An automatic porometer recording from eight leaves

Automated system for following stomatal behaviour of plants in growth cabinets

Allaway W. G., Mansfield T. A.(1969)

Canadian Journal of Botany 47: 1995–1998 – https://doi.org/10.1139/b69-292

https://cdnsciencepub.com/doi/10.1139/b69-292

Abstract

An automatic porometer recording from eight leaves is described. The apparatus has enabled experiments lasting several days to be performed, with porometer readings every half-hour from each leaf. The porometer cups apparently did not alter the behavior of stomata even after long attachment, and readings were closely correlated with measured stomatal apertures on the upper surface of amphistomatous leaves.

In situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

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原位荧光显微镜结合图像分析的叶气孔计数

Contagem estomática in situ por microscopia de fluorescência e análise de imagem combinada.

Conteo estomático in situ mediante microscopía de fluorescencia y análisis de imagen combinado.

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Epicuticular Phenolics Over Guard Cells: Exploitation for in situ Stomatal Counting by Fluorescence Microscopy and Combined Image Analysis

Karabourniotis G., Tzobanoglou D., Nikolopoulos D., Liakopoulos G. (2001)

George Karabourniotis, Despina Tzobanoglou, Dimosthenis Nikolopoulos, Georgios Liakopoulos,

===

Annals of Botany 87(5): 631–639 – https://doi.org/10.1006/anbo.2001.1386

https://academic.oup.com/aob/article/87/5/631/2588449?login=false

Abstract

Guard cells emit an alkali-induced, blue fluorescence upon excitation by ultraviolet radiation (emission maximum energy at 365 nm). Fluorescence emission of guard cells was brighter than that of the neighbouring epidermal cells in a number of wild and cultivated plants including conifers, but the relative fluorescence intensity and quality was species-dependent. Three representative plants possessing stomatal complexes which differed morphologically were studied: Olea europaea , Vicia faba and Triticum aestivum . Immersing leaves of these plants in chloroform for 30 s (thereby removing epicuticular waxes) significantly reduced the intensity of the fluorescence emitted by guard cells. This indicates that guard cell fluorescence could be due to either an increased concentration of fluorescing compounds (probably wax-bound phenolics), or a thicker cuticular layer covering the guard cells. Given that the alkali-induced blue fluorescence of the guard cells is a common characteristic of all plants examined, it could be used as a rapid and convenient method for in situ measurements of the number, distribution and size of stomatal complexes. The proposed experimental procedure includes a single coating of the leaf surface by, or immersion of the whole leaf in, a 10% solution of KOH for 2 min, washing with distilled water, and direct observation of the leaf surface under the fluorescence microscope. Fluorescence images were suitable for digital image analysis and methodology was developed for stomatal counting using Olea europaea as a model species. 

Implications for global gross primary productivity (GPP) modeling and estimation, as integrating stomatal metrics can enhance predictions of plant growth and resource usage worldwide

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全球总初级生产力(GPP)建模和估计的含义在于,整合气孔度量可以增强对全球植物生长和资源利用的预测。

Implicações para a modelagem e estimativa da produtividade primária bruta (GPP) global, uma vez que a integração de métricas estomáticas pode aprimorar as previsões do crescimento das plantas e do uso de recursos em todo o mundo.

Implicaciones para la modelización y estimación de la productividad primaria bruta (GPP) global, ya que la integración de métricas estomáticas puede mejorar las predicciones del crecimiento de las plantas y del uso de recursos en todo el mundo.

