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Water Deficit Treatment and Measurement in Apple Trees
苹果树的水分亏损处理和测量   

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BMC Plant Biology
Jul 2014

Abstract

Water is considered perhaps the most limiting factor for plant growth and productivity (Boyer, 1982), and climate change predicts more frequent, more severe and longer drought periods for a significant portion of the world in coming years. Unfortunately, drought resistance is particularly difficult to measure due in part to the complexity of the underlying biology that contributes to a plant’s ability to cope with water limitations. For example, water deficit is frequently examined by detaching leaves or withholding water for a set period of time prior to tissue collection. Such approaches may elucidate the early stages of drought response but are generally not physiologically relevant for maintenance of drought resistance over a longer period. A more realistic approach is to impose a gradual water limitation with a sustained soil moisture level, particularly in the case of woody perennials. We describe here a protocol that imposes a long-term water deficit under controlled laboratory conditions that allow a molecular biology approach to understanding how woody plants survive severe water limitations. Representative data can be found in Artlip et al. (1997) and Bassett et al. (2014).

Keywords: Malus x domestica (苹果), Tissue culture (组织文化), Water limitation (水的限制), Climate change (气候变化), Pressure bomb (压弹)

Materials and Reagents

  1. Woody plants (seedlings, grafted, or own-rooted)
  2. Potting soil (MetroMix 360; SunGro Horticulture)
  3. Water source for watering plants
  4. ‘Play’ sand (can be obtained from any ‘home and garden’ store) washed three times with hot tap water and dried
  5. Slow release fertilizer (Osmocote, Scott’s Miracle-Gro Products, catalog number: 19-6-12 N-P-K )
  6. MS salts (Phytotechnology Laboratories, catalog number: M524 )
  7. Sucrose (Phytotechnology Laboratories, catalog number: S391 )
  8. Agar (Fisher Scientific, catalog number: BP1423 )
  9. Myo-inositol (Sigma-Aldritch, catalog number: I3011 )
  10. IBA (Sigma-Aldritch, catalog number: I5386 )
  11. Nutrient solution (MiracleGro, Scott’s Miracle-Gro Products, catalog number: 24-8-16 N-P-K )
  12. Rooting medium (see Recipes)

Equipment

  1. 1 gallon plastic pots (or desired size to accommodate plants)
  2. Plastic beaker for measuring soil
  3. Aluminum foil (optional)
  4. Small ruler
  5. Oasis rooting cubes (Smithers-Oasis LC-1 Horticubes, catalog number: 5240 )
  6. Conviron TC16 tissue culture chamber (Conviron)
  7. Greenhouse or Growth chamber for maintaining appropriate light, temperature and photoperiod (equivalent to Conviron, models: PGV36 or PGW36 )
  8. Scale appropriate for weighing pots with plants, (e.g. Weigh-Tronix, model: 830 )
  9. Scholander pressure bomb (SoilMoisture Equipment Corp., model: 3005 Series )

Procedure

  1. Plants
    1. Own-rooted ‘Royal Gala’.
      Explants were propagated in tissue culture as described by Norelli et al. (1988) and Ko et al. (2002), with root induction as described by Bolar et al. (1998).
    2. Upon root formation (~ one month), the seedlings were transferred to Oasis rooting cubes, and maintained in a Conviron TC16 tissue culture chamber for one month (24 °C, 70% r. h., 20 h light, 70 μmol photons/m2/s PPFD), with watering as needed and nutrient solution application weekly per the manufacturer’s recommendation.

  2. Growth conditions
    1. 5-L pots with equal volumes of Metromix 310 (horticultural vermiculite, Canadian Sphagnum peat moss, processed bark ash, composted pine bark) or equivalent product, and washed sand.
    2. The trees were grown in a glasshouse with supplemental lighting (High Pressure Sodium lamps) to maintain the day length at 16 h, and a maximum-minimum temperature range 35 °C to 20 °C. Trees were watered daily, with weekly application of nutrient solution, with supplemental application of Osmocote every two months at the indicated rate of 10 g.
    3. Age and size: Trees were in the glasshouse for a total of 8 months, with final stem diameter measurements ranging from 0.5 cm to slightly more than 1.0 cm, and heights varying from 1 to 2 m. It is preferable to select trees as close to the same size (stem diamter or height) as possible.

