Method for screening drought-resistant gene transgenic cotton

Abstract

The invention discloses a method for screening drought-resistant gene transgenic cotton in the technical field of biology, which comprises the following steps: step 1, setting an experimental group and a control group, the experimental group being a plurality of transgenic cotton seeds to be tested, and the control group being a plurality of common cotton seeds; 2, cotton physiological water demand is calculated, and a plurality of irrigation gradients are set; 3, determining the germination potential and germination rate of the cotton seeds of the experimental group and the control group, and transplanting normally germinated cotton seedlings; 4, irrigation control pots are prepared, cotton seedlings of the experimental group and the control group are planted in the irrigation control pots placed in the greenhouse, illumination and temperature are controlled to be consistent, and cotton plants of the experimental group and the control group are irrigated according to the irrigation gradient; 5, counting the survival rate of the cotton plants of the experimental group and the control group under different irrigation gradients, and controlling the boll setting number in the quality comparison group to be consistent when the cotton grows to the flowering and boll setting period; 6, picking the cotton, and detecting and comparing the yield and quality characters.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening drought-resistant gene-transformed cotton. Background Art

[0002] Drought has become the main cause of plant yield reduction and the main stress suffered by plants during their growth and development. Drought has many effects on plants, including water deficit in plants and imbalance in water metabolism. It may cause plant cells to dehydrate, the hydration of protoplasm to decrease, the dispersion of colloid particles to decrease, the protoplasm to change from sol state to gel state, and the physiological function of protoplasm to be destroyed; under drought conditions, the stomata of plant leaves may be closed to reduce water evaporation. However, this will also lead to a reduction in the supply of carbon dioxide required for photosynthesis, thereby reducing the efficiency of photosynthesis. At the same time, drought may also inhibit the formation of chlorophyll, further affecting the progress of photosynthesis; under drought conditions, the respiration of plants may be enhanced to decompose more organic matter to provide energy. This will lead to an increase in the consumption of organic matter in plants, which will in turn affect their growth and development, so it is particularly important to enhance the drought resistance of crops.

[0003] Scientists use genetic engineering technology to introduce drought-resistant genes (such as bZIP transcription factors and NAC transcription factors) into cotton. After these genes are expressed in cotton, they can regulate the gene expression of plants under adverse conditions such as drought, thereby improving the drought resistance of plants. For example, the differential expression of bZIP transcription factors may play an important role in cotton's response to drought stress, while NAC transcription factors are involved in the expression of response genes such as drought and high salt, and play an important role in plant resistance to abiotic stresses such as drought and high salt. However, after the target gene is introduced, the harvested transgenic cotton seeds need to undergo costly genetic engineering screening, and these screenings can hardly guarantee the drought resistance of transgenic cotton, let alone determine whether growth, development, yield, and quality traits have changed.

[0004] Therefore, it is necessary to propose a method for screening transgenic cotton with drought-resistant genes that can accurately screen and verify the drought resistance of transgenic cotton and identify the yield and quality traits of transgenic cotton. Summary of the invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a method for screening transgenic drought-resistant cotton, which accurately identifies the drought resistance of transgenic cotton by synchronous germination rate determination and irrigation gradient experiment with ordinary cotton, and compares the yield and quality traits of cotton with different irrigation gradients and combinations after fruiting, and identifies the actual impact of transgenic drought-resistant genes on cotton growth and development under drought conditions. This method can obtain more intuitive and systematic screening and identification results, and can be used as a screening and evaluation method for whether transgenic drought-resistant cotton is suitable for actual production.