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Measuring stomatal and guard cell metrics for plant physiology and growth using StoManager1

Wang J., Renninger H. J., Ma Q., Jin S. (2024)

Jiaxin Wang, Heidi Renninger, Qin Ma, Shichao Jin,

Shichao Jin’s Lab

===

Plant Physiology DOI: 10.1093/plphys/kiae049

https://www.researchgate.net/publication/377386976

Abstract

Automated guard cell detection and measurement are vital for understanding plant physiological performance and ecological functioning in global water and carbon cycles. Most current methods for measuring guard cells and stomata are laborious, time-consuming, prone to bias, and limited in scale. We developed StoManager1, a high-throughput tool utilizing geometrical and mathematical algorithms and convolutional neural networks to automatically detect, count, and measure over 30 guard cell and stomatal metrics, including guard cell and stomatal area, length, width, stomatal aperture area/guard cell area, and orientation, stomatal evenness, divergence, and aggregation index. Combined with leaf functional traits, some of these StoManager1-measured guard cell and stomatal metrics explained 90% and 82% of tree biomass and intrinsic water use efficiency (iWUE) variances in hardwoods, making them significant factors in leaf physiology and tree growth. StoManager1 demonstrates exceptional precision and recall (mAP@0.5 over 0.96), effectively capturing diverse stomatal properties across over 100 species. StoManager1 facilitates the automation of measuring leaf guard cells, enabling broader exploration of stomatal control in plant growth and adaptation to environmental stress and climate change. This has implications for global gross primary productivity (GPP) modeling and estimation, as integrating stomatal metrics can enhance predictions of plant growth and resource usage worldwide. Easily accessible open-source code and standalone Windows executable applications are available on a GitHub repository (https://github.com/JiaxinWang123/StoManager1) and Zenodo (https://doi.org/10.5281/zenodo.7686022).

Semi-automatic detection of stomatal regions from a fluorescence microscopic image of Arabidopsis leaf surface cell contours

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从拟南芥叶表面细胞轮廓的荧光显微图像中半自动检测气孔区域

Detecção semi-automática de regiões estomáticas a partir de uma imagem microscópica de fluorescência dos contornos das células da superfície foliar de Arabidopsis.


Detección semiautomática de regiones estomáticas a partir de una imagen microscópica de fluorescencia de los contornos de las células de la superficie de la hoja de Arabidopsis.

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CARTA-based semiauto-matic detection of stomatal regions on an Arabidopsis cotyledon surface

Higaki T., Kutsuna N., Hasezawa S. (2014)

Takumi HigakiNatsumaro KutsunaSeiichiro Hasezawa,

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo

===

Plant Morphol 26: 9–12 – https://doi.org/10.5685/plmorphol.26.9

https://www.jstage.jst.go.jp/article/plmorphol/26/1/26_9/_article

Abstract

Recent advances in imaging equipment have enabled the acquisition of many kinds of bioimages in huge numbers. With the acquisition of such imagery, computer assistance becomes increasingly important for image inspection. To provide an automated and versatile bioimage classification system, we have developed an active learning algorithm combined with a genetic algorithm and self-organizing map named Clustering-Aided Rapid Training Agent (CARTA). Using CARTA, similar images can be drawn from many images. Applying this feature of CARTA, we are developing a framework for the detection of similar cellular architectures in wide-field fluorescence microscopic images. In this article, we describe an example case of semi-automatic detection of stomatal regions from a fluorescence microscopic image of Arabidopsis leaf surface cell contours.

Statistical analysis revealed that the stomatal density of the dwarf plants was higher than that of the giant plants, but the maximum stomatal aperture area, average stomatal aperture area, total number of stomata, and average leaf area were lower than those of the giant plants

============


统计分析显示,侏儒植物的气孔密度高于巨型植物,但最大气孔开度面积、平均气孔开度面积、气孔总数和平均叶片面积均低于巨型植物。


A análise estatística revelou que a densidade estomática das plantas anãs era maior do que a das plantas gigantes, mas a área máxima da abertura estomática, a área média da abertura estomática, o número total de estômatos e a área média da folha eram menores do que as das plantas gigantes.

El análisis estadístico reveló que la densidad estomática de las plantas enanas era mayor que la de las plantas gigantes, pero el área máxima de apertura estomática, el área promedio de apertura estomática, el número total de estomas y el área promedio de la hoja eran inferiores a los de las plantas gigantes.

