CN114646727A - Method for identifying water-saving and drought-resisting function of plant - Google Patents
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Abstract
The invention discloses an identification method of water-saving and drought-resisting functions of plants, which comprises the following steps: selecting the same batch of plant seeds which are subjected to pregermination, culturing to a specific growth stage, and selecting the plants at the specific growth stage for phenotype identification; dynamically evaluating the water-saving drought-resisting genetic phenotype according to the phenotype identification result; wherein the culturing to a specific growth stage comprises: water culture in a laboratory to a seedling stage, soil culture in a laboratory to a seedling stage and field culture to a tillering stage; wherein said phenotypic identification comprises: carrying out transpiration phenotype identification on laboratory water culture seedlings, carrying out drought phenotype identification on laboratory soil culture seedlings, and carrying out physiological phenotype identification on field tillering stage seedlings. The method systematically evaluates the water-saving drought-resisting phenotype of the plant in the seedling stage from two aspects of heredity and physiology, and can accurately reflect the water-saving drought-resisting function of the plant.
Description
Technical Field
The invention relates to an identification method for a water-saving and drought-resisting function of a plant, belonging to the technical field of agricultural biology.
Background
The global warming and the drought caused by water resource shortage are the main problems facing the agricultural production at present, the water quantity required by the agricultural production in China is large, and the agricultural water accounts for two thirds of the total water consumption in China. Therefore, the water-saving and drought-resisting problems of plants, particularly crops, are always concerned by society. Water conservation and drought resistance are two distinct but interrelated concepts. The plant water conservation refers to the capability of effectively utilizing water, reducing water consumption and having higher water utilization efficiency in the plant growth and development process. Drought resistance of plants can be divided into three aspects: drought avoidance, drought tolerance and restored drought resistance. The water-saving drought-resisting means that the plants are prevented or delayed from suffering from drought by reducing water loss, reducing water consumption and preserving water, and is similar to drought avoidance in drought-resisting. The water-saving drought-resistant crops can save water, reduce water consumption, preserve water to avoid drought, ensure normal growth of plants and have no influence on yield after the plants are watered again when the plants are moderately arid and lack water or are in stage drought. Reducing water loss is an important link for water conservation and drought resistance of plants. Transpiration is the main way for plants to consume water, and stomatal transpiration is the main form of plant leaf transpiration. The plants can control the transpiration of the stomata by adjusting the opening and closing of the stomata and adjust the utilization of water.
Compared with the classical drought-resistant research, the concept of water conservation and drought resistance is provided later. The division of drought resistance was proposed internationally from 1972, and the joint evaluation of drought resistance and water use efficiency was started until 1999, and water conservation and drought resistance was proposed until 2004. The water-saving drought-resistant character of the plant is very complex, and relates to a plurality of important physiological domains, more than 98 percent of articles in the current water-saving drought-resistant research are concentrated in the fields of cultivation and breeding, and the research improves the water-saving drought-resistant cultivation technology to obtain water-saving drought-resistant crop varieties; however, the research on the aspects of the excavation of water-saving drought-resistant genes, the identification of gene functions, mechanism and molecular mechanism is few, and although genes related to transpiration are found, the water-saving drought-resistant function of the genes is not systematically explored.
The existing water-saving drought-resistant function identification of plants is mainly carried out in the aspects of cultivation and breeding, and wastes time and labor.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the identification method of the water-saving and drought-resisting function of the plant, which can identify the water-saving and drought-resisting function of the plant in the seedling stage from the two aspects of heredity and physiology. In order to achieve the purpose, the invention is realized by adopting the following technical scheme:
the invention provides a method for identifying the water-saving and drought-resisting function of a plant, which comprises the following steps:
selecting the same batch of plant seeds which are subjected to pregermination, culturing to a specific growth stage, and selecting the plants at the specific growth stage for phenotype identification;
dynamically evaluating the water-saving drought-resisting function according to the phenotype identification result;
wherein the culturing to a specific growth stage comprises: water culture in a laboratory to a seedling stage, soil culture in a laboratory to a seedling stage and field culture to a tillering stage;
wherein said phenotypic identification comprises: carrying out transpiration phenotype identification on laboratory water culture seedlings, carrying out drought phenotype identification on laboratory soil culture seedlings, and carrying out physiological phenotype identification on field tillering stage seedlings.
Further, the dynamic evaluation of the water-saving drought-resisting function comprises the following steps:
preliminarily evaluating the water-saving drought-resisting genetic phenotype according to the transpiration phenotype identification of the laboratory water-cultured seedlings;
verifying the water-saving drought-resisting genetic phenotype for a long time according to the drought phenotype identification of the soil-cultured seedlings in a laboratory;
and (4) detecting the water-saving drought-resisting physiological phenotype in real time according to the physiological phenotype identification of the seedlings in the field tillering stage.
