1. A method for rapidly identifying rice cadmium sensitivity by using a fluorescent staining technology is characterized by comprising the following steps: the method comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, soaking the in-vitro leaves for 6-48 h, carrying out stress treatment on the in-vitro leaves in the cadmium solution, releasing a large amount of Reactive Oxygen Species (ROS) from mesophyll cells, and reacting the reactive oxygen species with fluorescein under the catalysis of enzyme to form a compound with fluorescence;

s2, observing by using microscope

Taking out the in vitro leaves in the test tube, and sequentially placing the leaves under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the difference of the cadmium sensitivity of the rice mesophyll cells among genotypes can be distinguished, and the more the fluorescence spots in the leaves are, the larger the fluorescence area is, the more ROS released by the mesophyll cells is, the stronger the sensitivity to cadmium ions is, and the worse the cadmium resistance is; conversely, the stronger the cadmium tolerance of the mesophyll cells.

2. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claim 1, which is characterized in that: the method for identifying the cadmium sensitivity of the rice has another mode and comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, carrying out stress treatment on the in-vitro leaves in the cadmium solution, enabling the hydrogen peroxide distribution characteristics of mesophyll cells to show obvious difference among genotypes, putting the in-vitro leaves into the cadmium solution with different concentrations for treatment for 12-48 h, taking out the leaves, soaking the leaves in a reaction tank containing reduced Dichlorofluorescein (DCF) for incubation for 1-10 h, oxidizing non-fluorescent acetate groups in the DCF by hydrogen peroxide in the leaves, and dyeing the in-vitro leaves after generating the dichlorofluorescein with charges;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more green fluorescent spots in the leaves, the more hydrogen peroxide generated by the leaves in a cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; conversely, the less green fluorescent spots in the leaf, the greater the cadmium tolerance of the mesophyll cells.

3. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claim 1, which is characterized in that: the method for identifying the cadmium sensitivity of the rice has another mode and comprises the following steps:

s1 stress treatment

Cutting the in vitro leaves of different rice genotypes, respectively putting the in vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in vitro leaves are submerged, carrying out stress treatment on the in vitro leaves in the cadmium solution, wherein O2-distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in vitro leaves in the cadmium solution with different concentrations for treatment for 6-48 h, taking out the in vitro leaves, soaking the in a reaction tank containing Nitrogen Blue Tetrazole (NBT) for incubation for 1-10 h, reducing the nitrogen blue tetrazole into blue methylhydrazone, having a maximum absorption peak at 560nm, and then dyeing the in vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescent microscope to observe the size and distribution characteristics of light spots in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more blue spots in the leaves, the more O2 & lt- & gt generated by the leaves in the cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; conversely, the less blue fluorescent spots in the leaf, the stronger the cadmium tolerance of the mesophyll cells.

4. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claims 1-3, wherein the method comprises the following steps: the cutting length of the in vitro leaves of different rice genotypes is 3 cm.

5. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claims 1-3, wherein the method comprises the following steps: the cadmium solution is cadmium chloride solution, wherein the concentration of cadmium is 0.9, 4.5, 9.0, 13.5 and 18.0 mu mol.L^-1One or more of (a).

6. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claim 2, which is characterized in that: the detailed dyeing treatment operation is that dichlorodihydrofluorescein diacetate is selected from a reactive oxygen species ROS analysis kit, the in vitro leaves are dyed, and the incubation time is 1-10 h.

7. The method for rapidly identifying the cadmium sensitivity of rice by using the fluorescent staining technology as claimed in claim 3, which is characterized in that: the detailed dyeing treatment operation is to select a nitroblue tetrazolium (NBT) dyeing kit, dye the leaves in vitro and incubate for 1-10 h.

Background

Cadmium is harmful heavy metal with the widest global distribution and the greatest biological toxicity, but the cadmium resistance of rice is stronger, and rice with the cadmium exceeding the standard can be produced even if the rice is planted in clean soil with the Cd content of less than 0.3 mg.kg < -1 >. Therefore, a large amount of capital is invested in China to support the breeding work of the rice genotype with low cadmium accumulation, the cadmium content of the rice is highly positively correlated with the cadmium resistance of the rice, the cadmium resistance genotype can enrich a large amount of cadmium in nutritive organs and grains, and the rice genotype sensitive to cadmium generally has the characteristic of low cadmium accumulation.

At present, the identification of the cadmium-resistant capability of the rice genotype is mainly completed by comparing the difference of indexes such as the survival rate of rice seedlings, the growth amount, the cadmium content in rice and the like among the genotypes in a cadmium stress environment, but the rice growth period is long, and the rainfall and the photo-thermal resource change in the growth period are large, so that the accuracy of a test result is poor, the error is large, and the efficiency is low.

