A method for assisting identification of drought resistance of cotton
By performing drought stress treatment on cotton in an incubator and using transmission electron microscope to observe the endoplasmic reticulum structure, the specific thread structure is used as the basis for identification of drought resistance, the problem of drought resistance identification in the prior art is solved, and the drought of cotton is achieved Effective identification under coercion.
Patent Information
- Application Number
- CN202210378817.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-04-12
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2042-04-12
AI Technical Summary
The prior art is difficult to effectively assist in identifying the drought resistance of cotton, especially the specific markers of plant endoplasmic reticulum structure changes under drought stress.
By culturing cotton in an incubator to the first-center stage of three leaves, natural drought stress treatment was performed, and the endoplasmic reticulum structure of cotton leaves was observed using transmission electron microscope. The specific thread structure was used as the basis for identification of drought resistance, and the soil moisture content and light conditions were strictly controlled. , combine glutaraldehyde fixing solution and potassium permanganate aqueous solution to fix the leaves, and observe the changes in the thread structure of the endoplasmic reticulum.
The auxiliary identification of cotton drought resistance under drought stress was achieved. The strength of drought resistance was judged by the number of thread structures, and the identification method of closely related endoplasmic reticulum structural changes and drought stress was provided, which improved the identification efficiency of drought resistance varieties.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for auxiliary identification of drought resistance of cotton, belonging to the technical field of biomedicine. Background Art
[0002] Drought is one of the main factors limiting the growth, development and distribution of higher plants in the biosphere. Soil drought and atmospheric dryness can both lead to plant water loss. It is generally believed that the morphological characteristics of various parts of plants show different plasticity in response to drought stress. These differences are mainly manifested in leaf size, leaf morphology, root development, etc. The endoplasmic reticulum is an important site for protein processing. Adverse conditions will lead to endoplasmic reticulum stress, exacerbating the accumulation of unfolded and misfolded proteins. Summary of the invention
[0003] The purpose of the present invention is to provide a method for assisting identification of drought resistance of cotton. The present invention assists identification by changes in the endoplasmic reticulum structure under drought stress: the emergence of a whorl structure, and the drought stress of cotton in the present invention is closely related to the endoplasmic reticulum ultrastructure.
[0004] The method for assisting in identifying drought resistance of cotton provided by the present invention comprises the following steps:
[0005] Cotton was cultured in an incubator until it reached at least the three-leaf and one-heart stage, and then subjected to natural drought stress. When the soil moisture content dropped to 1-2%, samples were taken and the endoplasmic reticulum structure of the cotton leaves was observed using a transmission electron microscope. If a circular whorl structure appeared, the cotton was drought-resistant. Normally grown seedlings were used as a control.
[0006] Specifically, the drought resistance of the cotton is determined according to the number of the whorl structures present: the more the whorl structures there are, the worse the drought resistance is.
[0007] In the above method, the culture conditions are:
[0008] Lighting 12-16h, temperature 25-28℃;
[0009] Darkness 8-12h, temperature 23-25℃.
[0010] In the above method, the soil moisture content is measured by drying and weighing method;
[0011] For example: use a 0.1g accurate balance to weigh the weight of the soil sample, record it as the wet weight of the soil sample M, dry the soil sample in a 105℃ oven for 8h to constant weight, measure the dried soil sample immediately after drying, record it as the dry weight of the soil sample Ms;
[0012] Soil moisture content = (mass of the aluminum box and soil sample before drying - mass of the aluminum box and soil sample after drying) / (mass of the aluminum box and soil sample after drying - mass of the empty aluminum box after drying) × 100%.
[0013] In the above method, the sampling process is as follows: the leaf is cut into multiple leaf pieces with a length and width of 1 to 5 mm, all from the same leaf, and then immediately placed in a fixing solution;.
[0014] When taking samples, the movements should be gentle, the instruments should be sharp, a new blade should be replaced each time the leaf is cut, and squeezing of the tissue should be avoided.
