CN111157575A - Method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of hemerocallis fulva - Google Patents
Method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of hemerocallis fulva Download PDFInfo
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Abstract
The invention discloses a method for screening drought-resistant hemerocallis fulva based on the leaf surface temperature of the hemerocallis fulva, which comprises the following steps of: s1: primary screening: measuring the leaf surface temperature of different hemerocallis and sequencing the leaf surface temperature of each hemerocallis; s2: further determination: and (4) selecting the hemerocallis with the highest or the lowest hemerocallis in the hemerocallis leaf surface temperature sequence in the step (S1), measuring the cell ploidy, the stomatal conductance, the relative water content of the leaves, the water loss rate of the leaves in vitro and the stomatal size and density of the leaves, and determining and screening drought-resistant hemerocallis varieties through the survival capability of the hemerocallis under the natural drought stress test and the growth and development condition after the rehydration test. The drought-resistant screening method provided by the invention is simple and low in workload, avoids natural stress experiments on a large amount of hemerocallis, and only needs temperature preliminary screening and then further confirmation experiments.
Description
Technical Field
The invention belongs to the field of flower screening, and particularly relates to a method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva.
Background
The hemerocallis are ancient called the ornamental hemerocallis, are perennial root herbs, are mostly native to China, are mainly collected by germplasm resources companies, plantations and gardening enthusiasts in the United states, are produced in China and Japan, are obtained by hybridization breeding, are easy to hybridize among hemerocallis, and have great variation ranges in the sizes, colors, flower forms, flowering periods and the like of certain kinds of flowers, so that the ornamental hemerocallis have the advantages of long ornamental value, high ornamental value, various varieties, rich and gorgeous colors, strong stress resistance, strong adaptability and the like, are increasingly paid attention to people as ornamental plants, and have wide development prospects. Since 1974, China has complex terrain, changeable climate and overall situation in China, and a large amount of land is between arid and semiarid, the breeding of drought-resistant hemerocallis variety is one of important targets of hemerocallis breeding. The method needs a long measuring period, consumes a large amount of human resources, has large workload, uses chemical agents to measure the physiological state of the plant in a large scale, has high capital consumption and easily causes irreversible land pollution.
Disclosure of Invention
The invention aims at the problems in the prior art and provides a method for screening drought-resistant hemerocallis on the basis of leaf surface temperature, a drought-resistant variety is preliminarily screened on the basis of the leaf surface temperature of the hemerocallis, and then whether the variety is drought-resistant or not is further determined.
In order to solve the problems, the technical scheme of the invention is as follows:
a method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva is characterized by comprising the following steps of:
s1: primary screening: measuring the leaf surface temperature of different hemerocallis and sequencing the leaf surface temperature of each hemerocallis;
s2: further determination: and (4) selecting the hemerocallis with the highest or the lowest hemerocallis in the hemerocallis leaf surface temperature sequence in the step (S1), measuring the cell ploidy, the porosity conductivity, the relative water content of the leaves, the water loss rate of the leaves in vitro and the pore size and density of the leaves, and screening drought-resistant hemerocallis varieties through the survival capability and the growth and development conditions after a rehydration test under the natural drought stress test of the hemerocallis.
Further, the method for determining the ploidy of the hemerocallis fulva cells comprises the following steps: use of: a10 mg sample of fresh, tender leaf tissue of Hemerocallis fulva was placed in a petri dish, 400. mu.l of nuclear extraction buffer was added, the tissue was minced using a razor blade, the tissue was cut for 60-180 seconds to ensure uniform mincing of the Hemerocallis fulva tissue, 1600. mu.l of DAPI staining solution was added, the mixture was left at 20 ℃ for 20min and the sample was filtered at 38. mu.m, and measured using a flow cytometer.
Further, the cell nucleus buffer extract comprises 100mmol/L citric acid, Tween-80 with volume fraction of 0.5% pH 3, 400mmol/L heptahydrate and disodium hydrogen phosphate; for flow cytometry measurements, the voltage was set at 400mV and the flow rate was set at 0.4. mu.l/s.
Further, measuring the stomata of the hemerocallis fulva leaves by adopting a scanning electron microscope, selecting the leaves of the hemerocallis fulva in a normal physiological state in the sun-facing direction at 9-11 am under natural illumination, shearing the upper half parts of the leaves, removing the leaf tips, putting the leaves into the plant tissue fixing liquid, then placing the leaves in an ice box for storage, and measuring the data of the stomata of the hemerocallis fulva scanning electron microscope.
Further, the plant tissue fixing solution is a mixed solution of 65% absolute ethyl alcohol, 6% acetic acid and 5% formaldehyde.
Preferably, if the plant tissue fixing solution is stored for a long time, the plant tissue fixing solution further comprises 4-6 ml of glycerin to prevent evaporation and material hardening, and the plant tissue fixing solution is used as a storage solution.
