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A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase. 利用 NanoLuc 荧光素酶快速准确地评估 PROTACs 在细胞中降解 pan-Akt 的方法。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-03-05 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae014
Xiaojun Ji, Lei Miao, Yebin Wu, Tingli Zhao, Yaxuan Si, Xiaoyun Tan, Qiuhua Zhou, Rui Zuo, Junjie Pei, Jian Wu, Changyou Ma, Zhongjun Ma, Dan Xu

Proteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.

蛋白水解靶向嵌合体(PROTAC)是一种蛋白质降解技术,近年来越来越多地被用于新药研发。Akt 是一种经典的丝氨酸/苏氨酸激酶,近年来它在激酶之外的作用逐渐受到关注,成为 PROTAC 的靶向蛋白之一。目前,用于评估细胞内蛋白质降解的方法很多,但各有利弊。本研究旨在利用 NanoLuc 荧光素酶法研究 PROTACs(MS21 和 MS170)评估细胞内泛 Akt 蛋白降解的可行性。在对该方法和经典的 Western 印迹法在不同细胞中的应用进行了全面比较,并测试了多批次实验的稳定性之后,我们发现 NanoLuc 荧光素酶法是一种高准确性、高稳定性、低成本、易操作的方法,可用于评估 PROTACs 在细胞内降解 pan-Akt 蛋白的情况,且周期短、细胞扩展性强。鉴于这种方法的诸多优点,我们假设它可以扩展到评估 PROTACs 对更多靶蛋白的降解。总之,NanoLuc荧光素酶是一种适用于早期蛋白质降解筛选PROTAC化合物的方法。
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引用次数: 0
An improved method for measuring catalase activity in biological samples. 测量生物样本中过氧化氢酶活性的改进方法。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-03-05 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae015
Mahmoud Hussein Hadwan, Marwah Jaber Hussein, Rawa M Mohammed, Asad M Hadwan, Hawraa Saad Al-Kawaz, Saba S M Al-Obaidy, Zainab Abbas Al Talebi

Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.

过氧化氢酶(CAT)是一种重要的酶,它能将过氧化氢(H2O2)分解成水和氧气,从而保护生物大分子免受氧化损伤。CAT 存在于所有需氧微生物、动物和植物中。然而,正常人的尿液中没有这种物质,但在病理尿液中可以检测到。因此,CAT 检测有助于发现此类尿液。本研究介绍了一种测定 CAT 活性的新型分光光度法,该方法具有简便、灵敏、特异和快速的特点。该方法是将含酶样品与精心选择浓度的 H2O2 一起孵育一段时间。随后,加入含有硫酸亚铁铵(FAS)和磺胺水杨酸(SSA)的溶液以终止酶的活性。这时会形成一种独特的褐红色硫代水杨酸亚铁复合物。这种复合物的形成是 FAS 与存在的任何残留过氧化物反应的直接结果。这导致与 SSA 配位后生成铁离子。复合物的最大吸光度为 490 纳米。这种先进的方法无需使用浓酸来阻止 CAT 的活动,因此更安全、更易于操作。与标准硫氰酸亚铁法的对比分析表明,两者的相关系数为 0.99,表明新方法具有可比性和可靠性。总之,本研究展示了一种利用比色皿或微孔板评估 CAT 活性的简单可靠的方法。这种不受干扰的方案可轻松用于研究和临床分析,并具有相当高的准确性和精确性。
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引用次数: 0
Pairing a bioinformatics-focused course-based undergraduate research experience with specifications grading in an introductory biology classroom. 在生物学入门课堂上,将以生物信息学为重点的课程为基础的本科生研究体验与规范评分相结合。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-28 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae013
Melinda A Yang, Kylie Korsnack

