• High-Sensitivity Camera — A Tool for Detecting Ultra-Weak Light Signals

The system uses a high-sensitivity, high-resolution research-grade CCD camera with ultra-high quantum efficiency, maximizing sensitivity for detecting weak light signals. The camera is optimized for fluorescent and bioluminescent imaging of plants in vivo and is applied in areas such as plant stress resistance and breeding research, gene expression regulation studies, plant biological rhythm analysis, and photoperiodism and photoinduction research.

• High-Speed Lens Technology

The use of an ultra-fast fixed-focus lens (aperture F< 0.9) significantly increases light signal acquisition speed, reduces exposure time, and minimizes noise from prolonged imaging, effectively improving the signal-to-noise ratio.

• Full-Spectrum Fluorescence Imaging — Seven-Channel Excitation in the UV-IR Range

The system is equipped with two LED boards featuring high-power excitation sources, providing seven excitation channels and full spectral coverage. Combined with high-quality filters, this minimizes interference from background fluorescence and sample autofluorescence, meeting the needs of any in vivo fluorescence imaging experiments with various label types.

• Wide-Format 400 mm Imaging — Accommodating Various Samples and High-Throughput Screening

The BIOVIVO™ G series in vivo plant imaging system provides a field of view up to 400×400 mm with stepless adjustment at 1 mm precision. This enables continuous observation of the complete plant life cycle—from seed germination and seedling growth to mature plants—facilitates high-throughput screening of mutant lines (simultaneous imaging of up to 16 potted plants), and supports research on large plants (samples ≥ 60 cm in height).

• Comprehensive Environmental Control for Plants — Temperature, Humidity, Lighting

Inside the light-tight chamber, temperature and humidity are controlled. The system features a temperature-regulated stage with a range of 10–45°C for temperature stress studies.

A combination of 8 spectral bands (UV-visible-near IR) simulates sunlight; the intensity and duration of individual spectra and their combinations are freely adjustable. Light intensity reaches 26,000 lux (at 400 mm distance)—ideal for studying plant biological rhythms and photoperiodism.

• Professional Intelligent Software for Image Acquisition, Processing, and Analysis

Biovivo LabEasy™ software is designed to maximize simplicity in in vivo imaging experiments: a simple and intuitive interface, quick learning curve; one-click acquisition of high-quality images; convenient image processing, rapid background removal and signal identification; precise quantitative ROI analysis; multi-image analysis; multispectral fluorescence analysis function for separating multiple fluorescent signals and effectively removing background noise; support for dual-color and multi-color fluorescent labeling imaging.

• Applications of In Vivo Plant Imaging Systems

1. Research on plant stress resistance and breeding
2. Study of protein interactions using transient expression in tobacco
3. Research on gene expression regulation in plants in vivo
4. Study of signal transduction pathways in plants in vivo
5. Research on plant growth and development
6. Plant phenotyping
7. Study of plant biological rhythms
8. Research on plant photoperiodism
9. Study of light-induced processes in plants
10. Research on plant diseases and immune responses

As early as the first two decades of the 20th century, and even earlier in the 19th century, scientists discovered that focusing on relatively simple organisms could partially unravel the mysteries of developmental phenomena. For example, in uncovering the laws of heredity in nature, Mendel used peas as experimental material, while Morgan chose the fruit fly Drosophila. In their studies, peas and Drosophila had fewer cells, their structure was more homogeneous, and changes were easier to observe.

As a result of evolution, cellular life forms exhibit significant similarities in fundamental developmental patterns, and many basic life mechanisms are similar across various biological species on Earth. Therefore, it is possible to use species at relatively low steps of the biological complexity ladder to study general developmental patterns. This is especially true when common characteristics of morphogenesis and changes are found in organisms with different developmental features, allowing for the establishment of universal principles of development. Studying such organisms helps us uncover universal patterns of life phenomena and understand the general laws governing the living world. Thus, these specially selected organisms were named “model organisms.” The choice of a specific organism as a model depends primarily on the scientific task the researcher sets, as well as on finding the species most suitable for solving this task.

