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Measuring Stomatal Density in Rice
水稻叶片气孔密度的测定   

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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
Sep 2012

Abstract

The number of stomata on leaves is known to be affected by various environmental factors and intrinsic developmental program. Stomatal density and stomatal index are generally used as indicators of the leaf development and the plant growth. This protocol describes an easy, non-destructive method for preparing imprints of the rice leaf surface that is suitable for observation and counting of stomata. Researchers can process many leaf samples at once in the field or in the green house distance from the laboratory.

Keywords: Rice (水稻), Stomatal density (气孔密度), CO2 (CO2), Stomata (气孔), Respiration (呼吸)

Materials and Reagents

  1. Microscope cover glasses (Matsunami Glass, 24 x 40 mm No.1)
    Note: Though tougher glass slides (1-1.2 mm thickness) are safer, you should use cover glasses if the objective lens of the microscope is designed for standard 0.17 mm glass thickness. Only special objective lenses that have long working distances (ELWD, LWD) will allow observation through a thick glass slide. Alternatively objectives designed for use without a cover glass (NCG, NC) will enable direct observation of imprints placed on the slide glasses. I use large (24 x 40 mm) glasses for making imprints covering large leaf area. Larger glass is also useful when you set it on the microscope stage.
  2. Instant glue (Aron Alpha Super Set) (Toagosei Co., catalog number: EA936A-5 )
    Note: Aron Alpha Super Set includes liquid glue and accelerator. Aron Alpha is sold as Krazy Glue in North America and Cyanolit in Europe.
  3. Accelerator for instant glue
    Note: While I use the genuine accelerator made by Toagosei, third-party accelerators developed for Aron Alpha (Krazy Glue) will work fine. You can observe stomata without the accelerator, but the imprints may be less clear.

Equipment

  1. Light microscope with equipment for photomicrography
  2. Eyepiece micrometer and stage micrometer
    Note: These are indispensable for measurement of field of view unless your microscope can automatically calculate scale bars.

Procedure

  1. Select the leaves for observation. Make sure that they are not wet with rain or dew (Figure 1). Imprints can be taken from both upper (adaxial) and lower (abaxial) surfaces. Unless there are particular reasons, widest (middle) region of mature leaf blade should be selected as a target area. Avoid thick major vain for making smooth imprints. 


    Figure 1. Typical healthy leaf of rice.

  2. Apply several drops of the accelerator to the cover glass (Figure 2) and wait until they are thoroughly dry (2-3 min, at room temperature) (Figure 3).


    Figure 2. Place a drop of accelerator on a cover glass.


    Figure 3. Dried accelerator on the cover glass.

  3. Immediately after applying a drop of instant glue to the surface of the leaf, press the accelerator side of the cover glass on the leaf for about 30 seconds (Figures 4 and 5).


    Figure4. Instant glue dropping on the leaf surface.


    Figure 5. Drying glue mixture.

  4. Remove the cover glass from the leaf gently. Make sure that the imprint is on the cover glass (Figure 6). If glue mixture is completely dried, obtained imprint will be sturdy and durable. When a healthy leaf is used, only a remnant will be left on the leaf surface.


    Figure 6. Removed imprints on the cover glasses (left).


    Figure 7. A cover glass placed on the microscope stage. In this case, imprint is placed on the upper surface of the cover glass for imaging on inverted microscope.

  5. Observe imprints under the light microscope (Figure 7). Stomata in rice are formed in rows or files that are parallel to the sides of the leaf (Figure 8) (Hoshikawa, 1989). Therefore, finding stomata is relatively easy. Take photographs of the magnified image, calculate captured leaf area by using the micrometer or by the build-in imaging software, and then count all stomata within the printed image. I routinely counted stomata within a 0.42 mm2 of leaf area.


    Figure 8. Typical imprint image of medial leaf reagion. Red circles indicate positions of stomata.

    Note: Since imaging through a microscope gives a very shallow depth of field, only a very narrow region of the picture may be in focus at a time. To solve the focus problem, you can use autofocusing microscope or photo-processing software. I routinely use Keyence BZ-9000 microscope with optical software (Keyence, Osaka, Japan). ImageJ software (http://rsb.info.nih.gov/ij/) with appropriate plug-ins (e.g. Stack Focuser, Extended Depth of Field) also generates reasonable in-focus composite images Figure 9).


    Figure 9. Focus stacking. Left are the three source images at different focal depths. Right is a composite image generated by ImageJ and the Stack Focuser plug-in (http://rsb.info.nih.gov/ij/plugins/stack-focuser.html).

  6. Imprints can be stored without any sealing treatment for long time at room temperature. I could get clear images even after 2 years.

Acknowledgments

I am grateful to Ryoko Kaji for her technical assistance. This work was supported by the Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (P&P), a Grant-in-Aid for Scientific Research on Innovative Areas (No. 21114002) and the Ministry of Education, Science and Culture of Japan (No. 22570045).

References

  1. Hoshikawa, K. (1989). The growing rice plant: an anatomical monograph. Tokyo: Nobunkyo xvi, 310p.-illus.. ISBN 245913836.
  2. Kusumi, K., Hirotsuka, S., Kumamaru, T. and Iba, K. (2012). Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein. J Exp Bot 63(15): 5635-5644.

