我们感谢东洋大学应用生物科学系的 Koudai 先生和群马县农业技术中心谷口先生的技术援助。我们还感谢 Shosaku Kashiwada 教授和东洋大学 Maheswari Rajagopalan 博士的建议。这项研究由东洋大学生命与环境科学研究中心部分支持。

注: 本研究使用催吐剂 (ipecacuanha), 因为它有助于分析内源性激素。
1. 诱导催吐剂不定芽的生长条件
<ol><li>制备无激素的 B5 培养基, 调整为 pH 值为 5.710, 并添加0.2% 结冷胶胶。热处理消毒。</li>
	<li>将25毫升的蒸压介质倒入无菌培养皿 (90 毫米 x 20 毫米)。</li>
	<li>在不育的丙烯酸板上, 用一把22刀片的外科手术刀切开8毫米茎的催吐剂苗, 并放置在培养基上 (图 1)。</li>
	<li>无激素 B5 培养基的培养在24摄氏度以下15µmol m−2 s−1的光在14小时 light/10-h 黑暗周期5周。</li>
	<li>在每个段中标识区域 I (顶端) 到 IV (基底) (图 1B)。每周一次显微镜下计算每个区域中不定芽的数量 > 0.3 毫米长度。</li>
</ol>2. 激素的提取和纯化
<ol><li>在四2毫升的样品管中放入5毫米氧化锆珠。</li>
	<li>在不育的丙烯酸板上使用外科手术刀将片段切成区域 I 至 IV (每长度2毫米)。</li>
	<li>收集八段的每个区域在不同的取样管 (10-30 毫克新鲜重量)。</li>
	<li>称重, 然后冻结液氮中的样品。</li>
	<li>用珠基均质机粉碎冷冻样品。</li>
	<li>在1毫升乙腈中悬浮被粉碎的样品, 其中含有生长素和中正的每个内部标准的 500 pg (d5-IAA, d5-, d5-tZR, d6-iP, d6-知识产权) 使用涡旋混合器。</li>
	<li>保持在4°c 为1小时, 然后离心机在 3500 x g在室温下5分钟。</li>
	<li>在含有 1% (v/v) 乙酸的 80% (v/v) 乙腈中清洗颗粒。离心机再次在 3500 x g室温下5分钟。将上清液 (从步骤2.7 和 2.8) 组合在一次性玻璃管中。</li>
	<li>添加600µL 含 1% (v/v) 乙酸的水, 每联合上清, 并蒸发的乙腈使用真空选矿厂。</li>
	<li>平衡亲水性-亲脂平衡 (HLB) 柱筒, 适用于每乙腈, 甲醇和含 1% (钒/钒) 乙酸的水的1毫升。</li>
	<li>每平衡 HLB 墨盒应用一个样本解决方案。</li>
	<li>用含有 1% (v/v) 醋酸的1毫升水清洗墨盒。</li>
	<li>洗脱所有激素与2毫升 80% (v/v) 乙腈含有 1% (v/v) 乙酸在玻璃管。</li>
	<li>将洗脱液中的乙腈蒸发, 用真空浓缩器在含有 1% (v/v) 乙酸的水中获得萃取物。 注: 不要完全干燥。</li>
	<li>平衡混合模式, 强阳离子交换 (MCX) 柱盒通过应用1毫升乙腈, 1 毫升甲醇, 0.5 毫升0.1 米 HCl, 和1毫升水含 1% (v/v) 乙酸。</li>
	<li>每个平衡 MCX 墨盒应用一个示例解决方案。</li>
	<li>用含有 1% (v/v) 醋酸的1毫升水清洗墨盒。</li>
	<li>洗脱 IAA 与2毫升 30% (v/v) 乙腈含有 1% (v/v) 乙酸在玻璃管。</li>
	<li>用2毫升 80% (v/v) 乙腈清洗墨盒, 含 1% (v/v) 乙酸。</li>
	<li>用2毫升水和1毫升水清洗墨盒, 含5% 水氨。</li>
	<li>洗脱中正与2毫升 60% (v/v) 乙腈含有5% 水氨的玻璃管。</li>
	<li>使用真空浓缩器和存储在−30°c, 直到 LC-ms/毫秒分析, 蒸发每个荷尔蒙分数的溶剂。</li>
</ol>3. IAA 和中正的 LC-ms/毫秒分析
<ol><li>将每个激素提取物溶解在600µL 甲醇的管中, 将溶液转移到螺钉颈部总回收瓶中。使用真空浓缩器蒸发溶剂。</li>
	<li>将20µL 30% (v/v) 乙腈中的 IAA 分数和20µL 含 1% (v/v) 乙酸的 CK 分数溶解在螺钉颈总回收瓶中。</li>
	<li>以高效液相色谱系统为例, 分析了三元四极 MS 系统正离子模式下的样品。 注意: 我们设置了表 1中列出的高效液相色谱条件。<ol>
<li>对于 IAA 洗脱, 使用 5%-50% 溶剂 b 的二进制梯度超过7分钟, 然后增加98% 溶剂 b 和保持1分钟, 然后平衡2分钟在5% 溶剂 b 通过巢注射。</li>
		<li>对于 CK 洗脱, 使用 2%-40% 溶剂 b 的二进制梯度超过5分钟, 40%-70% 溶剂 b 在7分钟, 然后增加95% 溶剂 b 和举行1分钟, 然后平衡2分钟在2% 溶剂 b 通过巢注射。</li>
		<li>设置离子源的电喷雾电离 (ESI)-MS 参数, 如表 2所示。</li>
	</ol>
</li>
	<li>使用多重反应监测 (MRM) 转换来量化表 3中列出的每个分析物。</li>
	<li>将内源 IAA 和 CK 水平与未标记比率与氘标记标准的标准曲线进行量化。</li>
</ol>