In the early 20th century, Morgan chose the fruit fly Drosophila melanogaster as his research subject. Based on this work, the chromosomal theory of heredity was established, laying the foundation for classical genetics and initiating the use of Drosophila as a model organism. Scientists not only confirmed Mendel’s laws in Drosophila but also discovered sex-linked inheritance of the white-eye mutation, formulated the principle of linear gene arrangement on chromosomes, and the law of linkage and crossing over. In 1933, Morgan was awarded the Nobel Prize for these achievements. The sequencing of the Drosophila genome, completed in 2000, solidified its status as a crucial model animal for studying genetics and developmental biology.

Since 1965, scientist Sydney Brenner introduced the nematode Caenorhabditis elegans into the field of molecular and developmental biology research. In 1983, the complete cell lineage of the worm from fertilized egg to adult was described, marking a milestone in the history of developmental biology. Subsequently, C. elegans found widespread application in studies of embryonic development, sex determination, apoptosis, behavior, and neurobiology, and became a crucial model organism in the study of aging and lifespan.

Zebrafish possess characteristics such as high fecundity, external fertilization and development, transparent embryos, a short sexual maturity cycle, small size, and ease of maintenance, making them an ideal choice for studying mechanisms of embryonic development, gene functions, and disease pathogenesis. The African clawed frog (Xenopus laevis) has large and abundant oocytes, which are convenient for microsurgical manipulations. Furthermore, they can be used to create biologically active cell-free systems suitable for biochemical analysis, ensuring their indispensable role in developmental biology research.

The main model animal is, undoubtedly, the laboratory mouse! Due to their small size, docile nature, ease of maintenance, and the similarity of developmental processes and anatomical tissue structure to humans, mice began to be used in anatomy and animal experiments as early as the 17th century. As a result of long-term selection and breeding under artificial conditions, thousands of independent outbred and inbred lines have been developed to date. The laboratory mouse has become the most important model organism for deciphering human gene functions and studying human diseases, as well as the most thoroughly studied mammal in the world for scientific experiments. Moreover, mouse genes exhibit a high degree of homology with human genes: 99% of human genes have corresponding genes in the mouse genome. The mouse is widely used as a model organism in biomedical research and is the most detailed studied mammal for experiments in the modern world.

Above were presented model organisms from the animal kingdom, which we commonly refer to as model animals. Are there also model plants? The answer is affirmative. In modern botanical research, Arabidopsis thaliana, tobacco, tomato, rice, and others are most frequently used as model plants, with each serving its specific research purposes.

Arabidopsis thaliana opens the list of model plants. It belongs to the division of angiosperms, the class of dicotyledons, and is a representative of the mustard family. This plant itself does not possess great economic value. However, its small size, high seed productivity, and short life cycle; clearly distinguishable morphological features, convenient mutant phenotypes for observation, and self-pollination make it an exceptionally convenient object. The genome of Arabidopsis thaliana is considered small for higher plants (about 125 million base pairs and 5 pairs of chromosomes), and its genes are characterized by a high degree of homozygosity. Treatment with physicochemical factors leads to a high frequency of mutations, making it easy to obtain mutants with defects in various metabolic functions. Thanks to this, Arabidopsis thaliana has become an ideal model organism for studying the genetics, cellular development, and molecular biology of flowering plants, earning it the title of “Drosophila of the plant world” among scientists.

The history of Arabidopsis thaliana research dates back to the 16th century. In 1943, Laibach thoroughly substantiated its advantages as a model organism, which contributed to the first International Conference on Arabidopsis thaliana in Germany in 1965. However, its active use as a model organism began approximately in the last two decades of that century. In 1986, the Meyerowitz laboratory first reported the cloning of an Arabidopsis thaliana gene. In 1988, the first RFLP map of its genome was published. In the following few years, reports of gene cloning of mutants with T-DNA insertions and gene cloning based on genetic maps followed. In 2000, the work on determining the complete nucleotide sequence of its genome was completed, making Arabidopsis thaliana the first plant with a fully sequenced genome.