简介

已知叶子上的气孔数量受到各种环境因素和内在发育程序的影响。 气孔密度和气孔指数通常用作叶发育和植物生长的指标。 该协议描述了一种用于制备适于观察和计数气孔的稻叶表面的印记的容易的,非破坏性的方法。 研究人员可以在实验室的场地或温室距离内一次处理许多叶片样品。

关键字:水稻, 气孔密度, CO2, 气孔, 呼吸

材料和试剂

  1. 显微镜护罩眼镜(Matsunami Glass,24 x 40 mm No.1)
    注意:虽然更坚固的玻璃载玻片(厚度为1-1.2 mm)更安全,但如果显微镜的物镜设计为标准的0.17 mm玻璃厚度,则应使用保护玻璃。只有具有长工作距离(ELWD,LWD)的特殊物镜才允许通过厚玻璃片观察。或者,被设计用于没有盖玻片(NCG,NC)的物镜将能够直接观察放置在载玻片上的印记。我使用大(24×40毫米)眼镜制作覆盖大叶面积的印记。更大的玻璃也是有用的,当你把它放在显微镜载物台上。
  2. 速溶胶(Aron Alpha Super Set)(Toagosei Co.,目录号:EA936A-5)
    注意:Aron Alpha超级套装包括液体胶和加速器。 Aron Alpha在北美和Cyanolit在欧洲作为Krazy Glue销售。
  3. 速溶胶剂
    注意:虽然我使用由Toagosei制造的真正的加速器,为Aron Alpha(Krazy Glue)开发的第三方加速器将工作正常。你可以观察到气孔没有加速器,但印记可能不太清楚。

设备

  1. 带有显微照相设备的光学显微镜
  2. 目镜千分尺和平台千分尺
    注意:这些是视野测量不可或缺的,除非您的显微镜可以自动计算比例尺。

程序

  1. 选择叶子观察。 确保它们不会被雨水或露水弄湿(图1)。 可以从上(近轴)和下(后轴)表面获取印记。 除非有特殊原因,最宽 应选择成熟叶片的(中)区域作为目标区域。避免粗糙的主要徒劳,以获得平滑的印记。


    图1.米的典型健康叶片。

  2. 将几滴促进剂涂在玻璃盖上(图2),并等待直至完全干燥(室温下2-3分钟)(图3)。


    图 2.将一滴加速器放在保护玻璃上。


    图3.玻璃盖上的干燥加速器
  3. 在将一滴速溶胶施加到叶子表面后,立即将盖玻片的加速器侧按在叶子上约30秒(图4和5)。


    图 4.瞬间胶水滴在叶片表面。


    图 5.干燥胶水混合物。

  4. 轻轻地从玻璃盖上取下玻璃盖。确保刻印在盖玻片上(图6)。如果胶水混合物完全干燥,获得的印记将是坚固耐用的。当使用健康叶时,叶片表面上只剩下残余物。


    图 6.删除封面眼镜上的印记(左)。


    图 7.盖玻片放在显微镜镜台上。在这种情况下, 将压印放置在用于成像的盖玻片的上表面上  倒置显微镜
  5. 在光学显微镜下观察印记(图7)。水稻中的气孔形成为平行于叶的侧面的行或锉(图8)(Hoshikawa,1989)。因此,找到气孔相对容易。拍摄放大图像的照片,通过使用千分尺或内置成像软件计算捕获的叶面积,然后计数打印图像内的所有气孔。我常规地计算在0.42mm 2/d的叶面积内的气孔

    图8.中叶反应的典型印记图像。红色圆圈表示气孔的位置。

    注意:由于成像通过显微镜给出非常浅的景深,只有一个非常狭窄的区域的图片可能会在一个时间对焦。要解决对焦问题,可以使用自动对焦显微镜或照片处理软件。我常规使用Keyence BZ-9000显微镜用光学软件(Keyence,Osaka,Japan)。具有适当插件(例如,堆栈聚焦器,扩展景深)的ImageJ软件(http://rsb.info.nih.gov/ij/)也产生合理的聚焦合成图像/em> 9)。


    图 9.聚焦堆叠。左侧是三个不同的源图片 焦深。 Right是由ImageJ生成的合成图像 堆栈Focuser插件 (http://rsb.info.nih.gov/ij/plugins/stack-focuser.html)。

  6. 压印可以在室温下长时间保存而不进行任何密封处理。 我可以得到清晰的图像,即使在2年后。

致谢

我感谢Ryoko Kaji的技术援助。 这项工作得到了九州大学教育与研究发展项目(P& P),创新地区科学研究资助(第21114002号)和教育,科学和文化部 日本(No. 22570045)。

参考文献

  1. Hoshikawa,K。(1989)。 生长的水稻:解剖学专着。 Tokyo:Nobunkyo xvi,310p.-illus .. ISBN 245913836.
  2. Kusumi,K.,Hirotsuka,S.,Kumamaru,T。和Iba,K。(2012)。 在缺乏SLAC1的水稻突变体中增加的气孔导度引起的叶片光合作用,SLAC1是保卫细胞阴离子通道 蛋白质。 J Exp Bot 63 (15):5635-5644。
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引用:Kusumi, K. (2013). Measuring Stomatal Density in Rice. Bio-protocol 3(9): e753. DOI: 10.21769/BioProtoc.753.
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Rohma Nur Azmi
Gadjah Mada University
I just made this longitudinal section of rice leaf using this procedure, but I end up being confused to determine the stomata. Unfortunately I couldn't upload my photograph here. I really need help for this..
9/8/2014 4:13:19 AM Reply
Kensuke Kusumi
Department of Biology, Faculty of Sciences, Kyushu University, Japan

Sorry for late reply. If your problem still remains unsolved, send your photographs to the following address. I will look for stomata.
kkususcb@kyushu-u.org

9/18/2014 3:40:55 AM