Transgenic wheat molecular breeding method

Abstract

The invention discloses a transgenic wheat molecular breeding method, and relates to the technical field of wheat breeding, and the method comprises the following steps: selecting a flat land area which can be irradiated by sunlight for a long time in the daytime, turning the soil, then removing straws, weeds, stones and other sundries in the soil, finally applying a base fertilizer, deeply ploughing the land parcel, and finally applying the base fertilizer to the land parcel. Carrying out rotary grinding and crushing on the soil; the method comprises the following steps: firstly, selecting complete and excellent wheat seeds which grow for more than two years in the field, then treating the wheat seeds, hybridizing the treated wheat seeds with high-quality and high-yield wheat varieties to obtain first-generation wheat seeds, planting the first-generation wheat seeds to obtain second-generation wheat, editing target DNA fragments in receptor wheat genomes by using a gene editing method according to second-generation wheat hybrid seeds, and screening the second-generation wheat hybrid seeds. Gene edited wheat with changed target DNA fragments is obtained, target wheat with the grain hardness increased compared with that of receptor wheat is screened from the gene edited wheat, and therefore wheat grains with the moderate hardness can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of wheat breeding, in particular to a molecular breeding method for transgenic wheat. Background Art

[0002] Wheat caryopsis is one of the staple foods of human beings. After being ground into flour, it can be used to make bread, steamed buns, biscuits, noodles and other foods; after fermentation, it can be made into beer, alcohol, liquor, or biofuel. Therefore, it can be said that wheat is an indispensable substance in people's daily life. Wheat is a general term for wheat-related plants. It is a monocotyledonous plant and a grass plant that is widely planted in my country and even in the world. With the continuous development of science and technology and the continuous reform of agriculture, wheat varieties and planting methods are constantly innovating, but the existing genetically modified wheat still has some shortcomings. For example, the hardness of its wheat grains is not enough, which leads to certain effects when it is subsequently processed into other commodities. At the same time, the existing genetically modified wheat contains less selenium-rich elements and cannot provide the human body with an appropriate amount of selenium-rich elements. For this reason, we propose a molecular breeding method for genetically modified wheat. Summary of the invention

[0003] The purpose of the present invention is to provide a transgenic wheat molecular breeding method to solve the problems raised in the above background technology.

[0004] A molecular breeding method for transgenic wheat comprises the following steps: Step 1: Selection and preparation of planting site: Select a flat area with sufficient sunlight during the day, turn the soil, remove straw, weeds, stones and other debris from the soil, apply base fertilizer, plough the land, grind the soil, make ridges on the leveled land, form drainage ditches between ridges, and dig ditches in the ridges; Step 2: Processing of wheat seeds: First, select wheat seeds that have grown in the wild for more than two years and are intact and good, rinse them with water, put them in a container after natural air drying, and then soak and clean them in a sterile environment. The obtained wheat seeds are reserved for use and used as wheat female parents. Select high-quality and high-yield varieties suitable for local planting, resistant to pests and diseases, weak tillering, resistant to lodging, large ears, and high fruiting rate as wheat male parents, and hybridize the wheat male parent and the wheat female parent to obtain hybrid combination first-generation wheat seeds; Step 3: placing the first generation wheat seeds in an induction medium, maintaining the culture temperature between 23° C. and 27° C., the relative humidity between 80% and 90%, the light intensity between 1700 and 3100 Lux, and the light duration at 7 hours per day for 55 to 60 days, thereby obtaining young wheat seedlings; Step 4: Plant the young wheat seedlings at a staggered spacing of 0.3-0.6m. When the wheat matures, harvest them uniformly to obtain the second-generation wheat hybrid seeds. Step 5: The second-generation wheat hybrid seeds are subjected to gene editing to edit the target DNA fragment in the genome of the recipient wheat, thereby obtaining gene-edited wheat in which the target DNA fragment is changed, and the target wheat having increased grain hardness compared with the recipient wheat is screened from the gene-edited wheat; Step 6: Plant the wheat with increased hardness in step 5, and harvest it uniformly after it matures, so as to obtain the third-generation wheat hybrid seeds; Step 7: Use the second-generation wheat hybrid seeds as the female parent and the third-generation wheat hybrid seeds as the male parent to crossbreed with the female parent to obtain hybrid wheat seeds. Then select high-quality selenium-rich wheat seeds for crossbreeding with them. After sowing and maturity, you will get selenium-rich wheat seeds with hard and high grains.