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作者声明他们没有利益冲突。

为了确定参与器官激素的分布, 重要的是使用植物材料在无激素培养基上观察器官, 因为当激素被外部应用于外植体诱导芽或根, 它们影响整个外植体, 使得很难评价植物的内在可塑性的细胞分化和器官。不定芽可诱导其他植物的无激素培养基 (如石竹石竹L.11、 Aegle marmelos (l.) Corrêa12、 Bacopa 床子(l.) Pennell13, 藤苋菜。14和Kalanchoë长寿Poelln。15. 有可能在这些植物物种中应用该议定书。
我们提取乙腈中的 IAA、tZR、iP 和知识产权, 并通过固相萃取纯化。原方法使用三种类型的墨盒柱 (HLB, MCX 和弱阴离子交换 (蜡)), 因为所有激素被纯化 (包括赤霉素, 脱落酸, 茉莉酸和水杨酸)16。HLB 柱使用聚合物反相吸附剂, MCX 柱使用相同的阳离子交换组, 蜡柱使用相同的弱阴离子交换组。原方法 elutes 中正 (基本) 与60% 乙腈含水氨在 MCX 柱第二步, 然后 IAA (酸性) 与80% 乙腈含1% 醋酸在蜡柱上的最后一步。由于我们的重点是生长素和中正, 它相互作用 antagonistically 调节植物生长17, 简化协议只使用 HLB 和 MCX 列;IAA 是洗脱与30% 乙腈含有1% 乙酸在 HLB 塔的第一步。从样品准备到 LC/ms 分析需要两天时间。
乙腈溶剂不应被允许在植物激素纯化的弹药筒柱上干燥。如果有, 并用重悬样品在乙腈, 以防止激素成为粘在玻璃管和丢失的样品。在该协议中, 离子阱 MS 系统的检测限为 IAA 和中正10-30 毫克新鲜组织的 10 pg。为了分析较小的数量, 需要收集更多的样本或使用更高灵敏度的 MS。
激素分析是评价植物生长发育的重要技术。利用这种方法, 我们可以确定植物生长素和 CK 治疗的时机, 在最适宜的培养条件尚不清楚的植株中, 不定芽的形成。随着激素量化变得越来越重要, 本文介绍的 LC ms/毫秒协议将使对小样本的分析具有高灵敏度和分辨率。我们以前的方法的简化将有助于净化和分析, 并带来了高通用性和重现性。在今后的研究中, 该方法可应用于其他植物种间生长素和 CK 的动态变化。

