CN115839937A - Taxus chinensis stress detection method based on chlorophyll fluorescence imaging technology - Google Patents
Taxus chinensis stress detection method based on chlorophyll fluorescence imaging technology Download PDFInfo
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Abstract
The invention discloses a taxus chinensis stress detection method based on a chlorophyll fluorescence imaging technology, which mainly comprises the steps of collecting and preprocessing small branches with leaves of taxus chinensis, measuring a chlorophyll fluorescence parameter Fv/Fm value and collecting corresponding RGB images, applying a stress grading standard and the like. Because the chlorophyll fluorescence dynamics of different taxus chinensis germplasm conifers have obvious difference under different climatic environments all the year around, the health degree or the stress state of the taxus chinensis can be identified by screening specific parameters and analyzing imaging characteristics. The method has little influence on growth and quality traits of the taxus chinensis, and has the advantages of simple operation, good result repeatability and high identification efficiency; and has wide application prospect in the fields of taxus resource protection and utilization, environment-tolerant taxus variety breeding, mutant strain screening and the like, and provides technical basis for genetic improvement of taxus.
Description
Technical Field
The invention relates to a plant early stress diagnosis technology, in particular to a taxus chinensis stress detection method based on a chlorophyll fluorescence imaging technology.
Background
The Taxus chinensis is evergreen needle-leaved shrub or arbor of Taxus genus (Taxaceae), is a national grade I wild rare or endangered protective plant, has great material, ornamental and medicinal values, is especially specific diterpenoid substance taxol in trees, is considered as the most effective anticancer drug in first-line clinical, and has excellent curative effect on treating breast cancer, lung cancer, ovarian cancer and the like. However, the growth of the taxus chinensis is slow, the requirements on the habitat are very strict, even if environmental factors in a suitable living area change, the growth and development of the taxus chinensis can be influenced, even the survival of the taxus chinensis is threatened, and the cultivation and popularization of the taxus chinensis are severely restricted. Therefore, a scientific and effective detection method for the early stage stress state of the taxus chinensis is necessary to be established, which is the basis for reasonably developing taxus chinensis resources, quickly and accurately identifying and screening environment-tolerant taxus chinensis germplasm and improving the breeding efficiency of the taxus chinensis, and has important significance for the protection and utilization of the taxus chinensis.
Chlorophyll fluorescence technology is a powerful tool for studying the change of photosynthesis of plants in different environments. Based on the advantages of rapidness, sensitivity, non-destructive measurement and the like, the method is widely applied to the fields of plant stress resistance physiology and the like. After the plant is stressed, the fluorescence intensity of the leaves is changed before the phenotypic characteristics are changed. Due to the loss of photosynthetic activity, fluorescence information is correspondingly reduced, and in severe cases, there is almost no fluorescence information. Therefore, by researching the change of chlorophyll fluorescence dynamics and combining with an image processing technology, the healthy plants and stressed plants can be distinguished.
At present, although there have been attempts to explore the effects of different biotic or abiotic stresses on plants, such as temperature, moisture, plant diseases and insect pests, etc., by using chlorophyll fluorescence imaging technology. However, the method for detecting the taxus chinensis stress state is not reported at present, and the chlorophyll fluorescence parameters of different plants have different sensitivity degrees to environmental changes, so that proper parameters need to be screened for construction of taxus chinensis fluorescence imaging.
Disclosure of Invention
In view of the above, the invention aims to provide a taxus chinensis stress detection method based on a chlorophyll fluorescence imaging technology.
It should be noted that most of the methods published at present are directed at specific periods and specific stresses, and the recognition effect under natural or agricultural production conditions is general; and the survival state of the plants is directly influenced by seasonal variation. Therefore, it is necessary to analyze the influence of seasonal variation on the detection effect of the taxus stress state more comprehensively.
In addition, the prior art mostly carries out single-point or multi-point measurement aiming at broad-leaved plants, and the taxus chinensis is taken as a typical conifer species, after the taxus chinensis is stressed, the chlorophyll fluorescence parameters of different areas on the surface of a leaf or different conifers have spatial heterogeneity, and the single-point or multi-point measurement analysis of the single leaf and the leaf is insufficient; and the requirements of the taxus chinensis on the growth environment are strict, and a monitoring method prior to phenotypic symptoms is developed for detecting the early stress state of the taxus chinensis so as to promote the protection and utilization of the taxus chinensis.
