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パート1。植物栽培用固形培地の調製。 集中Murasigeと1％ショ糖および0.7％寒天を添加したスクーグ（MS）培地上で我々は通常、カルチャの植物。それは植物が健康を保つために必要なすべての成分が含まれています。 MSの1L、メディアのpH 5.7調製するための <ol><li> 1.5 - 2.0Lオートクレーブ可能なボトルに水800mlにMSの粉末2.15gを追加します。 </li><li>ボトルに攪拌棒を配置し、攪拌プレート上で培地を攪拌することにより、MSパウダーを溶かす。 </li><li>撹拌しながら、5.7にMS培地のpHを調整するために、滴、1N KOHを加える。 </li><li>ショ糖10 gを追加し、攪拌してください。 </li><li>寒天7gを追加* </li><li> 1000 mlに調製した培地の最終的な音量を調整し、オートクレーブで滅菌.* </li><li>場所は、攪拌プレート上で培地をオートクレーブ処理し、攪拌しながら、それを冷ます、その寒天が瓶の底に沈殿しないように。 〜60℃*に培地を冷やす</li><li> 100ミリメートルペトリ皿に注ぐ。 4でプレートを保つ℃に</li></ol> *ヒント1：寒天を溶解しようとしないでください。それは、オートクレーブ中に可溶化されます。 *ヒント2：オートクレーブ中に瓶の中に攪拌棒を保つには、後で必要になります。 *ヒント3：あなたが10秒間高圧蒸気滅菌瓶にあなたの手を保つことができるなら、それは媒体が十分冷えてからして、プレートを注いで開始できることを意味します。 パート2。植物材料と成長条件。 <ol><li> シロイヌナズナの種子を滅菌<ol><li>エッペンドルフ微量遠心チューブに種子100 mgを置き、70％エタノール1 mlを加える。よく混和して、2分間インキュベートする。 、種をスピンダウンし、上清を吸引除去する。 </li><li> 1.8％漂白剤溶液1mlを追加* 0.1％Tween - 20を含む。 10分間混ぜる。種子や吸引漂白剤溶液をスピンダウン。 </li><li>このステップでは、層流フードで行う必要があります。種に滅菌水1 mlを追加、よく混ぜる。水を吸引、種子をスピンダウン。このステップを5回繰り返します。 </li></ol></li><li>ステップ1プレート（100粒/プレート）で調製に種を広げる。 </li><li> 4℃で24〜48時間のためにシードのプレートを保持°層別化のための暗所で。 </li><li> 19分の23 ° Cの明/暗、温度体制で8時間light/16時間暗光周期（250マイクロモル、M -2 s -1の光合成光量子束密度時）で生育チャンバー内で14日間の植物を育て、75％の相対湿度。 </li></ol> *ヒント1：ブリーチ溶液を6％の次亜塩素酸ナトリウムを含む、0.1％の最終濃度にTween - 20を追加して、希釈家庭クロロックスがアップされています。 第3部。 シロイヌナズナ実生からのプロトプラストの単離。 <ol><li>濾過滅菌TVL溶液15mlで新鮮なカミソリの刃と14日齢の実生のスライス2グラム。我々は、無菌使い捨てペトリ皿に植物材料を切る。 </li><li> 200mlのビーカーに刻んだ組織を転送する、フィルター滅菌した酵素液、スワール酵素溶液で組織のミックスは、パラフィルムとアルミ箔でカバーできるようにビーカーの20mlを加える。 </li><li> 16〜18時間室温で暗所で35 rpmで植物組織を振る</li><li>チーズクロスの8層、W5ソリューションのプリウェットを通してふるいに50 mlのFalconチューブ中に放出されたプロトプラストを集める。 </li><li> W5溶液15mlで布を洗浄することによりチーズクロスつのより多くの時間からふるいプロトプラスト。 </li><li>糖勾配を乱さないように、W5溶液の10mlでプロトプラストを慎重にオーバーレイ、100 gで7分間遠</li><li>酵素液とW5ソリューション（図1B）の界面でのプロトプラストの10mlを収集し、新しい50 mlのFalconチューブに移す。 </li><li> 15ミリリットルW5溶液、60 gで5分間遠心分離を追加します。上清を取り除く。 </li><li> 60グラムで5分間遠、W5溶液の15ミリリットルでプロトプラストを懸濁することにより、酵素液から遊離プロトプラストを洗う</li><li> 1〜3ミリリットルW5溶液の上清、再懸濁しますペレットプロトプラストを削除します。 </li><li>血球計でカウント細胞によるプロトプラストの収量を評価する。 </li></ol>パート4。試薬： TVL：0.3 Mソルビトール、50mMのCaCl 2をフィルター滅菌し、-20℃で保存します。 酵素溶液：0.5 Mのショ糖、10mMのMES - ​​KOH [pHを5.7]、20mMのCaCl 2を 、40mMのKClを、1％セルラーゼ（小野塚R - 10）、1％Macerozyme（R10）。フィルター滅菌し、新鮮な使用。 W5解決策：0.1％（w / v）のグルコース、0.08％（w / v）の塩化カリウム、0.9％（w / v）の塩化ナトリウム、1.84％（w / v）のCaCl 2を 、2mMのMES - ​​KOH pHは5.7。ろ過滅菌し、室温で保管してください。 パート5：代表的な結果： パート1および2で説明したシロイヌナズナ培養条件を使用すると、プロトプラスト（図1A）を単離するのに適した健康な苗を、レンダリングします。プロトプラストは、ショ糖密度グラジエントによって精製されていますENTの遠心分離とW5と酵素のソリューション（図1B）のインターフェースで収集。第3部で説明したプロトプラストの単離法を用いて、我々は一般的に新鮮な苗1グラムから無傷のプロトプラストの5-10 10 6（図1C）を収穫。 <img src="/files/ftp_upload/1149/Fig1.jpg" alt="図1" /> 図1。シロイヌナズナ実生からのプロトプラストの単離。 、シロイヌナズナの14日齢の実生からの組織が ​​あるのは、変更手続き（チェンとHalkier、2000）によって収集し、プロトプラストに変換された。B、プロトプラストは、ショ糖密度勾配遠心法により精製し、酵素液の界面で収集していたW5 uffer（ 白矢印 ）。C、プロトプラストの明視野顕微鏡。顕微鏡はツァイスのAxioscope 2に加え、顕微鏡とインタフェース冷却CCDカメラを用いて収集した。