Haberlandt (1854-1945) 提出了 "全能" 的概念, 在成熟植物中, 植物细胞可以分化、分化和再生整个植物, 甚至在它们的前向特定细胞类型的差异1。在组织培养中, 能否诱导植物再生是由外部在生长培养基中应用激素的组合和浓度决定的。Skoog 和米勒发现, 在含有高中正比植物生长素的培养基上, 烟草愈伤组织可诱导不定芽, 而不定根可在含有低比值2 的培养基上诱导。自此发现以来, 组织培养已广泛用于经济重要作物的繁殖和转基因植物的再生3。不定芽可诱导从其他组织以外的茎尖分生, 如叶, 根, 和节间。在大多数植物中诱导不定芽需要激素治疗。然而, 最佳的浓度和组合不同的物种和组织之间作为外植体。因此, 大量的努力去确定激素的最佳浓度和组合的实验。
Carapichea ipecacuanha(Brot)l. 安德森 (催吐剂) 是一种药用植物, 含有吐根碱和 cephaeline 等生物碱, 主要在根4 中。根提取物用作祛痰、催吐剂和 amoebicide5。虽然催吐剂自然生长在巴西的热带雨林中, 但它不愿意在文化中设置种子, 在日本的种子储藏过程中, 发芽率下降, 气候较冷的6。相反, 它传播的组织培养, 在节间不定的拍摄形成是最有效的方法7,8。有趣的是, 不定芽可以诱导在这个物种没有激素治疗8。
不定芽形成于茎节顶端区域的表皮上, 不愈伤组织, 但不在基底区域9。这种差异表明茎段的组织极性, 这可能是在 phytohormonal 调节。催吐剂培养系统允许一个独特的机会来分析在不定芽形成期间内源激素水平的变化。在这里, 我们介绍了分析一个生长素 (indole-3-acetic 酸 (IAA)) 和四中正 (isopentenyl 腺嘌呤 (iP), isopentenyl 腺嘌呤核苷 (iPR),反式-玉米 () 和反式-玉米核苷 (tZR) 的内生水平的方法。在茎段中通过使用 LC/ms。

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		<col />
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			<td height="21" style="height:21px;width:216px;">[2H5]indole-3-acetic acid</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">031 1531</td>
			<td style="width:167px;">Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]trans-zeatin</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0301</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[2H5]trans-zeatin riboside</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0311</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H6]N6-isopentenyl adenine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0161</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">[2H6]N6-isopentenyl adenosine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0171</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">indole-3-acetic acid</td>
			<td style="width:176px;">Wako</td>
			<td>098 00181</td>
			<td>standard for LC-MS/MS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trans-zeatin</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">Z0876 5MG</td>
			<td>standard for LC-MS/MS</td>
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			<td height="21" style="height:21px;">trans-zeatin riboside</td>
			<td style="width:176px;">Wako</td>
			<td>262 01081</td>
			<td>standard for LC-MS/MS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenine</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">D7674 1G</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenosine</td>
			<td style="width:176px;">ACROS ORGANICS</td>
			<td style="width:119px;">22648 1000</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">acetonitrile hypergrade for LC-MS LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.00029.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Water for chromatography LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.15333.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:176px;">SHIMADZU</td>
			<td style="width:119px;">Prominence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS</td>
			<td style="width:176px;">Sciex</td>
			<td style="width:119px;">3200QTRAP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis HLB 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">WAT094225</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">screw neck total recovery vial</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002805</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000274</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Acquity UPLC BEH C18, 2.1x100 mm</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002350</td>
			<td>UPLC column</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Proshell 120 EC-C18, 2.1x50 mm</td>
			<td style="width:176px;">Agilent</td>
			<td style="width:119px;">699775-902</td>
			<td>UPLC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">DHS1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser II</td>
			<td style="width:176px;">QIAGEN</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 4</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Savant SpeedVac/Refregerated vapor trap</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SPD111/RVT4104</td>
			<td>vacuum concentrartor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable glass tobe (13x100 mm)</td>
			<td style="width:176px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile petri dish</td>
			<td style="width:176px;">INA OPTICA</td>
			<td style="width:119px;">I-90-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