Further, the method for carrying out transpiration phenotype identification on laboratory hydroponic seedlings comprises the following steps:
planting seedlings of different strains on small plates with proper sizes, planting the same seedlings with a certain number at intervals on each plate, independently placing the seedlings in a transparent square pot with the length of 11.5 multiplied by 8.5 multiplied by 18cm, filling nutrient solution and weighing;
placing in an incubator with enough illumination, constant temperature of 30 deg.C and constant humidity of 60% for 3 days, normally culturing, observing water level change in the transparent square basin, marking water level with black line, and weighing;
at least three biological replicates per strain were performed;
calculating the water consumption which can be used for preliminarily evaluating the water-saving drought-resisting genetic phenotype, wherein the calculation formula is as follows: water consumption =0 days nutrient weight-3 days later nutrient weight.
Preferably, hydroponic seedlings of at least three groups of platelets are cultured for transpiration phenotyping to ensure at least three biological replicates are performed.
Further, the method for carrying out drought phenotype identification on the soil-cultured seedlings in the laboratory comprises the following steps:
planting seedlings of different strains in small pots of 10 × 10 × 8.5cm containing the same weight of soil, planting the same seedlings with a certain number at intervals in each small pot, independently placing each six small pots of each strain in a water storage disc of 32 × 23 × 8.2cm which is suitable in size and closed after water is saturated, adding water with the same volume, culturing in an incubator with enough illumination, constant temperature and 30 ℃ and constant humidity of 60%, continuously weighing every day, and not watering any more at the same time to carry out drought stress;
adding water to recover the culture, and counting the survival rate;
at least three biological replicates per strain were performed;
calculating the soil water content of each pot of seedlings which can be used for verifying the water-saving drought-resistant hereditary phenotype for a long time, and calculating the formula: soil water content = (small basin and water storage tray before drying and soil sample mass-small basin and water storage tray after drying and soil sample mass)/(small basin and water storage tray before drying and soil sample mass-dry empty small basin and water storage tray mass) × 100%.
Preferably, at least three sets of plantlets of earth-cultivated seedlings per line are subjected to drought phenotypic identification to ensure at least three biological replicates.
Further, the step of water saturation is: immersing each small pot seedling in water to absorb enough water, draining off water at the same time until each small pot seedling does not drip any more, and then putting the small pot seedlings into a dry water storage tray.
Further, the identification of physiological phenotype of seedlings in field growth period includes: stomatal conductance, transpiration rate, photosynthesis efficiency, leaf surface temperature and stomatal observation.
Further, the environment for physiological phenotype identification of seedlings in the field growth period is as follows: 9:00-11:00 am on a sunny day of 6-8 months or a greenhouse with enough illumination and constant temperature of 30 ℃ and constant humidity of 60%.
Furthermore, when physiological phenotype identification is carried out on seedlings in the field growth period, 10 or more leaves with the same growth vigor and good growth state are selected for each strain to carry out measurement and stomata observation sampling.
Further, the measuring and vent observation sampling comprises: and scanning, observing and photographing the leaf stomata by adopting an environment scanning electron microscope, counting the opening, density and length of the stomata, and counting at least three hundred stomata of each strain.
Furthermore, the time for measuring the leaves and observing and sampling the stomata of the seedlings in the field growth period when the physiological phenotype identification is carried out on the seedlings in the field growth period is less than one minute.
Compared with the prior art, the identification method for the water-saving and drought-resisting functions of the plants, provided by the embodiment of the invention, has the following beneficial effects:
the method disclosed by the invention is used for carrying out transpiration phenotype identification on the laboratory hydroponic seedlings to preliminarily evaluate the water-saving drought-resisting genetic phenotype, carrying out drought phenotype identification on the laboratory soil-cultured seedlings to verify the water-saving drought-resisting genetic phenotype for a long time, carrying out physiological phenotype identification on the seedlings in the field growth period to detect the water-saving drought-resisting physiological phenotype in real time, systematically and dynamically evaluating and identifying the water-saving drought-resisting phenotype of the plant seedling period from two aspects of heredity and physiology, accurately reflecting the water-saving drought-resisting function of the plant in a small range, saving time and labor, and conveniently researching the gene function identification, mechanism and molecular mechanism, so that the method has important significance for relieving the water resource crisis, developing water-saving agriculture, resisting drought, and guaranteeing national grain safety, ecological safety and social sustainable development.