Further, the method for measuring the porosity conductance comprises the following steps: under natural illumination of 9-11 am, selecting sunny side of daylily leaf in normal physiological state, measuring stomatal conductance (Gs), and setting the measurement light intensity at 1000 μmol-2s-1。
Further, a high-precision infrared thermometer is adopted to measure the surface temperature of the hemerocallis fulva leaves.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects:
according to the invention, the temperature of the leaf surface of the plant can be reduced according to the transpiration effect, and a method capable of rapidly screening drought-resistant hemerocallis fulva varieties in a large scale is established. Sunlight irradiates the day lily leaves, and most of energy is converted from light energy to heat energy. The temperature of the day lily leaf surface is increased, so that the plants can emit moisture through the stomatal transpiration to prevent the day lily leaf from being burnt, and the heat energy is absorbed in the process that the water is changed into the water vapor, so that the temperature of the day lily leaf surface is reduced. Researches show that the transpiration of plants depends on the stomatal transpiration. Theoretically, the hemerocallis under the same physiological state have large stomatal density, high stomatal conductance and remarkable transpiration effect, low leaf surface temperature, rapid water loss and weak drought resistance, and vice versa. Therefore, the drought-resistant hemerocallis variety is preliminarily screened based on the leaf surface temperature, and then the drought-resistant hemerocallis variety is confirmed by measuring the indexes of the hemerocallis variety such as cell ploidy, stomatal conductance, water content of leaves, water loss of leaves in vitro and the like and further by the survival ability under the natural drought stress test and the growth and development condition after the rehydration test. Therefore, the screening method provided by the invention does not need to perform a large number of drought stress experiments, adopts temperature to perform primary screening, and then further performs confirmation experiments.
Drawings
FIG. 1 is the aerial part growth morphology of the "Chongzi flower" variety after 0d, 7d, 14d, 21d, 28d and rehydration 7d simulated drought stress experiment in example 1;
FIG. 2 is the aerial part growth pattern of the "Chonglihua" variety after 0d, 7d, 14d, 21d, 28d and rehydration 7d simulated drought stress experiment in example 2;
FIG. 3 is the aerial growth patterns of the "afternoon" variety after 0d, 7d, 14d, 21d, 28d and rehydration 7d simulated drought stress experiments in example 3;
FIG. 4 is the aerial part growth pattern of the "X-12" variety after 0d, 7d, 14d, 21d, 28d and rehydration 7d simulated drought stress experiment in example 4;
FIG. 5 shows the growth patterns of aerial parts of the "small hugs" variety after 0d, 7d, 14d, 21d, 28d and rehydration 7d in the experiment of simulating drought stress in example 5.
Detailed Description
The following describes in detail a method for screening drought-resistant hemerocallis fulva based on the foliar temperature of the hemerocallis fulva according to the present invention with reference to the accompanying drawings and specific examples. Advantages and features of the present invention will become apparent from the following description and from the claims.
Example 1
A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva comprises the following steps:
(1) the central console of the glass greenhouse displays the specific data of the experimental environment, the indoor temperature is 29.4 ℃, the indoor humidity is 95.2%, the carbon dioxide concentration is 1329PPM, the illumination intensity is 15.7KL, and the experimental environment is kept consistent.
The method comprises the steps of measuring the leaf surface temperature of all kinds of hemerocallis in the glass greenhouse by using a precise infrared thermometer, selecting five hemerocallis of the same kind with the same physiological state and similar physiological state for the same kind, measuring, and averaging the temperature of three points of the measured leaf surface of each hemerocallis as the leaf surface temperature of a single hemerocallis. And screening and sorting by using excel software, and screening the hemerocallis variety with the highest or lowest leaf surface temperature.
Example one is one of the hemerocallis varieties with the highest leaf surface temperature: 'Chongzihua', the measured leaf surface temperature was 32.6 ℃ (decimal place retains one bit).
(2) The method for determining the chromosome ploidy of the hemerocallis cell comprises the following steps: taking a 10mg fresh and tender day lily leaf tissue sample, placing the sample in a culture dish, adding 400 mu l of nuclear extraction buffer solution, cutting the tissue by using a sharp blade, cutting for 60-180 seconds to ensure that the day lily tissue is uniformly cut, adding 1600 mu l of DAPI staining solution, standing at 20 ℃ for 20min, filtering the sample by using 38 mu m (400 meshes), measuring by using a flow cytometer, setting the voltage to be 400mV, adjusting the threshold to be maximum, and setting the flow rate to be 0.4 mu l/s, and measuring the variety of the day lily with a Mengzi flower' as a diploid.