[0006] In order to achieve the above object, the technical scheme of the present invention is as follows: a method for screening transgenic drought-resistant cotton, step 1: setting an experimental group and a control group, the experimental group is a number of transgenic cotton seeds to be tested, and the control group is a number of ordinary cotton seeds;

[0007] Step 2: Calculate the single physiological water requirement of cotton, use the calculated standard physiological water requirement as the intermediate value, and set several irrigation gradients;

[0008] Step 3: Determine the germination potential and germination rate of cotton seeds in the experimental group and the control group, and transplant the cotton seedlings that germinate normally;

[0009] Step 4: preparing irrigation control pots, which are used to plant cotton and quantitatively control the irrigation amount of cotton in the pots. The irrigation control pots are placed in a greenhouse, and cotton seedlings of the experimental group and the control group are planted in the irrigation control pots. The light and temperature are controlled to be consistent, and the cotton plants of the experimental group and the control group are irrigated according to the irrigation gradient;

[0010] Step 5: Count the survival rates of cotton plants in the experimental group and the control group under different irrigation gradients, randomly divide the surviving cotton plants in the experimental group and the control group into a yield comparison group and a quality comparison group, and control the boll number in the quality comparison group to be consistent when the cotton grows to the flowering and boll stage;

[0011] Step 6: Pick the cotton from the yield comparison group and the quality comparison group under different irrigation gradients to test and compare the yield and quality traits.

[0012] The principle of the basic plan is: to identify the transformation efficiency and drought resistance of transgenic cotton by measuring the germination rate and survival rate under different irrigation gradients, to ensure that the flower buds under drought conditions are fully developed by controlling the number of bolls, and to judge the potential impact of drought genes on cotton production quality and the feasibility of their application in actual production through cotton quality testing and comparison.

[0013] The beneficial effects of the basic scheme are: 1. By setting up experimental and control groups and multiple irrigation gradients, this method can scientifically evaluate the growth performance and drought resistance of transgenic cotton under different water conditions. At the same time, by accurately measuring germination potential and germination rate, survival rate and cotton quality, the accuracy and reliability of the experimental results are ensured.

[0014] 2. This method not only focuses on the drought resistance of transgenic cotton, but also examines the fiber quality of cotton under drought conditions by controlling the number of bolls, eliminating the impact of the difference in the number of bolls per plant on fiber quality under different irrigation gradient conditions, and assesses the potential impact of drought genes on cotton production quality by detecting cotton quality traits. This comprehensive investigation helps to gain a deeper understanding of the adaptability and production performance of transgenic cotton under different environmental conditions.

[0015] 3. This method is carried out in a greenhouse, which can better simulate the actual production environment and control conditions such as light and temperature, making the experimental results more valuable for practical application. By comparing the yield and quality traits of cotton under different irrigation gradients, it can provide a scientific basis for the planting and irrigation management of transgenic cotton and guide agricultural production practices.

[0016] 4. This method innovatively evaluates the impact of drought genes on cotton yield and quality traits, as well as the feasibility of application in actual production, by introducing transgenic technology and setting irrigation gradients. This innovative research method helps promote the progress and development of agricultural science and technology, and provides new ideas and technical support for the sustainable development of the cotton industry.

[0017] 5. The results of this method not only help to comprehensively evaluate the drought resistance of transgenic cotton, but also provide reference for the research and application of drought resistance genes in other crops. At the same time, this program also emphasizes the importance of irrigation management in agricultural production and provides a scientific basis for optimizing irrigation strategies and improving water resource utilization efficiency.

[0018] Furthermore, the calculation of the single physiological water requirement of cotton in step 2 uses the following formula:

[0019]

[0020] Where ET0 is the reference evaporation rate, R n is the net radiation of the crop surface, G is the soil heat flux, T is the daily average temperature, u2 is the wind speed, Δ is the slope of the saturated water vapor pressure curve, γ is the humidity constant, and e s is the saturated water vapor pressure, e a is the actual water vapor pressure.

[0021] The actual crop water requirement is derived from the following formula:

[0022] ET=K c ET0

[0023] Where ET is the actual evaporation, K c is the crop coefficient.

[0024] The beneficial effect of the basic scheme is that by calculating the single physiological water requirement of cotton (i.e. the actual crop evapotranspiration ET), the irrigation needs of cotton can be determined more accurately. This helps to avoid over-irrigation and under-watering problems, thereby improving the accuracy of irrigation gradients.

