这项工作得到了聊城大学研究基金（318012028）和山东省自然科学基金（ZR2020MC034）的支持。

1. 植物生长条件与根茎培养 
<ol><li>播种 注：野生大豆种子采自中国聊城阳谷县;美洲南瓜和 南 瓜当地品种银苏的种子是从市场上购买的。<ol><li>收集野生大豆，美洲南瓜和当地 南瓜品种的种子，将它们播种在1厘米深的蛭石中，并彻底浇水。将 20 颗种子种植在 8 厘米 x 11 厘米 x 9 厘米的塑料盒中。在24±2°C的生长室中以约70%相对湿度进行16小时光照/ 8小时黑暗循环培养植物。 注意：播种前必须打破野生大豆种子的外套。如果研究问题集中在植物和微生物之间的相互作用上，种子、蛭石、塑料盒和水在使用前必须进行消毒。</li>
		<li>根茎曲霉K599的活化和培养 注意：根茎A. rhizogenes K599菌株有一个二元载体pRed13052 携带红色荧光报告基因 DsRed2。
		<ol><li>从-80°C冰箱中取出根 茎曲霉 菌株K599，并在固体LB培养基（用50mg / L卡那霉素和50mg / L链霉素）上在28°C下活化细菌48小时。</li>
			<li>挑选菌株K599的单个克隆，并将其在1mL含液体抗生素的LB培养基中培养12小时。</li>
			<li>将500μL细菌悬浮液均匀地散布在含有固体抗生素的LB培养基上，然后在28°C下孵育24小时。</li>
		</ol></li>
	</ol></li>
</ol>2. 一步法 A . 根茎介导的毛根转化方法
<ol><li>下胚轴切口<ol><li>7天后（将种子的播种定义为第0天），幼苗子叶刚刚展开（图1A），使用无菌的锋利手术刀切割约0.5-1厘米的下胚轴（图1B）。丢弃主根和部分下胚轴。 注意：请小心使用手术刀。</li>
	</ol></li>
	<li>K599接种<ol><li>用K599细菌涂覆下胚轴切口（图1C）。</li>
		<li>将幼苗种植在潮湿的蛭石中（图1D）。</li>
		<li>通过根 茎介导的毛根转化感染每个物种的 30 株植物。在四分之一强度（0.25x）Gamborg B-5碱性培养基中用5mL重悬的K599细菌悬浮液（OD600 = 0.5-0.6）浇灌每株植物（图1E）。</li>
		<li>用高度透明的塑料袋（图1F）盖住花盆，并将它们放入生长室中。</li>
	</ol></li>
</ol>3.毛根生产
<ol><li>接种后种植植物~12天，将在切口部位诱导并产生新的毛根。15天后，毛根长度通常达到2-5厘米（图2）。</li>
	<li>为了确定产生的毛根是否是转基因的，检查依赖于K599中转化载体的报告基因的表达。<ol><li>使用化学发光成像系统检测报告基因DsRed2的表达，绿色激发光在540nm处，发射波长为600nm（图2B，图2D和图2F）。</li>
	</ol></li>
</ol>

一步法毛状根转化方法推进根相关生物学

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作者没有利益冲突需要声明。

一步法根茎介导的毛根法是比下胚轴注射法更简单、更有效的复合植物生产方法。一步法ARM方法显著提高了毛根转化效率，缩短了产生毛根的时间，增加了毛根的数量，减少了所涉及的工作量。改进的转化方案最适合研究豆科植物与根瘤菌之间以及植物与丛枝菌根真菌之间的共生关系。这可以归因于这样一个事实，即在产生毛根后不需要移植复合植物，这避免了移植过程中发生的非接种菌株污染。此外，一种植物（南瓜）的转化效率为100%。
以下原因可以解释为什么一步法ARM方法在毛根转化中比下胚轴注射方法更有效。首先，虽然去除了主根，但保持了幼苗蒸腾作用。因此，蒸腾作用拉动促进了根 际真菌对 切口中下胚轴细胞的侵袭。其次，下胚轴切口引起的伤口面积大于下胚轴注射法引起的伤口面积，因此，更多的植物细胞被 根茎曲霉感染。最后，去除主根后，将下胚轴切口埋在黑暗潮湿的蛭石中，这是产生根的有利环境18。