(3) The method for measuring the porosity conductance comprises the following steps: under the natural illumination of 9-11 am, selecting the sunny side of the leaves of Hemerocallis fulva of 'Menispermaceae' variety for measurement, and measuring the stomatal conductance (Gs), wherein the measured light intensity is set to be 1000 mu mol-2.s-1Five biological repeated experiments are carried out, and the measurement results are averaged, wherein the porosity conductance of the hemerocallis fulva of the 'Menispermaceae' variety is 0.046mol H2O m-2s-1。
(4) Method for determining relative water content: cutting three mature leaves of hemerocallis fulva (with the same physiological state) of the variety of the 'branchlet flowers' at the relative water content RWC of 8:00-10:00 in the morning, and quickly packaging in a plastic bag. And meanwhile, the sample is placed in an ice box and is rapidly brought back to the laboratory, and the laboratory is kept at a constant temperature and is protected from light. Taking out the experimental material from the plastic, wiping off the water on the leaves, weighing the materials respectively by using an 1/10000 electronic balance, recording initial data, namely the fresh weight Wf of the sample, putting the sample into a centrifuge tube to absorb water for 24 hours, weighing the saturated weight Wt, then putting the leaves into an oven at 70 ℃ to dry for 48 hours, weighing the dry weight Wd, calculating the relative water content RWC of the hemerocallis fulva variety (Wf-Wd) | (Wt-Wd), repeating the experiment for three times, and taking an average value.
When the hemerocallis are not subjected to drought stress treatment, the RWC content is 90.8 percent; after the day lily is subjected to drought treatment for 7 days, the RWC is measured to be 83.4 percent again; the RWC was found to be 73.8% at the end after day lily drought treatment for 14 days.
(5) The method for measuring the water loss rate of the in-vitro blade comprises the following steps: 3 leaves are respectively cut from hemerocallis fulva with small scissors and small tweezers and are placed on weighing paper for weighing. Avoiding direct sunlight, placing the leaves on weighing paper under indoor weak light, weighing the leaves for 5 times at different time (0 h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h and 5h respectively), repeating the experiment for three times to calculate the water loss rate of the hemerocallis leaves of different varieties, and recording the water loss data (0.00%, 1.59%, 1.89%, 3.17%, 3.57%, 4.76%, 6.35% and 7.94%) of the hemerocallis leaves.
(6) The method for measuring the density of the pores by a scanning electron microscope comprises the following steps: under the natural illumination of 9-11 points, selecting a sunny day lily leaf in a normal physiological state, shearing the upper half part of the leaf, removing the leaf tip, putting the leaf tip into plant tissue fixing liquid, then placing the leaf tip into an ice box for storage, measuring day lily stomatal data by using a Hitachi scanning electron microscope (Hitachi S-3400N), and calculating the stomatal data by using ImageJ software. (data summary Table 1)
(7) Selecting the screened 'fierce flower' to carry out a drought stress experiment, fully watering the experimental material on the same day as the beginning of the experiment, placing the experimental material in a glass greenhouse to keep a relatively constant external environment, then continuously watering for 28d, photographing and recording the morphological parts of the hemerocallis after 0d, 7d, 14d, 21d, 28d and rehydration after 7d, observing the survival capability and the growth and development conditions after rehydration under the drought experiment, referring to fig. 1, the growth morphologies of the hemerocallis after 0d, 7d, 14d, 21d, 28d and rehydration 7d from left to right, observing and knowing that the morphological parts of the 'fierce flower' are in the ground, the leaf withering degree is slight, the hemerocallis can be quickly recovered after rehydration, and the drought-tolerant hemerocallis variety is judged by various factors such as comprehensive air permeability, relative water content, in-vitro leaf water loss rate and drought stress experiment results.
Example 2
A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva comprises the following steps:
(1) the central console of the glass greenhouse displays the specific data of the experimental environment, the indoor temperature is 29.4 ℃, the indoor humidity is 95.2%, the carbon dioxide concentration is 1329PPM, the illumination intensity is 15.7KL, and the experimental environment is kept consistent.
The method comprises the steps of measuring the leaf surface temperature of all kinds of hemerocallis in the glass greenhouse by using a precise infrared thermometer, selecting five hemerocallis of the same kind with the same physiological state and similar physiological state for the same kind, measuring, and averaging the temperature of three points of the measured leaf surface of each hemerocallis as the leaf surface temperature of a single hemerocallis. And screening and sorting by using excel software, and screening the hemerocallis variety with the highest or lowest leaf surface temperature.
Example two is one of the hemerocallis varieties with the highest leaf surface temperature: ' Inula `, the leaf surface temperature was measured to be 32.8 deg.C (one decimal place).
(2) Method for determining ploidy of hemerocallis cells: placing 10mg of fresh and tender day lily leaf tissue sample in a culture dish, adding 400 μ l of nuclear extraction buffer, cutting the tissue with a sharp blade, cutting for 60-180 seconds to ensure that the day lily tissue is uniformly cut, adding 1600 μ l of DAPI staining solution, standing at 20 ℃ for 20min, filtering the sample with 38 μm (400 meshes), measuring on a flow cytometer, setting the voltage at 400mV, adjusting the threshold value to the maximum value, setting the flow rate at 0.4 μ l/s, and obtaining the measurement result as diploid.