[0025] Furthermore, in step three, a germination test method is used to determine the germination potential and germination rate of cotton seeds.

[0026] The beneficial effects of the basic scheme are: the germination test method can directly and accurately evaluate the quality of cotton seeds. By measuring the germination potential and germination rate, the germination ability and speed of seeds under suitable conditions can be understood. The germination potential reflects the germination of seeds in a short period of time, while the germination rate reflects the germination potential of seeds in a longer period of time. These two indicators together constitute an important basis for evaluating seed quality. At the same time, a germination moisture gradient experiment can also be set up to test the drought resistance of transgenic cotton seeds during the germination period.

[0027] Furthermore, in step 4, fluorescent lamps and thermostats are provided in the greenhouse, wherein the fluorescent lamps are used to supplement the light in the greenhouse, and the thermostats are used to control the temperature in the greenhouse.

[0028] The beneficial effect of the basic scheme is that the fluorescent lamps and thermostats in the greenhouse can optimize the growth environment and provide a more comfortable and stable environment for the growth of cotton plants. This optimized production environment is conducive to the growth and development of cotton plants, reduces the interference and influence of the external environment on the plants, and thus improves the stability and accuracy of the screening experiment.

[0029] Furthermore, the irrigation control basin in step 4 includes a basin body, a plurality of through holes are opened at the bottom of the basin body, the through holes are covered with a leak-proof net, a lower annular track member is fixedly connected to the upper extension of the basin body, the lower annular track member is connected to a liquid supply pipe, an upper annular track member is slidably connected to the top of the lower annular track member, an annular cavity is formed between the lower annular track member and the upper annular track member, a plurality of nozzles are connected to the upper annular track member, and a baffle is fixedly connected to one side of the nozzle head, and the baffle faces the inside of the basin body.

[0030] The beneficial effects of the basic scheme are: 1. A plurality of nozzles connected by the upper annular track member can achieve uniform spraying of water, ensuring that the bottom of the cotton plant in the pot body obtains sufficient water.

[0031] 2. The baffle fixedly connected on one side of the nozzle head can be directed toward the inside of the basin to further control the direction and range of water spraying, ensuring that the water can be accurately sprayed onto the roots and leaves of the cotton plants.

[0032] 3. The water flow sprayed from the sprinkler hits the baffle and the reaction force of the water spray can drive the bumper and the upper annular track to rotate on the lower annular track, making irrigation more uniform and automated.

[0033] Furthermore, in step five, the bud removal and flower thinning method is used to control the number of bolls per cotton plant in the quality comparison group to be no more than 10.

[0034] The beneficial effects of the basic scheme are: 1. During the growth process, cotton plants consume a lot of nutrients to support the development of flower buds. By controlling the number of bolls by removing the buds and thinning the flowers, the plant can be prevented from producing too many flower buds, thereby reducing unnecessary nutrient consumption under drought stress conditions. This allows the plant to concentrate more nutrients on the remaining strong flower buds and fruits, improve the quality of cotton and the yield of single boll, lay a good foundation for the subsequent detection and comparison of cotton quality traits, and improve the accuracy and reliability of the experiment.

[0035] 2. Too many flower buds will make the branches and leaves of cotton too dense, affecting the ventilation and light conditions inside the plant. Through the bud thinning method, some flower buds and branches and leaves can be removed to improve the ventilation and light conditions inside the plant. This is conducive to reducing the occurrence of diseases and pests, improving the resistance of the plant, and is also conducive to the coloring and ripening of the fruit.

[0036] Furthermore, in step six, the detection of the yield and quality of the harvested cotton includes single boll weight, total boll weight per plant, lint percentage, fiber length, fiber strength, micronaire value and fiber uniformity.

[0037] The beneficial effect of the basic program is: through the detection and comparison of various cotton yield and quality traits, the impact of drought-resistant genes on the yield and quality of transgenic cotton under the premise of drought resistance is evaluated, so as to evaluate the feasibility of applying transgenic cotton in actual production.