根茎农杆菌是根茎科革兰氏阴性土壤细菌。 A. rhanzogenes可以通过伤口感染几乎所有的双子叶植物，少数单子叶植物和单个裸子植物，在受感染的植物中产生毛根。细菌携带Ri（发根）质粒，Ri质粒的T-DNA携带opine合成基因和rol基因（根位点基因）。Ri质粒的T-DNA进入植物细胞并整合到宿主染色体中后，rol基因的表达诱导毛根的产生1。携带靶基因的植物二元表达载体转化为根茎曲霉，转化后的根茎曲霉用于感染植物。转基因根可以在受感染的植物中诱导，产生含有转基因根和非转基因茎和芽的复合植物。通常，复合植物可以在14-20天内获得。根茎介导的毛根转化在双子叶植物中一般不受基因型的限制2。根茎感染植物产生的毛根具有生长速度快、遗传稳定、操作方便等特点。由根茎A. rhizogene介导的毛根转化目前广泛用于研究与根相关的生物学。此外，毛根的转化也可用于验证和优化CRISPR/Cas9系统3,4,5和蛋白质亚细胞定位的靶点编辑效率。因此，毛根转化是研究植物基因功能、代谢工程以及根与根际微生物相互作用的重要工具6,7,8。
通过毛根转化获得的含有转基因根的复合植物已在双子叶植物中广泛生产，特别是在豆科植物中。将下胚轴注射根茎曲霉的传统方法已被用于生产复合莲花9，大豆10，番茄11，甘薯12和许多其他植物5,8。下胚轴注射方法效率低下，可能导致幼小或微小的下胚轴植物死亡。因此，通过切断胚根，用根茎涂覆幼苗切口，然后将下胚轴置于无菌培养基上进行生根栽培来改进该方法13。但是，这些步骤是在无菌环境中进行的，并且操作步骤相对繁琐。特别是，由此产生的复合植物需要移植，这增加了工作量。在以前的工作中，在黄瓜、大豆、粳米莲、芥芷和番茄2,14,15,16,17中建立了一步法根茎介导的（ARM）毛根转化。去除主根和部分下胚轴，在剩余下胚轴的切口部位涂覆转化根茎，然后将幼苗种植到湿润的无菌蛭石中。栽培12天后，切口部位产生毛根。一步法 ARM 方法非常高效，产生毛根所需的时间更少。形成毛根后移植也是没有必要的。由于无需移植即可避免微生物污染，因此一步法ARM方法在研究植物与微生物之间的相互作用时特别有用，例如豆科植物与根瘤菌之间的共生固氮，以及植物与丛枝菌根真菌之间的共生。本文提供了详细的一步法根茎介导的毛根转化方案，并以野生大豆、美洲茄和南瓜中生产的复合植物为例。使用该协议，研究人员可以顺利地执行一步式ARM转换。

<table><tbody><tr><td>kanamycin</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A506636</td><td></td></tr><tr><td>LB medium</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B540113</td><td></td></tr><tr><td>plastic box</td><td>LiaoSu</td><td></td><td>8 cm x 11 cm x 9 cm</td></tr><tr><td>pumpkin</td><td></td><td></td><td>local variety Yinsu</td></tr><tr><td>streptomycin</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A610494&amp;nbsp;</td><td></td></tr><tr><td>Tanon-5200Multi machine</td><td>Tanon Co., Ltd., China</td><td>5200Multi</td><td>chemiluminescence imaging system</td></tr><tr><td>tomato</td><td></td><td></td><td>local variety Zhongshu4</td></tr><tr><td>wild soybean</td><td></td><td></td><td>collected in Yanggu County, Liaocheng, China</td></tr></tbody></table>

an efficient and reproducible method for producing composite plants by agrobacterium rhizogenes-based hairy root transformation

使用根茎农杆菌介导的毛根转化生产具有转基因根和非转基因茎芽的复合植物是研究 根相关生物学的有力工具。毛根转化在广泛的双子叶植物和几种单子叶植物中建立，并且几乎独立于基因型。传统的下胚轴注射根 茎得到 复合植株的方法效率低下、耗时、费力，且经常导致嫩小下胚轴植株死亡。此前建立了以根 茎曲霉 介导的高效一步法毛根转化，无需在产生毛根后进行移植。本研究切除部分下胚轴和主根，下胚轴切口部位涂有 根茎曲霉， 然后将下胚轴种植在无菌蛭石中。栽培12天后，下胚轴切口扩大，诱导新的毛根。本文提供了由 根茎介导的一步转化方法的详细方案，其有效性通过生产野生大豆、 美洲茄和南瓜的复合植物来证明。

