This work was supported by Biotech Risk Assessment Grant Program competitive grand no. 2006-39454-17438 to A. Snow, K. Mercer, and H. Alexander from the United States Department of Agriculture, National Institute of Food and Agriculture. Experiments using this method were conducted at and supported by the University of Kansas Field Station, a research unit of the Kansas Biological Survey and the University of Kansas. The authors would like to thank P. Jourdan and E. Regnier for helpful reviews on earlier versions of this manuscript. Additionally, this work was aided by the contributions of the staff at the University of Kansas Field Station, Waterman Farm at the Ohio State University (OSU), the USDA Ornamental Plant Germplasm Center at OSU, and the Seed Biology Lab in the Department of Horticulture and Crop Science at OSU, especially E. Renze, S. Stieve, A. Evans, and E. Grassbaugh, for technical support.

1. 여러 종 (種) 또는 단일 종의 제어 십자가에서 종자를 수집 참고 : 산모 (시드 일으키기) 부모 야생, 하이브리드, 작물 유형을 사용하여 종 Helianthus의 annuus (해바라기)에서 15 크로스 타입에서이 예를 사용 씨앗을. <ol><li> 성장시기의 끝에, 라벨 가방 성숙한 씨앗 머리를 수집합니다. 표준 형식 (즉, 산모 X의 아버지)에서 부모의 십자가 형으로 표시 봉투에 왕겨와 장소 씨앗에서 청소 씨. </li><li> 동일한 부모의 크로스 유형의 같은 수의 씨앗 같은 양을 사용하여 큰 봉투에 함께 벌크 씨. 예를 들어, 같은 어머니와 아버지의 부모와 함께 10 크로스 타입 100 씨앗. 식별 (크로스 타입) 정보 봉투 레이블. 이 대형 봉투 2 장에 매장 스트립 작성에 사용 시드 주인입니다. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody&gt; <tr><td></td><td colspan="3"> 모자 부모 </td></tr><tr><td> 아버지의 부모 </td><td> 야생 : WxW </td><td> F 1 : WXC </td><td> 자르기 : CXC </td></tr><tr><td> 야생 : WxW </td><td> 0 % WxW </td><td> 25 % F 1 XW † </td><td> 50 % CXW † </td></tr><tr><td> BC : WxF1 또는 F1xW </td><td> 12.5 % WxBC </td><td> 37.5 % F 1 XBC </td><td> 62.5 % CxBC </td></tr><tr><td> F 1 : WXC </td><td> 25 % WxF 1 † </td><td> 50 % F 1 XF 1 </td><td> 75 % CXF 1 † </td></tr><tr><td> F 2 : F 1 XF 1 </td><td> 25 % WxF 2 </td><td> 50 % F 1 XF (2) </td><td> 75 % CXF 2 </td></tr><tr><td> 자르기 : CXC </td><td> 50 % WXC † </td><td> 75 % F 1 XC † </td><td> 100 % CXC </td></tr></tbody></table> 표 1. 부모의 크로스 t손 수분. 해바라기 작물 야생 하이브리드 크로스 타입에서 생산 씨네마 스테이션은 시드 매장 실험에 사용하기 위해 손 수분으로 생산되었다. 모든 크로스 유형의 경우, 산모의 부모가 먼저 나열하고 아버지의 부모는 두 번째 목록. † 표시 크로스 유형은 동일한 % 작물 대립하지만 서로 다른 산모 부모와 함께 상호 교차 형 쌍의 일부입니다. 페이스, BA 등 표는 이전에 발표되었다. (2015) 15. 2. 사용자 지정 종자 매장 스트립 만들기 참고 :이 예를 들어, 우리는 세 가지 제거 날짜 트리트먼트와 15 복제가 있었다, 그래서 우리는 총 45 스트립이 필요합니다. 이 예는 스트립 당 15 구획 그래서, 우리는 15, 7 × 10cm 구획을 수용하기에 충분한 미세 메쉬 폴리 에스테르 직물 또는 모기장이 필요합니다. 그림 1을 참조하십시오. <ol><li> 각 씨 스트립에 대한 직물의 20 × 105cm 스트립을 절단하여 시작합니다. </li><li> 다음 반으로 직물을 배, 측정 및 구획을 표시합니다. HIG를 사용하여시간 - 온도 접착제 총, 하나의 짧은 (10cm) 끝을 밀봉. 