Disclosure of Invention

(1) Solves the technical problem

Aiming at the defects of the prior art, the invention provides a method for rapidly identifying rice cadmium sensitivity by using a fluorescent dyeing technology, and solves the problems of poor accuracy, large error and low efficiency in rice cadmium sensitivity detection of the prior art.

(2) Technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: a method for rapidly identifying rice cadmium sensitivity by using a fluorescent staining technology comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, soaking the in-vitro leaves for 6-48 h, carrying out stress treatment on the in-vitro leaves in the cadmium solution, releasing a large amount of Reactive Oxygen Species (ROS) from mesophyll cells, and reacting the reactive oxygen species with fluorescein under the catalysis of enzyme to form a compound with fluorescence;

s2, observing by using microscope

Taking out the in vitro leaves in the test tube, and sequentially placing the leaves under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the difference of the cadmium sensitivity of the rice mesophyll cells among genotypes can be distinguished, and the more the fluorescence spots in the leaves are, the larger the fluorescence area is, the more ROS released by the mesophyll cells is, the stronger the sensitivity to cadmium ions is, and the worse the cadmium resistance is; conversely, the stronger the cadmium tolerance of the mesophyll cells.

Preferably, the method for identifying the cadmium sensitivity of the rice comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, carrying out stress treatment on the in-vitro leaves in the cadmium solution, enabling the hydrogen peroxide distribution characteristics of mesophyll cells to show obvious difference among genotypes, putting the in-vitro leaves into the cadmium solution with different concentrations for treatment for 12-48 h, taking out the leaves, soaking the leaves in a reaction tank containing reduced Dichlorofluorescein (DCF) for incubation for 1-10 h, oxidizing non-fluorescent acetate groups in the DCF by hydrogen peroxide in the leaves, and dyeing the in-vitro leaves after generating the dichlorofluorescein with charges;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more green fluorescent spots in the leaves, the more hydrogen peroxide generated by the leaves in a cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; conversely, the less green fluorescent spots in the leaf, the greater the cadmium tolerance of the mesophyll cells.

Preferably, the method for identifying the cadmium sensitivity of the rice comprises the following steps:

s1 stress treatment

Cutting the in vitro leaves of different rice genotypes, respectively putting the in vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in vitro leaves are submerged, carrying out stress treatment on the in vitro leaves in the cadmium solution, wherein O2-distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in vitro leaves in the cadmium solution with different concentrations for treatment for 6-48 h, taking out the in vitro leaves, soaking the in a reaction tank containing Nitrogen Blue Tetrazole (NBT) for incubation for 1-10 h, reducing the nitrogen blue tetrazole into blue methylhydrazone, having a maximum absorption peak at 560nm, and then dyeing the in vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescent microscope to observe the size and distribution characteristics of light spots in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more blue spots in the leaves, the more O2 & lt- & gt generated by the leaves in the cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; conversely, the less blue fluorescent spots in the leaf, the stronger the cadmium tolerance of the mesophyll cells.

Preferably, the cut length of the in vitro leaves of different rice genotypes is 3 cm.

Preferably, the cadmium solution is a cadmium chloride solution, wherein the cadmium concentration is one or more of 0.9, 4.5, 9.0, 13.5 and 18.0 mu mol.L-1.

Preferably, the detailed dyeing treatment operation is to select dichlorodihydrofluorescein diacetate of a Reactive Oxygen Species (ROS) analysis kit and dye the in vitro leaves for 1-10 h.

Preferably, the detailed dyeing treatment is to select a nitroblue tetrazolium (NBT) dyeing kit, dye the in vitro leaf blade, and incubate for 1-10 h.

(3) Advantageous effects

The invention provides a method for rapidly identifying rice cadmium sensitivity by using a fluorescent staining technology. The method has the following beneficial effects:

according to the method, the cadmium sensitivity of the rice with different genotypes is intuitively and accurately judged by utilizing the correlation between the quantity of the active oxygen generated by the in-vitro leaves in the cadmium stress environment and the fluorescence intensity, the required materials are few, the detection time is short, the working efficiency is high, the method has a very wide application prospect, and has an important reference value for quickly identifying the cadmium resistance potential of other plants; and a large number of genotypes sensitive to cadmium resistance are screened according to the leaf fluorescence intensity identification technology, so that the scale of field tests can be effectively reduced, the time and the labor are saved, the screening efficiency of the low-cadmium genotype rice is greatly improved, the step of breeding new varieties of the low-cadmium rice is accelerated, and the method is worthy of vigorous popularization.

