A quick and efficient seedling raising method for cotton young embryo in vitro culture

Abstract

This application relates to the field of cotton tissue culture technology, and in particular to a rapid and efficient method for in vitro culture of cotton embryos for seedling formation. The method includes: pre-treatment and sterilization of detached cotton bolls for embryo extraction; induction of the embryos with indolebutyric acid (IBA) followed by inoculation in L-mAUH liquid medium with shaking culture in the dark; and finally, transfer to S-mAUH solid medium for seedling formation under light. The shaking culture and optimized medium synergistically promote nutrient absorption by the embryos. This method significantly improves the seedling formation rate and true leaf emergence rate, achieving a seedling formation rate of 89.4% in 6 days. Furthermore, the cultivated seedlings exhibit strong root development, with significantly increased root volume and surface area, effectively solving the problems of long seedling formation cycle, low seedling formation rate, and weak root system in existing technologies, thus greatly shortening the cotton breeding cycle.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, and more specifically, relates to a rapid and efficient method for in vitro culture of cotton embryos to produce seedlings. Background Technology

[0002] Cotton, belonging to the Malvaceae family and the Gossypium genus, is an important multi-purpose economic crop widely cultivated worldwide. Superior varieties are the core foundation of cotton production, while variety improvement is the key to increasing yield, quality, and resistance. To achieve the breeding goals of high yield, high quality, and multiple resistances, conventional breeding techniques such as hybridization and backcrossing, as well as molecular breeding methods such as tissue culture and transgenics, are widely used in breeding practice. However, both conventional and molecular breeding generally face the common problem of long breeding cycles, especially distant hybridization, which requires even higher time costs. Therefore, improving breeding efficiency and shortening the breeding cycle has become a key focus in cotton breeding research.

[0003] In vitro cotton embryo culture technology improves the developmental environment of young embryos through tissue culture, providing them with the nutrients necessary for growth, thereby promoting direct germination and development into healthy plants. This technology can significantly reduce the time required for cotton to develop from flowering to seed maturity, and is an effective way to shorten the cotton generation cycle and accelerate the breeding process.

[0004] Currently, commonly used culture media for cotton embryo culture include Mauney medium, BT medium, MS medium, and CEM medium. Although these media and associated culture methods have been applied to some extent in cotton breeding, limitations such as long embryo culture cycles and low seedling rates are still prevalent in practice. These shortcomings directly limit the effectiveness of embryo culture technology in accelerating cotton genetic improvement. Therefore, how to further optimize the composition of embryo culture media and improve culture methods to shorten seedling time and significantly improve seedling rate is a pressing technical problem that needs to be solved in the rapid development of the cotton industry. Summary of the Invention

[0005] To address the technical problems of long seedling formation cycle, low seedling rate, and weak root development in existing cotton embryo in vitro culture, this invention provides an in vitro culture method and a special culture medium to improve the seedling formation rate of cotton embryos.

[0006] The present invention provides an in vitro culture method for improving the seedling rate of cotton embryos, comprising the following steps: (1) Pretreatment: take cotton bolls 25-28 days after flowering and dry them in an environment with a temperature of 28±2℃ and a relative humidity of 40%~50% for 3 days; (2) Sterilization and embryo removal: after surface sterilization of the cotton bolls, cut open the carpels and remove the embryos; (3) Hormone induction: soak the embryos in an indolebutyric acid (IBA) solution with a mass concentration of 40mg / L for 2h; (4) Liquid shaking culture: inoculate the induced embryos into sterile liquid culture medium L-mAUH and shake them in the dark at 45r / min at 28℃ for 12h; (5) Solid culture for seedling formation: transfer the shaken embryos to sterile solid culture medium S-mAUH and place them in a light culture room for alternating light culture until seedlings are formed.

[0007] The cultivation conditions in the light culture chamber in step (5) are as follows: an 11-hour light / 13-hour dark cycle is adopted; the temperature during the light stage is controlled at 28±2℃, the light intensity is 1500-2500 lux, and the relative humidity is 40%~50%; the temperature during the dark stage is controlled at 25±2℃, and the relative humidity is 40%-50%.

[0008] The sterile liquid culture medium L-mAUH described in this invention comprises the following components and contents: potassium nitrate 950.0 mg / L, ammonium nitrate 825.0 mg / L, calcium chloride 220.0 mg / L, magnesium sulfate 185.0 mg / L, potassium dihydrogen phosphate 85.0 mg / L, disodium ethylenediaminetetraacetate 37.25 mg / L, ferrous sulfate 27.8 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, boron... The following are the concentrations of the following substances: acid 6.2 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, inositol 100.0 mg / L, nicotinic acid 1.0 mg / L, thiamine hydrochloride 10.0 mg / L, pyridoxine hydrochloride 1.0 mg / L, indolebutyric acid 0.1 mg / L, 6-benzylaminopurine 0.1 mg / L, and sucrose 20000.0 mg / L.

