Name: estimation of crystalline cellulose content of plant biomass using the updegraff method
Uploaded: 2021-05-15T14:00:00
Description: Watch this Scientific Journal Video about Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method at JoVE.com

我们感谢植物与土壤科学与棉花公司对这项研究的部分支持。

1. 实验准备
<ol><li>将干燥的植物材料磨成细粉末。</li>
	<li>蛋白质溶解缓冲区 （PSB）： 准备 1 M 三重奏 （pH 8.8）， 0.5 M 乙酰胺乙酰氨基酸 （EDTA） （pH 8.0） 的库存解决方案，并将其高压灭菌。从这些库存解决方案中制作新鲜的PSB缓冲，最终浓度为50m Tris、0.5 m EDTA 和 10% 硫酸钠 （SDS） 无菌水中。</li>
	<li>准备100mL的70%乙醇（v/v）：70mL的100%乙醇和30mL的无菌水。</li>
	<li>准备100mL的甲醇：氯仿在1：...

<ol>
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棉纤维是用棉籽生产的天然纤维。棉纤维是一种单细胞，纤维素含量约95%，纤维素含量高，在纺织行业具有广泛的应用。由于棉纤维含有约95%的纤维素，我们使用棉根组织来演示水晶纤维素含量的估计。棉根组织中等丰富的结晶纤维素含量，是一种常见的植物生物量。晶体纤维素含量所需的总细胞壁量仅为5毫克，因此可用于估计不同发育阶段/组织特定纤维素?...

