我们感谢清华大学的廖玉乐博士提供PVX-LIC载体。这项工作得到了德克萨斯州A&M AgriLife Research的启动基金和美国农业部国家食品和农业研究所的Hatch Project TEX0-1-9675的支持。

1. 种植马铃薯植物。
<ol><li>用Murashige和Skoog（MS）培养基加上Gamborg维生素（MS基盐混合物，Gamborg维生素，30g / L蔗糖，3.5 g / L琼脂，pH = 5.7）在培养管（25 x 150 mm）中繁殖马铃薯植物。将试管置于生长室中，温度为20-22°C，16小时光照/ 8小时黑暗光周期，光强度为120μmol/ m2∙s1。 注意：新芽和根通常在植物的1-2周内发育。每月用新鲜的MS / Gamborg维生素培养基繁殖植物。</li>
	<li>四周后，将体外植物移植到土壤中，并在20-22°C，16小时光照/ 8小时黑暗光周期和光强度120μmol/ m2∙s1下的温室中生长。 注：根叶新长的植物适合移栽。在前3-4天内保持新鲜移植植物的水分。</li>
</ol>2. 构造 VbMS 向量。
<ol><li>设计并克隆短串联TM分子（STTM， 图1）22，53 用于感兴趣的miRNA。 注：根据实验数据或从miRbase数据库获取miRNA序列54，55，56，57，58，59。本研究中使用的miR166序列之前已经描述过60。<ol>
<li>通过将不匹配序列（通常为5'-CTA-3'）插入到miRNA的反向补体序列中来设计TM模块，该序列位于与miRNA的第10-11个 核苷酸相对应的位点。 注意：例如，Stu-miR160序列是5'-UGCCUGGCUCCCUGUAUGCC-3'61，其中第10-11个核苷酸以粗体显示。反向补体序列（在脱氧核苷酸中）为5'-GGCATACAGGGAGCCAGGCA-3'，不匹配凸起插入位点以粗体显示。TM分子序列（脱氧核苷酸）应为5'-GGCATACAGG-CTA-GAGCCAGGCA-3'。</li>
		<li>设计用于克隆STM片段的引物（图1）。使用具有 48-nt 间隔序列的 DNA 寡核苷酸作为 PCR 克隆的模板。正向引物由LIC1连接子（5'-CgACgACAAgACCgT-3'），上述为TM分子设计的正向序列以及48-nt间隔物的部分5'序列（5'-GTTTTGTTGTTATGGT-3'）组成。反向引物由LIC2连接子（5'-gAggAgAagAgCCgT-3'），TM分子的反向补体序列和48-nt间隔物（5'-ATTCTTCTTCTTTAGACCAT-3'）的3'序列的部分反向补体组成。 注意：48-nt间隔序列是5'-GTTGTTGTTGTGTTCTAATTTAAATAATATGGTCTAAGAAGAAGAAT-3'。例如，对于STTM-miR160，前向引物应为5'-CgACgACAAgACCgT-GGCATACAGG-CTA-GAGCCAGGCA-GTTGTTGTTGTTGTTGGT-3';反向引物应为5'-gAggAgAagAgCCgT-TGCCTGGCTC-TAG-CCTGTATGCC-ATTCTTCTTCTTTAGACCAT-3'（图1）。</li>
		<li>使用合成的通用48-nt垫片作为模板和高保真DNA聚合酶，通过PCR扩增50μL体积的STTM片段。<ol>
<li>通过将每个引物0.5 μL（40 μM）、0.5 μL48-nt间隔物（40 μM）、5 μL 10x PCR缓冲液、1 μL dNTP混合物（每个10 mM）、0.1 μL高保真DNA聚合酶（10 U/μL）和43 μL ddH2O混合至总体积为50 μL来设置PCR反应。 94°C 3分钟，32次循环94°C持续45秒，60°C循环45秒，72°C循环60秒。</li>