  3. Water deficit
    1. Trees are placed in a Conviron PGV36 growth chamber (Conviron) at 25 °C day (16 h)/18 °C night (8 h) with light at 500 μmol photons/m2/s PPFD.
    2. The pot is then watered to saturation and weighed one to two hours later.
    3. To mimic field conditions where water is lost to the atmosphere by evapotranspiration (evaporation from the soil plus transpiration by the plant), the soil surface is left exposed. To measure transpiration alone, a moisture barrier, typically aluminum foil, is placed over the pot and around the stem of the tree.
    4. Water is withheld and the pot weighed at the same time of day on a daily basis. Control plants are watered to saturation (90-95% pot weight) every other day. Care should be taken not to over water the controls as this can cause ‘flooding’ symptoms, i.e., wilting, chlorosis etc.
    5. Water deficit should be imposed by withholding water until the pot masses are 45% of the saturated mass and maintained at this level for two weeks by adding back water to the 45% level; control trees are rewatered to the saturated weight. Sufficient trees should be used for statistical analysis (to be determined by the user). We typically used four replicates per apple line, per treatment and an equivalent number of control trees.
    6. Water status of plants should be determined in order to quantify the water deficit. Common measurements include water potential (Scholander pressure bomb with leaves, branches or stems; Scholander et al., 1964), leaf size (Bassett et al., 2011), leaf number (Figure 2) or relative water content (RWC; Sinclair and Ludlow, 1985), although Kramer (1990) argued that water potential is a preferable measure owing to its being commensurable with atmospheric or soil water measurements. This measurement should be done pre-dawn or with foil-covered leaves. A water status of -1.9 MPa can be achieved with peach trees (Artlip et al., 1997). Researchers in the field (e.g., Hsiao, 1973) consider a water potential of this magnitude to impose a severe stress on the plant.
    7. It is important to note that plant size relative to pot size is important (large plant in a small pot is not equivalent to a large plant in a large pot) to prevent plants from becoming root-bound. We found that 5 L pots were ideal to grow trees to 1 m prior to selection for the drought experiments. It should be emphsized that different cultivars or geographical populations can differ in their water use efficiency or dehydration tolerance. Obviously, if a lesser degree of stress is desired, more water added back would increase the water status of the tree.

Representative data



Figure 1. Young apple trees of cultivar Royal Gala from drought experiment. T1C: control trees two weeks after the beginning of the experiment. These trees were watered to saturation (90-95% pot weight). Arrow shows new leaf unrolling at shoot apex. T1E: experimental trees two weeks after being held at 40% saturation. Filled arrow marks dying shoot apex; open arrow shows leaf curling which is a common indicator of dehydration. Photographs were taken from Bassett et al. (2011) with permission from the American Society for Horticultural Science.


Figure 2. Typical water deficit experiment in the growth chambers

Apple trees ~ 1 m tall were selected, tagged and placed in the Conviron growth chamber. The figure shows trees with and without foil placed on the pots. Red tags represent water deficit treatment; blue tags represent well-watered controls. A. Water deficit treatment and controls without foil; B. Water deficit treatment with foil covering pot surfact.


Figure 3. Leaf number of ‘Royal Gala’ trees subjected to a severe drought. The number of leaves along three regions of the stem were counted as follows: young leaves (1-2 cm long) at the top of the plant, leaves (~4-6 cm long) along the middle of the plant, and leaves (~7-8 cm long) along the lower portion of the plant. Pots of water-restricted plants were maintained with (WUE + foil) or without (WUE - foil) foil covers for the duration of the experiment. Controls were well watered with no foil on the pots. The number of leaves was counted every other day for two weeks. The greatest difference in leaf loss due to water deficit is seen in the youngest leaves just beginning expansion, whereas oldest leaves having expanded before the water deficit was imposed show the least difference.

