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Rapid Induction of Water Stress in Wheat
小麦中水分胁迫的快速诱导   

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参见作者原研究论文

本实验方案简略版
Journal of Experimental Botany
May 2013

Abstract

Traditional water stress evaluation studies in wheat are time consuming and can take up to several months to finish. A rapid phenotypic screening for water stress is important for accommodating time-bound water stress studies such as transient gene silencing studies in wheat. This method explains a procedure to induce water stress in young wheat plants within three weeks.

Materials and Reagents

  1. Wheat seeds
  2. Permanent marker pen
  3. Green house handling gloves

Equipment

  1. Plastic pots and potting mixture
  2. Volumetric beaker (500 ml)
  3. Petri dishes (11 cm diameter)
  4. Growth chamber
  5. Light incubators/Green house facility
  6. Weighing scale

Procedure

  1. We have to calculate the field capacity (FC) of the experimental set up to maintain proper water stress. FC is the amount of water in the soil remaining after water is removed by gravity following water saturation. For the purpose of estimating the FC for the experimental set up.
    1. Plastic pots holding 500 g of potting mixture are fully saturated with water, drained by gravity for 3 h and weighed.
    2. These pots are allowed to fully dry over a period of 12 days. These pots are weighed again and the difference in weight constitutes the water held by soil in pot after gravitational drainage. This constitutes the approximation of FC for these pots. An average reading from 10 pots is assumed as the field capacity for this experimental setup (in our case FC was 245 g).
  2. Once we establish the FC for our system, achieving 50% FC is the goal for inducing water stress
    1. 50% FC corresponds to half of the amount of water (measured in weight) for the calculated FC. This FC is achieved in our pots by maintaining the weight of the pots at a level equal to weight of dried down pots (with soil) plus half of the measured FC (measured as weight of water) calculated for these pots. Control plants were kept at 100% FC. In this experiment 100% FC can be achieved by adding the water equal the calculated FC to the dried down pots. All the pots are periodically (twice a day) weighed to maintain the water level (50% FC and 100% FC).
  3. Preparation of wheat plants
    1. Sterilization and germination of wheat seeds: The wheat seeds are placed in petri dishes, covered with disinfecting (5% (w/v) sodium hypochlorite) for 15 min, stirred, drained, and washed four times with sterile deionized water. These seeds are placed on moist filter paper in petri dishes. Store these petri dishes in a dark place (preferably in a growth chamber) with stable room temperature (~25 °C).
    2. These seeds that germinated (~48 h) are then transplanted (3 seeds in a pot) into pots holding 500 g of potting mixture in a temperature-controlled growth room at 22–25 °C and relative humidity of 60% with a 12 h photoperiod with light intensity ranging from 300 to 400 μE/m2/s. Prophylactic measures (disease free seeds, clean water for treatment, pest and pathogen free environment etc) are taken to maintain the plants disease and pest free.
    3. The ideal age to start this experiment is found to be 3-5 leaf stage in wheat (~15 days from germination initiation). The sample size (number of plants) is determined by the experimental design. This experiment used 24 plants in each treatment.
  4. For this experiment, two subsets of plants (well watered set and water stressed set) are maintained.
    1. One set of plants (well watered set) is maintained at 100% field capacity (FC).
    2. The second set of plants is water stressed plants. Water stress is imposed on this set of plants by withholding water until 50% FC weight is achieved. Soil moisture regimes are monitored gravimetrically by weighing the pots every day. It was found that withholding water continuously for ~3 days could achieve a water level of 50% FC in our experimental setup.
  5. No other enclosure for pots are necessary as evapotranspiration from the pots under study was found to be statistically similar in all pots as conditions in the two treatments were identical (plant growth stage, pot size and growing conditions), except the treatment (water stress).
  6. Withholding water up to 50% FC is found to induce the water stress phenotype in the experimental plants placed in 500 g of potting mix within the time frame of three weeks.
  7. The 100% field capacity (FC) plants would give the control phenotype for well watered plants and 50% field capacity (FC) plants would show the water stressed phenotype (Figure 1).


    Figure 1. The phenotype of well watered (100% field capacity) and water stressed (50% field capacity) plants after 7 days of stress induction. Plastic covers are used to prevent evaporation from high light during photography.

  8. Stunted growth and chlorosis are observed in water stressed plants in comparison with well watered plants. To confirm and measure the water status in the plant, leaf relative water content (RWC) is estimated according to the method of Ekanayake et al., (1993).

Acknowledgments

This protocol is adapted from Ekanayake et al. (1993) and Manmathan et al. (2013).

References

  1. Ekanayake, I., De Datta, S. and Steponkus, P. (1993). Effect of water deficit stress on diffusive resistance, transpiration, and spikelet desiccation of rice (Oryza sativa L.). Ann Bot 72(1): 73-80.
  2. Loresto, G., Chang, T. and Tagumpay, O. (1976). Field evaluation and breeding for drought resistance. Philippine J Crop Sci 1(1): 36-39.
  3. Manmathan, H., Shaner, D., Snelling, J., Tisserat, N. and Lapitan, N. (2013). Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance. J Exp Bot 64(5): 1381-1392.