(3) The method for measuring the porosity conductance comprises the following steps: under the natural illumination of 9-11 am, selecting sunny side of leaves of Hemerocallis fulva of 'Chongli' variety for determination, and determining the air hole conductivity (Gs), wherein the determination light intensity is set to 1000 μmol-2.s-1Five biological repeated experiments are carried out, and the measurement results are averaged, wherein the porosity conductance of the hemerocallis fulva of the 'Mengzi' variety is 0.028mol H2O m-2s-1。
(4) Method for determining relative water content: cutting three mature leaves of Hemerocallis fulva (same physiological state) of the variety of Zhanhua in the morning with a relative water content RWC of 8:00-10:00, and rapidly packaging in a plastic bag. And meanwhile, the sample is placed in an ice box and is rapidly brought back to the laboratory, and the laboratory is kept at a constant temperature and is protected from light. Taking out the experimental material from the plastic, wiping off the water on the leaves, weighing the materials respectively by using an 1/10000 electronic balance, recording initial data, namely the fresh weight Wf of the sample, putting the sample into a centrifuge tube to absorb water for 24 hours, weighing the saturated weight Wt, then putting the leaves into an oven at 70 ℃ to dry for 48 hours, weighing the dry weight Wd, calculating the relative water content RWC of the hemerocallis fulva variety (Wf-Wd) | (Wt-Wd), repeating the experiment for three times, and taking an average value.
When the hemerocallis are not subjected to drought stress treatment, the RWC content is 90.7%; after the day lily is subjected to drought treatment for 7 days, the RWC is measured to be 81.7 percent again; after the day lily is subjected to drought treatment for 14 days, the RWC is finally measured to be 75.6%.
(5) Measuring the water loss rate of the in vitro blade: 3 leaves are respectively cut from the hemerocallis fulva' by using small scissors and small tweezers and are placed on weighing paper for weighing. Avoiding direct sunlight, placing the leaves on weighing paper under indoor weak light, weighing the leaves for 5 times at different time (0 h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h and 5h respectively), repeating the experiment for three times to calculate the water loss rate of the leaves of different varieties of hemerocallis, and recording the water loss data (0.00%, 2.13%, 4.26%, 5.67%, 7.09%, 8.51%, 9.93%, 10.64% and 12.77%) of the leaves of the Chinese daylily after the leaves are withered.
(6) The method for measuring the density of the pores by a scanning electron microscope comprises the following steps: under the natural illumination of 9-11 points, selecting sun-facing leaves of hemerocallis fulva in a normal physiological state, shearing the upper half leaves, removing the leaf tips, putting the leaves into plant tissue fixing liquid, then placing the leaves in an ice box for storage, measuring the hemerocallis stomatal data by using a Hitachi scanning electron microscope (Hitachi S-3400N), and calculating the stomatal data (the data are summarized to the table I) by using ImageJ software.
(7) Selecting the screened 'rushing flower' to carry out a drought stress experiment, fully watering the experimental material on the day of the experiment, placing the experimental material in a glass greenhouse to keep a relatively constant external environment, then not watering for 28 days, photographing and recording the growth forms of the hemerocallis fulva after 0d, 7d, 14d, 21d, 28d and rehydration 7d, observing the growth and development conditions of the hemerocallis fulva after drought experiment and rehydration, referring to fig. 2, from left to right, judging the morphology of the hemerocallis a above the ground part, the degree of leaf withering is slight, the hemerocallis a can be quickly recovered after rehydration, and judging the variety of the hemerocallis resistant to drought due to various factors such as comprehensive air pore conductivity, relative water content, in-vitro leaf water loss rate and drought stress experiment results.
Example 3
A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva comprises the following steps:
(1) the central console of the glass greenhouse displays the specific data of the experimental environment, the indoor temperature is 29.4 ℃, the indoor humidity is 95.2%, the carbon dioxide concentration is 1329PPM, the illumination intensity is 15.7KL, and the experimental environment is kept consistent.
The method comprises the steps of measuring the leaf surface temperature of all kinds of hemerocallis in the glass greenhouse by using a precise infrared thermometer, selecting five hemerocallis of the same kind with the same physiological state and similar physiological state for each variety to measure, and taking an average value of the temperature of three points of the measured leaf surface of each hemerocallis as the leaf surface temperature of a single hemerocallis. And (4) screening and sorting by using excel software, and screening the hemerocallis variety with the highest or lowest leaf surface temperature.
Example three is a screened hemerocallis variety with the lowest blade surface temperature: after the noon, the leaf surface temperature was measured to be 29.1 ℃ (decimal place one bit).
(2) Method for determining ploidy of hemerocallis cells: a10 mg sample of fresh, tender day lily leaf tissue is placed in a petri dish, 400. mu.l of nuclear extraction buffer is added, the tissue is minced with a sharp blade, the tissue is cut for 60-180 seconds to ensure that the day lily tissue is evenly minced, 1600. mu.l of DAPI staining solution is added, the mixture is placed for 20min at 20 ℃, a sample is filtered by 38 μm (400 meshes), the sample is measured on a flow cytometer, the voltage is set to 400mV, the threshold value is adjusted to the maximum value, the flow rate is set to 0.4. mu.l/s, and the result of measurement is tetraploid.