접착제가 약간 있지만 완전히 냉각되면, 시일을 생성하기 위해 함께 직물 측면을 누른다. 주의 : 피부와 접촉이 장기간 인 경우 고온 접착제 및 접착제 총 팁 화상을 입을 수 있습니다. </li><li> 각 구획 시점에서 개방 짧은 끝에 접힌 가장자리에서 접착제 라인. 구획 사이의 구멍없이 씰을 보장하기 위해 접착제 라인을 따라 함께 두 패브릭면을 누릅니다. 구획 사이에 이주 씨는 실험 오차에 기여한다. 참고 : 이제 구획이 만들어 졌는지, 펼쳐진 가장자리 씨와 자신의 식별 정보 (크로스 타입 이름)을받을 오픈 준비 남아 있어야한다. </li><li> 무작위로 각 스트립에 대한 구획 씨 크로스 유형을 지정합니다. <ol><li> 종자의 균일 한 수 (각 구획 20이 예에서 사용되는)과 종자 구획을 입력합니다. 그들이 할 수 있도록 라벨, 컷에 맞는 플라스틱 정원 다트에 산업 영구 마커를 사용매장을 견딜. 주 : 대체 라벨링 방법이 사용될 수있다. </li><li> 각 구획 시드 및 라벨로 채워진 후, 고온 접착제 총을 즉시 밀봉 구획. 전체 시드 스트립이 완전히 밀폐 될 때까지 진행합니다. 복제, 블록 및 / 또는 치료 정보 첫 번째 칸에 전체 스트립 레이블. 참고 : 우리는 시간 제거, 블록을 가지고 있었고, 블록 내에서 스트립을 복제하기 때문에 우리의 연구를 위해, 우리는이 모든 정보를 포함. </li><li> 다음 지구로 이동하기 전에 필요에 따라 구획 및 현장 접착제 사이의 간격을 확인합니다. </li></ol></li></ol><img alt="그림 1" src="/files/ftp_upload/54663/54663fig1.jpg" /> 각 구획에 대한 크로스 타입 식별을 보여주는 매장 스트립의 그림 1. 매장 씨 스트립 개략도. 예. 산모의 부모는 두 번째 나와있는 아버지의 부모와 먼저 나열됩니다. 색상이 다른 산모의 혈통을 표시작물에 대한 F 1 하이브리드 블루, 야생 노란색 및 빨간색. <a href="http://ecsource.jove.com/files/ftp_upload/54663/54663fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 3. 필드에서 시드 스트립 묻어 <ol><li> 집 스트립 지정된 깊이 플롯을 파. 분석의 줄거리 등 (이 예에서, 크로스 타입) 메인 플롯 및 구획 ID로 사용되는 스트립은 임의로 삭제 날짜를 허용하도록 배열 된 개별 블록 스트립을 할당한다. </li><li> 지구와 스트립을 커버. 컷 금속 메쉬 하드웨어 천에 묻혀 스트립을 통해 플롯과 장소보다 10cm 더 클 수 있습니다. 지구와 가장자리와 커버 천의 가장자리를 확보하기 위해 U 핀을 사용합니다. 참고 :이 덮개가내는 씨앗 포식자를 제외입니다. 또 다른 옵션으로, 머서 등. (13)는 건설 및 필드 삶을에 강한 고퍼 압력에 의한 종자 스트립을 포위 하드웨어 천 상자를 묻혀기.) </li><li> 스트립 위치 및 제거 날짜가 분명 서면 기록을 보완하기 위해 구분하는 색깔 측량의 플래그를 배치합니다. </li></ol> 4. 스트립을 발굴하고 종자를 평가 <ol><li> 주어진 제거 날짜에 할당 씨 스트립을 발굴. 그들은 시드 평가가 진행됩니다 실험실에 도달 할 때까지 서늘하고 습한 환경에서 스트립을 유지한다. 이것은 젖은 신문에 스트립 포장 및 운송 또는 특급 배송 쿨러에 배치하여 수행 할 수 있습니다. </li><li> 스트립 충분히 그 외부 이물질 것 모호하지 씨앗 깨끗해질 때까지 물에 스트립에서 진흙과 흙을 씻어. </li><li> 한 번에 오픈 한 구획을 잘라 가위를 사용합니다. 발아 씨앗을 계산하고이를 제거합니다. 분명히 죽은 씨앗 (예를 들어, 부패, 또는 부패 씨앗)를 계산합니다. 또한, 발아 휴면, 죽은 씨앗을 구별하기 위해 표시, 젖은 페트리 크기 블로터 종이에 발아 씨앗을 놓습니다. <ol><li> 발아와 배양 접시를 놓습니다25 ° C / 10 ° C 12 시간 주 / 야간 : 표준 발아 조건에 대한 성장 챔버 세트에 씨앗. 씨앗 일주가 발아 할 수 있습니다. , 가능한 이러한 계산하지만, 현장 조건에서 발아하지 않았기 때문에 발아 않는 모든. </li></ol></li><li> 성장 챔버의 처리 후, 검사는 테트라 졸륨 클로라이드 (TZ) (13)를 사용하여 생존력에 대해 발아 종자 남아. 이를 위해 AOSA 테트라 졸륨 테스트 핸드북 (14)를 사용하여 연구 종에 대한 TZ 적절한의 퍼센트 솔루션을 구성한다. 예를 들어, 1 % 용액이 사용된다. <ol><li> 반으로 발아 씨앗을 잘라 절반을 배치 배아와 배젖은 씨앗을 충당하기에 충분한 TZ 솔루션을 포함하는 배양 접시에서 볼 수 있었다 (씨앗이 괜찮 떠있다). </li><li> 30 ° C로 설정 인큐베이터에 TZ 배양 접시를 놓고 3 시간을 기다립니다. 원하는 경우 더 높은 온도, 빠른 반응 발생합니다,하지만 위험이 overstaining입니다 있습니다. </li></ol></li> &lt;리&gt;는 인큐베이터에서 배양 접시를 제거하고 평가합니다. 흠 씨가 죽은하면서 배아에 빨간 염색 한 씨앗, germinable 있습니다. 구별 한계 케이스 및 특정 종에 대한 세부 사항에 대한 AOSA 테트라 졸륨 테스트 안내서를 참조하십시오. </li></ol>