Drawings

FIG. 1 is a schematic diagram illustrating the effect of cadmium stress on the quantity and distribution characteristics of hydrogen peroxide in leaves according to the present invention;

FIG. 2 is a schematic diagram showing the effect of cadmium stress on the quantity and distribution characteristics of O2-in leaves.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, 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 first embodiment is as follows:

as shown in fig. 1-2, an embodiment of the present invention provides a method for rapidly identifying rice cadmium sensitivity by using a fluorescent staining technique, including the following steps:

s1 stress treatment

Cutting the detached leaves of different rice genotypes, respectively putting the detached leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the detached leaves are submerged, soaking the detached leaves for 48 hours, carrying out stress treatment on the detached leaves in the cadmium solution, releasing a large amount of Reactive Oxygen Species (ROS) from mesophyll cells under the catalysis of enzyme,

reacting the active oxygen with fluorescein to form a compound with fluorescence;

s2, observing by using microscope

Taking out the in vitro leaves in the test tube, and sequentially placing the leaves under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the difference of the cadmium sensitivity of the rice mesophyll cells among genotypes can be distinguished, and the more the fluorescence spots in the leaves are, the larger the fluorescence area is, the more ROS released by the mesophyll cells is, the stronger the sensitivity to cadmium ions is, and the worse the cadmium resistance is; on the contrary, the stronger the cadmium resistance of mesophyll cells, the more intuitively and accurately judges the cadmium sensitivity of rice with different genotypes by utilizing the correlation between the quantity of the active oxygen generated by the leaves in vitro and the fluorescence intensity in the cadmium stress environment, the less materials are needed, the detection time is short, the working efficiency is high, the application prospect is very wide, and the important reference value is realized for the rapid identification of the cadmium resistance potential of other plants.

In yet another embodiment, the method for identifying cadmium sensitivity in rice comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, carrying out stress treatment on the in-vitro leaves in the cadmium solution, wherein the hydrogen peroxide distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in-vitro leaves into the cadmium solution with different concentrations for treatment for 48h, taking out the leaves, soaking the leaves in a reaction tank containing reduced Dichlorofluorescein (DCF) for incubation for 10h, oxidizing non-fluorescent acetate groups in the DCF by hydrogen peroxide in the leaves to generate the dichlorofluorescein with charges, and then dyeing the in-vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more green fluorescent spots in the leaves, the more hydrogen peroxide generated by the leaves in a cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; on the contrary, the less green fluorescent spots in the leaves, the stronger the cadmium resistance of mesophyll cells, the cadmium sensitivity of different genotypes is identified by the technical system based on the fluorescent intensity and distribution characteristics in the rice leaves, only a small amount of rice leaves are needed without the limitation of the size of rice, the cadmium resistance of the rice can be quickly and accurately identified in a shorter time, and the method has the advantages of high efficiency, good repeatability, low cost and the like.

In yet another embodiment, the method for identifying cadmium sensitivity in rice comprises the following steps:

s1 stress treatment

Cutting the in vitro leaves of different rice genotypes, respectively putting the in vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in vitro leaves are submerged, carrying out stress treatment on the in vitro leaves in the cadmium solution, wherein O2-distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in vitro leaves in the cadmium solution with different concentrations for treatment for 48 hours, taking out the leaves, soaking the leaves in a reaction tank containing Nitrogen Blue Tetrazole (NBT) for incubation for 10 hours, wherein the nitrogen blue tetrazole is reduced into blue methylhydrazone, a maximum absorption peak is formed at 560nm, and then dyeing the in vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescent microscope to observe the size and distribution characteristics of light spots in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more blue spots in the leaves, the more O2 & lt- & gt generated by the leaves in the cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; on the contrary, the less the blue fluorescent spots in the leaves, the stronger the cadmium resistance of mesophyll cells, and the screening of a large number of genotypes sensitive to cadmium resistance according to the leaf fluorescence intensity identification technology can effectively compress the scale of field experiments, save time and manpower, greatly improve the screening efficiency of low-cadmium genotype rice and accelerate the step of breeding new varieties of low-cadmium rice.

The cutting length of the in vitro leaves of different rice genotypes is 3 cm.

The cadmium solution is a cadmium chloride solution, wherein the concentration of cadmium is one or more of 0.9, 4.5, 9.0, 13.5 and 18.0 mu mol.L < -1 >.

The detailed operation of the staining treatment is that dichlorodihydrofluorescein diacetate is selected as a reactive oxygen species ROS analysis kit, the in vitro leaves are stained, and the incubation time is 10 h.

The detailed operation of the staining treatment is to select a nitroblue tetrazolium (NBT) staining kit, stain the excised leaf and incubate for 10 h.