不定芽形成是重要的技术, 对重要作物的繁殖和转基因植物的再生。在大多数物种中诱导不定芽需要激素治疗。不定芽能否诱发是由生长素与细胞分裂素 (CK) 水平的平衡决定的。大量的努力, 以确定最佳的浓度和组合激素在每个组织作为外植体和在每个植物物种。在催吐剂中, 无激素处理可诱导培养基中茎段的不定芽。这使得催吐剂的内在可塑性可以被评估。为了诱导催吐剂中的不定芽, 我们在15µmol m−2 s−1的茎段中培养了24摄氏度, 在14小时 light/10-h 黑循环中, 在无激素 B5 培养基上固化0.2% 结冷胶胶5周。通过液相色谱-串联质谱 (LC ms) 测定了不定芽形成过程中的激素动态, 并对 indole-3-acetic 中的内源性酸和中正进行了检测。这种方法可以简单地分析内源性 indole-3-acetic 酸和中正水平。可用于研究其他植物器官中内源生长素和 CK 的动态变化。

在 1st周, 没有形成不定芽。在 2nd周, 出现了小芽。在 3rd和 4th周, 芽数增加主要在顶端区域 (I 和 II) (图 2A)。在 5th周, 芽的数量在区域 i 和 5 (图 2B) 中大约为7。相比之下, 第三和 iv. 区只形成了几枝芽。
在培养之前, IAA 水平略高在区域 I (4.1 pg/毫克, 新鲜的重量 (FW)) 比在区域 II IV (~ 2.5 pg/毫克固件;图 3)。在 1st周, IAA 水平在区域 IV (11.4 pg/mg fw) 中大大增加, 在区域 I III (1.5-2.2 pg/毫克固件) 中略有下降。在 2nd周, 区域 IV 中的 iaa 水平下降至4.4 页/毫克 FW, 表明在基底区域的 iaa 积累是瞬态的。经过5周的培养, IAA 浓度梯度出现, 从区域 I 到区域 iv 的水平增加。
在区域性之前, 只有大多数中正的跟踪级别 (图 3)。在 1st周, tZR 级别增加到第二个区域中的 13.8 pg/mg 固件, 以及第三区域的 18.1 pg/mg 固件。在5周的文化中, 水平逐渐下降。另一方面, 在文化背景下, "区域"、"知识产权" 和 "知识产权" 的水平只略有变化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56902/56902fig1.jpg"> 图 1: 不定射形成催吐剂的制备.(A) 茎段 (8 毫米长) 从干净的工作台上的再生催吐剂中切割。(B) 第一个节间用于不定射形成。(C) 在培养皿中培养25毫升无激素的 B5 培养基以诱导不定芽。刻度条 = 1 厘米.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56902/56902fig2.jpg"> 图 2: 在茎段上形成的不定芽的分布.(A) 在0到5周的文化之后形成的不定芽。刻度条 = 5 毫米 (B) 段被分成四个区域 (I IV), 每个区域的不定芽数量在5周计数。数据是指电子扫描电镜 (n = 3)。在每个实验中使用了十段。N.F. = 未找到。此数字已从小池et . 中进行了修改。9<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56902/56902fig3.jpg"> 图 3: 茎段中激素水平的时间过程分析.段分为四个区域 (IV.)。各区域内源 IAA 和中正 (tZR、iP 和知识产权) 均以 LC-ms/毫秒为量化。由于 iP 和知识产权级别非常低, 因此在同一图形中插入了一个放大的图形。数据是指电子扫描电镜 (n = 3)。在每个实验中使用了八段。此数字已从小池et . 中进行了修改。9<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56902/56902fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>列</td>
			<td>IAA: ACQUITY BEH C18 φ2.1×100 1.7 µm</td>
		</tr>
<tr>
<td></td>
			<td>中正: Poroshell EC-C18 φ2.1×50 2.7 µm</td>
		</tr>
<tr>
<td>温度</td>
			<td>40ºc</td>
		</tr>
<tr>
<td>移动阶段</td>
			<td>溶剂 A: 蒸馏水 + 0.05% 乙酸</td>
		</tr>
<tr>
<td></td>
			<td>溶剂乙: 乙腈 + 0.05% 乙酸</td>
		</tr>
<tr>
<td>流量</td>
			<td>0.35 毫升/分</td>
		</tr>
<tr>
<td>注塑量</td>
			<td>18µl</td>
		</tr>
</tbody></table>表 1: IAA 和 CK 分析中的 HPLC 条件。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>窗帘气 (天文单位) *</td>
			<td>10/40 **</td>
		</tr>
<tr>
<td>碰撞气体 (天文单位) *</td>
			<td>5/3 **</td>
		</tr>
<tr>
<td>离子喷涂电压 (V)</td>
			<td>5500</td>
		</tr>
<tr>
<td>温度 (ºC)</td>
			<td>600</td>
		</tr>
<tr>
<td>离子源气体 1 (天文单位) *</td>
			<td>30</td>
		</tr>
<tr>
<td>离子源气体 2 (天文单位) *</td>
			<td>40/80 **</td>
		</tr>
<tr>