(3) The method for measuring the porosity conductance comprises the following steps: selecting sunny side of leaves of Hemerocallis fulva of 'afternoon' variety under natural illumination at 9-11 am, measuring air hole conductivity (Gs), and setting light intensity at 1000 μmol-2.s-1Five biological repeated experiments are carried out, and the measurement results are averaged, wherein the air hole conductance of the hemerocallis fulva of the 'afternoon' variety is 0.059mol H2O m-2s-1。
(4) Method for determining relative water content: cutting three mature leaves of Hemerocallis fulva of 'afternoon' (same physiological state) with relative water content RWC at 8:00-10:00 in the morning, and rapidly packaging in plastic bags. And meanwhile, the sample is placed in an ice box and is rapidly brought back to the laboratory, and the laboratory is kept at a constant temperature and is protected from light. Taking out the experimental material from the plastic, wiping off the water on the leaves, weighing the materials respectively by using an 1/10000 electronic balance, recording initial data, namely the fresh weight Wf of the sample, putting the sample into a centrifuge tube to absorb water for 24 hours, weighing the saturated weight Wt, then putting the leaves into an oven at 70 ℃ to dry for 48 hours, weighing the dry weight Wd, calculating the relative water content RWC of the hemerocallis fulva variety (Wf-Wd) | (Wt-Wd), repeating the experiment for three times, and taking an average value.
When the hemerocallis are not subjected to drought stress treatment, the RWC is 87.9 percent; after the day lily is subjected to drought treatment for 7 days, the RWC is measured to be 83.9 percent again; the RWC was finally determined to be 70.7% after day lily drought treatment for 14 d.
(5) Measuring the water loss rate of the in vitro blade: 3 leaves are respectively cut from the hemerocallis midday 'afternoon' by small scissors and small tweezers and are placed on weighing paper for weighing. Avoiding direct sunlight, placing the leaves on weighing paper under indoor weak light, weighing the leaves for 5 times at different time (0 h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h and 5h respectively), repeating the experiment for three times to calculate the water loss rate of the leaves of different varieties of hemerocallis, and recording the water loss data (0.00%, 1.60%, 3.20%, 4.80%, 7.20%, 8.00%, 9.60%, 11.20% and 12.00%) of the leaves after the leaves wither.
(6) The method for measuring the density of the pores by a scanning electron microscope comprises the following steps: under the natural illumination of 9-11 points, selecting a sunny day lily leaf in a normal physiological state, shearing the upper half part of the leaf, removing the leaf tip, putting the leaf tip into plant tissue fixing liquid, then placing the leaf tip into an ice box for storage, measuring day lily stomatal data by using a Hitachi scanning electron microscope (Hitachi S-3400N), and calculating the stomatal data by using ImageJ software. (data summary table one)
(7) Selecting the screened 'afternoon' to carry out a drought stress experiment, fully watering the experimental material on the day of the experiment, placing the experimental material in a glass greenhouse to keep a relatively constant external environment, then continuously watering for 28 days without watering, photographing and recording the morphological parts of the hemerocallis on the hemerocallis ground after 0d, 7d, 14d, 21d and 28d and rehydration for 7d, referring to FIG. 3, from left to right, the hemerocallis are at 0d and 7d, 14d, 21d, 28d and rehydration 7d, observing the survival ability of the hemerocallis under the drought test and the growth and development conditions after rehydration, and observing that the morphology of the overground part of the hemerocallis at noon and the withered degree of the leaves are serious and account for one half of the whole hemerocallis, recovering the hemerocallis after manual pruning of the withered branches after rehydration, and judging the variety of the hemerocallis which are not drought-tolerant by combining multiple factors such as porosity conductivity, relative water content, water loss rate of the leaves in vitro, drought stress test results and the like.
Example 4
A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva comprises the following steps:
(1) the central console of the glass greenhouse displays the specific data of the experimental environment, the indoor temperature is 29.4 ℃, the indoor humidity is 95.2%, the carbon dioxide concentration is 1329PPM, the illumination intensity is 15.7KL, and the experimental environment is kept consistent.
The method comprises the steps of measuring the leaf surface temperature of all kinds of hemerocallis in the glass greenhouse by using a precise infrared thermometer, selecting five hemerocallis of the same kind with the same physiological state and similar physiological state for each variety to measure, and taking an average value of the temperature of three points of the measured leaf surface of each hemerocallis as the leaf surface temperature of a single hemerocallis. And (4) screening and sorting by using excel software, and screening the hemerocallis variety with the highest or lowest leaf surface temperature.
Example one is the screened hemerocallis variety with the lowest blade surface temperature: 'X-12', the foliar temperature was measured to be 29.1 deg.C (one bit retained in decimal places).
(2) Method for determining ploidy of hemerocallis cells: use of: a10 mg sample of fresh, tender day lily leaf tissue is placed in a petri dish, 400. mu.l of nuclear extraction buffer is added, the tissue is minced with a sharp blade, the tissue is cut for 60-180 seconds to ensure that the day lily tissue is evenly minced, 1600. mu.l of DAPI staining solution is added, the mixture is placed for 20min at 20 ℃, a sample is filtered by 38 μm (400 meshes), the sample is measured on a flow cytometer, the voltage is set to 400mV, the threshold value is adjusted to the maximum value, the flow rate is set to 0.4. mu.l/s, and the result of measurement is tetraploid.