		</ol>
</li>
		<li>通过乙醇沉淀纯化STTM片段。向PCR产物中加入2.5体积的乙醇和1/10体积的3M乙酸钠（pH = 4.0）。剧烈混合并以14，000× g 离心10分钟。除去上清液并用1mL 70%乙醇冲洗沉淀。干燥沉淀并将其溶解在20μL的ddH 2 O中。 注：使用DNA电泳检查STTM PCR产物的扩增。</li>
		<li>通过将2.5μL纯化的STTM PCR产物，0.5μL10x T4 DNA聚合酶缓冲液，0.05μL1M二硫脑油醇（DTT），0.25μL100mM dATP，0.1μLT4 DNA聚合酶（3 U / μL）和1.6μLdH2O混合至总体积为5μL，在冰上建立T4 DNA聚合酶反应。 并在75°C下处理产物20分钟以灭活T4 DNA聚合酶。</li>
	</ol></li>
	<li>准备基于 PVX 的 VbMS 构造。<ol>
<li>在100μL的体积中消化5μgPVX-LIC质粒38和2.5μLSmaI（20 U / μL）。 </li>
		<li>向消化后的PVX-LIC产品中加入等体积的苯酚：氯仿：异丙醇（25：24：1，pH = 6.7/8.0）并剧烈混合。以14，000× g 离心10分钟，并将上清液转移到新的离心管中。加入等体积的氯仿：异丙醇（24：1）并剧烈涡旋。以14，000× g 离心10分钟。 注：PVX-LIC 载体包含用于克隆的 LIC 盒。PVX-LIC载体的LIC盒含有 ccdB 基因和耐氯霉素基因，需要使用含有卡那霉素（50μg/ L）和氯霉素（15μg/ L）的LB培养基在 大肠杆菌 菌株DB3.1中维持/繁殖。</li>
		<li>将上清液转移到新的离心管中。加入2.5体积的乙醇和1/10体积的3M乙酸钠（pH = 4.0）并剧烈混合。以14，000× g 离心10分钟，除去上清液。</li>
		<li>用1mL 70%乙醇冲洗沉淀并剧烈涡旋。以14，000× g 离心10分钟，除去上清液。干燥沉淀并用100μLddH 2O溶解消化的PVX-LIC质粒。</li>
		<li>通过将2.5μL消化的PVX-LIC载体DNA，0.5μL10x T4 DNA聚合酶缓冲液，0.05μL1 M DTT，0.25μL100mM dTTP和0.1μLT4 DNA聚合酶（3 U / μL）混合在冰上建立T4 DNA聚合酶反应。将混合物在37°C下孵育15分钟，并在75°C下处理产物20分钟至20分钟灭活T4 DNA聚合酶。</li>
	</ol></li>
	<li>使用 LIC 反应将 STTM 序列克隆到 PVX-LIC 载体中。混合T4 DNA聚合酶处理的STTM PCR产物（5μL）和T4 DNA聚合酶处理的PVX-LIC质粒（5μL）。在70°C下孵育5分钟，以0.1°C / s的斜坡冷却至22°C，并使用PCR机器在22°C下保持30分钟。 注意：将孵育时间延长至4°C过夜，以实现更高的LIC克隆效率。</li>
	<li>将5μL的LIC反应产物转化为 大肠杆菌 DH5α，并在含有50μg/ mL卡那霉素的LB板上生长62，63。通过PCR选择阳性菌落，并使用PVX-LIC载体的克隆引物和通用引物，然后进行测序。<ol>
<li>使用 PVX-LIC 正向引物 （5'-GTGTTGGCTTGCAAACTAGAT-3'） 结合用于 STTM 克隆的反向引物进行菌落 PCR，以鉴定阳性克隆。PCR条带的大小应为~300 bp。 注：通过终止器循环测序验证STTM片段的序列64，65。</li>
	</ol></li>