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Automated estimation of stomatal number and aperture in haskap (Lonicera caerulea L.)

Meng X., Nakano A., Hoshino Y. (2023)

Xiangji Meng, Arisa Nakano, Yoichiro Hoshino,

===

Planta 258(4): – DOI: 10.1007/s00425-023-04231-y

https://www.researchgate.net/publication/373713827

Abstract and figures

Main conclusion This study developed the reliable Mask R-CNN model to detect stomata in Lonicera caerulea. The obtained data could be utilized for evaluating some characters such as stomatal number and aperture area. Abstract The native distribution of haskap (Lonicera caerulea L.), a small-shrub species, extends through Northern Eurasia, Japan, and North America. Stomatal observation is important for plant research to evaluate the physiological status and to investigate the effect of ploidy levels on phenotypes. However, manual annotation of stomata using microscope software or ImageJ is time consuming. Therefore, an efficient method to phenotype stomata is needed. In this study, we used the Mask Regional Convolutional Neural Network (Mask R-CNN), a deep learning model, to analyze the stomata of haskap efficiently and accurately. We analyzed haskap plants (dwarf and giant phenotypes) with the same ploidy but different phenotypes, including leaf area, stomatal aperture area, stomatal density, and total number of stomata. The R-square value of the estimated stomatal aperture area was 0.92 and 0.93 for the dwarf and giant plants, respectively. The R-square value of the estimated stomatal number was 0.99 and 0.98 for the two phenotypes. The results showed that the measurements obtained using the models were as accurate as the manual measurements. Statistical analysis revealed that the stomatal density of the dwarf plants was higher than that of the giant plants, but the maximum stomatal aperture area, average stomatal aperture area, total number of stomata, and average leaf area were lower than those of the giant plants. A high-precision, rapid, and large-scale detection method was developed by training the Mask R-CNN model. This model can help save time and increase the volume of data.

Making fresh preparations of a plant or animal for observation stomata for biology teachers

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制作植物或动物的新鲜样本,以供生物老师观察气孔。

Preparar amostras frescas de uma planta ou animal para observar os estômatos, destinadas a professores de biologia.

Preparando preparaciones frescas de una planta o animal para la observación de los estomas, destinado a profesores de biología.

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Bimbingan Teknik Preparasi Jaringan Epidermis Tumbuhan untuk Pengamatan Stomata kepada Guru Biologi

Amintarti S., Zaini M. i., Ajizah A. (2022)

Sri Amintarti, M. Zaini, Aulia Ajizah,

Program Studi Pendidikan Biologi Fakultas Keguruan dan Ilmu PendidikanUniversitas Lambung Mangkurat Banjarmasin, Indonesia

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Bubungan Tinggi Jurnal Pengabdian Masyarakat 4(2): 377 – DOI: 10.20527/btjpm.v4i2.4795

https://www.researchgate.net/publication/362371226

Abstract:

Making fresh preparations of a plant or animal is a skill that a biology teacher must possess because practicum is a learning activity that cannot be separated from biological theories/concepts. The appearance of preparation depends on the skill during preparation. To build preparation skills for observation stomata for biology teachers, the solution is to provide technical guidance for preparing plant epidermal tissue for leaf stomata observation. The preparation technique refers to Budi & Dhea (2018), Amintarti(2020) and Nugraha (2020). The technical guidance method is by 1) Delivering material about epidermal tissue in plants, 2) Showing a video of epidermal tissue preparation to observe stomata, and 3) The practice of making epidermal tissue preparations for stomata observation. The activity was attended by 35 high school biology teachers/equivalents in Tanah Laut Regency, which was carried out offline at SMAN 1 Pelaihari. The activity results showed that 77.8% of participants strongly agreed that the method provided by the team was very practical for making stomatal observation preparations. As many as 33.3% of the guidance participants have successfully prepared a very good appearance of stomata images under a microscope. As many as 66.6% have also succeeded in making preparations with rudimentary stomatal images. This success made 100% of the participants state that they would practice it in classroom learning.

