I. 비주얼 화면 및 역방향 유전자 접근 표피 돌연변이를 확인합니다. 모델 식물 Arabidopsis thaliana의에서 표피의 돌연변이가 &quot;베어링하지 왁스&quot;를 의미하는 &quot;eceriferum&quot;돌연변이,라고합니다. 그들은 영상 화면에서 수, 즉, 그냥 돌연변이보고에 의해, 당신은 돌연변이가 녹색으로 어두운 반짝 스템을하면서 야생 타입 식물의 꽃이 핌 줄기가 약간 흰 것을 볼 수 있습니다. 영상 화면이 유형의 격리되었다 돌연변이 중 하나는이, eceriferum4 또는 cer4이라고합니다. 재미있는 표피의 돌연변이를 발견의 또 다른 방법은 역방향 유전학 접근이다 : 관심의 선택 후보 유전자 그리고 그 유전자를 공부하고 공장 라인 중단. 이러한 돌연변이 TDNA 우리가 지금 MAH1 전화 것이,이 경우, 시토크롬 P450 관심의 유전자를 방해했습니다. PCR 결과 (동영상에 표시된 것과 같은) TDNA 삽입 관심의 유전자에있는 것을 확인할 수 있습니다. 이 유전자 특정 primers는 야생 형 유전자를 증폭하기 위해 사용하는 경우, 우리는 제품을 볼 수 있지만, TDNA가 인터럽트를하는 경우,이 돌연변이에서와 같이, 거기에는 제품이 없습니다. 반대로, TDNA 및 유전자 특정 primers를 사용하여 제품은 돌연변이에서 볼 수 있습니다. 우리는 유전자의 모자 아버지 사본을 모두 방해 아르 homozygous 돌연변이 라인을 찾습니다. 유전자의 한 복사본은 TDNA에 의해 방해와 다른 사본이 아직 와일드 타입이다면, PCR 결과는 혼합 수 있습니다. II. Cutlicle 왁스의 화학 성분을 확인하는 가스 크로마 토그래피를 사용하여. 우리는 가스 크로마 토그래피를 사용하여 왁스의 화학 성분을 분석. 첫째, 수용성 왁스 화합물은 클로로포름에 디핑하여 식물 표면에서 제거됩니다. 그들이 가열 가스의 흐름을 통해 발송됩니다 어디 그런 다음 샘플은 가스 크로마 토그래피 칼럼에 주입하고 있습니다. 다른 화합물은 화합물을 분리, 컬럼의 벽에 더 많은 이하 스틱, 그리고 그들이 칼럼의 다른 끝에있는 다른 후 하나를 나와. 화합물은 불꽃 이온화 검출기를 통해 통과로 그 다음, 우리는 봉우리를 참조하십시오. 각 피크의 유지 시간이 서로 다른 화합물에 대한 특성이며, 질량 분광법에서, 우리는 Arabidopsis 왁스의 각각의 구성 요소를 확인합니다. 주요 봉우리 현재 C29 alkane, C29 케톤과 C29 차 알코올의 수 있습니다. 작은 봉우리는 이차 알콜, 지방산, aldehydes 및 에스테르입니다. 표준에 상대적인 봉우리 아래에있는 지역은, 얼마나 많은 화합물이 존재하는 우리에게 알려줍니다. 이러한 표피의 돌연변이에서 가스 chromatograms에서 볼 때, 하나는 mah1 보조 알콜과 ketones이 부족하면서 cer4은 기본 알콜과 에스테르를 결여하는 방법 통보하실 수 있습니다. 이러한 phenotypes 우리 공장에 대한 관심의 유전자의 기능에 대해 많은 걸 알 수 있습니다. 예를 들어, 우리가 cer4 라인 변이되었던 유전자 알고 전에 표현형은 biosynthetic 경로의 기본 알코올 지점에서 문제가 중이랍니다. 그 유전자의 분자 ID가 Kunst 연구실에서 공부 후, 그것은 그것이 화학 표현형 잘 맞는 지방산 아실 reductases의 유전자 가족의 일부임을 발견했다. III. 표피 결정의 구조를 확인하는 Cryo - SEM을 사용하여 그들은 어두운 녹색, 광택 표현형 또는 줄기에있는 야생 - 타입 약간 흰 모양이있다면 우리가 Arabidopsis 표피의 돌연변이를 연구하면, 우리는 볼 수 있습니다. 우리는 식물의 표면에 표피보고 시각 표현형의 근거를 확인하기 위해 스캔 전자 현미경을 사용합니다. 우리는 SEM이 작동 진공을 필요로하고 그들을 냉동 보관하지 않고, 샘플가 건조하고 붕괴 것이 때문에 표피가 그대로 유지하기 위해 식물을 냉동 및 액체 질소 온도에서 그들을보고, cryo - SEM을 사용합니다. 여기 cer4 및 mah1 샘플은 야생 형 컨트롤과 함께 해부하고 SEM의 스텁에 냉동. 샘플은 현미경의 추위 단계로 전학하는 경우, 당신은 야생 형 Arabidopsis의 줄기 표피의 표면이 결정으로 덮여있다는 것을 알 수 있습니다. 그것은 줄기에게 그 약간 흰 모양을 제공합니다 그 결정을 분산하는 빛이다. 여기 cer4 줄기는, 그것이 광택 보였 기억나요? 그것은 표면에 크리스탈이 부족. 그들은 식물의 표피에서 순수한 왁스 화합물을 분리하고 그들은 실험실에서 recrystallize 때 수정이 식물에서 찾은 것과 같은 모양을 형성 어디에 사람들이 실험을 완료했습니다. Arabidopsis에서, 우리는 결정이 화합물의 혼합물로 구성되어 있으며 상황이 더 복잡합니다 있다고 믿습니다. 예를 들어, 화학 분석 cer4에 기본 알콜 및 파생 에스테르만을 작은 구성 요소가 누락되는 우리를 보여주는 아직 CE에서 결정R4 줄기 가고 있습니다. mah1의 경우에는, 그 시토크롬 P450 효소에 돌연변이의 효과는 돌연변이 식물 왁스, 보조 알콜 및 ketones의 두 가지 주요 구성 요소를 부족, 아직 mah1 식물에있는 크리스탈가 정상적으로 형성한다는 것입니다. Arabidopsis 표피의 돌연변이에 대한 자세한 내용하려면로 이동하십시오 <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.arplant.59.103006.093219" target="_blank">식물 생물학의 연례 검토</a> .