	<li>从经过验证的克隆中分离PVX-STTM质粒，并将其转化为农杆菌菌株GV3101，GV2260或EHA10562，63。通过 PCR 验证农杆菌菌落。 注：使用 PVX-LIC 的正向引物和 STTM 克隆的反向引物通过 PCR 确认 农杆菌菌 落。</li>
</ol>3. 在马铃薯植物中执行基于 PVX 的 VbMS 测定。
<ol><li>将4周龄的体外马铃薯植物移植到土壤中。移植的植物将在3-4天后进行VbMS测定。</li>
	<li>用含有PVX-STTM质粒的 农杆菌 接种马铃薯植物。 注意：对于马铃薯中的VbMS测定，同时进行 农杆菌介导的浸润和牙签刮擦接种。<ol>
<li>将含有 PVX-STTM 载体的阳性转化剂选取并接种到含有 50 μg/mL 卡那霉素和 50 μg/mL 利福平的 50 mL 液体 LB 中。在28°C培养箱中以220rpm生长16小时，直到OD 600 = 1.0。</li>
		<li>同时，将阳性的农杆菌菌落划到至少两个含有50μg/ mL卡那霉素和50μg/ mL利福平的新LB板上，并在28°C下生长1天。包括 PVX-LIC38，66 载体作为对照，将 PVX-GFP67，68 作为监测病毒传播。</li>
		<li>通过在室温下以3，400×g离心10分钟收集农杆菌液体培养物。重悬具有等体积浸润缓冲液（10 mM MgCl2，10 mM MES和200μM乙酰汗根，pH = 5.6）的农杆菌细胞，并调节至OD 600 = 1.0。在室温下孵育6小时。</li>
		<li>用1mL无针注射器将 农杆菌 培养物浸润到完全膨胀的叶子的轴侧。<ol>
<li>翻转并用一只手握住叶子，然后用一根手指从浸润部位的轴侧支撑叶片。用另一只手将注射器垂直到叶表面，并将 农杆菌 培养物浸入层的轴侧。</li>
		</ol>
</li>
		<li>从LB板中刮下 农杆菌培养物 ，并用牙签刮擦浸润的马铃薯植物的前一个或两个节间的茎表面。轻轻划伤茎的表皮。避免刺穿茎，这可能会对植物造成严重损害。</li>
	</ol>
</li>
	<li>在温室中以16小时光照/ 8小时黑暗光周期和120μmol/ m2∙s1 的光强度在22°C下生长浸润的植物。 注意：由miRNA沉默引起的表型通常出现在接种后2-4周（图2， 图3）。浸润后 VbMS 表型通常需要 10–20 天才能出现。VbMS表型取决于特定miRNA和靶基因的特性、生长条件和马铃薯品种。</li>
</ol>4. 执行表达式分析。
<ol><li>当表型出现在接种后2-4周时，用剪刀从VbMS植物中收集具有表型的组织，例如芽，叶，花或根，以及来自对照植物的组织。从收集的组织中分离总RNA。</li>
	<li>通过电泳检查RNA质量62，63 ，并通过用分光光度计测量OD260 吸光度来量化RNA浓度。</li>
	<li>使用茎环实时逆转录聚合酶链反应 （RT-PCR） 分析 miRNA 表达。<ol>
<li>对于特定的miRNA，设计一个茎环逆转录引物。茎环RT引物包含一个通用的5'主链和一个3'6-nt的特异性miRNA延伸。设计一个5'通用骨架（5'-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGAC-3'），形成茎环结构。（大写对应于反向重复序列，小写对应于循环区域）。</li>