Notes

  1. Most fruit trees are grafted onto a commercial rootstock. Use of grafted trees will reflect influences from both the scion and rootstock unless roots can be propagated from buds or stems of the desirable cultivar.
  2. ePure water is ≥ 18 Mohm-cm.

Recipes

  1. Rooting medium (pH 5.6)
    1. Ingredients                                               per 1 L
      ePure water
      1 L
      MS Salts
      2.15 g
      Thiamine-HCl (0.4 g/100 ml stock)
      1 ml
      Myo-inositol (100 mg/ml stock)
      1 ml
      Sucrose
      20 g
      Agar
      7 g
      Hormone
      IBA
      For root induction
      2.5 mg/L
      For root elongation
      0.0 mg/L
    2. Preparation
      Add 25-50% of water to a beaker on hotplate with constant stirring
      Add MS salts and sucrose to beaker
      Add agar and melt until completely dissolved
      Add remaining water
      Add Thiamine-HCl, Myo-inositol and IBA if needed
      Calibrate pH meter and adjust pH to 5.6 with 1 M NaOH
      Dispense into small baby food jars and autoclave for 8-10 min

Acknowledgments

This work was funded by USDA through the Agricultural Research Service as part of the in-house appropriated funding. We would like to acknowledge the contribution and excellent technical assistance of Sharon Jones.

References

  1. Artlip, T. and Wisniewski, M. (1997). Tissue-specific expression of a dehydrin gene in one-year-old Rio Oso Gem'Peach trees. J Amer Soc Hort Sci 122(6): 784-787.
  2. Bassett, C. L., Baldo, A. M., Moore, J. T., Jenkins, R. M., Soffe, D. S., Wisniewski, M. E., Norelli, J. L. and Farrell, R. E., Jr. (2014). Genes responding to water deficit in apple (Malus x domestica Borkh.) roots. BMC Plant Biol 14: 182.
  3. Bassett, C. L., Glenn, D. M., Forsline, P. L., Wisniewski, M. E. and Farrell, R. E. (2011). Characterizing water use efficiency and water deficit responses in apple (Malus× domestica Borkh. and Malus sieversii Ledeb.) M. Roem. HortScience 46(8): 1079-1084.
  4. Bolar, J. P., Norelli, J. L., Aldwinckle, H. S. and Hanke, V. (1998). An efficient method for rooting and acclimation of micropropagated apple cultivars. HortScience 33(7): 1251-1252.
  5. Boyer, J. S. (1982). Plant productivity and environment. Science 218(4571): 443-448.
  6. Hsiao, T. C. (1973). Plant responses to water stress. Annu Rev Plant Physiol 24(1): 519-570.
  7. Ko, K., Norelli, J. L., Reynoird, J.-P., Aldwinckle, H. S. and Brown, S. K. (2002). T4 lysozyme and attacin genes enhance resistance of transgenic 'Galaxy' apple against Erwinia amylovora. J Amer Soc Hort Sci 127(4): 515-519.
  8. Kramer, P. J. (1990). A brief history of water measurement in measurement techniques in plant science. In: Hashimoto, Y., Nonami, H., Kramer, P. J. and Strain, B. R. (eds). Academic Press, pp.45-68.
  9. Norelli, J., Aldwinckle, H. and Beer, S. (1988). Virulence of Erwinia amylovora strains to Malus sp. Novole plants grown in vitro and in the greenhouse. Phytopathology. 78:1292-1297.
  10. Scholander, P. F., Hammel, H. T., Hemmingsen, E. A. and Bradstreet, E. D. (1964). Hydrostatic pressure and osmotic potential in leaves of mangroves and some other plants. Proc Natl Acad Sci U S A 52(1): 119-125.
  11. Sinclair, T. and Ludlow, M. (1985). Who taught plants thermodynamics? The unfulfilled potential of plant water potential. Aust J Plant Physiol 12(3): 213-217.