Introducing bioinformatics-focused concepts and skills in a biology classroom is difficult, especially in introductory biology classrooms. Course-based Undergraduate Research Experiences (CUREs) facilitate this process, introducing genomics and bioinformatics through authentic research experiences, but the many learning objectives needed in scientific research and communication, foundational biology concepts, and bioinformatics-focused concepts and skills can make the process challenging. Here, the pairing of specifications grading with a bioinformatics-focused CURE developed by the Genomics Education Partnership is described. The study examines how the course structure with specifications grading facilitated scaffolding of writing assignments, group work, and metacognitive activities; and describes the synergies between CUREs and specifications grading. CUREs require mastery of related concepts and skills for working through the research process, utilize common research practices of revision and iteration, and encourage a growth mindset to learning-all of which are heavily incentivized in assessment practices focused on specifications grading.

在生物课堂上引入以生物信息学为重点的概念和技能是很困难的,尤其是在生物入门课堂上。以课程为基础的本科生研究经历(CURE)可以促进这一过程,通过真实的研究经历介绍基因组学和生物信息学,但科学研究和交流、基础生物学概念以及生物信息学概念和技能所需的许多学习目标可能会使这一过程具有挑战性。本文介绍了基因组学教育合作组织开发的规范分级与以生物信息学为重点的 CURE 的搭配情况。研究探讨了规格分级的课程结构如何促进写作作业、小组合作和元认知活动的支架;并描述了 CURE 与规格分级之间的协同作用。CUREs 要求掌握研究过程中的相关概念和技能,利用修改和迭代等常见的研究实践,并鼓励以成长的心态进行学习--所有这些在注重规格评分的评估实践中都得到了极大的激励。
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引用次数: 0
Correction to: Long amplicon nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples. 更正:长扩增片段纳米孔测序受感染葡萄叶片样本中的灰葡萄孢和其他真菌物种。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-23 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae010

[This corrects the article DOI: 10.1093/biomethods/bpad042.].

[此处更正了文章 DOI:10.1093/biomethods/bpad042]。
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引用次数: 0
Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification. 利用锁定核酸环介导等温扩增技术快速进行 IDH1-R132 基因分型检测。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-21 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae012
Kristian A Choate, Edward J Raack, Paul B Mann, Evan A Jones, Robert J Winn, Matthew J Jennings

While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

虽然单核苷酸变体(SNV)的检测对评估人类健康和疾病非常重要,但大多数基因分型方法都需要核酸提取步骤和漫长的分析时间。在这里,我们介绍了一种利用锁定核酸(LNA)整合到自退火环路引物中的方案,通过环路介导等温扩增(LAMP)对五种异柠檬酸脱氢酶 1 R132(IDH1-R132)变体进行等位基因鉴别。最初使用纯化的合成 DNA 对该基因分型面板进行了评估,以证明其具有特异性 SNV 识别能力。使用已知 IDH1-R132 突变状态的胶质瘤肿瘤裂解物进行的其他评估表明,该基因分型板无需核酸提取纯化步骤,在大约 35 分钟内就能达到特异性。这种基于 LNA-LAMP 的基因分型检测方法可以检测纯化核酸或组织匀浆中的单碱基差异,包括在背景野生型 DNA 过多的情况下出现的相关变体。LNA-LAMP 基于 pH 值的比色指示器便于对反应进行直观判读,我们还展示了利用吸光度比值将其转化为终点格式的成功案例,从而为区分阳性反应和阴性反应提供了另一种客观的方法。重要的是,LNA-LAMP 基因分型面板具有高度的可重复性,没有观察到假阳性或假阴性结果。
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引用次数: 0
Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection. 基于多探针策略的高灵敏度 TaqMan 方法的开发:在 ASFV 检测中的应用。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-19 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae011
Shuxiang Ding, Tianren Shen, Zixuan Feng, Sujing Diao, Yan Yan, Zhenkun Du, Yulan Jin, Jinyan Gu, Jiyong Zhou, Min Liao, Weiren Dong