[0015] In the above method, the fixing solution is a mixed solution of glutaraldehyde fixing solution and potassium permanganate aqueous solution, and the pH value is 6.8-7.2;
[0016] In the fixing solution, the volume content of the glutaraldehyde fixing solution is 48-50%, and the volume content of the potassium permanganate aqueous solution is 50-52%;
[0017] The fixative solution is placed in a 4°C refrigerator for later use;
[0018] In the above method, a Hitachi 7800 transmission electron microscope was used for observation.
[0019] The method of the present invention discovers the special endoplasmic reticulum whorl structure under drought stress of cotton, and its application in assisting identification of drought resistance of cotton also belongs to the protection scope of the present invention.
[0020] The present invention observes the endoplasmic reticulum structure under cotton drought stress by transmission electron microscopy, and closely links cotton drought stress with the endoplasmic reticulum structure. The endoplasmic reticulum-specific whorls can be used as a sign of cotton drought stress in the endoplasmic reticulum structure, and the number of whorls in the endoplasmic reticulum under different drought levels can be used as an aid in identifying the drought resistance of varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The endoplasmic reticulum structure of cotton material leaves treated differently in Example 1 of the present invention.
[0022] Figure 2 The endoplasmic reticulum structure of cotton material leaves treated differently in Example 2 of the present invention. DETAILED DESCRIPTION
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0025] The cotton materials used in the following examples are from the National Cotton Germplasm Resource Medium-term Bank, which are well-known public varieties that can be purchased from the market or introduced from corresponding breeding units or the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.
[0026] Example 1, based on Material 1: Changes in the endoplasmic reticulum structure of S1
[0027] Material 1: normal growth, natural drought, and rewatering after drought for 48 hours, three treatments in total.
[0028] 1) Sandy soil planting material 1, in an incubator with a light intensity of 16 hours and a temperature of 28°C, a dark intensity of 8 hours and a temperature of 25°C, the cotton was cultured to the three-leaf and one-heart stage and subjected to natural drought stress treatment, and sampling was performed when the soil moisture content dropped to about 2%. After natural drought, the seedlings that were rehydrated for 48 hours and grew normally were used as the control group, and the samples were prepared for observation of the endoplasmic reticulum structure using a transmission electron microscope.
[0029] 2) Relative moisture content of soil: A soil sample weighing about 70 g was dried in an aluminum box and dried in an oven at 105°C for 8 h. The relative moisture content of the soil was calculated and found to be 0.99%.
[0030] 3) The fixative used is 2% glutaraldehyde fixative and 1% potassium permanganate aqueous solution, the volume content of glutaraldehyde fixative is 50%, and the volume content of potassium permanganate aqueous solution is 50%; mix well and place in a 4°C refrigerator for later use, and the pH value is 7.0.
[0031] 4) When sampling, take 5 leaf blocks of approximately 2mm×2mm from a single leaf. The sampling should be quick and accurate. The blade used should be changed every time a small leaf block is cut to avoid tissue damage. The sample should be quickly placed in the prepared 2ml centrifuge tube containing 1.5ml of fixative.
[0032] 5) All prepared samples need to be vacuumed for 20 minutes to allow the leaves to sink to the bottom of the test tube, and ice bags and ice boxes are prepared for sample delivery.
[0033] 6) A Hitachi 7800 transmission electron microscope was used to observe the changes in the endoplasmic reticulum structure of cotton leaves under drought stress.
[0034] The endoplasmic reticulum structure of leaves of material 1 treated differently Figure 1 shown.
[0035] CK (left) in the picture is a normally growing cotton leaf in the three-leaf and one-heart stage. The structure of the endoplasmic reticulum can be clearly seen from the picture. The red arrow points to the endoplasmic reticulum, which is a flat linear structure at this time.
[0036] The natural drought in the figure (middle picture) shows cotton samples taken at the three-leaf and one-heart stage under natural drought stress and the ultrastructure of the cotton leaves was observed using a transmission electron microscope. At this time, the soil moisture content was about 2%. It can be seen from the figure that the linear endoplasmic reticulum indicated by the red arrow and the circular endoplasmic reticulum whorl structure indicated by the blue arrow can be seen. It can be found that the endoplasmic reticulum whorl structure coexists with the normal linear or flat endoplasmic reticulum structure.