A taxus chinensis stress detection method based on a chlorophyll fluorescence imaging technology comprises the following steps:
(1) Collecting small branches with leaves of the taxus chinensis, wherein the small branches with leaves of the taxus chinensis comprise small branches with full-developed and mature leaves in different collection time and different growth seasons;
(2) Wiping water and stains on the surfaces of the small branches with leaves of the taxus chinensis in the step (1) with filter paper or gauze, placing the filter paper or gauze in a self-sealing bag for extruding air, and placing the self-sealing bag in an opaque ice box for dark treatment;
(3) Measuring a chlorophyll fluorescence parameter Fv/Fm (maximum photon yield of a PSII reaction center) value of the small branches with leaves of the taxus chinensis by using a modulation fluorescence imaging system, and collecting corresponding RGB images;
(4) Determining the stress degree of the taxus chinensis germplasm to be detected according to the following grading standards:
Fv/Fm is more than or equal to 0.8, the leaves of the whole small branches in the RGB image are in uniform orange-red color and are in a healthy state;
Fv/Fm is 0.74-0.8, the leaves of the whole twig in RGB image are orange yellow, it is the mild stress state; the local leaves are yellow green and in a local moderate stress state;
Fv/Fm is 0.70-0.74, the leaves of the whole twig in RGB image are yellow green, it is the moderate stress state; the local leaves are blue-green and in a local severe stress state;
Fv/Fm is less than 0.7, the leaves of the whole small branch in the RGB image are blue-green, and are in a severe stress state.
Further, in step (1), the different collection times are preferably in a clear day with clear weather, and the time interval is from 9 to 11 am; the different growing seasons can be determined according to actual needs.
Preferably, the method collects the small branches with leaves of the taxus chinensis monthly as the test samples.
The small branches with full-spread mature leaves preferably have annual small branches at the tops of 3-7 th-level branches from the top of the plant, the collection length is 3-5 cm, and each small branch can be guaranteed to contain 15-20 needle leaves.
Further, in the step (2), the dark treatment time is at least 20 minutes, which can be determined according to actual needs.
Preferably, the invention combines the storage, transportation and dark treatment of the sample for 20 to 60 minutes, and can ensure the accuracy of the subsequent measurement of the excised leaf.
Further, in the step (3), operating the chlorophyll fluorescence imaging system according to the specifications of the instrument instruction, measuring the chlorophyll fluorescence parameter Fv/Fm value of the sample to be tested, and collecting the corresponding RGB image.
Preferably, the invention sets the RGB image color mode corresponding to Fv/Fm value to 0-255, and sets the RGB image color intensity value corresponding to Fv/Fm value to 0.35-0.9, which can present obvious grading effect when identifying the coercion degree;
furthermore, the chlorophyll fluorescence parameter Fv/Fm value obtained by measurement in the step (3) is a comprehensive value of the whole small branch sample with leaves; preferably, the invention detects each material by repeating the sample for 3 times, and calculates the average value, thereby obtaining the Fv/Fm value of the material to be detected.
Further, in the step (4), the grading standard is used for the comprehensive identification results of different Taxus species Fv/Fm values and corresponding RGB images under different weather conditions all the year round, and is universal for Taxus lobular branches under different growth environments.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the chlorophyll fluorescence imaging technology is applied to the detection of taxus germplasm stress, the chlorophyll fluorescence imaging is detected by adopting the small branches with leaves of the taxus, and the specific parameters and the imaging characteristics are optimized aiming at the taxus germplasm, so that the method has the advantages of small wound caused by plants, quick operation process, good result repeatability and good identification effect on the stress states of the taxus chinensis in different seasons and different growth environments.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a representative RGB image of Fv/Fm of Taxus media with leaves for 1-12 months without setting of unified imaging mode.
FIG. 2 is a representative RGB image of Fv/Fm of Taxus chinensis var mairei with leaves from 1 month to 12 months without setting of unified imaging mode.
FIG. 3 shows Fv/Fm values of Taxus media with leaves for 1-12 months and corresponding representative RGB images.