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

建立针对各种病原微生物的高灵敏度检测方法一直备受关注。本研究首先系统地研究了多探针策略,然后建立了高灵敏度的 TaqMan 实时荧光定量 PCR(qPCR)方法来检测非洲猪瘟病毒(ASFV)。简而言之,我们设计了四种基于非洲猪瘟病毒 B646L 基因的探针,并研究了探针在单一 TaqMan qPCR 检测中的不同组合对检测灵敏度的影响。通过已建立的 TaqMan qPCR 方法,检测到的 ASFV 基因拷贝/μl 低至 0.5-5。此外,水样中携带 B646L 的质粒可通过超滤浓缩 1000 倍,从而实现对痕量病毒核酸的高灵敏度检测。此外,该方法与其他常见的临床猪病毒(如 PCV2、PCV3、PCV4、PEDV、PDCoV、CSFV、PRRSV 和 PRV)没有交叉反应。在检测173份临床猪血清样本时,所开发的方法与WOAH(世界动物卫生组织)推荐方法的重合率为100%。这项研究为提高痕量病原微生物的检测灵敏度和基于 TaqMan 的 qPCR 分析的适用灵敏度提供了一种综合策略。
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引用次数: 0
A methodological primer of extracellular vesicles isolation and characterization via different techniques. 通过不同技术分离和表征细胞外囊泡的方法论入门。
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-13 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae009
Farhang Aliakbari, Noah B Stocek, Maxximuss Cole-André, Janice Gomes, Giovanni Fanchini, Stephen H Pasternak, Gunna Christiansen, Dina Morshedi, Kathryn Volkening, Michael J Strong

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.