[0037] The rehydration in the figure (right) shows that cotton was rehydrated for 48 hours after drought stress. The samples were cut and placed in a fixative for fixation. The ultrastructure of the cotton leaves was observed using a transmission electron microscope. After rehydration, a more obvious endoplasmic reticulum whorl structure can be seen (indicated by the blue arrow). At the same time, there are also linear or flat endoplasmic reticulum structures. It is speculated that the disappearance of the endoplasmic reticulum whorls is a long process during the rehydration process after drought stress. Through rehydration, a clearer endoplasmic reticulum whorl structure was unexpectedly discovered.
[0038] Example 2, based on Material 2: Changes in the endoplasmic reticulum structure of S4
[0039] Material 2: cotton material with poorer drought resistance than Material 1. Only the endoplasmic reticulum structure under normal growth and drought conditions was observed. The treatment method was the same as that of Material 1.
[0040] The endoplasmic reticulum structure of leaves of material 2 treated differently Figure 2 shown.
[0041] CK (left) in the picture is a normally growing cotton leaf in the three-leaf and one-heart stage. The structure of the endoplasmic reticulum can be seen from the picture. The red arrow points to the endoplasmic reticulum, which is a flat linear structure at this time.
[0042] The natural drought in the figure (right) is a natural drought stress treatment of cotton at the three-leaf and one-heart stage. The ultrastructure of cotton leaves was observed by transmission electron microscopy. At this time, the soil water content was about 2%. From the figure, we can see the linear endoplasmic reticulum pointed by the red arrow and the circular endoplasmic reticulum spiral structure pointed by the blue arrow. It can be found that the endoplasmic reticulum spiral structure coexists with the normal linear or flat endoplasmic reticulum structure. Compared with material 1, there are more endoplasmic reticulum spiral structures under drought treatment.
[0043] The above results show that after drought stress treatment, a concentric circle structure of the endoplasmic reticulum appears, which is referred to as the endoplasmic reticulum whorl structure in the present invention, and a clear endoplasmic reticulum whorl structure can still be seen after 48 hours of rehydration treatment. The changes in the endoplasmic reticulum structure seen closely link the endoplasmic reticulum with cotton drought stress, and the materials with poor drought resistance have more whorls, which can be used to assist in the identification of drought-resistant varieties.
Claims
1. A method for assisting in identifying drought resistance of cotton, comprising the following steps: Cotton was cultured in an incubator until it reached at least the three-leaf and one-heart stage, and then subjected to natural drought stress. When the soil moisture content dropped to 1-2%, samples were taken and the endoplasmic reticulum structure of the cotton leaves was observed using a transmission electron microscope. If a ring-shaped whorl structure appeared, the cotton was drought-resistant.
2. The method according to claim 1, characterized in that: The drought resistance of the cotton is determined based on the number of the whorl structures present: the more whorl structures there are, the worse the drought resistance is.
3. The method according to claim 1 or 2, characterized in that: The culture conditions are: Lighting 12~16h, temperature 25~28℃; Darkness 8~12h, temperature 23~25℃.
4. The method according to claim 1 or 2, characterized in that: The soil moisture content is measured by drying and weighing method.
5. The method according to claim 1 or 2, characterized in that: The sampling process is as follows: the leaves are cut into multiple leaf pieces with a length and width of 1 to 5 mm, and then placed in the fixative.
6. The method according to claim 5, characterized in that: The fixing solution is a mixture of glutaraldehyde fixing solution and potassium permanganate aqueous solution, and the pH value is 6.8-7.
2.
7. The method according to claim 1 or 2, characterized in that: The samples were observed using a Hitachi 7800 transmission electron microscope.
8. Application of endoplasmic reticulum whorl structure in assisting identification of cotton drought resistance.