Drawings
FIG. 1 is a flow chart of a method for identifying the water-saving and drought-resisting function of a plant provided by the invention;
FIG. 2 shows the evaluation of the transpiration phenotype provided in example 1 of the present inventionOsPIL15Mapping of overexpression and knock-out strains to wild type;
FIG. 3 is a drought phenotype signature provided in example 2 of the present inventionCenteringOsPIL15Mapping of overexpression and knock-out strains to wild type;
FIG. 4 shows the physiological phenotype determination provided in example 3 of the present inventionOsPIL15A comparison graph of stomatal conductance, transpiration rate, photosynthesis efficiency and leaf surface temperature of the over-expressed and knocked-out strain and wild type;
FIG. 5 shows the identification of physiological phenotype provided in example 3 of the present inventionOsPIL15Overexpression is compared to stomata observations of the physiological phenotype of the knockout line and the wild type.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The examples of the present invention all used wild type rice of Nipponbare as a control,OsPIL15nipponbare of overexpression and knockout strains is used as an experimental material.
Example 1:
the embodiment of the invention provides a transpiration phenotype implementation method of an identification method of a plant water-saving drought-resisting function.
Step 1: selection of Wild Type (WT) andOsPIL15over-expressed material (OE 5, OE6, OE 10) and knock-out material (15-2, 15-3) were cultured to four-leaf one-heart stage.
Step 2: selecting four-leaf one-heart Wild Type (WT) and WT with identical growth vigorOsPIL15Overexpression material and knock-out material 45 strains each. Different rice strains are placed in transparent square pots with the same size, the transparent square pots are filled with nutrient solution and placed in an incubator for 3 days, normal culture is carried out at 28 ℃, water level change in the transparent square pots is observed, water level is marked by black lines, and Wild Type (WT) and transpiration phenotype of over-expression and knockout materials are photographed and analyzed. The results are shown in figures 2A and B,OsPIL15the water level of the over-expressed line was higher than that of the wild-type control, indicating thatOsPIL15The transpiration rate of the over-expressed line was lower than the wild-type control;OsPIL15the mutant lines are reversed. Results displayOsPIL15Affecting the transpiration rate of rice and showing thatOsPIL15May have the functions of water saving and drought resistance.
And step 3: the strains were subjected to a weighing experiment on the same day when the nutrient solution was added and within the same time period after three days, and the water consumption was calculated, with the results shown in FIG. 2C,OsPIL15the water consumption of the over-expression strain is obviously lower than that of the wild type control, and the water consumption of the knockout strain is opposite, further showing thatOsPIL15The function of water saving and drought resistance.
Example 2:
the embodiment of the invention provides a drought phenotype implementation method of an identification method of a water-saving and drought-resisting function of a plant.
Step 1: selection of Wild Type (WT) andOsPIL15over-expressed material (OE 5, OE6, OE 10) and knock-out material (15-2, 15-3) were cultured to the four-leaf one-heart stage, with 12 seedlings per line, seeded in the same pot.
Step 2: selecting four-leaf one-heart Wild Type (WT) and WT with consistent growth vigorOsPIL15Over-expression material and knock-out material for soil-culture seedling, after water saturation, putting each strain in six small pots separately into a closed water storage tray, adding water with the same volume, continuously weighing every day, and simultaneously not watering any more, and carrying out drought stress.
And step 3: after six days of drought stress, water is added for recovery culture, and the survival rate is counted, the result is shown in fig. 3A and B, and after 5 days of drought stress, leaf of the wild plant begins to appear wilting phenomenon; most leaves of the over-expression plants are still not wilted and keep in a stretching state; knockout plants have shown severe wilting. Most of wild plants die after 10 days of re-watering, and only a few plants survive; the over-expression plants can basically recover to the growth state before drought stress; and the knockout plant dies substantially all; meanwhile, the statistical survival rate also shows that the over-expression plant survival rate is remarkably higher than that of the wild type, and the knockout is opposite. The same results as the previous transpiration phenotype further indicateOsPIL15The function of water saving and drought resistance.
And 4, step 4: calculating the soil water content of each strain, and calculating the formula: soil water content = (small basin and water storage basin before drying and soil sample mass-small basin and water storage basin after drying and soil sample mass)/(small basin and water storage basin before drying and soil sample mass-drying emptyQuality of the pots and impoundment pots) 100%, and the results are shown in fig. 3C, where OsPIL15 over-expressed the soil water content of the three lines higher than the wild-type control, while the knockout line had soil water content lower than the wild-type control, further indicating thatOsPIL15Affecting rice transpiration.