		<li>形成环路的5'骨架序列的一部分用作后续PCR扩增的反向引物（骨架序列中的粗体斜体序列）。向茎环引物添加一个与感兴趣的miRNA的3'端反向互补的6-nt延伸序列，以提供逆转录的特异性（补充图1A）。 注意：设计Chen等人69和Erika Varkonyi-Gasic等人描述的茎环逆转录引物，如70，71，72所述。例如，Stu-miR160的茎环逆转录引物被设计为5'-GTCTCCTCTGGTGCagggtccggtattcGCACCAGAGGAGACGGCATA-3'。马铃薯miR165/166家族的茎环逆转录引物被设计为5'-GTCTTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGGGGG（A/G）A-3'。（粗体大写序列提供了特定miRNA逆转录的特异性）。</li>
		<li>通过在冰上混合50-200ng总RNA，1μL茎环逆转录引物（100μM），2μL10x缓冲液，0.2μLRNase抑制剂（40 U / μL），0.25μLdNTPs（每个10mM）和1μL逆转录酶（200 U / μL）来设置逆转录反应。将不含核酸酶的ddH 2 O加入到20μL的总体积中。</li>
		<li>使用脉冲逆转录程序执行逆转录。将逆转录反应混合物在16°C孵育30分钟，进行30°C的温度循环30秒，42°C30 s和50°C共1 s的温度循环，共60个循环，并通过在85°C下孵育5分钟来灭活逆转录酶。</li>
		<li>对于miRNA表达的实时PCR分析，请根据miRNA序列设计正向引物，但不包括与上述设计的茎环逆转录引物重叠的序列。在前向引物的5'处添加3-7-nt延伸，以优化长度，熔化温度和GC含量（补充图1）。 注意：通用反向引物是5'-GTGCAGGGTCCGAGGT-3'。例如，Stu-miR160的茎环逆转录引物被设计为5'-CGGCTGCCTGGCTCC-3'。miR165/166家族的茎环逆转录引物是5'-CGGCTCGGACCAGGCTT-3'。粗体序列用作引物优化的5'扩展。用于miRNA的茎环逆转录引物和实时荧光定量PCR引物可以使用miRNA引物设计工具73进行设计。</li>
	</ol></li>
	<li>通过标准逆转录聚合酶链反应 （RT-PCR） 合成靶标 mRNA 的 cDNA。将逆转录反应混合物在37°C下孵育60分钟，并通过加热至85°C灭活逆转录酶5分钟。 注意：（1）如果靶标mRNA未知，则使用psRNA靶向程序74 预测靶标mRNA。（2）mRNA的通用逆转录引物是锚定引物5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT</li>
	<li>为目标 miRNA 和靶 mRNA 设置实时荧光定量 PCR 反应。在冰上混合0.5μL模板cDNA，5μL2x实时PCR缓冲液与SYBR绿色，0.05μL每个正向和反向引物（40μM）和4.4μLdH 2 O，总体积为5μL。</li>
	<li>在95°C下孵育3分钟，在95°C下循环40次3秒，60°C孵育30秒。执行随后的熔融曲线分析，如下所示：在95°C下孵育15 s，在20°C / s的斜坡下冷却至60°C，在60°C下保持60 s，在0.2°C / s的斜坡上加热至95°C，并在95°C下保持15 s。使用ΔΔCt方法分析Ct值76，77并绘制具有标准误差的均值。 注：（1）靶标mRNA的实时荧光定量PCR引物可以按所述设计75。（2）马铃薯 多泛素10 基因可作为马铃薯正常化的内部对照。马铃薯 多泛素10 基因的前向引物为5'-ATGTTGCCTTTTTTGTGTGGTTG-3'，反向引物为5'-TTATTTATTCACATAACACGACAGTC-3'。（3）对于实时荧光定量PCR分析，污染和引物二聚体的形成可能会产生假阳性结果。为了监测非特异性扩增并增加实时荧光定量 PCR 分析的责任，建议在实时荧光定量 PCR 测定中包括不带模板和逆转录酶的对照。</li>
</ol>