——————–

Abstrak: Pembuatan preparat segar suatu tumbuhan atau hewan merupakan ketrampilanyang harus dimiliki oleh seorang guru biologi karena praktikum merupakan kegiatanpembelajaran yang tidak bisa dipisahkan dengan teori / konsep-konsep biologi. Bagusatau tidaknya tampilan suatu preparat tergantung pada ketrampilan saat preparasi. Untukmembangun ketrampilan preparasi pengamatan stomata kepada para guru biologi makasolusinya dengan memberikan bimbingan teknis preparasi jaringan epidermis tumbuhanuntuk pengamatan stomata daun. Teknik preparasi mengacu pada Budi & Dhea (2018),Amintarti (2020) dan Nugraha (2020) Metode bimbingan teknis dengan cara: 1)Menyampaikan materi tentang jaringan epidermis pada tumbuhan; 2) Menayangkan videopreparasi jaringan epidermis untuk mengamati stomata; dan 3) Praktek pembuatanpreparat jaringan epidermis untuk pengamatan stomata. Kegiatan diikuti oleh 35 orangguru biologi SMA/sederajat di Kabupaten Tanah Laut yang dilaksanakan secara luringbertempat di SMAN 1 Pelaihari. Hasil kegiatan menunjukkan 77,8% pesertamenyatakan sangat setuju bahwa cara yang diberikan oleh Tim sangat praktis untukmembuat preparat pengamatan stomata. Sebanyak 33,3% peserta bimbingan telahberhasil membuat preparat dengan tampilan gambar stomata di bawah mikroskop yangsangat bagus, dan sebanyak 66,7% juga telah berhasil membuat preparat dengan tampilangambar stomata yang belum sempurna. Keberhasilan ini membuat 100% pesertamenyatakan mempraktekkannya pada pembelajaran di kelas. Dan 88,9% dari pesertabersedia untuk menginformasikannya kepada guru-guru biologi lain yang tidak mengikutikegiatan bimbingan teknis ini.Kata kunci: Bimbingan Teknis; Preparasi Jaringan Epidermis;

A novel perspective for stomatal study conducive to accelerating the application of stomatal circadian rhythm in wheat breeding

=================

一个新颖的角度对气孔研究有利于加速气孔昼夜节律在小麦育种中的应用。


Uma perspectiva inovadora para o estudo estomático, propícia para acelerar a aplicação do ritmo circadiano estomático na criação de trigo.

Una nueva perspectiva para el estudio de los estomas propicia para acelerar la aplicación del ritmo circadiano estomático en la cría de trigo.

===============

StomataTracker: Revealing circadian rhythms of wheat stomata with in-situ video and deep learning

Sun Z., Wang X., Song Y., Li Q., Song J ., Cai J., Zhou Q., Zhong Y., Jin S., Jiang D. (2023)

Zhuangzhuang Sun, Xiao Wang, Yunlin Song, Qing Li, Jin Song, Jian Cai, Qin Zhou, Yingxin Zhong, Shichao Jin, Dong Jiang,

Nanjing Agricultural University

===

Computers and Electronics in Agriculture 212: 108120 – DOI: 10.1016/j.compag.2023.108120