Tomato, or love apple, is an annual or perennial herbaceous plant of the nightshade family, genus Solanum. The plant height is 0.6–2 meters, its entire surface is covered with sticky glandular hairs, and it has a strong characteristic odor. The stem tends to lodge. Leaves are pinnately compound or pinnately dissected. The peduncle length is 2–5 cm, the inflorescence usually consists of 3–7 flowers. The calyx and corolla are wheel-shaped. The fruit is a juicy fleshy berry, oblate-spherical or almost spherical, with yellow seeds. The flowering and fruiting period occurs in summer and autumn. Due to its relatively small genome size, short life cycle, ability to self-pollinate, well-established genetic transformation system, and clear genetic basis, as well as a more distant relationship with other model plants (such as Arabidopsis thaliana or rice), tomato has become a classic model organism for studying fruit development and ripening processes.

Rice, as one of the most important cereal crops in the world, plays an increasingly significant role as a model plant for studying cereals. This is due to its relatively small genome, known genomic sequence, well-established Agrobacterium-mediated genetic transformation system, and high degree of homology (genetic similarity) with other cereal crops. Tobacco is not only an important economic crop but also one of the most valuable materials for scientific research. Many innovative studies have been conducted on tobacco. A significant portion of botanical knowledge, such as photoperiodism, plant nutrition, photosynthesis, photorespiration, organic metabolism, as well as virus and transgenic organism research, has been obtained from tobacco. No other plant has been studied as deeply by science as tobacco. It is a model organism for molecular biology and genetic engineering and has rightfully earned the title of “laboratory mouse of the plant kingdom.”

In addition to the aforementioned model animals and plants, the family of model organisms, of course, also includes microorganisms. Escherichia coli and yeast are key materials for studying the genetics of microorganisms and are widely used as model organisms in microbiological research. In the future, as research in life sciences develops, the family of model organisms will undoubtedly continue to grow.

Agrobacterium-Mediated Plant Genetic Transformation

The Agrobacterium-mediated transformation method is a highly efficient genetic transformation method characterized by a wide range of suitable biological material, high transformation frequency, large percentage of single-copy insertions, and transformant stability, opening broad prospects for its application.

Agrobacterium is a gram-negative bacterium widely distributed in soil. Under natural conditions, it is capable of chemotactically infecting damaged sites in most dicots and some gymnosperms, causing crown gall formation or “hairy” roots. Cells of Agrobacterium tumefaciens and Agrobacterium rhizogenes contain a T-DNA region. After penetrating a plant cell through a wound, Agrobacterium can integrate this T-DNA into the plant genome. Thus, Agrobacterium represents a natural plant genetic transformation system. By inserting a target gene into the modified T-DNA region, the infectious process of Agrobacterium can be used to transfer and integrate a foreign gene into plant cells. Then, using cell and tissue culture technologies, transgenic plants are regenerated. Under laboratory conditions, this method has successfully transformed organisms that are not natural hosts of Agrobacterium, such as fungi, monocots, gymnosperms, and even animal cells. Compared to traditional methods such as PEG-mediated protoplast transformation, electroporation, or the LiAC method, Agrobacterium-mediated transformation has the following advantages: wide range of suitable material, high transformation frequency, large percentage of single-copy insertions, and transformant stability. This is one of the most widely applied methods.

Tobacco Genetic Transformation Technology

I. Preparation

1. Tobacco plants grown in sterile culture (for 30 days)
2. Forceps, scissors, scalpels, Petri dishes (12 cm diameter) with 5-6 layers of filter paper

II.Transformation Process

(1) Preparation of bacterial suspension

The day before the experiment, select a single Agrobacterium colony intended for transformation and inoculate into 10 ml of LB medium (with rifampicin and kanamycin added at 50 mg/L each), then culture overnight.