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The authors have nothing to disclose.

여기에서 우리는 현장에서 미리 선택된 기간에 종자 발아, 휴면 및 다양한 종자 주식의 사망률을 관찰 시드 매장 스트립을 사용하는 방법을 제시한다. 각각의 패킷은 개별 패킷의 생성을 통해 스트립 실 구조 (1)의 속도에 놓여보다는 스트립을 사용하는 장점; 및 (2)를 용이하게하고, 패킷을 생략 또는 실수를 제거하는 위험이없이 하나의 동작으로 복수의 구획을 제거하는 속도. 토양이 어려운 현?...

이 방법의 전체 목표는 안정적으로 현장 조건 하에서 시간이 지남에 따라 시드 생존율을 평가하는 것이다. 토양 종자 은행은 일시적으로 또는 몇 년 1,2 지속 수도 있고 토양 표면에, 표면 쓰레기 내에서, 또는 토양 프로필 내에서 분산 분산, 가능한 아직 발아 씨앗의 예약입니다. 여기에 제시된 것과 유사한 씨의 매장 방법은 수십 종을 사용하여 17 년간의 연구에 적용되었을 때, 가능한 씨앗 3 시험 종의 다양한에서 발견되었다. 종자 휴면 생존을 모종의 조건의 적절한 조합 4 발생할 때까지 발아 종자하는 블록이다. 휴면 출시에 대한 외부 트리거가 발아 수 있습니다 때까지 대기 남은 씨앗이 낮은 겨울 온도, 양분 제한, 또는 계절 가뭄 등의 가혹한 조건을 살아남을 허용 할 수 있습니다. 휴면 출시에 대한 트리거는 f를 왼쪽으로 확장 감기, 화합물에 노출 다를 수 있습니다분노, 또는 동물 위산 5 마모 또는 연락처를 통해 종피의 물리적 공격. 발아 큐는 장군 또는 특정 종 수 종종 과거의 자연 선택의 결과 바와 같이, 부적응 종자 발아는 부적절한 시간에 발생하고, 씨앗이나 모종 사망 또는 불량 묘목의 성장을 초래할 수있는 것입니다. 휴면이 휴면 해제 (예를 들어, 물리적 휴면 생리적 휴면)의 메커니즘에 기초한 유형들로 분류되었지만, 6 종 휴면는 적어도 하나의 식물 생물학 주제 이해 남아있다. 따라서, 개별 씨앗 또는 관련 생태 학적 조건에서 씨앗의 그룹의 상태 평가를 허용 현장 연구는 단순히 실험실에서 표준 발아 검사에 의존하는 것보다 더 높은 설명력을 가지고있다. 알려진 종자의 특성을 악용 수면의 메커니즘에 대한 통찰력을 제공 할 수 있습니다. 종자 휴면의 제어는 공동생리 학적 및 형태 학적 요인의 유전 적 제어를 포함 mplex. 휴면 메커니즘의 폭의 완벽한 이해는 아직 밝혀지지 하였지만, 일반적인 모델은 두 식물 호르몬 Gibberellic 산 (GA) 및 아브시 스산 (ABA) (7) 사이의 피드백 관계를 수반 나왔다. ABA는 휴면 상태를 유지하는 역할을하면서 수면에 대한 생리 학적 성분과 씨앗이 일반화 된 모델에서, GA는 휴면 릴리스에 대한 신호 역할을한다. 모성 유전 적 효과뿐만 아니라 모체 생성 조직과 발달 신호 (8)를 통해 휴면 및 크기와 같은 다른 종자 특성을, 영향을 미칠 수 모체 성장 환경을 제공합니다. 모계 생리 학적 단서와 함께 시간, 수면 상태를 유지할 수있다 외부 구조 (또는 종자 코팅)을 생성합니다. 모계 유래 된 종자 코팅은 어머니 식물의 유전자에 의해 제어되기 때문에, 그들은 씨의 실제 핵 유전 메이크업을 반영하지 않을 수 있습니다. 우리는 우리가에드 작물 야생 하이브리드의 배열에서 Helianthus의 annuus의 수과는 9,10. 따라서, 다양한 종, 크로스 타입, 또는 유전자형을 포함하는 연구 설계가에 대한 정보를 수집합니다 수 종자 특성에 이러한 산모 대 배아 유전 적 영향을 애타게 교차 생태와 종자 휴면, 발아과 생존의 유전학. 종자 발아 및 생존 표현형 인구 역학에 영향을 미칠 수있는 방법의 중요한 예는 작물 야생 하이브리드 영역에서 볼 수 있습니다. 재배 식물의 순화 동안 선택은 대부분의 휴면을 제거하고 성장시기 이외의 생존을 위해 시드의 능력을 감소시킨다. 그러나 작물 야생 하이브리드 영역에서의 재배와 야생 유형 간의 유전자 이동, 또는 하이브리드, 종자 은행 역학에 대한 잠재적 영향과, 야생 인구에 작물 대립 유전자 (또는 유전자 변형)을 재 도입 할 수 있습니다. 재배와 야생 친척 사이의 잡종은 잠재적으로 소유 할 수있다 작물 야생 하이브리드 영역에서 발견재배 (예를 들어, 겨울) (11)의 외부 조건을 살아남을 것으로 예상 몇 표현형과 중간 휴면 표현형의 다양한. 이 논문의 목적은 우리가 현장 조건 하에서 자연 변화를 조사하기 위해 상이한 시간 기간에서 발아 휴면 및 시드 유형의 범위의 생존을 평가할 수 시드 매장 스트립 방법을 사용하는 방법을 보여주고있다. 우리는 종자 특성에 산모와 태아 유전 적 영향에 관심이 있기 때문에 우리의 예에서 우리는 15 작물 야생 하이브리드 크로스 유형에서 해바라기 씨앗을 고용했다.