use of arabidopsis eceriferum mutants to explore plant cuticle biosynthesis

식물 표피 물 절약의 기본 역할을 식물에 밀랍 외부 실정입니다뿐만 아니라, 병원성 미생물의 항목에 대한 중요한 장벽입니다. 표피는 &quot;cutin&quot;라는 힘든 가교 폴리머 및 보호 왁스 층 바다표범 식물 표면 구성되어 있습니다. 표피의 밀랍 층 아이비 잎에 빛나는 영화 또는 포도 또는 왁스에있는 산란 결정을 밝힐 수 양배추 리프 감사의 표면에 먼지가 외부 덮개으로 나타나는 많은 식물에 분명합니다. 표피가 이해, 지상파 환경에 식물의 필수 적응하기 때문에 식물 표피 형성에 관련된 유전자는 농업과 임업 모두에서 응용 프로그램을하고 있습니다. 오늘, 우리는 순방향 및 역방향 유전학 방법으로 식별 식물 표피의 돌연변이의 분석을 보여 드리죠.

Watch this Scientific Journal Video about Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis at JoVE.com

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

식물 표피 물 절약의 기본 역할을뿐만 아니라, 병원성 미생물의 항목에 대한 중요한 장애가 식물에 밀랍 외부 덮개입니다. 이 비디오에서는, 우리는 순방향 및 역방향 유전학 방법으로 식별 식물 표피의 돌연변이의 분석을 보여줍니다.

식물 표피의 생합성을 탐구 Arabidopsis eceriferum 돌연변이의 이용

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation, but is also an important barrier against the entry of ...

use-arabidopsis-eceriferum-mutants-to-explore-plant-cuticle

Research

JoVE Journal

Biology

12.8K 조회수.  University of British Columbia - UBC. 식물 표피 물 절약의 기본 역할을뿐만 아니라, 병원성 미생물의 항목에 대한 중요한 장애가 식물에 밀랍 외부 덮개입니다. 이 비디오에서는, 우리는 순방향 및 역방향 유전학 방법으로 식별 식물 표피의 돌연변이의 분석을 보여줍니다.

비디오: Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

에서 초기 단계의 배아의 효율적이고 신속한 분리<em&gt; 애기 장대</em&gt; 씨앗

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

연구

생물학

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

유전자 탐구하는 종피 침구 분석<em&gt; 체외</em&gt; 어떻게 배젖 컨트롤 종자 발아에<em&gt; 애기 장대</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

측정 공간적 칼슘<sup&gt; 2 +</sup애기 장대 식물에서&gt; 신호

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

식물 표피 지질 폴리 에스테르 단량체의 분리 및 성분 분석

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

의 측면 루트 유도 성 시스템<em&gt; 애기 장대</em&gt;과 옥수수

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

발광 시계 기자와 애기 장대 원형질체 트랜의 일주기 표현형의 신속한 분석

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

애기 씨의 높은 처리 살 균에 대 한 표준화 된 방법

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...