6种番茄叶表皮制片方法比较

doi:10.11923/j.issn.2095-4050.cjas2021-0093

摘要/Abstract

摘要：

结合番茄叶片结构特点,采用6种表皮制片技术对番茄叶片下表皮进行装片,并对不同方法的制片过程和观察效果进行比较研究。结果表明,番茄叶片薄,表皮为单层细胞,其上着生有丰富表皮毛,下表皮与疏松的海绵组织相邻;表皮撕取法和透明胶带粘取法可以真实地观测气孔大小、密度等静态性状指标和气孔开度等动态行为;因番茄叶柔软,刮片法对操作要求极高,而解离法不但用时长且效果不佳,都不适宜番茄叶下表皮制片;可撕拉型指甲油印迹法能获得气孔指数和气孔开度的印痕,但对叶片有一定损坏,而硅胶-指甲油双阶段印迹法对叶片没有损伤,可以多次印迹同一叶片相同部位,连续观测气孔运动及发育等动态行为。研究结果可为不同研究选择适合的番茄叶表皮制片方法提供依据。

关键词: 番茄, 表皮制片, 气孔, 显微观察, 方法比较与改良

Abstract:

This paper adopted six kinds of leaf epidermis slice techniques to treat lower epidermis of tomato leaves considering their structural characteristics, and compared the preparation process and observation effect of different techniques. The results show that tomato leaves are thin, the epidermis is single-layer cell with abundant epidermal hairs, and the lower epidermis is adjacent to the loose spongy tissue. Static characteristics, such as dynamic behavior of stomatal size, density and stomatal aperture, could be observed by skin peeling method and cellophane tape sticking method. Due to the softness of tomato leaves, the scraping method is extremely demanding to operate, and dissociation method is time-consuming and has poor effect, both the techniques are not suitable for the slice production of tomato leaf epidermis. Tearable nail oil blotting method could obtain the imprint of stomatal index and stomatal aperture, while the leaves would be damaged certainly. Silica gel-nail polish two-stage blotting method has no damage to the leaves, the same part of the same leaf could be imprinted continuously without scathe, and in this way, the dynamic behavior like stomatal movement and stomatal development could be observed continuously. The results could provide a basis for selecting suitable tomato leaf epidermis preparation methods for different research purposes.

Key words: Tomato, Leaf Epidermis Slice Techniques, Stoma, Microscopic Observation, Method Comparison and Improvement

中图分类号:

Q94-3

宋艳波, 杨云浩, 段鹏军, 王嘉浩, 魏丝晴, 张亚龙. 6种番茄叶表皮制片方法比较[J]. 农学学报, 2022, 12(1): 45-52.

SONG Yanbo, YANG Yunhao, DUAN Pengjun, WANG Jiahao, WEI Siqing, ZHANG Yalong. Comparison Study of 6 Kinds of Tomato Leaf Epidermis Slice Techniques[J]. Journal of Agriculture, 2022, 12(1): 45-52.

图/表 6

图1. 番茄叶片下表皮印迹和叶横切结构 A.表皮毛,B.叶片横切结构,40×,标尺=75 μm

图2. 撕取表皮法和透明胶带粘取法制片对比 A.表皮撕取法制片,B.透明胶带粘取法制片,A、B为40×,a、b为640×,标尺=50 μm

图3. 刮片法和解离法成片对比 A.刮片法成片,B.解离法成片,A、B为40×,a、b为640×,标尺=50 μm

图4. 印迹法成片对比 A.可撕拉型指甲油印迹法成片,B.不可撕拉型指甲油印迹法成片,A、B为40×,a、b为640×,标尺= 50 μm

图5. 硅胶-指甲油二次印迹成片对比 A.硅胶-指甲油印迹法成片,B.硅胶-指甲油二次印迹法成片（间隔30 min）,A、B为40 ×,a、b为640 ×,标尺= 50 μm

方法	时间/个	难度	取样面积占叶面积百分数/%	定位性	清晰度	气孔/表皮毛真实性	气孔密度/开度与指数观测
表皮撕取法	0.5~2	+++	0.9~2.5	++	++	+++	+++
透明胶带法	3~6	+	>90	+++	+++	+++	+++
指甲油印迹法	30	+	>90	+++	++	+	++
硅胶-指甲油印迹法	40	+	>90	+++	++	+	++
解离法	40	++	0.5~1.5	+	++	++	++
刮片法	0.5~2	+++	0.7~2.0	++	+	+++	+