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reliable method for assessing seed germination, dormancy, and mortality under field conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

다양한 어머니의 혈통과 작물 대립 유전자 비율 (표 1)와 크로스 유형 %가, 발아 발아에서 제거 날짜에 걸쳐 차이, 죽은 씨앗 (그림. 2, 3). 세 번째 제거 (봄)에 의해 발아 모든 씨앗이 진정으로 휴면 것으로 밝혀졌다 동안 발아 씨앗 TZ 테스트를 사용하여, 우리는 두 번째 제거 (이른 봄) (표 2)에서 일부 진정으로 ...

Watch this Scientific Journal Video about Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions at JoVE.com

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

여기에서 우리는 장사 표시 씨 스트립 및 테트라 졸륨 클로라이드 (TZ) 생존 능력 테스트를 사용하여 필드 조건에서 종자 생존, 발아 및 휴면을 평가하기위한 프로토콜을 제시한다.

이 필드 조건에서 종자 발아, 휴면 및 사망률을 평가하기위한 신뢰할 수있는 방법

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

reliable-method-for-assessing-seed-germination-dormancy-mortality

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

10.4K 조회수.  Ohio State University.  여기에서 우리는 장사 표시 씨 스트립 및 테트라 졸륨 클로라이드 (TZ) 생존 능력 테스트를 사용하여 필드 조건에서 종자 생존, 발아 및 휴면을 평가하기위한 프로토콜을 제시한다. 

비디오: Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

Protocols for Robust Herbicide Resistance Testing in Different Weed Species

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

A robust and flexible approach to confirm herbicide resistance in weed populations is presented. This protocol allows the herbicide resistance levels to be inferred and applied to a wide range of weed species and herbicides with minor adaptations.

다른 잡초 종의 강력한 제초제 저항 테스트를위한 프로토콜

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented ...

연구

환경과학

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

커널 Bioassays를 사용하여 곰팡이 콜로니, Sporogenesis 및 Mycotoxins 생산의 부량

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

면역학 및 감염병학

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

초적혈구 멜라노가스터의생존능력을 배아에서 성인으로 평가하는 직접적이고 간단한 방법

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

발생학

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 ...

생물학

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 ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration 분석 및 기정을 사용 하 여 Teliospore 발 아 조사

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

헬리안투스 아누스의 대규모 열화상 스크리닝에 의한 새로운 식물 증발 규제 기관 식별

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

병아리콩에서 건조 루트 썩은 질병 에세이 : 상세한 방법론

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

애기장염 씨앗의 표면 살균을 위한 높은 처리량, 견고하고 매우 시간 유연한 방법

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

균종속영양 식물 조직의 곰팡이 집락화 및 종자의 공생 발아를 해석하기 위한 현미경 기술

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...