https://www.researchgate.net/publication/373107547

Abstract and figures

Plant stomata are essential channels for gas exchange between plants and the environment. The infrared gas-exchange system has greatly accelerated the studies of stomatal conductance (g s). Nevertheless, due to the lack of in-situ monitoring techniques, the behavior of stomata themselves remains poorly understood, especially in nocturnal environmental conditions. Here, a deep-learning-based stoma tracking pipeline (StomataTracker) was first proposed to continuously monitor stoma traits from unprecedentedly long-term, continuous, and non-destructive video data. Compared to the semi-automatic method (ImageJ), the open-source StomataTracker could greatly improve the extraction efficiency from 207 s to 1.47 s of stomatal traits, including stomatal area, perimeter, length, and width. The R 2 adjusted of the four stomatal traits ranged from 0.620 to 0.752. In addition, the rhythm of wheat stomata opening in a completely dark environment was first reported from long-term video data. The closed time of stoma at night was negatively correlated with stomatal traits, and the R ranged from − 0.583 to − 0.855. The heterogeneity of stomatal behavior also highlighted that smaller stomata have the rhythm pattern of longer closure time at night. Overall, our study provides a novel perspective for stomatal study, and it is conducive to accelerating the application of stomatal circadian rhythm in wheat breeding.

A first step towards improving the measurements provided by stomata analysis tools, that will in turn help plant biologists to advance their understanding of dynamics in plants

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通过改进气孔分析工具提供的测量数据,可以帮助植物学家更好地理解植物动态的第一步。

Um primeiro passo para melhorar as medidas fornecidas pelas ferramentas de análise de estômatos, que por sua vez ajudarão os biólogos de plantas a avançar em sua compreensão da dinâmica das plantas.

Un primer paso hacia la mejora de las medidas proporcionadas por las herramientas de análisis de estomas, que a su vez ayudará a los biólogos vegetales a avanzar en su comprensión de la dinámica de las plantas.

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Stomata segmentation using deep learning

Angulo M. A., Casado-Garcia A., Heras J. (2023)

Miguel Alonso Angulo, Ángela Casado-García, Jónathan Heras,

Universidad de La Rioja (Spain)Mathematics and Computation

===

Conference: 7th workshop on Computer Vision in Plant Phenotyping and Agriculture at International Conference on Computer Vision (ICCV) 2021 – At: Online –

Abstract

Stomata are pores in the epidermal tissue of leaf plants formed by specialised cells called guard cells, which regulate the stomatal opening. Stomata facilitate gas exchange, being pivotal in the regulation of processes such as pho-tosynthesis and transpiration. The analysis of the number and behaviour of stomata is a task carried out by studying microscopic images; and, nowadays, this task is mainly conducted manually, or using programs that can count and determine the position of stomata but are not able to determine their morphology. In this paper, we have conducted a study of 10 deep learning algorithms to segment stom-ata from several species. The model that achieves the best Dice score, with a value of 96.06%, is obtained with the DeepLabV3+ algorithm, whereas the model that provides the best trade-off between inference time and Dice score was trained using the ContextNet architecture. This is a first step towards improving the measurements provided by stomata analysis tools, that will in turn help plant biologists to advance their understanding of dynamics in plants.

The study would help to develop a stomatal image-based user interface to identify species even without expert taxonomic knowledge and could be particularly useful in fields such as pharmacology, conservation biology, forestry, and environmental science

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该研究将有助于开发基于气孔图像的用户界面,即使没有专业的分类知识也能识别物种,并且在药理学、保护生物学、林业和环境科学等领域尤其有用。


O estudo ajudaria a desenvolver uma interface de usuário baseada em imagens estomáticas para identificar espécies mesmo sem conhecimento taxonômico especializado e poderia ser particularmente útil em campos como farmacologia, biologia da conservação, silvicultura e ciências ambientais.

El estudio ayudaría a desarrollar una interfaz de usuario basada en imágenes estomáticas para identificar especies incluso sin conocimientos taxonómicos expertos y podría ser particularmente útil en campos como la farmacología, la biología de la conservación, la silvicultura y la ciencia ambiental.