Example 3:
the embodiment of the invention provides a physiological phenotype implementation method of an identification method of a plant water-saving drought-resisting function.
Step 1: selection of Wild Type (WT) andOsPIL15over-expressed material (OE 5, OE6, OE 10) and knock-out material (15-2, 15-3) were cultured in the field to the five-leaf one-heart stage.
Step 2: the Wild Type (WT) and the Wild Type (WT) were determined at 9:00-11:00 am on a sunny day by using a portable photosynthetic apparatus Li-6400 (LI-COR Inc, Lincoln, Nebraska, USA),OsPIL15And (3) overexpressing and knocking out the stomatal conductance, transpiration rate and photosynthesis efficiency of the first unfolded leaf beside the heart leaf of the strain. 10 leaves with consistent growth and good growth state are selected for each strain to measure, and the measurement is carried out for three times respectively. Calculating the water utilization efficiency through the photosynthetic rate and the transpiration rate, wherein the water utilization efficiency calculation formula is as follows: the water use efficiency = photosynthesis efficiency/transpiration rate results show that, as shown in figures 4A, B, C and D,OsPIL15the transpiration rate and the stomatal conductance of the over-expression strain are both obviously lower than those of a contrast, the photosynthesis efficiency of the over-expression strain is lower than that of the contrast or is equal to that of the contrast,OsPIL15the transpiration rate, stomatal conductance and photosynthesis efficiency of the knockout strain are all remarkably higher than those of the contrast. In addition to this, the present invention is,OsPIL15the water utilization efficiency of the over-expression strain and the knockout strain is not reduced compared with that of a wild type; meanwhile, the leaf surface temperature of the transgenic rice and the wild type control was measured by using a thermal imaging camera FLIR E40, as shown in FIGS. 4E and F,OsPIL15the temperature of the over-expression strain leaves is very significantly higher than the control, and the temperature of the knock-out strain leaves is very significantly lower than the control. Because the higher the blade temperature, the lower the transpiration rate; the lower the blade temperature, the higher the transpiration rate, and therefore, the above results are in conjunction withOsPIL15The results of the transpiration phenotype observed by the transgenic lines are consistent, which indicates thatOsPIL15By reducing the porosity conductance and transpiration rate, the method improvesOsPIL15Water-saving drought resistance of over-expression strain.
And 3, step 3: fixing the wild type of the five-leaf one-heart stage andOsPIL15and (3) overexpressing and knocking out the first unfolded leaf beside the heart leaf of the strain, preparing the slice, and observing the stomatal aperture, stomatal density and stomatal length of each strain leaf by adopting an environment scanning electron microscope. As shown in fig. 5A, we classified the three stomata morphologies of rice into the following three types: fully open, partially open and fully closed, and statistics were taken of the ratio of the three pore morphologies. As shown in the figure 5B of the drawings,OsPIL15the completely opened stomata of the overexpression strain is lower than that of the wild type control, and the completely opened stomata of the knockout strain is higher than that of the wild type control;OsPIL15the over-expressing line had a completely closed stomata higher than the wild-type control, and the knockout line had a completely closed stomata lower than the wild-type control.OsPIL15The partially open stomata of the over-expressed and knockout strain were not significantly different compared to the wild-type control. In addition, as shown in FIGS. 5C and D,OsPIL15compared with wild type control, the stomatal length and stomatal density of the transgenic line and the knockout line have no significant difference. The above results show thatOsPIL15The transpiration rate is adjusted to play a role in saving water and resisting drought by influencing the opening and closing of the air holes of the rice.
The above examples 1-3 prove that the method for identifying the water-saving and drought-resisting function of the plant provided by the invention systematically evaluates the water-saving and drought-resisting phenotype of the plant at the seedling stage from two aspects of genetics and physiology, can accurately reflect the water-saving and drought-resisting function of the plant, and has important significance and wide application prospect.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for identifying the water-saving and drought-resisting function of a plant is characterized by comprising the following steps:
selecting the same batch of plant seeds which are subjected to pregermination, culturing to a specific growth stage, and selecting the plants at the specific growth stage for phenotype identification;
dynamically evaluating the water-saving drought-resisting function according to the phenotype identification result;
wherein the culturing to a specific growth stage comprises: water culture in a laboratory to a seedling stage, soil culture in a laboratory to a seedling stage and field culture to a tillering stage;
wherein said phenotypic identification comprises: carrying out transpiration phenotype identification on laboratory water culture seedlings, carrying out drought phenotype identification on laboratory soil culture seedlings, and carrying out physiological phenotype identification on field tillering stage seedlings.