(2) Infection of tobacco with Agrobacterium

1. Preparation of co-cultivation medium: MS medium + NAA (0.1 mg/L) + 6-BA (1.5 mg/L).
2. Using scissors and forceps, cut well-developed leaves from sterile tobacco plants and place them in a glass Petri dish.
3. In the Petri dish, cut the leaves with a scalpel into segments approximately 0.5–1 cm². Quickly run the blade across the leaf, cutting off pieces, trying to obtain clean cuts. Transfer the cut leaf segments with forceps into Agrobacterium suspension at OD 0.4–0.6 and incubate for 8–10 minutes.
4. Drain the suspension and transfer the leaf segments with forceps onto filter paper in a Petri dish to remove excess bacterial liquid. Allow the leaf surfaces to dry for some time in a laminar flow hood.
5. Place the leaf segments with removed suspension onto co-cultivation medium and culture in darkness at 25°C for three days.

(3) Differentiation and selection of transformed tobacco cultures

1. Preparation of medium: MS + 1.5 mg/L 6-BA + 0.1 mg/L NAA + 30 mg/L hygromycin + 100 mg/L cefazolin.
2. Transfer the leaf segments after three days of co-cultivation onto the prepared medium, ensuring their close contact with the medium surface and leaving some distance between segments. Cultivation is conducted under light at 25°C.
3. After 2–3 weeks of cultivation under light, tobacco shoots form. Cut the shoots and transfer them to rooting and growth enhancement medium (MS + 0.1 mg/L 6-BA + 0.01 mg/L NAA + 30 mg/L hygromycin + 100 mg/L cefazolin).
4. After approximately one week, select tobacco shoots that have formed roots and transfer them to rooting medium (MS + 30 mg/L hygromycin + 100 mg/L cefazolin).
5. Perform PCR analysis of tobacco shoots to confirm transformation.

In vivo imaging technology is becoming an increasingly important tool in phytopathology and plant-pest interaction research, allowing for the deciphering of pathogen infection mechanisms, host immune responses, and the dynamics of plant-pest relationships. Due to its non-invasive nature, real-time dynamic observation capabilities, and high resolution, this technology enables researchers to track pathogen spread, host plant defense responses, and pest behavior – from the molecular to the organ level. Its applications and the challenges it faces are discussed in detail below from several perspectives:

I. Overview of In Vivo Imaging Technology

In vivo imaging technology allows for the dynamic visualization of pathogens, pests, and associated physiological processes in plants through the application of fluorescent markers, bioluminescence, or optical probes in combination with microscopic or macroscopic imaging systems (such as IVIS, confocal microscopy). Commonly used methods include:

• Fluorescence imaging: labeling pathogens (e.g., GFP-tagged pathogenic fungi), plant immune signaling molecules (e.g., ROS probes), pest behavior (e.g., tracking insect feeding paths with fluorescent tags).
• Bioluminescence imaging: using pathogen-specific promoters to drive luciferase expression (e.g., PR1::LUC reporter system activated upon pathogen infection).
• Chlorophyll fluorescence imaging: assessing damage to the photosynthetic apparatus after pathogen infection (e.g., reduction in Photosystem II efficiency during Phytophthora infection).
• Near-infrared imaging: penetrating leaf tissues to track pathogen colonization in deeper layers (e.g., fluorescent labeling of bacteria in xylem).

II. Applications in Phytopathological Research

1. Dynamic Tracking of Pathogen Infection Process

Pathogen Colonization and Spread:

• Using GFP/RFP-tagged pathogens (e.g., Colletotrichum, Phytophthora) for real-time observation of their spread from penetration points (stomata, leaf wounds) into host plant tissues.
• Applying time-lapse imaging to identify systemic pathogen movement through xylem or phloem (e.g., movement of Xanthomonas bacteria through the vascular system).

Visualization of Infection Structures:

• Confocal microscopy observation of the formation of pathogen attachment structures (e.g., appressoria and haustoria of rust fungi) and their interaction with the plant cell wall.

2. Analysis of Host Immune Response

Monitoring Early Defense Signals:

• Using fluorescent probes (e.g., H2DCFDA) for real-time detection of reactive oxygen species (ROS) bursts induced by pathogen-associated molecular patterns (PAMPs).
• Applying calcium probes (e.g., GCaMP) to record calcium signaling oscillations triggered by pathogen infection (e.g., Ca²⁺ peaks induced by bacterial flagellin).