[0038] Furthermore, the comparison of cotton yield and quality traits in step six includes comparing cotton yield and quality traits under different irrigation gradients and the difference in cotton yield and quality traits between the experimental group and the control group under the same irrigation gradient.

[0039] The beneficial effects of the basic scheme are: 1. By comparing the cotton yield and quality shape under different irrigation gradients, the impact of irrigation amount on key quality indicators such as cotton boll weight, lint percentage, fiber length, fiber strength, micronaire value and fiber uniformity can be intuitively demonstrated. This helps to understand how drought conditions affect the growth and development of cotton and how these effects translate into differences in cotton quality.

[0040] 2. The performance of the experimental group (transgenic cotton) under different irrigation gradients can reflect the expression efficiency of drought genes in cotton plants. If the experimental group shows higher survival rate, higher yield and better quality under drought conditions, then this can be regarded as evidence of effective expression of drought genes.

[0041] 3. Comparing the cotton quality of the experimental group and the control group under the same irrigation gradient can verify the effect of drought genes in actual production. If the experimental group shows better cotton yield or quality under the same irrigation conditions, then this can prove that drought genes are feasible in actual production.

[0042] Furthermore, fluorescent lamps supplement the greenhouse lighting to between 8000Lux and 70000Lux.

[0043] The beneficial effect of the basic solution is that the natural light in the greenhouse may be uneven, and the addition of fluorescent lamps can significantly improve this problem. By properly arranging the position and number of fluorescent lamps, the light in the greenhouse can be made more uniform, providing a more stable light environment for plant growth.

[0044] Furthermore, the thermostat controls the greenhouse temperature to remain between 26°C and 32°C.

[0045] The beneficial effects of the basic scheme are: ensuring the stability of the temperature in the greenhouse to ensure the rapid growth of each group of cotton plants, shortening the experimental cycle, improving experimental efficiency, reducing the impact of temperature changes on the growth of experimental plants, reducing errors within the experiment, and improving the accuracy of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a schematic diagram of the method for screening transgenic drought-resistant cotton in an embodiment of the present invention.

[0047] Figure 2 It is an axonometric view of an irrigation control basin in an embodiment of the present invention.

[0048] Figure 3 It is a front cross-sectional view of the irrigation control basin in the embodiment of the present invention.

[0049] The figure marks in the drawings of the specification include: 1. basin body; 2. through hole; 3. anti-leakage net; 4. liquid supply pipe; 5. lower annular track member; 6. upper annular track member; 7. nozzle; 8. baffle. DETAILED DESCRIPTION

[0050] The following is further described in detail through specific implementation methods:

[0051] Example 1

[0052] Basically as attached Figure 1 Shown: A method for screening transgenic drought-resistant cotton.

[0053] Step 1: Set up an experimental group and a control group, the experimental group is a number of genetically modified cotton seeds to be tested, and the control group is a number of ordinary cotton seeds;

[0054] Step 2: Calculate the single physiological water requirement of cotton. Use the calculated standard physiological water requirement as the intermediate value and set several irrigation gradients. The calculation of the single physiological water requirement of cotton uses the following formula:

[0055]

[0056] Where ET0 is the reference evaporation rate, R n is the net radiation of the crop surface, G is the soil heat flux, T is the daily average temperature, u2 is the wind speed, Δ is the slope of the saturated water vapor pressure curve, γ is the humidity constant, and e s is the saturated water vapor pressure, e a is the actual water vapor pressure.

[0057] The actual crop water requirement is derived from the following formula:

[0058] ET=K c ET0

[0059] Where ET is the actual evaporation, K c The crop coefficient is generally between 0.82-1.06;

[0060] Step 3: Determine the germination potential and germination rate of cotton seeds in the experimental group and the control group. Take cotton seeds that have been soaked and absorbed enough water, maintain the temperature at 25°C-30°C, the percentage of germination on the third day is the germination potential, and the percentage of germination on the ninth day is the germination rate, and transplant the cotton seedlings that germinate normally;

[0061] Step 4: Prepare irrigation control pots, which are used to plant cotton and quantitatively control the irrigation amount of cotton in the pots. The irrigation control pots are placed in a greenhouse, and cotton seedlings of the experimental group and the control group are planted in the irrigation control pots. The light and temperature are controlled to be consistent. Fluorescent lamps are used to supplement the greenhouse light to between 8000 Lux and 70000 Lux. The air conditioner controls the greenhouse temperature to be between 26°C and 32°C, and the cotton plants of the experimental group and the control group are irrigated according to the irrigation gradient.