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

植物遺伝子の機能解析で、その使用のためにこの手順を記述した仮特許は、技術企業と商業化コーネルセンター（CCTEC）、2008年6月に提出されています。レシートは50341/015001ない：Vatamaniuk、[OK]を、そして斎、植物遺伝子の迅速な機能解析のためのZ.方法。

無傷のプロトプラストを高収率を確保するためには、健康な植物で、起動に非常に重要です。プロトプラストを単離するための溶液をろ過滅菌して使用してください。プロトプラストが壊れやすいことに注意してください。したがって、プロトプラストを処理する際に、ピペットまたはそれはそれらを制動される精力的に以来、渦、それらを混在させないでください。代わりに、徐々に遠心?...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
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<td>Agar</td>
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<td>Kreckeler Scientific</td>
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<td>VWR</td>
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<td>Axioskop2 Plus Microscope</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

プロトプラストは細胞壁が酵素的に除去あった植物細胞で​​ある。異なる植物組織からのプロトプラストの単離は、最初の40年以上前に報告された<sup&gt; 1</sup&gt;それ以来このような細胞内タンパク質の局在、無傷の細胞小器官の分離と二本鎖RNA干渉（RNAi）による遺伝子不活性化標的としての細胞プロセスの様々な、研究するために適応されています<sup&gt; 2月5日</sup&gt;。プロトプラストの分離プロトコルのほとんどは、成熟したシロイヌナズナの葉組織（例えば、35日齢の植物）を使用してください<sup&gt; 2月4日</sup&gt;。我々は、14日齢のシロイヌナズナの苗を用いることにより、既存のプロトコルを変更。この手順では、14日齢の実生の1グラムは、5 10が得られた<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt;少なくとも96時間そのまま残りますプロトプラスト。苗からのプロトプラストの収量は、成熟したシロイヌナズナの葉から調製品と同等ですが、代わりに35〜36日から、プロトプラストの単離は、15日間で完了します。これは成熟した植物を使用するときにプロトプラストを単離するために必要な時間と成長室のスペースを減少させることができ、そのままプロトプラストを必要と下流の研究を促進します。

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

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

このビデオでは、シロイヌナズナの14日齢の実生の組織から無傷のプロトプラストを単離するための手順を示しています。分離されたプロトプラストは、少なくとも96時間のためにそのまま残るし、代わりに一ヶ月の成熟植物の苗から分離されていることを考えると、この手順は、無傷プロトプラストを必要とするアッセイを迅速に処理されます。

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 ...

isolation-protoplasts-from-tissues-14-day-old-seedlings-arabidopsis

Research

JoVE Journal

Biology

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

35.9K ビュー.  Cornell University. このビデオでは、シロイヌナズナの14日齢の実生の組織から無傷のプロトプラストを単離するための手順を示しています。分離されたプロトプラストは、少なくとも96時間のためにそのまま残るし、代わりに一ヶ月の成熟植物の苗から分離されていることを考えると、この手順は、無傷プロトプラストを必要とするアッセイを迅速に処理されます。

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

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物プロトプラストの蛍光活性化細胞選別

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

気-液クロマトグラフィー（GLC）とあいまって薄層クロマトグラフィー（TLC）によりシロイヌナズナポーラーグリセロ脂質プロファイリング

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

からのタグ付き核の親和性に基づく分離<em&gt;ショウジョウバエ</em遺伝子発現解析のための&gt;組織

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

発光時計レポーターとシロイヌナズナプロトプラストトランスフェにおける概日表現型の迅速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

異なる光質におけるシロイヌナズナ成長挙動の解析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.