FIG. 4 shows Fv/Fm values of Taxus chinensis var mairei with leaves for 1-12 months and corresponding representative RGB images.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings of the specification, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention should not be construed as being limited thereto, and the insubstantial modifications and variations of the present invention as shown in the above-described summary of the invention are considered to fall within the scope of the invention by those skilled in the art.
The technical solution of the present invention will be further described with reference to the following specific examples.
Example 1
1. Collection of small branches with leaves of taxus chinensis
The Taxus germplasm adopted by the invention is respectively Taxus media (Taxus media) and Taxus mairei (Taxus mairei). The yew with leaves and small branches for trial use are respectively collected in the germplasm resource garden of yew in the basalt Nanjing Zhongshan botanical garden in the basalt region in Jiangsu province, nanjing City, 1-12 months in 2021. The environmental factor changes during the observation period are shown in table 1.
As can be seen from Table 1, with the change of seasons, the temperature, the illumination and the precipitation are changed to different degrees, so that the growth and development of the Taxus chinensis are influenced by the potential environmental stress. Selecting a clear and cloudless sunny day in each month (10-15 days), collecting current-year-old twigs with full-spread and mature leaves at the tops of 3-7 th-level branches from the top of the plant downwards in 9-11 am, wherein the collection length is 3-5 cm, and each twigs is guaranteed to contain 15-20 needle leaves;
TABLE 1 environmental factor data from 1 to 12 months 2021
2. Pretreatment of yew with leaves and twigs
Collecting a taxus chinensis small branch sample with leaves every month, immediately wiping off water and stains on the surface of the sample by using prepared filter paper or gauze, placing the sample in a self-sealing bag for extruding air, placing the sample in an opaque ice box for storage, and completing the processes of sample transportation and dark treatment at the same time, wherein the time is 20-60 minutes, so as to ensure the accuracy of the subsequent measurement of the isolated sample.
3. The dark-processed sample is flatly placed on a placing table of a detection chamber of a Chlorophyll Fluorescence imaging system, the Chlorophyll Fluorescence imaging system used in the embodiment of the invention is a modulated Fluorescence imaging system Chlorophyll Fluorescence-Imager (CFI, technology, UK), the Chlorophyll Fluorescence imaging system is operated according to the specifications of an instrument instruction, and Chlorophyll Fluorescence parameters including Fv/Fm, fv '/Fm' (effective light quantum yield of PSII reaction center), NPQ (non-photochemical quenching), qP (photochemical quenching) and qP (photochemical quenching) are automatically calculated and obtained by using built-in software Fluor Imager (version 2.2)
(actual photon yield of PSII reaction center) and RGB image of corresponding parameters.
As shown in Table 2, fv '/Fm ' and Fv ' were found by analyzing the dynamic change of different chlorophyll fluorescence parameters of different yew germplasms throughout the year
The variation amplitude under different environmental conditions is less than Fv/Fm, and the index sensitivity is weaker; NPQ and qP are affected by the activity of the photosynthetic machinery and do not exhibit a consistent trend of change when indicating different environmental stresses; has good effectAnd (4) selectively determining Fv/Fm as a sensitive index indicating the stress state of the taxus chinensis.
TABLE 2 monthly dynamic changes in chlorophyll fluorescence parameters of two Taxus chinensis species
In addition, representative RGB images of Fv/Fm of Taxus media and Taxus chinensis var mairei without the unified imaging mode setting are shown in FIGS. 1 and 2. As can be seen in fig. 1 and 2, neither the health nor heterogeneity of the leaves can be accurately distinguished.
And setting a fluorescence imaging mode for matching parameter grading according to annual change results of different taxus germplasms Fv/Fm. Preferably, the color mode of the RGB image is set to be 0-255, the color intensity value of the RGB image is set to be 0.35-0.9, and obvious grading effect is presented when the stress degree of the taxus chinensis is identified. 3 repeated samples are set for each material, and the average value is calculated to obtain the Fv/Fm value of the material.
4. Determining the stress degree of the taxus chinensis to be tested according to the following grading standards:
Fv/Fm is more than or equal to 0.8, the leaves of the whole small branches in the RGB image are in uniform orange-red color and are in a healthy state;
Fv/Fm is 0.74-0.8, the leaves of the whole twig in RGB image are orange yellow, it is the mild stress state; the local leaves are yellow green and in a local moderate stress state;
Fv/Fm is 0.70-0.74, the leaves of the whole twig in RGB image are yellow green, it is the moderate stress state; the local leaves are blue-green and in a local severe stress state;
Fv/Fm is less than 0.7, the leaves of the whole small branch in the RGB image are blue-green, and are in a severe stress state.