表1. 6种方法制片对比

参考文献 27

[28]	宋杰, 李树发, 李世峰, 等. 遮阴对高山杜鹃叶片解剖和光合特性的影响[J]. 广西植物, 2019, 39(6):94-103.
[29]	姜成东, 蔡胜忠, 卢业凌. 杧果叶片气孔观察方法研究[J]. 热带农业科学, 2008(3):23-24.
[30]	王培, 陈玉蓉, 方仁, 等. 用保卫细胞鉴定花粉植株倍性方法的研究[J]. 中国农业大学学报, 1989, 6(2):141-146.
[31]	师长海, 李玉欣, 董宝娣, 等. 禾本科植物叶片表皮气孔观察的样品制备方法改良[J]. 植物生理学通讯, 2010, 46(4):395-398.
[32]	陈娜, 王婧. 白及叶片气孔观察方法的研究[J]. 安徽农业科学, 2015, 43(25):80-82.
[33]	楼柏丹, 姚岚. 几种观察植物表皮气孔方法的比较[J]. 生物学教学, 2015, 40(9):42-43.
[34]	封涛, 胡东. 单子叶植物叶片气孔观察方法改良的研究[J]. 植物研究, 2008(1):82-84.
[35]	黄新敏, 何生根, 李红梅, 等. 月季'卡罗拉'切花的气孔观察及特征[J]. 仲恺农业工程学院学报, 2013, 26(4):8-12.
[36]	苏云松, 郭华春, 杨雪兰. 马铃薯叶片气孔观察方法的比较研究[J]. 中国马铃薯, 2009, 23(1):37-39.
[37]	WU S, ZHAO B. Using clear nail polish to make arabidopsis epidermal impressions for measuring the change of stomatal aperture size in immune response[M]. Methods Mol Biol, 2017:1578-1578.
[38]	ZWIENIECKI Maciej A, HAANING Katrine S, BOYCE C Kevin, et al. Stomatal design principles in synthetic and real leaves[J]. Journal of the royal society interface, 2016, 13:1-7.
[39]	DRAKE P L, FROEND R H, FRANKS P J. Smaller, faster stomata: Scaling of stomatal size, rate of response, and stomatal conductance[J]. Journal of experimental botany, 2012, 64(2):495-505. doi: 10.1093/jxb/ers347 URL
[40]	魏爱丽, 董惠文, 李雨春, 等. 小麦抗病性与气孔特性关系初探[J]. 作物杂志, 2010, 36(3):23-25.
[41]	MARTIN-Stpaul , DELZON S, COCHARD H. Plant resistance to drought depends on timely stomatal closure[J]. Ecology letters, 2017, 20(11):1437-1447. doi: 10.1111/ele.2017.20.issue-11 URL
[42]	TAHIR A, RASOULI F, CHEN Z H, et al. A comparative analysis of stomatal traits and photosynthetic responses in closely related halophytic and glycophytic species under saline conditions[J]. Environmental and experimental botany, 2020, 181(1):104300. doi: 10.1016/j.envexpbot.2020.104300 URL
[43]	ALI Kiani-Pouya, UTE Roessner, NIRUPAMA S, et al. Epidermal bladder cells confer salinity stress tolerance in the halophyte quinoa and Atriplex species[J]. Plant, cell & environment, 2017, 40(9):1900-1915.
[44]	MAFAKHERI A, SIOSEMARDEH A, BAHRAMNEJAD , et al. Effect of drought stress on yield, proline and chlorophyll contents in three chickpea cultivars[J]. Aust J crop Ssci, 2010, 4(8):580-585.
[45]	LAWSON T, TERASHIMA I, FUJITA T, et al. A Platform for Performing Photosynbook[A].// Coordination Between Photosynbook and Stomatal Behavior[M]. Springer international publishing AG, 2018:141-161.
[1]	SHUSEI Sato, SATOSHI Tabata, HIDEKI Hirakawa, et al. The tomato genome sequence provides insights into fleshy fruit evolution[J]. Nature: International Weekly Journal of Science, 2012, 485:635-641.
[2]	RamóN Maldonado-Torres, MORALES-Camacho J I, FERNANDO López-Valdez, et al. Assessment of techno-functional and nutraceutical potential of tomato (Solanum lycopersicum) seed meal[J]. Molecules, 2020, 25(18):4235-4235. doi: 10.3390/molecules25184235 URL
[46]	曾斌, 王庆亚, 唐灿明. 三个转Bt基因抗虫杂交棉杂种优势的解剖学分析[J]. 作物学报, 2008(3):496-505.
[3]	李君明, 项朝阳, 王孝宣, 等. “十三五”我国番茄产业现状及展望[J]. 中国蔬菜, 2021(2):13-20.
[4]	REIMERS Kristin J, KEAST Debra R. Tomato consumption in the United States and its relationship to the US department of agriculture food pattern: results from what we eat in America 2005-2010[J]. Nutrition today, 2016, 1(1):1-8.
[5]	ROMANO R, LUCA L D, MANZO N, et al. A new type of tomato puree with high content of bioactive compounds from 100% whole fruit[J]. Journal of food science, 2020, 85(10):3264-3272. doi: 10.1111/jfds.v85.10 URL
[6]	Perveen R, Suleria H, Anjum F M, et al. Tomato (Solanum lycopersicum) carotenoids and lycopenes chemistry; metabolism, absorption, nutrition, and Allied Health Claims- A comprehensive Review[J]. Critical reviews in food science and nutrition, 2015, 55(7):919-929. doi: 10.1080/10408398.2012.657809 URL