[0062] Step 5: Count the survival rates of cotton plants in the experimental group and the control group under different irrigation gradients, and randomly divide the surviving cotton plants in the experimental group and the control group into a yield comparison group and a quality comparison group. When the cotton grows to the flowering and boll stage, the number of bolls in the quality comparison group is controlled to be consistent and not more than 10;

[0063] Step 6: Pick cotton from the yield comparison group and the quality comparison group under different irrigation gradients for yield and quality shape detection and comparison, including single boll weight, number of bolls per plant, lint percentage, fiber length, fiber strength, micronaire value and fiber uniformity detection.

[0064] By setting up experimental and control groups and multiple irrigation gradients, this method can scientifically evaluate the growth performance and drought resistance of transgenic cotton under different water conditions. The accuracy and reliability of the experimental results are ensured by accurately measuring the germination potential and germination rate, survival rate and cotton quality. This method is carried out in a greenhouse, which can better simulate the actual production environment and control conditions such as light and temperature, making the experimental results more valuable for practical application. By dividing the surviving experimental and control groups into yield comparison groups and quality comparison groups and controlling the number of bolls per plant in the quality comparison group, the potential impact of excessive bolls on cotton quality traits under drought stress conditions is eliminated. By comparing the growth and development of cotton and the fruit yield and quality traits under different irrigation gradients, a scientific basis can be provided for the planting and irrigation management of transgenic cotton, guiding agricultural production practices. By introducing transgenic technology and setting irrigation gradients, this method innovatively evaluates the impact of drought genes on cotton production quality and the feasibility of application in actual production. The results of this method not only help to comprehensively evaluate the drought resistance (drought resistance, yield, quality) of transgenic cotton, but also provide reference and reference for the research and application of drought resistance genes in other crops.

[0065] Example 2

[0066] The difference from the above embodiment is that, as shown in the attached Figure 2 , Figure 3 As shown: the irrigation control basin in step 4 includes a basin body 1, a plurality of through holes 2 are opened at the bottom of the basin body 1, and the through holes 2 are covered with anti-leakage nets 3, which are used to prevent soil erosion, a lower annular track member 5 is welded on the upper extension of the basin body 1, and the lower annular track member 5 is connected to a liquid supply pipe 4, and an upper annular track member 6 is slidably connected to the top of the lower annular track member 5, and an annular cavity is formed between the lower annular track member 5 and the upper annular track member 6, and the upper annular track member 6 is connected to a plurality of nozzles 7, and a baffle 8 is welded on one side of the head of the nozzle 7, and the baffle 8 faces the inside of the basin body 1.

[0067] The specific implementation process is as follows: when irrigation is needed, the liquid enters the annular cavity formed between the lower annular track member 5 and the upper annular track member 6 along the liquid supply pipe 4, and is supplied to the nozzle 7 for spraying. The liquid flow sprayed by the nozzle 7 hits the baffle 8, flows into the soil and the root position of the plant in the basin body 1, and drives the nozzle 7 and the upper annular track member 6 to rotate under the influence of the reaction force, so that the sprayed liquid flow is more evenly sprinkled in the basin body 1, thereby improving the uniformity of irrigation.

