 <td>S37440</td>
 <td></td>
 </tr>
 <tr>
 <td >Mineral salts for buffers and media</td>
 <td>Fisher</td>
 <td>Various</td>
 <td>Various vendors sell these reagents</td>
 </tr>
 </tbody>
</table>

isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture

市販の生分解性マルチ（BDM）フィルムを植民地化し、農業の土壌に自生する菌類を分離し、プラスチックを分解する可能性について評価した。典型的には、プラスチックの製剤は公知であり、原料の供給源が利用可能である場合には、粉末状のプラスチックがクリアゾーンの視覚化によって決定される寒天ベースのメ​​ディアおよび分解中に懸濁させることができる。しかし、このアプローチでは不十分BDMS の in situ分解に模倣している。まず、BDMS、土壌マトリックス全体に小さな粒子として分散されていない。次に、BDMSは、純粋なポリマーとしてではなく、微生物の増殖に影響を及ぼす可能性の添加剤（ 例えば充填剤、可塑剤および染料）を含有する膜として市販されていない。本明細書に記載した手順を土壌埋め込み型マルチフィルムから得られた単離に用いた。発掘BDMSから取得真菌分離株はない炭素源eを含まない規定培地の上に置いた新しい、消毒BDMSの部分に成長のために個別にテストされました寒天をxcept。 BDMS上に成長した単離物がさらにBDMSが追加された唯一の炭素源であっ液体培地において試験した。約10週間後、菌のコロニー形成とBDM劣化を走査型電子顕微鏡により評価した。分離株はリボソームRNA遺伝子配列の分析によって同定した。このレポートは、真菌単離するための方法が記載されているが、細菌は、細菌のための適切な媒体を置換することにより、これらの方法を用いて単離した。我々の方法論は、プラスチック原料が使用可能どちらか不明ではありませんそのため無傷プラスチックフィルムまたは製品の内訳を調査研究のために有用であることが分かるはずです。しかし我々のアプローチは、BDM劣化の速度を比較するための定量的な方法を提供していません。

最近の研究24では、4人は、 &quot;生分解性&quot;というラベルの付いた3市販BDMSそれぞれ、プラスの実験映画と従来のプラスチック制御、マウントバーノン、ワシントン州では、2010年の春にトマトの生産のための腐葉土として土の上に配置されたを複製ノックスヴィル、テネシー州、そしてラボック、テキサス州。 2010年秋には、BDMフィルム正方形は4反復のプロットで腐葉土を風化それ?...

Watch this Scientific Journal Video about Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture at JoVE.com

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

&quot;生分解性&quot;というラベルの付いたプラスチックフィルムはmulchesとして農業用市販されている。耕作は、魅力的な処分方法を表しますが、フィールド条件下での劣化はあまり理解されています。本研究の目的は、フィールド埋葬後にプラスチックマルチフィルムを植民ネイティブ土壌菌類や細菌を単離する方法を開発することであった。

農業で使用する生分解性プラスチックマルチフィルムを分解するための可能性を持つネイティブ土壌微生物の分離

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

isolation-native-soil-microorganisms-with-potential-for-breaking-down

Research

JoVE Journal

Biology

30.4K ビュー.  Western Washington University. "生分解性"というラベルの付いたプラスチックフィルムはmulchesとして農業用市販されている。耕作は、魅力的な処分方法を表しますが、フィールド条件下での劣化はあまり理解されています。本研究の目的は、フィールド埋葬後にプラスチックマルチフィルムを植民ネイティブ土壌菌類や細菌を単離する方法を開発することであった。

ビデオ: Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

の複合植物の世代ウマゴヤシtruncatula根粒形成のアッセイに使用

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

ハイスループットシークエンシングによりマイクロRNA定量のための低分子RNA分子の高効率ライゲーション

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

可視化と系統特異的マーカーと間葉系細胞脂肪細胞の微分の潜在性の定量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

X 線マイクロ断層レントゲン写真撮影に基づいてトウモロコシの維管束のミクロン スケール表現型解析技術

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice (Oryza sativa ssp.)

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously applied herbicides has presented the need for additional management methods. Allelopathy is a physiological process that some plant species possess that provide the plant with an advantage over its neighbors. Allelopathic crop varieties would be equipped with the ability to suppress the growth of surrounding competitors, thus reducing potential yield loss due to weed interference. This paper focuses on the construction and operation of a stair-step assay used for the screening of the allelopathic potential of a donor species (Oryza sativa) against a receiver weed species (Echinochloa crus-galli) in a greenhouse setting. The structure described in this paper serves as a stand for the plant samples and incorporates a timed watering system for the accumulation and distribution of allelochemicals. Allelochemicals produced by the plant roots are allowed to flow downward through a series of four pots separately into a collection tank and recycled back to the top plant through electric pumps. This method of screening provides an avenue for the allelochemicals from the donor plant to reach receiver plants without any resource competition, thus allowing quantitative measurement of the allelopathic potential of the selected donor plant. The allelopathic potential is measurable through the height reduction of the receiver plants. Preliminary screening data for the effectiveness of this method demonstrated height reduction in the receiver species, barnyardgrass (E. crus-galli), and thus the presence of allelopathic residues from the donor plant, weedy rice (Oryza sativa).

Repeatable Stair-step Assay to Access the Allelopathic Potential of Weedy Rice Oryza sativa ssp.

Allelopathy has shown promise as a useful supplemental weed control strategy in cropping systems. To determine the allelopathic potential of a desired plant specimen, a stair-step screening method is provided.

ウィーディライスのアロパシーポテンシャルにアクセスするための繰り返し可能な階段ステップアッセイ(オリザサティバssp.

Weed competition contributes significantly to yield losses in cropping systems worldwide. The evolution of resistance in many weed species to continuously ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

植物における空間的および時間的組織特異的な遺伝子発現解析のためのレーザー捕捉マイクロ解離RNAシーケンシング

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多パラメータ灌流制御による原発性ラット肝細胞の単離

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

げっ歯類の頭部に定義された加速度を生成するための受動的頭部運動の適用

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...