简介

小麦的传统水胁迫评估研究是耗时的,可能需要几个月才能完成。 水胁迫的快速表型筛选对于适应时间有限的水胁迫研究如在小麦中的瞬时基因沉默研究是重要的。 该方法解释了在三个星期内在年轻小麦植物中诱导水分胁迫的程序。

材料和试剂

  1. 小麦种子
  2. 永久性标记笔
  3. 温室处理手套

设备

  1. 塑料盆和灌封混合物
  2. 容量烧杯(500ml)
  3. 培养皿(直径11厘米)
  4. 生长室
  5. 光孵化器/温室设施
  6. 称重秤

程序

  1. 我们必须计算实验设置的田间容量(FC),以保持适当的水分胁迫。 FC是在水饱和后通过重力除去水后剩余的土壤中的水量。为了估算实验设置的FC。
    1. 保持500g灌封混合物的塑料罐用水完全饱和,通过重力排干3小时并称重。
    2. 使这些盆在12天的时间内完全干燥。这些盆再次称重,重量差值构成重力排水后盆中由土壤保持的水。这构成了这些花盆的FC的近似值。从该实验装置的场容量(在我们的情况下FC为245g),假设10个罐的平均读数。
  2. 一旦我们为我们的系统建立FC,实现50%FC是诱导水分胁迫的目标
    1. 50%FC对应于计算的FC的水量的一半(以重量计)。通过将罐的重量维持在等于干燥罐(具有土壤)的重量加上对于这些罐计算的测量的FC(以水的重量测量)的一半的水平,在我们的罐中实现该FC。将对照植物保持在100%FC。在该实验中,通过将等于计算的FC的水加入干燥的锅中可以实现100%FC。所有的盆定期(一天两次)称重以保持水位(50%FC和100%FC)。
  3. 小麦植物的制备
    1. 小麦种子的灭菌和发芽:将小麦种子放在培养皿中,用消毒(5%(w/v)次氯酸钠)覆盖15分钟,搅拌,排干,并用无菌去离子水洗涤四次。将这些种子放在培养皿中的湿滤纸上。将这些培养皿存放在室温(〜25℃)稳定的黑暗地方(最好在生长室)。
    2. 然后将这些发芽的种子(约48小时)移入(在盆中3个种子)盆中,盆中在温度控制的生长室中在22-25℃和60%的相对湿度下用12小时保持500g盆栽混合物光周期的光强度范围为300至400μE/m 2/s/s。采取预防措施(无病种子,清洁的水进行处理,无病虫害和无病原的环境等)以维持植物疾病和病虫害。
    3. 发现开始该实验的理想年龄是小麦中的3-5叶期(从发芽开始约15天)。 样品大小(植物数量)由实验设计确定。 该实验在每次处理中使用24株植物
  4. 对于该实验,保持两个植物子集(良好浇水组和水应激组)。
    1. 一组植物(良好浇水组)保持在100%田间容量(FC)
    2. 第二组植物是水胁迫植物。 通过停留水直到达到50%FC重量,对该组植物施加水应力。 通过每天称量盆来通过重量分析监测土壤水分状况。 发现在我们的实验装置中,持续约3天的保持水可以达到50%FC的水位。
  5. 由于在两种处理中的条件相同(植物生长阶段,盆尺寸和生长条件),除了处理(水胁迫)之外,在所有盆中发现从所研究的盆中蒸发蒸发是相同的,所以不需要盆的其他封闭体。 。
  6. 在三周的时间范围内,在500g盆栽混合物中放置的实验植物中发现高达50%FC的保留水诱导水分胁迫表型。
  7. 100%田间容量(FC)植物将给予良好浇水植物的对照表型,50%田间容量(FC)植物将显示水胁迫表型(图1)。


    图1.胁迫诱导7天后充分浇水(100%田间容量)和水胁迫(50%田间容量)植物的表型。 在拍摄期间,塑料盖用于防止高光的蒸发。

  8. 在受水胁迫的植物中观察到生长不良和褪绿,与良好浇水的植物相比。为了确认和测量植物中的水状态,根据Ekanayake等人(1993)的方法估计叶相对含水量(RWC)。

致谢

该协议改编自Ekanayake等人(1993)和Manmathan等人(2013)。

参考文献

  1. Ekanayake,I.,De Datta,S。和Steponkus,P。(1993)。 水分亏缺胁迫对扩散阻力,蒸腾和小穗干燥的影响 稻(> L.)。 Ann Bot 72(1):73-80。
  2. Loresto,G.,Chang,T.and Tagumpay,O。(1976)。 抗旱的田间评估和育种菲律宾作物科学   1(1):36-39。
  3. Manmathan,H.,Shaner,D.,Snelling,J.,Tisserat,N.和Lapitan,N。(2013)。 小麦中拟南芥基因同源物的病毒诱导基因沉默鉴定基因 赋予改善的耐旱性。 64(5):1381-1392。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Manmathan, H. and Lapitan, N. L. (2013). Rapid Induction of Water Stress in Wheat. Bio-protocol 3(16): e867. DOI: 10.21769/BioProtoc.867.
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