[0009] The sterile solid culture medium S-mAUH of the present invention is based on the components of the sterile liquid culture medium L-mAUH, with the addition of 3500.0 mg / L of agar.

[0010] In summary, this application includes at least one of the following beneficial technical effects: 1. Significantly improves seedling rate and true leaf emergence rate: Through the method of combining hormone induction and liquid shaking culture described in this invention, the average seedling rate of cotton embryos after 6 days of culture can reach 89.4%, which is about 41.9 percentage points higher than that of conventional AUH medium; the average true leaf emergence rate after 12 days of culture reaches 71.6%, which is 38.2 percentage points higher than that of conventional medium.

[0011] 2. Promotes root development: This invention promotes nutrient absorption through shaking culture in L-mAUH medium, resulting in seedling root development indicators that are significantly better than those of conventional culture. Specifically, the total root length of seedlings increased by 259.55%, root volume increased by 200.00%, and root surface area increased by 151.09% compared to conventional culture.

[0012] 3. Shorten the breeding cycle: This method optimizes the culture medium composition and shaking culture parameters, enabling the cotyledons to unfold and the roots to elongate by 0.5-1 cm within 12 hours after inoculation. This effectively saves the time for cotton seed maturation and dormancy, providing an efficient technical path to accelerate the cotton breeding process. Attached Figure Description

[0013] Figure 1 A flowchart of an in vitro culture method for improving the seedling rate of cotton embryos provided by the present invention; Figure 2 This is a schematic diagram of the growth state of cotton embryos after liquid shaking culture in this invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, a rapid and efficient method for in vitro culture of cotton embryos for seedling formation is described in detail below with reference to specific embodiments and experimental data. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0015] The present invention provides an in vitro culture method for improving the seedling rate of cotton embryos. Its core logic lies in the physical environment induction of pretreatment, combined with precise hormone pretreatment and liquid shaking culture environment, to synergistically optimize nutrient composition, thereby solving the problems of long seedling cycle and poor root vitality in the existing technology.

[0016] In a preferred embodiment of the present invention, the specific operation steps of the in vitro culture method are as follows: Step 1, Pretreatment. Select cotton plants after flowering. The cotton bolls were harvested. The harvested bolls were then placed in an artificial climate chamber for drying. Environmental parameters were set as follows: temperature controlled at... Relative humidity controlled at The drying process lasts for a continuous period of time. This step, by simulating the natural physiological maturation and dehydration process of cotton bolls, initially activates the germination potential of the young embryos.

[0017] Step two, sterilization and boll removal. The dried cotton bolls are first treated with a volume percentage concentration of... Wipe the surface with an ethanol solution, then soak it in an ethanol solution with a mass percentage concentration of [missing information]. Sterilization in mercuric chloride solution Finally, rinse with sterile water. Next, on a clean bench, the cotton boll carpel is cut open with a scalpel, and the embryo is carefully peeled out, avoiding mechanical damage.

[0018] Step 3, Hormone Induction. Prepare hormones with a mass concentration of... A solution of indolebutyric acid (IBA) was used. The extracted embryos were completely immersed in this solution for treatment. IBA pre-induction can directly act on the root primordia cells of immature embryos, laying the physiological foundation for subsequent rapid root development.

[0019] Step 4, Liquid Shaking Culture. The induced embryos are inoculated into Erlenmeyer flasks containing sterile liquid culture medium L-mAUH. The Erlenmeyer flasks are placed in a full-temperature shaking incubator. Under the condition, the set speed is Dark shaking culture was performed for 12 hours. During this process, the embryos were in full contact with the culture medium, and their nutrient absorption efficiency was evaluated using the following formula for root water and nutrient flux: in, This represents the water absorption rate per unit surface area of ​​the embryo. The hydraulic conductivity coefficient of the cell wall. Due to the difference between internal and external pressure, The reflection coefficient, This is due to the osmotic pressure difference. Shaking cultivation effectively maintained a high level of... This accelerates the pumping of nutrients.

[0020] Step 5: Solid-state culture for seedling formation. The immature embryos after shaking culture are transferred to sterile S-mAUH solid culture medium. They are then placed in a light-alternating culture chamber for alternating light and dark culture until seedlings emerge and true leaves appear. The light-alternating cycle in the culture chamber is set as follows: 11 hours of light followed by 13 hours of darkness. Environmental parameters during the light stage: temperature... Light intensity is 1500-2500 lux, relative humidity is Dark phase environmental parameters: temperature relative humidity .

[0021] The specific component ratios of the sterile liquid culture medium L-mAUH used in this invention are shown in Table 1.

[0022] Table 1. Components and content of L-mAUH culture medium The sterile solid culture medium S-mAUH is based on the L-mAUH components mentioned above, with additional additives. Agar is used as a solidifying agent to adjust to Then it undergoes high-pressure sterilization.