纤维素是细胞壁的主要承重成分，存在于主细胞壁和辅助细胞壁中。细胞壁是环绕植物细胞的细胞外基质，主要由纤维素、木质素、血糖、果胶和基质蛋白组成。大约三分之一的植物生物量是纤维素1，它通过提供强度、刚性、细胞生长的速度和方向、细胞形状维护以及防止生物和非生物应激因素，在植物生长发育中发挥着重要作用。棉纤维含有95%纤维素2含量，而树木含有40%至50%的纤维素，这取决于植物种类和器官类型3。纤维素由纤维素的重复单位组成，纤维素是葡萄糖残留物的去除，由β-1，4糖体键4连接。纤维素乙醇是由葡萄糖从植物细胞壁5中存在的纤维素中提取的。纤维素纤维由几个微纤维组成，其中每个微纤维作为核心单位与500-15000葡萄糖单体1，6。纤维素同聚物由等离子膜嵌入纤维素合成酶复合物（CSC的）1，7合成。单个纤维素合成酶A（CESA）蛋白合成葡萄糖链和相邻的葡萄糖链通过氢键连接，形成晶体纤维素1，8。纤维素存在于几个晶体形式与两种主要形式，纤维素I+和纤维素I+作为原生形式9。在较高的植物中，纤维素存在于纤维素I+形式中，而低植物纤维素存在于I+形式10，11中。总的来说，纤维素在向植物细胞壁传授力量和刚性方面起着重要作用。
第一代生物燃料主要产自玉米淀粉、甘蔗糖和甜菜糖，它们是食物来源，而第二代生物燃料则主要生产来自非食品植物生物质细胞壁材料12的生物燃料。准确估计结晶纤维素含量不仅对纤维素生物合成和细胞壁动力学的基础研究具有重要意义，而且对应用生物燃料和生物制品研究也具有重要意义。开发和优化了多种植物生物量纤维素估算方法，上格拉夫法是纤维素估算中应用最广泛的方法。第一个报告的纤维素估计方法是由克罗斯和贝文在1908年13。该方法基于硫酸钠交替氯化和提取的原则。然而，由克罗斯和贝文方法的原始和修改协议获得的纤维素显示，除了大量的木质素和曼南14的微小部分污染。尽管对从纤维素部分去除木质素和半纤维素进行了多次修改，但交叉贝文方法保留了相当数量的曼南和纤维素。后来，库尔施纳的方法是利用硝酸和乙醇提取纤维素15。该方法指出，总木质素和75%的五氯苯甲酸被去除，但真正的纤维素结果与十字和贝文的氯化方法估计的结果相同。另一种方法（诺曼和詹金斯）是利用甲醇苯、硫酸钠和次氯酸钠提取纤维素16而开发的。这种方法还保留了一些部分木质素 （3%）和大量的五氯苯二酯导致在纤维素的准确估计。后来，基塞尔和塞米加诺夫斯基使用另一种方法，使用80%浓缩硫酸对纤维素进行水解，而水解减少的糖分则由贝特朗的方法17估计。根据基塞尔和塞米加诺夫斯基的方法开发的两种方法，瓦克斯曼和史蒂文斯18和萨洛14，19，也比早期的方法20少4-5%纤维素含量。
上格拉夫法是估计结晶纤维素含量最广泛的方法。1969年21年，乌普德格拉夫首次对纤维素的测量方法进行描述。Updegraff方法将库尔施纳法（使用硝酸）、基塞尔和塞米诺斯基方法（用硫酸将纤维素水解成葡萄糖单体）与一些修改相结合，并结合了维尔斯和西尔弗曼的炭热检测，用于对葡萄糖和结晶纤维素含量进行简单的色度估计。这种方法的原理是使用醋酸和硝酸（上格拉夫试剂）从同质化植物组织中去除半球素和木质素，从而留下耐醋酸/耐硝酸纤维素作进一步加工和估计。醋酸/耐硝酸纤维素用67%的硫酸处理，将纤维素分解成葡萄糖单体，释放的葡萄糖单体估计由炭酸检测21，23。对原来的上格拉夫方法进行了几次修改，以简化程序和纤维素估计由炭细检测24。从广义上讲，这种方法可以分为五个阶段。在第一阶段，工厂材料已准备就绪。在第二阶段，粗细胞壁与总生物量分离，因为纤维素是植物细胞壁的关键成分。后来，在第三阶段，纤维素通过使用上格拉夫试剂处理，从非纤维素细胞壁组件中分离出来。在第四阶段，通过硫酸处理，醋酸/耐硝酸纤维素被分解成葡萄糖单体。纤维素硫酸处理导致葡萄糖单体与硫酸反应形成5-羟基硫化物化合物。最后，在最后阶段，炭热产生一个绿色的蓝色复合物，沸腾与前一阶段25产生的毛皮化合物。这种基于炭铁的色度测量方法于1942年首次被德雷伍德使用。Anthrone 是一种在酸性条件下识别五氯蔗糖和六甲酸脱水产品（如 5-羟基紫外线）的毛皮化合物的染料。与五角大厦25相比，六角形的反应产生强烈的色彩和更好的反应。受约束葡萄糖的含量以光谱光度计吸收量测量，在620纳米，绿色蓝色复合物的强度与样品中的糖量成正比。测量的吸收值与葡萄糖标准曲线回归线进行比较，以计算样品的葡萄糖浓度。测量的葡萄糖含量用于估计植物生物量的纤维素含量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Chemical</td>
			<td>A18-500</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Anthrone</td>
			<td>Sigma Aldrich</td>
			<td>90-44-8</td>
			<td>For colorimetric assay</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>For centrifugation</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Mallinckrodt</td>
			<td>67-66-3</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>6381-92-6</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore Sigma</td>
			<td>EM-EX0276-4S</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1004-090</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma</td>
			<td>SKU A6283</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Heat block/ ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>13527550</td>
			<td>For controlled temperatures</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisherbrand</td>
			<td>150152633</td>
			<td>Used for drying plant sample</td>
		</tr>
		<tr>
			<td>Measuring Scale</td>
			<td>Mettler Toledo</td>
			<td>30243386</td>
			<td>For specific quantities</td>
		</tr>
		<tr>
			<td>Methanol 100 %</td>
			<td>Fisher Chemical</td>
			<td>A412-500</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Microplate (96 well)</td>
			<td>Evergreen Scientific</td>
			<td>222-8030-01F</td>
			<td>For anthrone assay</td>
		</tr>
		<tr>
			<td>Nitric acid</td>
			<td>Sigma Aldrich</td>
			<td>695041</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Polypropylene Microvials (2 mL) / screw capped tubes</td>
			<td>BioSpec Products</td>
			<td>10831</td>
			<td>For high temperatures</td>
		</tr>
		<tr>
			<td>Spectrophotometer(Multimode Detector)</td>
			<td>Beckmancoulter DTX880</td>
			<td>1000814</td>
			<td>For measuring absorbances</td>
		</tr>
		<tr>
			<td>Spex SamplePrep 6870 Freezer / Mill</td>
			<td>Spex Sample Prep</td>
			<td>68-701-15</td>
			<td>For grinding plant tissues into fine powder</td>
		</tr>
		<tr>
			<td>Sulphuric acid</td>
			<td>J.T.Baker</td>
			<td>02-004-382</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>151-21-3</td>
			<td>Used in the PSB buffer</td>
		</tr>
		<tr>
			<td>Tubes (2 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>&#160;1185-53-1</td>
			<td>Used in the PSB buffer</td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977</td>
			<td>Used in the protocol</td>
		</tr>
		<tr>
			<td>Vacuum dryer (vacufuge plus)</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>For drying samples</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisherbrand</td>
			<td>14-955-151</td>
			<td>For mixing</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Thermoscientific</td>
			<td>TSGP02PM05</td>
			<td>For temperature controlled conditions at specific steps</td>
		</tr>
		<tr>
			<td>Weighing Paper</td>
			<td>Fisher Scientific</td>
			<td>09-898-12A</td>
			<td>Used in the protocol</td>
		</tr>
	</tbody>
</table>

estimation of crystalline cellulose content of plant biomass using the updegraff method