我们提出了一种基于PVX的miRNA沉默系统，通过将STTM设计整合到PVX载体中来表征马铃薯中内源性miRNA的功能。VbMS系统被证明可以有效地沉默马铃薯中的miRNA165/166，马铃薯是跨植物物种的高度保守的miRNA家族。
TM方法已被开发用于干扰基于人工miRNA靶标模拟的miRNA的表达，该拟态旨在在miRNA互补序列内的预期切割位点处创建不匹配环，从而导致靶向miRNA的封存并阻止其活性<sup class="xr...

植物微RNA （miRNA） 的特征是 20–24 个核苷酸长、核编码的调节性 RNA1 ，在植物生物过程的几乎每个方面都起着重要作用，包括生长和发育2、3、光合作用和代谢4、5、6、7、激素合成和信号传导8、9、生物和非生物反应10，11，12，13，以及营养和能量调节14，15。植物miRNA的调节作用是良好编程的，通常在转录后水平上通过切割或翻译抑制靶标mRNA来实现。
在马铃薯中miRNA的鉴定、转录谱分析和靶标预测方面取得了巨大进展16、17、18、19、20、21。然而，由于缺乏高效和高通量的遗传方法，包括马铃薯在内的植物中miRNA的功能表征落后于其他生物体。通过标准功能丧失分析对单个miRNA进行功能分析具有挑战性，因为大多数miRNA属于具有相当遗传冗余的家族22。此外，单个miRNA可以控制多个靶基因23 ，几个不同的miRNA可以协同调节相同的分子途径24，25。这些特性使得难以表征特定miRNA或miRNA家族的功能。
miRNA的大部分功能分析在很大程度上依赖于具有明显局限性的功能增益方法。人工miRNA（amiRNA）方法利用内源性原代转录本（pri-miRNA）在高水平上产生miRNA，从而抑制靶基因表达26，27，28，29。然而，使用强本构35S启动子的活化标记和miRNA过表达通常会导致miRNA的表达升高，这些表达不代表体内条件，因此可能无法反映miRNA30的内源性功能。已经开发了一种替代方法，涉及表达在结合和/或切割位点中含有不易脱位突变的靶基因的miRNA抗性形式31，32，33。但是，由于转基因伪影，这种方法也可能导致对源自miRNA耐药靶基因的表型的误解。因此，应谨慎从这些功能获得研究中得出结论34。上述方法的另一个主要局限性是它们需要转换，这是劳动密集型和耗时的。此外，转基因依赖性方法几乎不适用于顽固的植物物种。因此，必须开发一种快速有效的功能丧失方法来解开miRNA的功能。
为了绕过转化过程的先决条件，通过将靶向模拟（TM）策略与病毒衍生载体相结合，建立了基于病毒的microRNA沉默（VbMS）。在VbMS系统中，人工设计的TM分子从病毒骨架瞬时表达，为解剖植物内源性miRNA的功能提供了一种功能强大，高通量和省时的工具35，36。VbMS最初是在边 楸 和番茄中与烟草拨浪鼓病毒（TRV）35，36，37 一起开发的，并且已经扩展到拟南芥，棉花，小麦和玉米，使用各种其他病毒表达系统，包括马铃薯病毒X（PVX）38，棉花屑病毒（ClCrV）39，黄瓜花叶病毒（CMV）40，41，42，中国小麦花叶病毒（CWMV）43和大麦条纹花叶病毒（BSMV）44，45。
马铃薯（Solanum tuberosum）是世界上第四大粮食作物，也是种植最广泛的非藜麦作物，主要是因为它具有高营养价值、高能量产量和相对较低的投入要求46。马铃薯的几个特点使其成为一种有吸引力的双子叶植物模型植物。它是一种营养繁殖的多倍体作物，具有高越交率，杂合性和遗传多样性。然而，迄今为止，还没有报告使用VbMS表征马铃薯中miRNA的功能。在这里，我们提出了一种基于结扎独立克隆（LIC）的马铃薯PVX的VbMS方法来评估miRNA在马铃薯植物中的功能38。我们选择了miR165/166家族来说明VbMS测定，因为miR165/166家族及其靶标mRNA和III类同源域/Leu拉链（HD-ZIP III）转录因子已被广泛表征22，47，48。 HD-ZIP III 基因是分生组织发育和器官极性的关键调节因子，miR165/166功能的抑制导致 HD-ZIP III 基因表达增加，导致顶端优势降低和叶极性异常模式等多向性发育缺陷22，35，38，41.与miRNA165/166沉默相关的易于评分的发育表型能够准确评估基于PVX的VbMS测定的有效性。
在这项研究中，我们证明了基于PVX的VbMS系统可以有效地阻断马铃薯中miRNA的功能。由于基于PVX的病毒诱导基因沉默（VIGS）系统已经在许多马铃薯品种中建立49，50，51，52，因此这种基于PVX的VbMS方法可能适用于广泛的二倍体和四倍体马铃薯品种。