简介

水被认为可能是植物生长和生产力的最大限制因素(Boyer,1982),气候变化预测未来几年世界上相当大一部分地区的更频繁,更严重和更长的干旱期。不幸的是,抗旱性特别难以测量,部分原因是基础生物学的复杂性,有助于植物应对水分限制的能力。例如,在组织收集之前,通常通过将叶子或保留水分离一段设定的时间来检查水缺乏。这些方法可以阐明干旱反应的早期阶段,但是通常在较长时期内维持抗旱性不是生理学相关的。更现实的方法是用持续的土壤水分水平强加逐步的水限制,特别是在多年生木本的情况下。我们在这里描述一个协议,在受控的实验室条件下施加长期缺水,允许分子生物学方法来了解木本植物如何生存的严重水限制。代表性数据可以在Artlip等人(1997)和Bassett等人(2014)中找到。

关键字:苹果, 组织文化, 水的限制, 气候变化, 压弹

材料和试剂

  1. 木本植物(幼苗,嫁接或自生根)
  2. 盆栽土(MetroMix 360; SunGro园艺)
  3. 浇水厂的水源
  4. "玩"沙子(可以从任何"家和花园"商店获得)用热自来水冲洗三次并干燥
  5. 缓释肥料(Osmocote,Scott's Miracle-Gro Products,目录号:19-6-12N-P-K)
  6. MS盐(Phytotechnology Laboratories,目录号:M524)
  7. 蔗糖(Phytotechnology Laboratories,目录号:S391)
  8. 琼脂(Fisher Scientific,目录号:BP1423)
  9. 肌醇(Sigma-Aldritch,目录号:I3011)
  10. IBA(Sigma-Aldritch,目录号:I5386)
  11. 营养液(MiracleGro,Scott's Miracle-Gro Products,目录号:24-8-16 N-P-K)
  12. 根系介质(参见配方)

设备

  1. 1加仑塑料盆(或适合植物的所需尺寸)
  2. 用于测量土壤的塑料烧杯
  3. 铝箔(可选)
  4. 小尺子
  5. Oasis生根立方体(Smithers-Oasis LC-1 Horticubes,目录号:5240)
  6. Conviron TC16组织培养室(Conviron)
  7. 温室或生长室保持适当的光,温度和光周期(相当于Conviron,型号:PGV36或PGW36)
  8. 适用于称量具有植物的盆栽的秤(例如,Weigh-Tronix,型号:830)
  9. Scholander压力炸弹(SoilMoisture Equipment Corp.,型号:3005系列)

程序

  1. 植物
    1. 自主的"皇家节目"。
      外植体在组织培养中繁殖   如Norelli等人(1988)和Ko等人所述。 (2002),with root 诱导,如Bolar等人(1998)所述。
    2. 根 形成(约一个月),将幼苗转移到Oasis生根   立方体,并保持在Conviron TC16组织培养室中   (24℃,70%相对湿度,20小时光照,70μmol光子/m 2 s/s PPFD),其中 根据需要浇水和营养溶液每周应用 制造商的建议

  2. 生长条件
    1. 5-L锅具有等体积的Metromix 310(园艺蛭石, 加拿大泥炭藓,加工的树皮灰,堆肥松树皮)或   等效产品和洗砂
    2. 树种在a 玻璃房与补充照明(高压钠灯) 保持16小时的白天长度和最大 - 最小温度范围   35℃至20℃。 树每天浇水,每周应用 营养溶液,每两个补充应用Osmocote 个月,指示的速率为10g。
    3. 年龄和尺寸:树木 在温室中总共8个月,具有最终茎杆直径 测量范围为0.5cm至略大于1.0cm, 高度从1到2m变化。 最好选择树近   到相同的尺寸(干直径或高度)