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Automated plant species identification from the stomata images using deep neural network: A study of selected mangrove and freshwater swamp forest tree species of Bangladesh

Dey B., Ahmed R., Ferdous J., Haque M. M. U., Khatun R.,Hasan F. E., Uddin S. N. (2023)

Biplob Dey, Romel Ahmed, Jannatul Ferdous, Mohammed Masum Ul Haque, Rahela Khatun, Faria Erfana Hasan, Sarder Nasir Uddin,

Shahjalal University of Science and Technology

===

Ecological Informatics 75: 102128 – DOI: 10.1016/j.ecoinf.2023.102128 – 

https://www.researchgate.net/publication/370766731

Abstract and figures

Stomatal traits of leaves are critical for regulating the exchange of gases between plant tissues and the atmosphere, and thus play a crucial role in the physiological activities of plants. The hypothesis of this study is that distinct stomatal features among different species grown in diverse habitats can serve as a potential marker for species identification. Leaf samples were collected from the mangrove forests of Sundarbans and the freshwater swamp forests of Ratargul in Bangladesh. In total, we examined 11 species from eight different families. We used deep convolutional neural network (DCNN) to automatically identify tree species from microscopic stomatal imprints, as there is currently no established protocol for this task. For model training, 80% (866 images) of the data was used for training the models. Our study observed significant variations in stomatal attributes such as length, width, and density among different species, families, and habitats. These variations could help in accurate species identification by machine learning approaches used in the present study. An empirical comparison was conducted among EfficientNetV2, Xception, VGG16, VGG19, MobileNetV2, ResNet50V2, Resnet152, DenseNet201, and NasNetLarge. We propose a novel approach called the “Normalized Leverage Factor” that utilizes accuracy, precision, recall, and f1-score to select the optimal model. This approach eliminates the non-uniformity of the scores. Although MobileNetV2 achieved an accuracy of 99.06%, our findings indicate that EfficientNetV2 is the optimal model for species identification. This is due to its higher normalized leverage factor (1.92) compared to MobileNetV2 (1.88). The findings demonstrate that plants of diverse habitats show a unique footprint of stomata that offers an innovative method of species identification using DCNN. The study would help to develop a stomatal image-based user interface to identify species even without expert taxonomic knowledge and could be particularly useful in fields such as pharmacology, conservation biology, forestry, and environmental science.

By modifying the classical object detection model YOLO-X via the introduction of a multi-head self-attention mechanism in the object detection model, we were able to accurately detect open and closed stomata

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通过在经典物体检测模型YOLO-X中引入多头自注意力机制的修改,我们能够准确检测到开放和闭合的气孔。

Ao modificar o modelo clássico de detecção de objetos YOLO-X através da introdução de um mecanismo de autoatenção multi-cabeça no modelo de detecção de objetos, conseguimos detectar com precisão estômatos abertos e fechados.


Al modificar el modelo clásico de detección de objetos YOLO-X mediante la introducción de un mecanismo de autoatención multi-cabeza en el modelo de detección de objetos, pudimos detectar con precisión estomas abiertos y cerrados.

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Microscopy image recognition method of stomatal open and closed states in living leaves based on improved YOLO-X

Li K., Cong S., Dai T., Zhang J., Liu J. (2023)

Kexin Li, Shijie Cong, Tianhong Dai, Jingzong Zhang, Jiuqing Liu,

Northeast Forestry University

===

Theoretical and Experimental Plant Physiology 35(10): - DOI: 10.1007/s40626-023-00296-y – 

https://www.researchgate.net/publication/374709725

Abstract

A plant’s stomata serve as the primary means of gas exchange between the plant and its surroundings. This process involves photosynthesis and transpiration. Guard cells enclose the stomata, and the plant controls the rate of transpiration by opening or closing them. Stomata behavior is crucial in determining plant health. Scientists use stomatal counting techniques to study the number of open and closed stomata and their density and distribution on the leaf surface. This study focused on recognizing open and closed stomata in the bottom epidermis of living Populus leaves. By modifying the classical object detection model YOLO-X via the introduction of a multi-head self-attention mechanism in the object detection model, we were able to accurately detect open and closed stomata. Our proposed stomatal detection model achieved accuracy and mAP of 97.1% and 96.9%, respectively. This new model will help researchers obtain stomatal information more efficiently and easily.