Dynamics of Immune Gene Expression:

• Utilizing fluorescent reporter systems (e.g., NPR1::GFP) to track the spatio-temporal characteristics of salicylic acid signaling pathway activation.
• Applying bioluminescence imaging (e.g., LUC driven by a WRKY transcription factor promoter) to quantify the intensity of immune gene induction.

3. Studying Pathogen-Host Interaction Mechanisms

Investigation of Effector Protein Functions:

• Tagging pathogen effector proteins (e.g., RXLR effectors in oomycetes) to visualize their intracellular localization in the host plant (e.g., targeting the nucleus or plasma membrane).
• Applying bimolecular fluorescence complementation (BiFC) to determine the interaction sites of effector proteins with host target proteins.

Assessment of Plant Disease Resistance:

• Combining chlorophyll fluorescence imaging with pathogen labeling for rapid screening of resistant plant mutant lines (e.g., Arabidopsis thaliana lines resistant to powdery mildew).

III. Applications in Pest Monitoring and Behavior Research

1. Tracking Pest Feeding Paths and Spread

Fluorescent Labeling of Insects:

• Feeding insects food containing fluorescent dyes (e.g., Cy5-labeled sucrose solution), followed by macro-fluorescence imaging to track feeding sites and movement paths of aphids, whiteflies, and other pests on the plant.

Research on Pathogen-Vector Insects:

• Using in vivo imaging to observe virus replication and transport in insect vectors (e.g., tobacco whitefly), for example, by labeling viral capsid proteins.

2. Dynamics of plant-pest interactions

Plant signals induced by insect damage:

• Using a GFP-labeled reporter system for the jasmonic acid (JA) signaling pathway (e.g., JAZ1::GFP) to monitor real-time systemic transmission of JA signals following insect damage.

Regulation of pest behavior:

• Application of near-infrared imaging to record the diurnal activity of nocturnal pests (e.g., armyworm caterpillars) and their response to plant volatiles.

3. Evaluation of the effectiveness of plant protection measures against pests and diseases

Visualization of pesticide mechanisms of action:

• Using fluorescently labeled pesticides (e.g., rhodamine B-labeled insecticides) to track their uptake and distribution in the plant.
• Application of in vivo imaging to assess the effectiveness of pest infection by biopesticides (e.g., entomopathogenic nematodes labeled with fluorescent dyes).

Functional validation of pest resistance genes:

• Combining CRISPR genome editing technology with fluorescence imaging to confirm the inhibitory effect of pest resistance genes (e.g., protease inhibitor genes) on insect digestion.

IV. Technology Advantages

1. Dynamic continuity: long-term tracking of disease development or pest damage on the same plant, eliminating the influence of individual differences between specimens.
2. High sensitivity: ability to detect infection at an early stage (e.g., pathogen behavior before infection, prior to penetration through stomata) or capture weak defense signals.
3. Multidimensional integration: simultaneous monitoring of interactions between pathogen, host plant physiology, and environmental factors (temperature and humidity).

V. Technology Challenges and Limitations

1. Tissue penetration limitations: imaging thick tissues (such as fruits or tubers) or insect chitinous coverings requires the use of near-infrared imaging or optoacoustic imaging.
2. Risk of label influence: fluorescent labeling of pathogens or insects may potentially alter their pathogenicity or behavior (e.g., GFP expression may affect fungal toxicity).
3. Complexity of natural environment modeling: differences between laboratory imaging conditions and the natural environment (e.g., in illumination, microflora) may affect the reliability of results obtained.
4. Data analysis challenges: processing massive arrays of dynamic data (pathogen movement trajectories, pest behavior patterns) requires the application of artificial intelligence algorithms, such as object tracking models and behavior classification.

VI. Prospective Development Directions

Development of new probes:

• Creation of pathogen-specific probes (e.g., fluorescently labeled nanobodies targeting specific effector proteins).
• Development of label-free imaging methods (e.g., Raman spectroscopy-based imaging for direct pathogen identification).

Integration of multimodal imaging:

• Combining methods such as micro-CT and fluorescence imaging for 3D reconstruction of the spatial distribution of pathogens in tissues (e.g., in nodules or stems).