  3. 缺水
    1. 将树木放置在Conviron PGV36生长室(Conviron)中,25℃ 天(16小时)/18℃夜晚(8小时)用500μmol光子/m 2/s PPFD的光照射。
    2. 然后将盆浇水至饱和并在一至两小时后称重。
    3. 模拟场地条件,其中水流失到大气中 蒸发(从土壤蒸发加蒸腾蒸发) 植物),土壤表面暴露。 测量蒸腾 单独地,将防潮层(通常为铝箔)放置在其上 罐和在树的词根附近。
    4. 保留水 锅每天在同一时间称重。 控制植物   每隔一天浇水至饱和(90-95%盆重)。 注意应该 不要过度控制水,因为这可能导致"洪水" 症状,即,枯萎,萎黄等。
    5. 缺水应该是 通过扣留水直到锅质量为45% 饱和质量,并通过加回来维持在该水平两个星期   水至45%水平; 控制树被重新润湿到饱和 重量。应使用足够的树进行统计分析(待定)  由用户确定)。我们通常每个苹果使用四次重复 线,每次处理和等量的对照树
    6. 应确定植物的水状况以便量化 缺水。常见的测量包括水势(Scholander 压力炸弹与叶,枝或茎; Scholander等人,1964), 叶片大小(Bassett等,2011),叶片数(图2)或相对值 水分含量(RWC; Sinclair和Ludlow,1985),尽管Kramer(1990) 认为水势是一个优选措施,因为它的存在 与大气或土壤水测量相称。这个 测量应在黎明前或用箔覆盖的叶子进行。水  可以用桃树实现-1.9MPa的状态(Artlip等人 1997)。该领域的研究者(例如,,Hsiao,1973)考虑水 这种大小的潜在对植物施加严重的压力。
    7. 重要的是注意植物大小相对于罐尺寸 重要的(大型植物在小锅里不等同于大型植物   在大盆中)以防止植物变成根系。 我们发现 5 L盆是理想的生长树到1 m之前选择   干旱实验。 应该强调不同的品种或   地理人口的水利用效率可能不同 脱水耐性。 显然,如果压力程度较小 期望,更多的水回加将增加水的状态 树。

代表数据



图1.来自干旱实验的栽培品种Royal Gala的年轻苹果树。 T1C:实验开始后两周的控制树。 将这些树木浇灌至饱和(90-95%盆重)。 箭头显示新 叶在茎尖处展开。 T1E:在40%饱和度下保持两周后的实验树。填充的箭头标记死亡芽顶端;开放箭头显示叶卷曲,这是脱水的常见指示物。照片是从美国园艺科学协会授权的Bassett等人(2011年)拍摄的。


图2.生长室中的典型缺水实验

选择约1m高的苹果树,标记并置于Conviron生长室中。该图显示了有和没有箔放置在花盆上的树。红色标签代表缺水处理;蓝色标签代表灌溉良好的控件。 A.缺水处理和控制无箔; B.用箔覆盖锅表面的缺水处理

图3.经受严重干旱的"皇家嘎拉"树的叶数。沿着茎的三个区域的叶数计数如下:幼叶(1-2cm长)在植物的顶部,沿植物中部的叶(约4-6cm长)和沿植物下部的叶(〜7-8cm长)。在实验期间,用(WUE +箔)或不具有(WUE-箔)箔盖保持盆限水植物。对照组充分浇水,在盆上没有箔。每隔一天计算叶数,持续两周。由于缺水引起的叶片损失的最大差异出现在刚刚开始扩大的最小叶片中,而在缺水之前已经扩大的最老叶片显示出最小的差异。