[0023] To verify the technical effect of the present invention, a control group (using conventional AUH culture medium and conventional solid inoculation method) was set up in this embodiment, and the seedling rate was compared. and true leaf growth rate Statistical calculations were performed. The calculation formula is as follows: in, For the number of seedlings, The number of individuals that have grown true leaves. This represents the total number of vaccinations.

[0024] Experimental results show that in the culture Subsequently, the average seedling survival rate of the method of the present invention reached The control group only ;nourish Subsequently, the true leaf growth rate of the method of the present invention reached... It was significantly better than the control group.

[0025] In terms of root development evaluation, root volume was measured and calculated using a root scanner. : in, The total length of the root. This represents the cross-sectional area. Test data is recorded in Table 2.

[0026] Table 2 Comparison of seedling root development indicators In summary, this invention achieves a significant improvement in the efficiency of cotton embryo in vitro culture by precisely controlling the pretreatment of cotton embryos, hormone induction, and unique liquid shaking culture process, combined with optimized L-mAUH medium components. This method not only significantly shortens the generation cycle of cotton breeding but also improves the transplant survival rate of in vitro seedlings by strengthening root development, demonstrating extremely high scientific research and application value.

[0027] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects.

[0028] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rapid and efficient method for in vitro culture of cotton embryos to promote seedling emergence, characterized in that, Includes the following steps: (1) Pretreatment: Take flowers after flowering The young cotton bolls were placed at a temperature of Relative humidity is Drying in the environment (2) Sterilization and embryo removal: After surface sterilization of the cotton bolls, the carpels of the cotton bolls are cut open to remove the embryos; (3) Hormone induction: The embryos are immersed in a solution with a mass concentration of Treatment in indolebutyric acid solution (4) Liquid shaking culture: The induced embryos were inoculated into sterile liquid culture medium L-mAUH, and in Under the condition of The cells were cultured under dark vibration at a rotation speed for a duration of [time missing]. (5) Solid culture seedlings: After shaking culture, the embryos are transferred to sterile solid culture medium S-mAUH and placed in a light culture room for alternating light culture until seedlings are formed.

2. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, The specific process of surface sterilization in step (2) is as follows: the dried cotton bolls are first sterilized with a volume percentage concentration of... Wipe the surface with an ethanol solution, then soak it in an ethanol solution with a mass percentage concentration of [missing information]. Sterilization in mercuric chloride solution Finally, rinse with sterile water. Second-rate.

3. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, During the liquid shaking culture process described in step (4), the nutrient absorption efficiency of the embryo is evaluated using the following formula: in, This represents the water absorption rate per unit surface area of ​​the embryo. The hydraulic conductivity coefficient of the cell wall. Due to the difference between internal and external pressure, The reflection coefficient, This refers to the osmotic pressure difference.

4. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, The conditions for alternating light culture in step (5) are as follows: using illumination / The cycle of darkness; the temperature during the light phase is The light intensity is The relative humidity is The temperature during the dark phase is The relative humidity is .

5. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, The components and contents of the sterile liquid culture medium L-mAUH are as follows: potassium nitrate ammonium nitrate Calcium chloride Magnesium sulfate Potassium dihydrogen phosphate Disodium ethylenediaminetetraacetate ferrous sulfate manganese sulfate Zinc sulfate Boric acid Potassium iodide Sodium molybdate Copper sulfate Cobalt chloride Inositol ,niacin Thiamine hydrochloride pyridoxine hydrochloride Indolebutyric acid 6-Benzylaminopurine and sucrose .

6. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 5, characterized in that, The sterile solid culture medium S-mAUH is based on the components of the sterile liquid culture medium L-mAUH, with additional additives. agar, and adjust to .

7. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, The seedling root development index after seedling establishment is measured by root volume. The evaluation is conducted using the following formula: in, The total length of the root. It represents the cross-sectional area.

8. The method for rapid and efficient in vitro culture of cotton embryos for seedling formation according to claim 1, characterized in that, The seedling survival rate of the method The calculation method is as follows: in, For the number of seedlings, Total number of inoculations; culture The average seedling survival rate after that was .

9. A special culture medium for the in vitro culture method according to claim 1, characterized in that, It includes sterile liquid culture medium L-mAUH and sterile solid culture medium S-mAUH; the components of the sterile liquid culture medium L-mAUH include: potassium nitrate. ammonium nitrate Calcium chloride Magnesium sulfate Potassium dihydrogen phosphate Disodium ethylenediaminetetraacetate ferrous sulfate manganese sulfate Zinc sulfate Boric acid Potassium iodide Sodium molybdate Copper sulfate Cobalt chloride Inositol ,niacin Thiamine hydrochloride pyridoxine hydrochloride Indolebutyric acid 6-Benzylaminopurine and sucrose The sterile solid culture medium S-mAUH, in addition to the components of the sterile liquid culture medium L-mAUH, contains... Agar.

10. The special culture medium according to claim 9, characterized in that, The sterile solid culture medium S-mAUH for .

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