Development of portable field devices:

• Creation of portable in vivo imaging systems enabling rapid diagnostics and early warning of diseases and pests directly in field conditions.

In vivo imaging technology provides a comprehensive toolkit for research in phytopathology and plant protection, covering the entire chain of events—from molecular interactions at the micro level to ecological relationships at the macro level. It makes a significant contribution to deciphering the mechanisms of disease and pest resistance, as well as to developing strategies for environmentally safe plant protection. Despite existing technological limitations, progress in probe development, imaging equipment improvement, and data analysis methods will continue to expand the boundaries of its application, providing key technological support for sustainable agricultural development.

In vivo imaging technology (Live Imaging) as a non-invasive method for dynamic real-time monitoring plays an important role in plant stress resistance research and breeding. By combining optical imaging, fluorescent labeling, genome editing, and other technologies, it allows researchers to visually observe physiological and biochemical processes, gene expression dynamics, and morphological structure changes in the plant without disrupting its natural growth. Below are specific application areas and advantages of this method in plant stress resistance research and breeding:

I. Application in Plant Stress Resistance Research

1. Real-time monitoring of stress-related gene expression

• Using fluorescent reporter genes (GFP, luciferase) to label genes involved in stress response (e.g., DREB, NAC, WRKY transcription factors) to track spatiotemporal patterns of their expression under drought, salinity, low temperature, and other stressors.
• Example: Observing through in vivo imaging the dynamic response of key genes in the abscisic acid (ABA) signaling pathway to analyze molecular mechanisms of plant response to water stress.

2. Dynamic visualization of physiological and biochemical processes

• Monitoring physiological parameters such as reactive oxygen species (ROS) accumulation, ion fluxes (e.g., Ca²⁺ fluctuations), pH changes, which allows identification of early signals of plant response to adverse conditions.
• Example: Application of fluorescent probes (e.g., H2DCFDA for ROS detection) combined with confocal microscopy for real-time observation of ROS accumulation bursts in root tips under salt stress.

3. Spatiotemporal analysis of stress response mechanisms

• Using in vivo imaging technology to track dynamic changes in plant organs (root tips, leaf stomata) under stress exposure, such as root system structural remodeling or stomatal opening/closing behavior.
• Example: application of micro-CT or laser scanning to analyze the process of three-dimensional root system structure remodeling under drought conditions.

4. Investigation of pathogen interactions and disease resistance

• Dynamic monitoring of the pathogen infection process and plant immune response (such as HR-mediated cell death, callose deposition) by labeling pathogens (e.g., fluorescently labeled pseudomonads) or plant immunity-related proteins.
• Example: in vivo imaging to reveal spatiotemporal patterns of PTI (PAMP-triggered immunity) activation in Arabidopsis thaliana.

II. Application in Plant Breeding

1. Phenomics and rapid screening of agronomically valuable traits

• High-throughput in vivo imaging systems (e.g., PhenoCam, hyperspectral imaging) combined with artificial intelligence-based analysis enable non-invasive screening of stress-resistant traits (drought tolerance, photosynthetic efficiency) in large plant populations.
• Example: rapid identification of rice varieties with high photosynthetic capacity using chlorophyll fluorescence imaging.

2. Optimization of root system architecture and nutrient use efficiency

• Application of X-ray computed tomography (X-ray CT) or magnetic resonance imaging (MRI) for dynamic monitoring of root growth to select genotypes with high water and nutrient uptake efficiency.
• Example: in wheat breeding, using in vivo imaging to select drought-tolerant varieties with deeply developed root systems.

3. Real-time validation of genome editing effectiveness

• Functional validation of stress resistance genes edited using CRISPR/Cas9 (e.g., OsNAC14) by in vivo imaging methods, for example, by observing transgenic plant survival under stress conditions or metabolite accumulation.
• Example: using fluorescently labeled promoters controlling salt tolerance gene expression for real-time monitoring of the correlation between their expression strength and plant phenotype under salt stress.

4. Dynamic investigation of seed development and germination

• In vivo imaging technology allows tracking embryo activity, storage substance metabolism, and stress responses during seed germination, providing a foundation for breeding varieties with high germination rates.