笔记

  1. 大多数果树被嫁接到商业砧木上。 嫁接树的使用将反映接穗和根茎的影响,除非根可以从期望的栽培种的芽或茎繁殖。
  2. 纯净水≥18 Mohm-cm。

食谱

  1. 生根培养基(pH 5.6)
    1. 成分                                              每1 L                      ;               
      ePure水
      1 L
      MS盐
      2.15克
      硫胺-HCl(0.4g/100ml母液)
      1 ml
      肌醇(100mg/ml母液)
      1 ml
      蔗糖
      20克
      Agar
      7克
      激素
      IBA
      用于根诱导
      2.5 mg/L
      对于根伸长
      0.0 mg/L
    2. 准备
      在电炉上在不断搅拌下将25-50%的水加入烧杯中 向烧杯中加入MS盐和蔗糖
      加入琼脂并融化直至完全溶解
      添加剩余水
      如果需要,加入硫胺素-HCl,肌醇和IBA
      校准pH计,并用1M NaOH将pH调节至5.6 分配到小的婴儿食品罐和高压灭菌器8-10分钟

致谢

这项工作由美国农业部通过农业研究服务作为内部拨款资金的一部分提供资金。 我们要感谢Sharon Jones的贡献和卓越的技术援助。

参考文献

  1. Artlip,T。和Wisniewski,M。(1997)。 一岁的Rio Oso Gem'Peach中脱水蛋白基因的组织特异性表达 J Amer Soc Hort Sci 122(6):784-787。
  2. Bassett,C.L.,Baldo,A.M.,Moore,J.T.,Jenkins,R.M.,Soffe,D.S.,Wisniewski,M.E.,Norelli,J.L.and Farrell,R.E.,Jr。(2014)。 基因对苹果缺水的反应( x domestica Borkh。)根。BMC植物生物学14:182.
  3. Bassett,C.L.,Glenn,D.M.,Forsline,P.L.,Wisniewski,M.E.and Farrell,R.E。(2011)。 描述苹果中的水分利用效率和水分亏缺反应(×malestica < em> Borkh。和 Malus sieversii Ledeb。)M. Roem。 HortScience 46(8):1079-1084。
  4. Bolar,J.P.,Norelli,J.L.,Aldwinckle,H.S.and Hanke,V。(1998)。 一种生根和驯化的有效方法 微繁的苹果栽培品种。 HortScience 33(7):1251-1252。
  5. Boyer,J.S。(1982)。 植物生产力和环境 科学 218(4571) :443-448。
  6. Hsiao,T.C。(1973)。 植物对水分胁迫的反应。 植物生理学 24(1):519-570
  7. Ko,K.,Norelli,J.L.,Reynoird,J.-P.,Aldwinckle,H.S.and Brown,S.K。(2002)。 T4溶菌酶和attacin基因增强转基因'Galaxy'苹果对抗Erwinia amylovora的抗性。 J Amer Soc Hort Sci 127(4):515-519。
  8. Kramer,P.J。(1990)。植物科学测量技术中水测量的简要历史。在:Hashimoto,Y.,Nonami,H.,Kramer,P.J.and Strain,B.R。(eds)。 Academic Press,pp.45-68。
  9. Norelli,J.,Aldwinckle,H。和Beer,S。(1988)。 欧文氏菌amylovora 菌株对 Malus的毒力 sp。 Novole植物在体外生长和在温室中生长。 Phytopathology 78:1292-1297。
  10. Scholander,P.F.,Hammel,H.T.,Hemmingsen,E.A。和Bradstreet,E.D。(1964)。 红树林和其他植物叶片的静水压和渗透势。 Proc Natl Acad Sci USA 52(1):119-125。
  11. Sinclair,T。和Ludlow,M。(1985)。 谁教植物热力学? 植物水势的未实现的潜力。 Aust J Plant Physiol 12(3):213-217。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bassett, C. L., Artlip, T. S. and Wisniewski, M. E. (2015). Water Deficit Treatment and Measurement in Apple Trees. Bio-protocol 5(3): e1388. DOI: 10.21769/BioProtoc.1388.
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