Example two:

as shown in fig. 1-2, an embodiment of the present invention provides a method for rapidly identifying rice cadmium sensitivity by using a fluorescent staining technique, including the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, soaking the in-vitro leaves for 6 hours, carrying out stress treatment on the in-vitro leaves in the cadmium solution, releasing a large amount of Reactive Oxygen Species (ROS) from mesophyll cells, and reacting the reactive oxygen species with fluorescein under the catalysis of enzyme to form a compound with fluorescence;

s2, observing by using microscope

Taking out the in vitro leaves in the test tube, and sequentially placing the leaves under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the difference of the cadmium sensitivity of the rice mesophyll cells among genotypes can be distinguished, and the more the fluorescence spots in the leaves are, the larger the fluorescence area is, the more ROS released by the mesophyll cells is, the stronger the sensitivity to cadmium ions is, and the worse the cadmium resistance is; on the contrary, the stronger the cadmium resistance of mesophyll cells, the more intuitively and accurately judges the cadmium sensitivity of rice with different genotypes by utilizing the correlation between the quantity of the active oxygen generated by the leaves in vitro and the fluorescence intensity in the cadmium stress environment, the less materials are needed, the detection time is short, the working efficiency is high, the application prospect is very wide, and the important reference value is realized for the rapid identification of the cadmium resistance potential of other plants.

In yet another embodiment, the method for identifying cadmium sensitivity in rice comprises the following steps:

s1 stress treatment

Cutting the in-vitro leaves of different rice genotypes, respectively putting the in-vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in-vitro leaves are submerged, carrying out stress treatment on the in-vitro leaves in the cadmium solution, wherein the hydrogen peroxide distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in-vitro leaves into the cadmium solution with different concentrations for treatment for 12h, taking out the leaves, soaking the leaves in a reaction tank containing reduced Dichlorofluorescein (DCF) for incubation for 1h, oxidizing non-fluorescent acetate groups in the DCF by hydrogen peroxide in the leaves to generate the dichlorofluorescein with charges, and then dyeing the in-vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescence microscope to observe the fluorescence intensity and distribution characteristics in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more green fluorescent spots in the leaves, the more hydrogen peroxide generated by the leaves in a cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; on the contrary, the less green fluorescent spots in the leaves, the stronger the cadmium resistance of mesophyll cells, the cadmium sensitivity of different genotypes is identified by the technical system based on the fluorescent intensity and distribution characteristics in the rice leaves, only a small amount of rice leaves are needed without the limitation of the size of rice, the cadmium resistance of the rice can be quickly and accurately identified in a shorter time, and the method has the advantages of high efficiency, good repeatability, low cost and the like.

In yet another embodiment, the method for identifying cadmium sensitivity in rice comprises the following steps:

s1 stress treatment

Cutting the in vitro leaves of different rice genotypes, respectively putting the in vitro leaves into different test tubes, dropwise adding an equal amount of cadmium solution into the test tubes until the in vitro leaves are submerged, carrying out stress treatment on the in vitro leaves in the cadmium solution, wherein O2-distribution characteristics of mesophyll cells show obvious difference among genotypes, putting the in vitro leaves in the cadmium solution with different concentrations for treatment for 6 hours, taking out the leaves, soaking the leaves in a reaction tank containing Nitrogen Blue Tetrazole (NBT) for incubation for 1 hour, reducing the nitrogen blue tetrazole into blue methylhydrazone, having a maximum absorption peak at 560nm, and then dyeing the in vitro leaves;

s2, observing by using microscope

The in vitro leaves in the reaction tank are placed under a fluorescent microscope to observe the size and distribution characteristics of light spots in the in vitro leaves, so that the cadmium resistance of the genotype can be rapidly and accurately judged, and the more blue spots in the leaves, the more O2 & lt- & gt generated by the leaves in the cadmium stress environment, the stronger the sensitivity of mesophyll cells to cadmium ions and the poorer the cadmium resistance; on the contrary, the less the blue fluorescent spots in the leaves, the stronger the cadmium resistance of mesophyll cells, and the screening of a large number of genotypes sensitive to cadmium resistance according to the leaf fluorescence intensity identification technology can effectively compress the scale of field experiments, save time and manpower, greatly improve the screening efficiency of low-cadmium genotype rice and accelerate the step of breeding new varieties of low-cadmium rice.

The cutting length of the in vitro leaves of different rice genotypes is 3 cm.

The cadmium solution is a cadmium chloride solution, wherein the concentration of cadmium is one or more of 0.9, 4.5, 9.0, 13.5 and 18.0 mu mol.L < -1 >.

The detailed operation of the staining treatment is that dichlorodihydrofluorescein diacetate is selected as a reactive oxygen species ROS analysis kit, the in vitro leaves are stained, and the incubation time is 1 h.

The detailed operation of the staining treatment is to select a nitroblue tetrazolium (NBT) staining kit, stain the excised leaf and incubate for 1 h.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.