<td>决议</td>
			<td>单位</td>
		</tr>
</tbody></table>表 2: 离子源的参数.* 任意单位。** IAA 分析/CK 分析。此表已从小池et . 中进行了修改。9
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>Q1 (m/z)</td>
			<td>Q3 (m/z)</td>
			<td>分割电位 (V)</td>
			<td>入口潜力 (V)</td>
			<td>碰撞能量 (V)</td>
		</tr>
<tr>
<td>iaa</td>
			<td>176</td>
			<td>130</td>
			<td>26</td>
			<td>6。5</td>
			<td>21</td>
		</tr>
<tr>
<td>d5-IAA</td>
			<td>181</td>
			<td>134</td>
			<td>36</td>
			<td>4。0</td>
			<td>23</td>
		</tr>
<tr>
<td>tz</td>
			<td>220</td>
			<td>136</td>
			<td>36</td>
			<td>5。0</td>
			<td>23</td>
		</tr>
<tr>
<td>d5-</td>
			<td>225</td>
			<td>137</td>
			<td>31</td>
			<td>5。0</td>
			<td>21</td>
		</tr>
<tr>
<td>tZR</td>
			<td>352</td>
			<td>220</td>
			<td>41</td>
			<td>5。5</td>
			<td>23</td>
		</tr>
<tr>
<td>d5-tZR</td>
			<td>357</td>
			<td>225</td>
			<td>51</td>
			<td>5。0</td>
			<td>21</td>
		</tr>
<tr>
<td>ip</td>
			<td>204</td>
			<td>136</td>
			<td>31</td>
			<td>9。0</td>
			<td>19</td>
		</tr>
<tr>
<td>d6-iP</td>
			<td>210</td>
			<td>137</td>
			<td>31</td>
			<td>5。5</td>
			<td>21</td>
		</tr>
<tr>
<td>知识产权</td>
			<td>336</td>
			<td>204</td>
			<td>31</td>
			<td>5。0</td>
			<td>21</td>
		</tr>
<tr>
<td>d6-知识产权</td>
			<td>342</td>
			<td>210</td>
			<td>36</td>
			<td>5。5</td>
			<td>21</td>
		</tr>
</tbody></table>表 3: IAA 和中正在 LC/ms 分析中的 MRM 过渡.此表已从小池et . 中进行了修改。9

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

催吐剂节间培养中内源生长素和细胞分裂细胞的量化

无激素治疗的催吐剂茎段可诱发不定芽。为了评价不定芽形成过程中的激素动态, 我们用 LC-ms/ms 测定了茎段内源性生长素和细胞分裂素。

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

Nanyang Technological University

Research

JoVE Journal

Environment

8.9K 次观看.  Toyo University. 无激素治疗的催吐剂茎段可诱发不定芽。为了评价不定芽形成过程中的激素动态, 我们用 LC-ms/ms 测定了茎段内源性生长素和细胞分裂素。

视频: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

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. ...

科研

环境科学

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

在排水过程中捕获农用运河中的流动加权水和悬浮颗粒

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

百里香精油应用于肉类干燥过程中微生物负荷的影响

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

基于同步辐射 x 射线衍射的岩石和矿物团聚体冷压缩应力分布

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

爬行动物皮肤脂类的化学分离、定量和分离

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

热光学有机/元素碳测量的分割点分析与不确定性定量

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

用于扫描电子显微镜的胚胎、蛋壳和真菌培养

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

具有腐蚀性烟气的台式光生物反应器中的微藻培养和生物量定量

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...