<table border="0" cellpadding="0" cellspacing="0" style="width:691px;" width="691">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">100 &#181;M dATP and 100 &#181;M dTTP</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">TQAC136</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">3 M Sodium acetate, pH 4.0.</td>
			<td style="width:188px;">Teknova, Hollister, CA 95023, USA</td>
			<td style="width:120px;">#S0297</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetosyringone</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D2666-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Chloroform</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0757-950ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dimethyl sulfoxide, DMSO</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">D0798-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DTT</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0281-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">E. coli DB3.1</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for maintenance of PVX-LIC and pTRV2e containing the ccdB gene</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">E. coli DH5&#945;</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td>for the destination constructs generated by LIC cloning</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed</td>
			<td>ICL Specialty Fertilizers, Summerville, SC 29483, USA</td>
			<td style="width:120px;">G99260</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA, USA</td>
			<td>7958960001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isoamyl alcohol</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0944-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Koptec Pure Ethanol &#8211; 200 Proof</td>
			<td style="width:188px;">Decon Labs, King of Prussia, PA 19406 , USA</td>
			<td style="width:120px;">V1005M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MES</td>
			<td style="width:188px;">TCI America, Portland, OR 97203, USA</td>
			<td style="width:120px;">M0606-250G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MgCl2</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">MFCD00149781</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">M-MuLV Reverse Transcriptase</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0253L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Nano-drop spectrometer: NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">ND-ONEC-W</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Bio-Rad MyCycler PCR System</td>
			<td style="width:188px;">Bio-Rad, Hercules, California 94547, USA</td>
			<td style="width:120px;">170-9703</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PCR machine: Eppendorf Mastercycler pro</td>
			<td style="width:188px;">Eppendorf, Hauppauge, NY 11788, USA</td>
			<td style="width:120px;">950030010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">pH meter</td>
			<td style="width:188px;">Sper Scientific, Scottsdale, AZ 85260, USA</td>
			<td style="width:120px;">Benchtop pH / mV Meter - 860031</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.</td>
			<td style="width:188px;">VWR Corporate, Radnor, PA 19087-8660, USA</td>
			<td style="width:120px;">VWRV0883-400ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phytagel: Gellan Gum</td>
			<td>Alfa Aesar, Tewksbury, MA 01876, USA</td>
			<td style="width:120px;">J63423-A1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVX VIGS vector: PVX-LIC</td>
			<td style="width:188px;"></td>
			<td style="width:120px;"></td>
			<td style="width:167px;">Zhao et al., 2016</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:188px;">ThermoFisher, Waltham, MA 02451, USA</td>
			<td style="width:120px;">4485697</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Real-time PCR reagent: KAPA SYBR&#174; FAST qPCR Master Mix (2x) Kit</td>
			<td style="width:188px;">Roche Sequencing and Life Science, Kapa Biosystems, 
			Wilmington, MA 01887, USA</td>
			<td style="width:120px;">7959389001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Restriction enzyme: SmaI</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td>R0141S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Reverse transcription reagents: qScript cDNA SuperMix</td>
			<td>Quanta BioSciences, Gaithersburg, MD 20877 , USA</td>
			<td style="width:120px;">95107-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNA extraction Kit: E.Z.N.A. Plant RNA Kit</td>
			<td style="width:188px;">Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td>
			<td style="width:120px;">SKU: D3485-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">RNase Inhibitor Murine</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0314L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNAzol RT</td>
			<td style="width:188px;">Sigma-Aldrich, St. Louis, MO 63103, USA</td>
			<td style="width:120px;">R4533</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Soil: Metro-Mix 360</td>
			<td>Sun Gro Horticulture, Agawam, MA 01001-2907, USA</td>
			<td style="width:120px;">Metro-Mix 360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">T4 DNA polymerase and buffer</td>
			<td>New England BioLabs, Ipswich, MA 01938-2723 USA</td>
			<td style="width:120px;">M0203S</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