(3) The method for measuring the porosity conductance comprises the following steps: under the natural illumination of 9-11 am, selecting the sunny surface of the leaves of Hemerocallis fulva of 'X-12' variety for measurement, and measuring the stomatal conductance (Gs), wherein the measured light intensity is set to be 1000 mu mol-2.s-1Five biological repeated experiments are carried out, and the measurement results are averaged, wherein the porosity conductance of the hemerocallis fulva of the 'X-12' variety is 0.085mol H2O m-2s-1。
(4) Method for determining relative water content: cutting three mature leaves of Hemerocallis fulva of 'X-12' variety (same physiological state) at a relative water content RWC of 8:00-10:00 in the morning, and rapidly packaging in plastic bags. And meanwhile, the sample is placed in an ice box and is rapidly brought back to the laboratory, and the laboratory is kept at a constant temperature and is protected from light. Taking out the experimental material from the plastic, wiping off the water on the leaves, weighing the materials respectively by using an 1/10000 electronic balance, recording initial data, namely the fresh weight Wf of the sample, putting the sample into a centrifuge tube to absorb water for 24 hours, weighing the saturated weight Wt, then putting the leaves into an oven at 70 ℃ to dry for 48 hours, weighing the dry weight Wd, calculating the relative water content RWC of the hemerocallis fulva variety (Wf-Wd) | (Wt-Wd), repeating the experiment for three times, and taking an average value.
When the hemerocallis are not subjected to drought stress treatment, the RWC content is 92.1 percent; after the day lily is subjected to drought treatment for 7 days, the RWC is measured to be 85.8 percent again; after the day lily is subjected to drought treatment for 14 days, the RWC is finally measured to be 78.8%.
(5) Measuring the water loss rate of the in vitro blade: 3 leaves are respectively cut from the hemerocallis fulva of 'X-12' by using small scissors and small tweezers and are placed on weighing paper for weighing. Avoiding direct sunlight, placing the leaves on weighing paper under indoor weak light, weighing the leaves at different time (0 h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h and 5h respectively), weighing for 5 times, repeating the experiment for three times, calculating the water loss rate of the hemerocallis leaves of the variety, and recording the water loss data (0.00%, 2.02%, 4.04%, 6.06%, 8.08%, 10.10%, 12.12%, 13.13% and 15.15%) of the x-12 leaves after the leaves wither.
(6) The method for measuring the density of the air holes by the scanning electron microscope comprises the following steps: under the natural illumination of 9-11 points, selecting a sunny day lily leaf in a normal physiological state, shearing the upper half part of the leaf, removing the leaf tip, putting the leaf tip into plant tissue fixing liquid, then placing the leaf tip into an ice box for storage, measuring day lily stomatal data by using a Hitachi scanning electron microscope (Hitachi S-3400N), and calculating the stomatal data by using ImageJ software. (data summary table one).
(7) Selecting the screened 'X-12' to carry out a drought stress experiment, fully watering the experimental material on the day of the experiment, placing the experimental material in a glass greenhouse to keep a relatively constant external environment, then continuously watering for 28 days, photographing and recording the morphological parts of the day lily after 0d, 7d, 14d, 21d, 28d and rehydration 7d, referring to figure 4, from left to right, the day lily is at 0d and 7d, 14d, 21d, 28d and rehydration 7d, observing the survival ability of the hemerocallis under the drought test and the growth and development conditions after rehydration, observing the shapes of the overground parts of 'X-12', ensuring the severe degree of leaf withering, ensuring that the withered branches and leaves occupy two thirds of the whole plant, needing artificial assistance, cutting off the occupation of the withering, being capable of recovering after replacing pot soil, and judging the hemerocallis which are not drought-resistant by combining various factors such as air hole conductivity, relative water content, in-vitro leaf water loss rate, drought stress test results and the like.
Example 5
1. A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva comprises the following steps:
(1) the central console of the glass greenhouse displays the specific data of the experimental environment, the indoor temperature is 29.4 ℃, the indoor humidity is 95.2%, the carbon dioxide concentration is 1329PPM, the illumination intensity is 15.7KL, and the experimental environment is kept consistent.
The method comprises the steps of measuring the leaf surface temperature of all kinds of hemerocallis in the glass greenhouse by using a precise infrared thermometer, selecting five hemerocallis of the same kind with the same physiological state and similar physiological state for each variety to measure, and taking an average value of the temperature of three points of the measured leaf surface of each hemerocallis as the leaf surface temperature of a single hemerocallis. And (4) screening and sorting by using excel software, and screening the hemerocallis variety with the highest or lowest leaf surface temperature.
Example one is the screened hemerocallis variety with the lowest blade surface temperature: 'little hug', the foliar temperature was measured to be 29.1 ℃ (decimal places retain one bit).