[7]	CHOKSI P M, JOSHI V Y. A Review on lycopene- extraction, purification, stability and applications[J]. International journal of food properties, 2007, 10(2):289-298. doi: 10.1080/10942910601052699 URL
[8]	LI N, WU X, ZHUANG W, et al. Tomato and lycopene and multiple health outcomes: Umbrella review[J]. Food chemistry, 2020, 343(3):1-40.
[9]	GóMezprieto M S, CAJA M M, HERRAIZ M, et al. Supercritical fluid extraction of all-trans-lycopene from tomato[J]. Journal of agricultural & food chemistry, 2003, 51(1):3-7.
[10]	CELMA A R, CUADROS F, F López-Rodríguez. Characterisation of industrial tomato by-products from infrared drying process[J]. Food & bioproducts processing, 2009, 87(4):282-291.
[11]	BERGOUGNOUX V. The history of tomato: From domestication to biopharming[J]. Biotechnology advances, 2014, 32(1):170-189. doi: 10.1016/j.biotechadv.2013.11.003 URL
[12]	ACHUO E A, PRINSEN E, HÖFTE M. Influence of drought, salt stress and abscisic acid on the resistance of tomato to Botrytis cinerea and Oidium neolycopersici[J]. Plant pathology, 2006, 55(2):178-186. doi: 10.1111/ppa.2006.55.issue-2 URL
[13]	普晓妍, 王鹏程, 李苏. 亚热带森林附生植物叶片气孔特征及其可塑性对光照变化的响应[J]. 广西植物, 2020, 1(1):1-12.
[14]	LAWSON Tracy, BLATT Michael R. Stomatal size, speed, and responsiveness impact on photosynjournal and water use efficiency[J]. Plant physiology, 2014, 164(4):1556-1570. doi: 10.1104/pp.114.237107 URL
[15]	DU M, ZHAI Q, LEI D, et al. Closely related NAC transcription factors of tomato differentially regulate stomatal closure and reopening during pathogen Attack[J]. The plant cell, 2014(7):7.
[16]	WIPHAWEE L, VU A L, AH-Fong A, et al. How does phytophthora infestans evade control efforts? Modern insight into the late blight disease[J]. Phytopathology, 2018, 108(1):1-9.
[17]	BIAN Z, ZHANG X, WANG Y, et al. Improving drought tolerance by altering the photosynthetic rate and stomatal aperture via green light in tomato (Solanum lycopersicum L.) seedlings under drought conditions[J]. Environmental and experimental botany, 2019, 167:1-30.
[18]	NAZIR F, FARIDUDDIN Q, HUSSAIN A, et al. Brassinosteroid and hydrogen peroxide improve photosynthetic machinery, stomatal movement, root morphology and cell viability and reduce Cu- triggered oxidative burst in tomato[J]. Ecotoxicology and environmental safety, 2020, 207:1-14.
[19]	KINOSHITA Toshinori, TOH Shigeo, TORII Keiko U. Chemical control of stomatal function and development[J]. Current opinion in plant biology, 2021, 60(1):102010. doi: 10.1016/j.pbi.2021.102010 URL
[20]	王萍, 邱念伟, 侯文雨, 等. “光和K⁺对气孔开度的影响”实验设计再优化[J]. 植物生理学报, 2020, 386(4):194-203.
[21]	HAWORTH M, SCUTT C P, DOUTHE C, et al. Allocation of the epidermis to stomata relates to stomatal physiological control: stomatal factors involved in the evolutionary diversification of the angiosperms and development of amphistomaty[J]. Environmental & experimental botany, 2018, 151(1):55-63.
[22]	TAO J J, CHEN S Y, ZHANG J S. Simple methods for screening and statistical analysis of leaf epidermal cells in dicotyledonous plants[J]. Bio-protocol, 2016, 6:1916.
[23]	LI F, CHEN X, ZHOU S, et al. Overexpression of SlMBP22 in Tomato Affects Plant Growth[J]. Plant science, 2020, 301(1):1-12.
[24]	BIAN Z, ZHANG X, WANG Y, et al. Improving drought tolerance by altering the photosynthetic rate and stomatal aperture via green light in tomato (Solanum lycopersicum L.) seedlings under drought conditions[J]. Environmental and experimental botany, 2019, 167:1-30.
[25]	FARBER M, ATTIA Z, WEISS D. Cytokinin activity increases stomatal density and transpiration rate in tomato[J]. Journal of experimental botany, 2016, 67(2):6351-6362. doi: 10.1093/jxb/erw398 URL
[26]	陈佰鸿, 李新生, 曹孜义, 等. 一种用透明胶带粘取叶片表皮观察气孔的方法[J]. 植物生理学通讯, 2004, 40(2):215-218.
[27]	温寿星, 郭祥桃, 黄镜浩, 等. 观察柑橘叶片表皮细胞与气孔结构的简易制片方法[J]. 东南园艺, 2016, 4(6):21-23.