The Fv/Fm values and RGB pictures of different Taxus germplasms are shown in FIG. 3 and FIG. 4.
According to the ranking criteria of the present embodiment, as can be seen from the test results (fig. 3 and 4),
the taxus media presents a healthy state in 2 months and 4-9 months, presents a mild stress state in 10 months and 11 months, presents a local moderate stress state in 3 months, presents a moderate stress state in 12 months and presents a severe stress state in 1 month;
the taxus mairei is in a healthy state in months 4, 5, 7 and 9, in a mild stress state in month 11, in a local moderate stress state in months 3, 6, 8 and 10, in a moderate stress state in months 2 and 12 and in a severe stress state in month 1;
in addition, the southern taxus chinensis has the phenomenon of green fading on the surface of the leaves in 1 month, and the accuracy of the classification standard is further verified.
Moreover, the tested taxus chinensis is a common cultivation material, and the implementation of the invention is not limited to the germplasm.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A taxus chinensis stress detection method based on a chlorophyll fluorescence imaging technology is characterized by comprising the following steps:
(1) Collecting small branches with leaves of the taxus chinensis, wherein the small branches with leaves of the taxus chinensis comprise small branches with full-developed and mature leaves in different collection time and different growth seasons;
(2) Wiping water and stains on the surfaces of the small branches with leaves of the taxus chinensis in the step (1) with filter paper or gauze, placing the filter paper or gauze in a self-sealing bag for extruding air, and then placing the self-sealing bag in an opaque ice box for dark treatment;
(3) Measuring a chlorophyll fluorescence parameter Fv/Fm (maximum photon yield of a PSII reaction center) value of the small branches with leaves of the taxus chinensis by using a modulation fluorescence imaging system, and collecting corresponding RGB images;
(4) Determining the stress degree of the taxus chinensis germplasm to be detected according to the following grading standards:
Fv/Fm is more than or equal to 0.8, the leaves of the whole small branches in the RGB image are in uniform orange-red color and are in a healthy state;
Fv/Fm is 0.74-0.8, the leaves of the whole twig in RGB image are orange yellow, it is the mild stress state; the local leaves are yellow green and in a local moderate stress state;
Fv/Fm is 0.70-0.74, the leaves of the whole twig in RGB image are yellow green, it is the moderate stress state; the local leaves are blue-green and in a local severe stress state;
Fv/Fm is less than 0.7, the leaves of the whole small branch in the RGB image are blue-green, and are in a severe stress state.
2. The method for detecting taxus chinensis stress based on the chlorophyll fluorescence imaging technology according to claim 1,
in the step (1), selecting a sunny day without clouds, and selecting a current-year-old new branch with fully developed and mature needle leaves at the top of a 3 rd to 7 th branch from the top of the plant in 9 to 11 am for collection, wherein the collection length is 3 to 5cm.
3. The method for detecting taxus chinensis stress based on the chlorophyll fluorescence imaging technology according to claim 1,
in the step (2), the storage, transportation and dark treatment of the sample in the ice box can be performed simultaneously for 20 to 60 minutes.
4. The method for detecting taxus chinensis stress based on the chlorophyll fluorescence imaging technology according to claim 1,
in the step (3), the measured chlorophyll fluorescence parameter Fv/Fm value is the comprehensive value of the whole leaf-carrying small branch sample; and 3 repeated samples are set for each material to be detected, and then the average value is calculated to obtain the Fv/Fm value of the material to be detected.
5. The method for detecting taxus chinensis stress based on the chlorophyll fluorescence imaging technology according to claim 4,
the RGB image color mode corresponding to the Fv/Fm value is set to be 0-255; the color intensity value of the RGB image corresponding to the Fv/Fm value is set to be 0.35-0.9.
6. The method for detecting taxus chinensis stress based on the chlorophyll fluorescence imaging technology according to claim 1,
in the step (4), the grading standard of the stress degree of the taxus chinensis germplasm is divided by taking the identification results of different taxus chinensis germplasms under different climatic conditions all year round as reference.
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