(2) Method for determining ploidy of hemerocallis cells: use of: placing 10mg of fresh and tender day lily leaf tissue sample in a culture dish, adding 400 μ l of nuclear extraction buffer, cutting the tissue with a sharp blade, cutting for 60-180 seconds to ensure that the day lily tissue is uniformly cut, adding 1600 μ l of DAPI staining solution, standing at 20 ℃ for 20min, filtering the sample with 38 μm (400 meshes), measuring on a flow cytometer, setting the voltage at 400mV, adjusting the threshold value to the maximum value, setting the flow rate at 0.4 μ l/s, and obtaining the measurement result as diploid.
(3) The method for measuring the porosity conductance comprises the following steps: under the natural illumination of 9-11 am, selecting the sunny surface of a day lily leaf of the 'small hug' variety for determination, determining the air hole conductance (Gs), setting the determination light intensity to be 1000 mu mol.m-2.s-1, performing five times of biological repeated experiments, averaging the measurement results, and determining the air hole conductance of the day lily of the 'small hug' variety to be 0.085mol H2Om-2s-1。
(4) Method for determining relative water content: cutting three mature leaves of hemerocallis fulva (with the same physiological state) of the 'small hug' variety at the relative water content RWC of 8:00-10:00 in the morning, and quickly packaging in a plastic bag. And meanwhile, the sample is placed in an ice box and is rapidly brought back to the laboratory, and the laboratory is kept at a constant temperature and is protected from light. Taking out the experimental material from the plastic, wiping off the water on the leaves, weighing the materials respectively by using an 1/10000 electronic balance, recording initial data, namely the fresh weight Wf of the sample, putting the sample into a centrifuge tube to absorb water for 24 hours, weighing the saturated weight Wt, then putting the leaves into an oven at 70 ℃ to dry for 48 hours, weighing the dry weight Wd, calculating the relative water content RWC of the hemerocallis fulva variety (Wf-Wd) | (Wt-Wd), repeating the experiment for three times, and taking an average value.
When the hemerocallis are not subjected to drought stress treatment, the RWC content is 88.2 percent; after the day lily is subjected to drought treatment for 7 days, the RWC is measured to be 74.2 percent again; at last 68.3% RWC was measured after 14 days of drought treatment of day lily.
(5) The water loss rate of the leaves in vitro is respectively cut from the day lily in small hugs by small scissors and small tweezers and 3 leaves are placed on weighing paper for weighing. Avoiding direct irradiation of sunlight, placing the leaves on weighing paper under indoor weak light, weighing the leaves at different time (0 h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h and 5h respectively), weighing for 5 times, repeating the experiment for three times, calculating the water loss rate of the leaves of different varieties of hemerocallis, and recording the water loss data (0.00%, 1.92%, 3.85%, 5.77%, 7.69%, 9.62% and 11.54%) of the leaves in small hugs after the leaves are withered.
(6) The method for measuring the density of the pores by a scanning electron microscope comprises the following steps: under the natural illumination of 9-11 points, selecting a sunny day lily leaf in a normal physiological state, shearing the upper half part of the leaf, removing the leaf tip, putting the leaf tip into plant tissue fixing liquid, then placing the leaf tip into an ice box for storage, measuring day lily stomatal data by using a Hitachi scanning electron microscope (Hitachi S-3400N), and calculating the stomatal data by using ImageJ software. (data summary table one)
(7) Selecting the screened small embrace to carry out a drought stress experiment, fully watering the experimental material on the day of the experiment, placing the experimental material in a glass greenhouse to keep a relatively constant external environment, then continuously watering for 28 days, photographing and recording the morphological parts of the day lily after 0d, 7d, 14d, 21d, 28d and 7d of rehydration, referring to FIG. 5, from left to right, the day lily is at 0d and 7d, 14d, 21d, 28d and rehydration 7d, observing the survival ability of the hemerocallis under the drought test and the growth and development conditions after rehydration, observing the shapes of the overground parts of the hemerocallis in small hugs, ensuring the severe degree of leaf withering, ensuring that withered branches and leaves occupy two thirds of the whole plant, needing artificial help, cutting off withering careers, being capable of recovering after replacing pot soil, and judging the hemerocallis which are not drought-tolerant by combining various factors such as air hole conductivity, relative water content, in-vitro leaf water loss rate, drought stress test results and the like.
The method for measuring the stomatal conductance in the above examples is to use the LI-6400XT portable photosynthesis measurement system of LI-COR, USA; in the method for measuring cell ploidy, the cell nucleus extract comprises 100mmol/L citric acid, Tween-80 with a volume fraction of 0.5% pH 3, 400mmol/L heptahydrate and disodium hydrogen phosphate; when the hemerocallis plant tissue is observed with an electron microscope, the plant tissue fixing solution used contains 65% absolute ethyl alcohol, 6% acetic acid, and 5% formaldehyde (formalin).