高效一步法根 茎介导的毛根转化 在接种工程K599后12天，在下胚轴切口部位产生毛根。根据二元载体中所含报告基因的表达确定转基因毛根。在自然（图2A、图2C和图2E）和绿色激发光（图2B、图2D和 图2F）下观察用复合野生大豆、美洲沙门氏菌和南瓜的报告基因DsRed2转化的转基因根。
当复合植物含有至少一个转基因根时，它被指定为转基因复合植物。3个品种各接种30株中，野生大豆28株、美洲南瓜18株、 南瓜 30株为转基因复合材料。因此，毛根转化效率分别为93.3%（大豆）、60%（美洲南瓜）和100%（南瓜）。对三种类型植物的比较表明，具有厚下胚轴的植物比具有薄下胚轴的植物产生更多的转基因毛根。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65688/65688fig01.jpg"> 图 1：一步法 A. 根茎介导的毛根转化。 （A）七日龄南瓜幼苗。（B）K599细菌溶液中下胚轴切口的顶端部分。（C）K599细菌团块涂覆下胚轴切口。（D）外植体种植在潮湿、无菌的蛭石中。（E） 用 5 mL 重悬的 K599 细菌溶液在四分之一强度 （0.25x） Gamborg B-5 碱性培养基中浇水。（F） 高透明塑料袋覆盖物。比例尺 = 1 厘米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65688/65688fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65688/65688fig02v2.jpg"> 图2：从一步法根茎介导的毛根转化中获得的复合植物。 （A，B）野生大豆，（C，D）美洲茄和（E，F）南瓜在（A，C，E）天然和（B，D，F）绿色激发光下复合植物的根。白色箭头表示转基因毛根;黑色箭头表示非转基因毛根。比例尺 = 1 厘米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65688/65688fig02largev2.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation at JoVE.com

作者聚焦：通过根瘤农杆菌介导的毛状根转化简化复合植物生产 

在这里，我们提供了由 根癌农杆菌 介导的一步转化方法的详细协议，以生产复合植物。

一种基于根 茎农杆菌毛根转化生产复合植物的高效且可重复的方法

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

我们的团队专注于基于根茎农杆菌的毛根转化。毛茸茸的根转化对于研究植物基因功能、代谢工程以及根与根际微生物之间的相互作用至关重要。本文提供了一种由根茎农杆菌介导的一步转化方法。毛根转化在各种双子叶和单子叶植物种中建立，并且几乎与基因型独立。与下胚轴注射方法相比，根茎农杆菌介导的一步法用于诱导毛根，在创建复合植物方面更简单、更有效。下胚轴注射方法效率低下，可能导致幼小或微小的下胚轴植物死亡。一步法根茎农杆菌介导的方法非常有效，需要更少的时间来产生毛根。而且，没有必要移植过去毛根的形成。α-征调控基因和β-征合成基因作为根茎农杆菌介导的毛根转化的病毒筛选标志物。

an-efficient-reproducible-method-for-producing-composite-plants

Nanyang Technological University

Research

JoVE Journal

Biology

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

1.9K 次观看.  Liaocheng University. 我们的团队专注于基于根茎农杆菌的毛根转化。毛茸茸的根转化对于研究植物基因功能、代谢工程以及根与根际微生物之间的相互作用至关重要。本文提供了一种由根茎农杆菌介导的一步转化方法。毛根转化在各种双子叶和单子叶植物种中建立，并且几乎与基因型独立。与下胚轴注射方法相比，根茎农杆菌介导的一步法用于诱导毛根，在创建复合植物方面更简单、更有?...

视频: 作者聚焦：通过根瘤农杆菌介导的毛状根转化简化复合植物生产 

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 ...

科研

环境科学

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化 （法戈皮鲁姆塔塔里库姆）

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

遗传学

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

高效的聚乙二醇（PEG）介导的转化苔小立碗藓</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

生物学

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

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

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

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

复合植物代苜蓿truncatula</em用于检测结瘤

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

转基因大豆毛状根的高效生产

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

作者聚焦:用于基因功能分析的大豆毛状根转化  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

大豆毛根转化基因功能分析

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

生物工程

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

一种基于根茎农杆菌毛根转化生产复合植物的高效且可重复的方法

副本

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此视频中的章节

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

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化（法戈皮鲁姆塔塔里库姆）

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

高效的聚乙二醇（PEG）介导的转化苔小立碗藓

复合植物代苜蓿truncatula

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

高级别瞬态表达重组蛋白的植物高效的农杆菌

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

一种基于根 茎农杆菌毛根转化生产复合植物的高效且可重复的方法

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摘要

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此视频中的章节

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

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化 （法戈皮鲁姆塔塔里库姆）

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

高效的聚乙二醇（PEG）介导的转化苔小立碗藓

复合植物代苜蓿truncatula

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

高级别瞬态表达重组蛋白的植物高效的农杆菌

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

一种基于根茎农杆菌毛根转化生产复合植物的高效且可重复的方法

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化（法戈皮鲁姆塔塔里库姆）