我们介绍了四种不同复杂程度的细胞培养衍生胞外囊泡 (EV) / 外泌体富集组分分离方案,目的是为研究人员提供易于操作的方法,这些方法可适用于各种实验室环境和地点的多种不同用途。这些方案主要基于聚合物沉淀、过滤和/或超速离心以及尺寸排阻色谱法(SEC),包括(i) 在条件培养基中添加聚乙二醇和氯化钠,然后进行低速离心;(ii) 对条件培养基进行超速离心;(iii) 通过 100 kDa 排阻滤器对条件培养基进行过滤;(iv) 使用标准商业试剂盒进行分离。必要时,在这些技术之后,还可以通过超速离心、蔗糖密度梯度离心或 SEC 等方法进一步纯化。用 HEK293 和 SH-SY5Y 细胞培养物生成含有外泌体的条件培养基。然后清除培养基中的细胞和碎片,用 0.2-µM 过滤器过滤,并在分离外泌体之前添加蛋白酶和 RNAse 抑制剂。纯化的外泌体可立即使用,也可在4°C下稳定保存(成像或下游使用完整的外泌体可保存一周),或在-80°C下长期保存,然后用于生化研究。我们的目的不是对这些方法进行比较,而是为它们提供描述指标,以便研究人员在各自的条件下选择适合自己工作的 "最佳方法"。
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引用次数: 0
An amplicon-based protocol for whole-genome sequencing of human respiratory syncytial virus subgroup a 基于扩增子的人类呼吸道合胞病毒 A 亚群全基因组测序方案
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-06 DOI: 10.1093/biomethods/bpae007
J. Vázquez-Pérez, Eber Martínez-Alvarado, Alberto Antony Venancio-Landeros, C. Santiago-Olivares, Fidencio Mejía-Nepomuceno, Enrique Mendoza-Ramírez, E. Rivera-Toledo
It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative RT-PCR. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130X to 200X. Eight samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8 and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.
研究人类呼吸道合胞病毒(hRSV)的完整基因组序列,便于进行基因组特征描述和高传播性或致病性变异体的鉴定。对病毒载量较低的呼吸道标本进行 hRSV 全基因组测序具有挑战性。在此,我们介绍了一种基于扩增片段的 hRSV A 亚群全基因组测序方案,该方案对来自鼻咽拭子和受感染细胞培养物的 24 个分离物进行了验证,经定量 RT-PCR 测定,这些分离物的周期阈值(Ct)从 10 到 31 不等。MinION 纳米孔为每个样本生成了 3200 到 5400 个读数,对超过 93% 的 hRSV-A 基因组进行了测序。每个等位基因的覆盖率从 130 倍到 200 倍不等。八个样本的 Ct 值分别为 20.9、25.2、27.1、27.7、28.2、28.8 和 29.6,测序结果显示病毒基因组的覆盖率超过 99.0%,表明即使在高 Ct 值的情况下基因组覆盖率也很高。由于引物是针对高度保守的区域设计的,因此该方案能够鉴定 hRSV A 亚群基因型。因此,它有望应用于该 hRSV 亚群的分子流行病学和监测。
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引用次数: 0
An amplicon-based protocol for whole-genome sequencing of human respiratory syncytial virus subgroup a 基于扩增子的人类呼吸道合胞病毒 A 亚群全基因组测序方案
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-06 DOI: 10.1093/biomethods/bpae007
J. Vázquez-Pérez, Eber Martínez-Alvarado, Alberto Antony Venancio-Landeros, C. Santiago-Olivares, Fidencio Mejía-Nepomuceno, Enrique Mendoza-Ramírez, E. Rivera-Toledo
It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative RT-PCR. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130X to 200X. Eight samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8 and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.
研究人类呼吸道合胞病毒(hRSV)的完整基因组序列,便于进行基因组特征描述和高传播性或致病性变异体的鉴定。对病毒载量较低的呼吸道标本进行 hRSV 全基因组测序具有挑战性。在此,我们介绍了一种基于扩增片段的 hRSV A 亚群全基因组测序方案,该方案对来自鼻咽拭子和受感染细胞培养物的 24 个分离物进行了验证,经定量 RT-PCR 测定,这些分离物的周期阈值(Ct)从 10 到 31 不等。MinION 纳米孔为每个样本生成了 3200 到 5400 个读数,对超过 93% 的 hRSV-A 基因组进行了测序。每个等位基因的覆盖率从 130 倍到 200 倍不等。八个样本的 Ct 值分别为 20.9、25.2、27.1、27.7、28.2、28.8 和 29.6,测序结果显示病毒基因组的覆盖率超过 99.0%,表明即使在高 Ct 值的情况下基因组覆盖率也很高。由于引物是针对高度保守的区域设计的,因此该方案能够鉴定 hRSV A 亚群基因型。因此,它有望应用于该 hRSV 亚群的分子流行病学和监测。
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引用次数: 0
Efficient regeneration of protoplasts from solanum lycopersicum cultivar Micro-Tom 从番茄茄属栽培品种 Micro-Tom 中高效再生原生质体
IF 3.6 Q2 Agricultural and Biological Sciences Pub Date : 2024-02-06 DOI: 10.1093/biomethods/bpae008
Yeong Yeop Jeong, Yoo-Sun Noh, Suk Weon Kim, P. Seo
Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from 3rd and 4th true leaves and cultured at an optimal density of 1 × 105 protoplasts/mL. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications such as genome engineering as well as basic research on plant regeneration in Solanaceae species.
原生质体再生已成为基因和基因组工程的关键平台。然而,我们缺乏可靠且可重复的方法来实现番茄(Solanum lycopersicum)栽培品种原生质体的高效再生。在此,我们优化了番茄栽培品种 Micro-Tom 的原生质体分离、小胼胝体增殖、芽再生和小植株建立的细胞和组织培养方法。从第 3 和第 4 片真叶中分离出的原生质体上涂有一层薄薄的藻酸盐,并以 1 × 105 个原生质体/毫升的最佳密度进行培养。我们确定了原生质体增殖、胼胝体形成、新芽再生和根再生的最佳培养基。再生的小植株表现出正常的形态生长和有性生殖。从原生质体到开花植株的整个再生过程在 5 个月内完成。优化后的原生质体再生平台可用于基因组工程等生物技术应用以及茄科植物再生的基础研究。
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引用次数: 0
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