纤维素是地球上光合作用产生的最丰富的聚合物，也是细胞壁的主要承重成分。细胞壁通过提供细胞生长的强度、刚度、速率和方向、细胞形状维护以及生物和非生物应激源的保护，在植物生长发育中发挥着重要作用。细胞壁主要由纤维素、木质素、血糖和果胶组成。最近，植物细胞壁成为第二代生物燃料和生物能源生产的目标。具体来说，植物细胞壁的纤维素成分用于纤维素乙醇的生产。生物量纤维素含量的估计对于基础和应用细胞壁研究至关重要。Updegraff方法简单、坚固，是估计植物生物量结晶纤维素含量的最广泛应用的方法。酒精不溶性粗细胞壁分数在治疗与厄普德格拉夫试剂消除血细胞素和木质素分数。后来，上格拉夫耐纤维素分量受到硫酸处理，将纤维素同聚合体水解成单体葡萄糖单位。使用各种葡萄糖浓度开发回归线，用于估计实验样本中纤维素水解后释放的葡萄糖量。最后，纤维素含量根据按彩色测定炭甲基检测的葡萄糖单体量进行估计。

在绿房子里种植的棉花植物被选为这项研究的人选。选择两种不同的棉花实验线进行纤维素含量的比较分析。对于每个实验线，根组织是从三个生物复制品中收集的。总共500毫克的组织是同质化的，其中20毫克用于粗细胞壁提取。后来，5毫克的粗细胞壁提取物用于上格拉夫试剂处理，从纤维素中去除血细胞素和木质素。纯纤维素通过硫酸处理进行水解，将纤维素分解成葡萄糖单位。吸收读数为620?...

Watch this Scientific Journal Video about Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method at JoVE.com

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

上格拉夫法是纤维素估计使用最广泛的方法。本次演示的主要目的是提供详细的上格拉夫协议，用于估算植物生物质样本中的纤维素含量。

使用上格拉夫方法估计植物生物质的晶体纤维素含量

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...