potato virus x-based microrna silencing (vbms) in potato.

基于病毒的microRNA沉默（VbMS）是一种快速有效的工具，用于植物中microRNA（miRNA）的功能表征。VbMS系统已被开发并应用于各种植物物种，包括 Nicotiana benthamiana，番茄，拟南芥，棉花和单子叶植物，如小麦和玉米。在这里，我们描述了一个详细的方案，使用基于PVX的VbMS载体来沉默马铃薯中的内源性miRNA。为了降低特定miRNA的表达，设计了目标miRNA的靶向模拟（TM）分子，将其整合到植物病毒载体中，并通过 农杆菌 浸润在马铃薯中表达，以直接与感兴趣的内源性miRNA结合并阻断其功能。

图2 显示了PVX-STTM165/166马铃薯植株（Katahdin），其叶组织从叶片的轴侧沿叶脉异位生长。还观察到更严重的表型，如喇叭形叶的形成。相比之下，在PVX控制工厂中没有观察到表型异常。这些结果表明，VbMS系统可有效抑制四倍体马铃薯植株的内源性miRNA功能，PVX-VbMS系统是确定特定miRNA或miRNA家族功能的强大遗传工具。
图3 显示了PVX-STTM...

Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

Potato Virus X-Based microRNA Silencing VbMS In Potato.

我们提出了基于马铃薯病毒X（PVX）的microRNA沉默（VbMS）系统的详细方案，以功能性地表征马铃薯中的内源性microRNA（miRNA）。将目标miRNA的靶向拟态（TM）分子整合到PVX载体中，并在马铃薯中瞬时表达，以沉默靶miRNA或miRNA家族。

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

potato-virus-x-based-microrna-silencing-vbms-in-potato

Research

JoVE Journal

Environment

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

3.0K 次观看.  Texas A&M University System. 我们提出了基于马铃薯病毒X（PVX）的microRNA沉默（VbMS）系统的详细方案，以功能性地表征马铃薯中的内源性microRNA（miRNA）。将目标miRNA的靶向拟态（TM）分子整合到PVX载体中，并在马铃薯中瞬时表达，以沉默靶miRNA或miRNA家族。

视频: Potato Virus X-Based microRNA Silencing VbMS In Potato.

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

科研

环境科学

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

EPA方法1615年测量和肠道病毒水中诺如病毒发生由文化部和RT-qPCR的的。 II。总可培养病毒试验

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

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.

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

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

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&ndash;Environment Interactions

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating a knowledge gap. To meet the challenge of improving crops to feed the growing global population, this gap must be bridged.
Physiological traits are considered key functional traits in the context of responsiveness or sensitivity to environmental conditions. Many recently introduced high-throughput (HTP) phenotyping techniques are based on remote sensing or imaging and are capable of directly measuring morphological traits, but measure physiological parameters mainly indirectly.
This paper describes a method for direct physiological phenotyping that has several advantages for the functional phenotyping of plant&#8211;environment interactions. It helps users overcome the many challenges encountered in the use of load-cell gravimetric systems and pot experiments. The suggested techniques will enable users to distinguish between soil weight, plant weight and soil water content, providing a method for the continuous and simultaneous measurement of dynamic soil, plant and atmosphere conditions, alongside the measurement of key physiological traits. This method allows researchers to closely mimic field stress scenarios while taking into consideration the environment&#8217;s effects on the plants&#8217; physiology. This method also minimizes pot effects, which are one of the major problems in pre-field phenotyping. It includes a feed-back fertigation system that enables a truly randomized experimental design at a field-like plant density. This system detects the soil-water-content limiting threshold (&#952;) and allows for the translation of data into knowledge through the use of a real-time analytic tool and an online statistical resource. This method for the rapid and direct measurement of the physiological responses of multiple plants to a dynamic environment has great potential for use in screening for beneficial traits associated with responses to abiotic stress, in the context of pre-field breeding and crop improvement.

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant&#8211;Environment Interactions

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant&#8211;environment interactions.

植物与环境相互作用实时生理表型的遥测重力平台

Food security for the growing global population is a major concern. The data provided by genomic tools far exceeds the supply of phenotypic data, creating ...