2. The method for identifying the water-saving drought-resistant function of the plant according to claim 1, wherein the dynamically evaluating the water-saving drought-resistant function comprises:
preliminarily evaluating the water-saving drought-resisting genetic phenotype according to the transpiration phenotype identification of the laboratory water-cultured seedlings;
verifying the water-saving drought-resisting genetic phenotype for a long time according to the drought phenotype identification of the soil-cultured seedlings in a laboratory;
and (4) detecting the water-saving drought-resisting physiological phenotype in real time according to the physiological phenotype identification of the seedlings in the field tillering stage.
3. The method for identifying the water-saving and drought-resisting function of the plant according to claim 1, wherein the method for carrying out transpiration phenotype identification on the laboratory water-cultured seedling comprises the following steps:
planting seedlings of different strains on small plates with proper sizes, planting the same seedlings with a certain number at intervals on each plate, independently placing the seedlings in a transparent square pot with the length of 11.5 multiplied by 8.5 multiplied by 18cm, filling nutrient solution and weighing;
placing in an incubator with enough illumination, constant temperature of 30 deg.C and constant humidity of 60% for 3 days, normally culturing, observing water level change in the transparent square basin, marking water level with black line, and weighing;
at least three biological replicates per strain were performed;
calculating the water consumption which can be used for preliminarily evaluating the water-saving drought-resisting genetic phenotype, wherein the calculation formula is as follows: water consumption =0 days nutrient solution weight-3 days later nutrient solution weight.
4. The method for identifying the water-saving and drought-resisting function of the plant according to claim 1, wherein the method for identifying the drought phenotype of the soil-cultured seedlings in a laboratory comprises the following steps:
planting seedlings of different strains in small pots of 10 × 10 × 8.5cm containing the same weight of soil, planting the same seedlings with a certain number at intervals in each small pot, independently placing each six small pots of each strain in a water storage disc of 32 × 23 × 8.2cm which is suitable in size and closed after water is saturated, adding water with the same volume, culturing in an incubator with enough illumination, constant temperature and 30 ℃ and constant humidity of 60%, continuously weighing every day, and not watering any more at the same time to carry out drought stress;
adding water to recover the culture, and counting the survival rate;
at least three biological replicates per strain were performed;
calculating the soil water content of each pot of seedlings which can be used for verifying the water-saving drought-resistant hereditary phenotype for a long time, and calculating the formula: soil water content = (small basin and water storage tray before drying and soil sample mass-small basin and water storage tray after drying and soil sample mass)/(small basin and water storage tray before drying and soil sample mass-dry empty small basin and water storage tray mass) × 100%.
5. The method for identifying the water-saving and drought-resisting function of the plant according to claim 4, wherein the water saturation step comprises the following steps: immersing each small pot seedling in water to absorb enough water, draining off water at the same time until each small pot seedling does not drip any more, and then putting the small pot seedlings into a dry water storage tray.
6. The method for identifying the water-saving and drought-resistant function of the plant according to claim 1, wherein the physiological phenotype identification of the seedlings in the field growth period comprises the following steps: stomatal conductance, transpiration rate, photosynthesis efficiency, leaf surface temperature and stomatal observation.
7. The method for identifying the water-saving and drought-resistant function of the plant according to claim 1, wherein the environment for performing physiological phenotype identification on the seedlings in the field growth period is as follows: 9:00-11:00 am on a sunny day of 6-8 months or a greenhouse with enough illumination and constant temperature of 30 ℃ and constant humidity of 60%.
8. The method for identifying the water-saving and drought-resistant function of the plant according to claim 1, wherein 10 or more leaves with the same growth vigor and good growth state are selected from each plant line for measurement and stomata observation and sampling when the physiological phenotype identification is carried out on the seedlings in the growth period in the field.
9. The method for identifying the water-saving drought-resistant function of the plant according to claim 8, wherein the measurement and stomata observation and sampling comprises: and scanning, observing and photographing the leaf stomata by adopting an environment scanning electron microscope, counting the opening, density and length of the stomata, and counting at least three hundred stomata of each strain.
10. The method for identifying the water-saving and drought-resistant functions of plants according to claim 8, wherein the time for measuring leaves and observing and sampling stomata when physiological phenotype identification is carried out on seedlings in the growth period of the field is less than one minute.
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| CN115927445A (en) * | 2022-07-13 | 2023-04-07 | 扬州大学 | Application of OsPIL15 gene in regulation and control of water conservation and drought resistance of rice |
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| CN115927445A (en) * | 2022-07-13 | 2023-04-07 | 扬州大学 | Application of OsPIL15 gene in regulation and control of water conservation and drought resistance of rice |
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