TABLE 1 summary of pore density data for each variety of hemerocallis
In conclusion, the method can reduce the temperature of the leaf surface of the plant according to the transpiration, and can be used for rapidly screening drought-resistant hemerocallis varieties in a large scale. Sunlight irradiates the day lily leaves, and most of energy is converted from light energy to heat energy. The temperature of the day lily leaf surface is increased, so that the plants can emit moisture through the stomatal transpiration to prevent the day lily leaf from being burnt, and the heat energy is absorbed in the process that the water is changed into the water vapor, so that the temperature of the day lily leaf surface is reduced. Researches show that the transpiration of plants depends on the stomatal transpiration. Theoretically, the hemerocallis under the same physiological state have large stomatal density, high stomatal conductance and remarkable transpiration effect, low leaf surface temperature, rapid water loss and weak drought resistance, and vice versa. Therefore, the drought-resistant hemerocallis variety is preliminarily screened based on the leaf surface temperature, and then the drought-resistant hemerocallis variety is confirmed by measuring the indexes of the hemerocallis variety such as cell ploidy, stomatal conductance, water content of leaves, water loss of leaves in vitro and the like and further by the survival ability under the natural drought stress test and the growth and development condition after the rehydration test. Therefore, the screening method provided by the invention does not need to perform a large number of drought stress experiments, adopts temperature to perform primary screening, and then further performs confirmation experiments.
The method of the invention performs feasible experiments from macroscopical (part of the ground morphology of the hemerocallis fulva) to microcosmic (the pores and the physiological state of the hemerocallis fulva), and obtains a great deal of experimental data. Therefore, the method has the advantages of accurate screening result, lower cost, shorter screening period and the like. The method can quickly, efficiently and accurately obtain drought-resistant high-quality germplasm resources in the face of large-scale and multi-variety screening experiments of the hemerocallis fulva, and has a certain reference value for researching and breeding the hemerocallis fulva drought resistance.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.
Claims (8)
1. A method for screening drought-resistant hemerocallis fulva based on leaf surface temperature of the hemerocallis fulva is characterized by comprising the following steps of:
s1: primary screening: measuring the leaf surface temperature of different hemerocallis and sequencing the leaf surface temperature of each hemerocallis;
s2: further determination: and (4) selecting the hemerocallis with the highest or the lowest hemerocallis in the hemerocallis leaf surface temperature sequence in the step (S1), measuring the cell ploidy, the stomatal conductance, the relative water content of the leaves, the water loss rate of the leaves in vitro and the stomatal size and density of the leaves, and determining and screening drought-resistant hemerocallis varieties through the survival capability of the hemerocallis under the natural drought stress test and the growth and development condition after the rehydration test.
2. The method for screening drought-resistant hemerocallis fulva based on foliar temperature of hemerocallis fulva according to claim 1, wherein the method for determining the ploidy of hemerocallis fulva cells in step 2 comprises: use of: a10 mg sample of fresh, tender leaf tissue of Hemerocallis fulva was placed in a petri dish, 400. mu.l of nuclear extraction buffer was added, the tissue was minced using a razor blade, the tissue was cut for 60-180 seconds to ensure uniform mincing of the Hemerocallis fulva tissue, 1600. mu.l of DAPI staining solution was added, the mixture was left at 20 ℃ for 20min and the sample was filtered at 38. mu.m, and measured using a flow cytometer.
3. The method for screening drought-resistant hemerocallis fulva based on foliar temperature of hemerocallis fulva of claim 2, wherein the cell nucleus buffer extract comprises 100mmol/L citric acid, Tween-80 with a volume fraction of 0.5% pH 3, 400mmol/L heptahydrate and disodium hydrogen phosphate; for flow cytometry measurements, the voltage was set at 400mV and the flow rate was set at 0.4. mu.l/s.
4. The method for screening drought-resistant hemerocallis fulva based on the leaf surface temperature of the hemerocallis fulva according to claim 1, wherein the stomata of the hemerocallis fulva leaves are measured by a scanning electron microscope, the sunward leaves of the hemerocallis fulva in a normal physiological state are selected under natural illumination at 9-11 pm, the upper half parts of the leaves are cut, the leaf tips are removed, the leaves are placed in a plant tissue fixing solution and then placed in an ice box for storage, and the data of the stomata of the hemerocallis fulva are measured by a scanning electron.
5. The method for screening drought-resistant hemerocallis fulva based on foliar temperature of hemerocallis fulva of claim 4, wherein the plant tissue fixative is a mixture of 65% absolute ethanol, 6% acetic acid and 5% formaldehyde.
6. The method for screening drought-resistant hemerocallis fulva based on foliar temperature of hemerocallis fulva according to claim 5, wherein the plant tissue fixing solution further comprises 4-6 ml of glycerol.
7. The method for screening drought-resistant hemerocallis fulva based on foliar temperature of hemerocallis in claim 1, wherein the method for measuring stomatal conductance in step 2 comprises: under natural illumination of 9-11 am, selecting sunny side of daylily leaf in normal physiological state, measuring porosity conductance, and setting light intensity at 1000 μmol-2s-1。
8. The method for screening drought-resistant hemerocallis fulva based on the leaf surface temperature of the hemerocallis fulva according to claim 1, wherein the leaf surface temperature of the hemerocallis fulva is measured by a high-precision infrared thermometer.
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Application publication date: 20200515 |