在这里，我们演示了上格拉夫方法，世界上最广泛使用的方法，用于估计结晶纤维素含量。纤维素基本上由葡萄糖单体组成，由β-1，4糖体键连接。我们把这个协议分为五个阶段。在第一阶段，我们准备植物生物质材料。在第二阶段，我们提取粗糙的细胞壁。在第三阶段，我们使用上格拉夫治疗，其中上皮草是由醋酸和硝酸，帮助从我们的样品去除血糖素和木质素。在第四阶段，我们通过硫酸使其进行酸水解，硫酸产生葡萄糖单体。最后，在第五阶段，我们使用炭铁检测来估计结晶纤维素含量。从温室收集植物材料，用水彻底清洗，空气干燥两天，分离组织，将组织转移到单独标记的容器中，在49摄氏度的孵化器中孵化10天。最后，用剪刀或刀片切成碎片。冻结组织，加载到砂浆和虫子细磨成均匀的粉末或替代使用冷冻机/磨坊。在这里，我们演示使用冷冻机/磨坊，所以我们加载我们的组织样品到这些研磨瓶，并把它们放在冷冻机/磨坊，并运行三个周期的10 CP速度。组织粉末收集在猎鹰管，如图所示。称20毫克的组织粉末，并转移到预称的两个mL管。添加一毫升蛋白质溶解缓冲器以去除蛋白质。旋涡。离心机在 15，000 RPM 下五分钟。丢弃超自然人。再重复此步骤两次。在保留的颗粒中加入一毫升无菌水。旋涡。离心机在 15，000 RPM 下五分钟。再重复此步骤两次。在保留的颗粒中加入一个 70% 乙醇的 ML。旋涡。转移到预热的70摄氏度区块，同时摇晃一小时。经过一个小时的孵化，离心机在15，000 RPM下5分钟。再重复一次此步骤。在保存的颗粒中，添加100%甲基酮的一毫升。旋涡。离心机在 15，000 RPM 下五分钟。加入一毫升氯仿甲醇。旋涡。离心机在 15，000 RPM 下五分钟。丢弃超自然，并添加一毫升的色酮。旋涡。在室温下孵化五分钟。离心机在 15，000 RPM 下五分钟。在 37 摄氏度的气温下重新丢弃超自然和空气干燥它们过夜或继续真空干燥。将样品放入真空干燥机，干燥两到三个小时，或直到颗粒完全干燥。重五毫克的细胞壁提取物从上一步获得。转移到一个两个mL螺丝帽管。在这一点上还包括积极的控制。为此目的使用两毫克的什么人滤纸。转移到两个mL螺丝帽管。在每个称重细胞壁提取物中加入 1.5 mL 的 Updegraff 试剂。在预热区块中以 100 摄氏度的漩涡和孵育 30 分钟。30分钟后孵化，转移到机架上。让它在长凳上冷却10分钟。离心机在 15，000 RPM 10 分钟。丢弃超自然，而不会干扰颗粒。加入一毫升无菌水。旋涡。离心机在 15，000 RPM 10 分钟。丢弃500微升的超自然人。添加一毫升的音调。旋涡。离心机在 15，000 RPM 下五分钟。丢弃超自然人的一毫升。添加一毫升的音调。旋涡。离心机在 15，000 RPM 下五分钟。完全丢弃超自然。添加一毫升的音调。旋涡。离心机在 15，000 RPM 下五分钟。丢弃超自然人。在37摄氏度下干燥过夜。经过一夜的孵化，转移到机架上。加入一升67%硫酸。旋涡。在25摄氏度下孵化一小时，同时摇晃。经过一个小时的孵化，确保颗粒完全溶解。转移到机架上。这是纤维素提取的最后一步。现在样品已准备好稀释样品。对于样品稀释，使用上一步每个样品的 10 微升，并添加 490 微升无菌水。对于所有实验样本重复此步骤。对于葡萄糖标准曲线准备，准备每毫升葡萄糖1毫克的新鲜库存，并准备零微克至200微克浓度，每毫升葡萄糖增加1毫克，并用无菌水将最终体积加到1毫克。在螺丝盖管中取下每个预制标准的 500 微升，并添加 1 mL 0.2% anthrone。现在样品已经准备好进行炭铁检测了。添加一毫升新准备的炭毒。立即通过漩涡混合并转移到冰上。以同样的方式重复所有标准和样品。然后转移到预热的100摄氏度区块10分钟。孵化10分钟后，转移到冰上，在冰上孵化5分钟。孵化后，在三个复制品中取取每个样本的 200 微升。以同样的方式重复标准和样品。一个装有96井的板子被装进了多模探测器。使用智能 Max Pro 程序，我们将滤镜设置设置设置为 620 纳米。板型到 96 井型。选择所有阅读区域。单击"确定"。读。阅读板。您将获得标准和样品的吸收读数，这些读数将收集在 Excel 表中，以便进一步分析和估计纤维素含量。葡萄糖标准曲线是利用 x 轴上每 mL 微克的葡萄糖浓度和 y 轴上 620 纳米的相应浓度的标准化吸收值来编制的。我们得到的回归线与m和c值在绘图散射图中，我们用来估计样品晶体纤维素含量。在第一步中，我们平均OD值为620纳米。然后，我们通过减去空白值使数据正常化。我们使用公式 x 计算葡萄糖浓度等于 OD 减去 c/m，其中我们从标准曲线中回归线的前一步插入 c 和 m 值。由于用于炭细检测的总体积为 500 微升，因此我们将其除以稀释因子 2。由于使用的样品体积只有10微升，我们将进一步除以10，以获得微克每微升葡萄糖浓度，这相当于毫克每毫升浓度。然后我们使用水分因子1.11，这是在这个过程中的水分增益。最后，我们通过将其与细胞壁重量（接近 5 毫克）除以并乘以 100 以获得百分比来计算晶体纤维素含量的百分比。然后，我们平均三个生物复制品的晶体纤维素含量，以获得单个样本的价值，这将与另一个实验线平均三个生物复制品进行比较。t 测试可用于测试其重要性。在表 A 中，我们显示浓度的表值和相应的 OD 值为 620 纳米，进一步用于绘制散射图和回归线，其中使用 C 中使用的 m 和 c 值来获取两个实验线之间的纤维素含量差异。最后，两个样本都显示出纤维素的差异。谢谢你的收看。

Research

JoVE Journal

Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

评估 乌西拉戈 病原性的快速高效方法，可在玉米和特奥辛特线上使用

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

科研

环境科学

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

单厂，无菌的缩影和结瘤的豆科植物生长<em&gt;苜蓿</em&gt;与根瘤菌共生体<em&gt;根瘤菌</em

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

木质纤维素生物质预处理低成本离子液体

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass

氨纤维膨胀（AFEX）预处理利诺纤维素生物量

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

利用酸性离子液体戊聚糖分析黄麻生物量及其转化为糖单体的新方法

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

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

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

循环介导等温扩增 (灯管) 法快速识别烟粉虱

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

BtM, a Low-cost Open-source Datalogger to Estimate the Water Content of Nonvascular Cryptogams

BtM, 一种低成本的开源数据采集器, 用于估算非血管化机制的含水量

Communities of nonvascular cryptogams, such as mosses or lichens, are an important part of the Earth's biodiversity, contributing to the regulation of the ...

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

木钻甲虫附着物扫描和透射电子显微镜的样品制备方法

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...