[0068] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0069] The above is only an embodiment of the present invention. The common sense such as the known specific structure and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for screening drought-resistant gene-transformed cotton, characterized in that: Step 1: Set up an experimental group and a control group, the experimental group is a number of genetically modified cotton seeds to be tested, and the control group is a number of ordinary cotton seeds; Step 2: Calculate the single physiological water requirement of cotton, use the calculated standard physiological water requirement as the intermediate value, and set several irrigation gradients; Step 3: Determine the germination potential and germination rate of cotton seeds in the experimental group and the control group, and transplant the cotton seedlings that germinate normally; Step 4: preparing irrigation control pots, which are used to plant cotton and quantitatively control the irrigation amount of cotton in the pots. The irrigation control pots are placed in a greenhouse, and cotton seedlings of the experimental group and the control group are planted in the irrigation control pots. The light and temperature are controlled to be consistent, and the cotton plants of the experimental group and the control group are irrigated according to the irrigation gradient; Step 5: Count the survival rates of cotton plants in the experimental group and the control group under different irrigation gradients, randomly divide the surviving cotton plants in the experimental group and the control group into a yield comparison group and a quality comparison group, and control the boll numbers of the experimental group and the control group in the quality comparison group to be the same when the cotton grows to the flowering and boll stage; Step 6: Pick the cotton from the yield comparison group and the quality comparison group under different irrigation gradients to test and compare the yield and quality traits.

2. The method for screening drought-resistant transgenic cotton according to claim 1, characterized in that: The calculation of cotton single physiological water requirement in step 2 uses the following formula: Where ET0 is the reference evaporation rate, R n is the net radiation of the crop surface, G is the soil heat flux, T is the daily average temperature, u2 is the wind speed, Δ is the slope of the saturated water vapor pressure curve, γ is the humidity constant, and e s is the saturated water vapor pressure, e a is the actual water vapor pressure. The actual crop water requirement is derived from the following formula: ET=K c ET0 Where ET is the actual evaporation, K c is the crop coefficient.

3. The method for screening drought-resistant transgenic cotton according to claim 2, characterized in that: In step three, the germination potential and germination rate of cotton seeds are determined using a germination test method.

4. The method for screening drought-resistant transgenic cotton according to claim 3, characterized in that: In step 4, fluorescent lamps and thermostats are installed in the greenhouse. The fluorescent lamps are used to supplement the light in the greenhouse, and the thermostat is used to control the temperature in the greenhouse.

5. The method for screening drought-resistant transgenic cotton according to claim 4, characterized in that: The irrigation control basin in step 4 comprises a basin body (1), a plurality of through holes (2) are formed at the bottom of the basin body (1), and the through holes (2) are all covered with anti-leakage nets (3). A lower annular track member (5) is fixedly connected to the upper extension of the basin body (1), and the lower annular track member (5) is connected to a liquid supply pipe (4). The top of the lower annular track member (5) is slidably connected to an upper annular track member (6), and an annular cavity is formed between the lower annular track member (5) and the upper annular track member (6). The upper annular track member (6) is connected to a plurality of nozzles (7), and a baffle (8) is fixedly connected to one side of the head of the nozzle (7), and the baffle (8) faces the inside of the basin body (1).

6. The method for screening drought-resistant transgenic cotton according to claim 5, characterized in that: In step 5, the bud removal and flower thinning method is used to control the number of bolls per cotton plant in the quality comparison group to be no more than 10.

7. The method for screening drought-resistant transgenic cotton according to claim 6, characterized in that: In step six, the yield and quality traits of the harvested cotton are tested, including single boll weight, number of bolls per plant, lint percentage, fiber length, fiber strength, micronaire value and fiber uniformity.

8. The method for screening drought-resistant transgenic cotton according to claim 7, characterized in that: The comparison of cotton yield and quality traits in step six includes comparing cotton yield and quality traits under different irrigation gradients and the difference in cotton yield and quality traits between the experimental group and the control group under the same irrigation gradient.

9. The method for screening drought-resistant transgenic cotton according to claim 8, characterized in that: Fluorescent lamps supplement greenhouse lighting to between 8000Lux and 70000Lux.

10. The method for screening drought-resistant transgenic cotton according to claim 9, characterized in that: The thermostat controls the greenhouse temperature to remain between 26℃-32℃.

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