[0005] Preferably, the land described in step 1 is a test site, and the test site needs to be artificially divided into multiple areas of similar size, and a greenhouse is laid in the area of ​​the test site, and a detachable connecting frame is provided between the two sides of the greenhouse and the ground.

[0006] Preferably, in step 4 and step 6, the land is watered by spraying when wheat is planted, and the topsoil is hoeed 3 to 5 days after watering to break up the compaction of the topsoil.

[0007] Preferably, the second generation seeds are placed in a container, and alcohol with a concentration of 6%-18% is added to the container, and the seeds are soaked for 3-6 hours. After completion, the second generation seeds are taken out of the container and air-dried, and finally placed in a diethyl sulfate solution and soaked for 3-5 hours, and the concentration of the diethyl sulfate solution is 0.05-0.5 mol / L.

[0008] Preferably, the land in step one needs to be covered with a nutrient soil layer before planting wheat, and the nutrient soil layer includes feces, wood ash, and chopped grass.

[0009] Preferably, during the planting period, regular surveys are conducted to record the different properties of each test material at various stages of wheat growth. The recording periods include pre-wintering, wintering, greening and seedling stages, and the recorded properties include cold resistance, disease resistance and yield.

[0010] Preferably, the depth of the furrowing is 9-12 cm, the spacing between adjacent furrows is 15 cm, and the deep plowing depth is not less than 28 cm.

[0011] Preferably, the wheat in step 4 and step 6 is fertilized with liquid fertilizer once on the 8th to 12th day after the wheat seedlings emerge, and then fertilized with liquid fertilizer again after 6 to 10 days, with the amount of fertilizer applied each time being 10 to 16 g / m2, and conventional seedling management is performed.

[0012] To achieve the above object, the present invention provides the following technical solutions: Compared with the prior art, the beneficial effects of the present invention are as follows: the transgenic wheat molecular breeding method firstly selects wheat seeds that have grown in the wild for more than two years and are complete and excellent, and then processes the wheat seeds and hybridizes them with high-quality and high-yield wheat varieties to obtain first-generation wheat seeds, and the first-generation wheat seeds are planted to obtain second-generation wheat, and the second-generation wheat hybrid seeds are edited by a gene editing method to edit the target DNA fragment in the genome of the recipient wheat to obtain gene-edited wheat with the target DNA fragment changed, and the target wheat with increased grain hardness compared with the recipient wheat is screened from the gene-edited wheat, so that wheat grains with moderate hardness can be obtained; and the wheat with increased hardness is planted, harvested uniformly after maturity, so as to obtain third-generation wheat hybrid seeds, and then hybridized and bred with the wheat seeds of the second-generation wheat hybrid seeds to obtain hybrid wheat seeds, and then high-quality selenium-enriched wheat seeds are selected to hybridize with them, and after sowing and maturity, selenium-enriched wheat seeds with high wheat grain hardness are obtained. DETAILED DESCRIPTION

[0013] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0014] Example 1 A molecular breeding method for transgenic wheat comprises the following steps: Step 1: Selection and preparation of planting site: Select a flat area with sufficient sunlight during the day, turn the soil, remove straw, weeds, stones and other debris from the soil, apply base fertilizer, plough the land, grind the soil, make ridges on the leveled land, form drainage ditches between ridges, and dig ditches in the ridges; Step 2: Processing of wheat seeds: First, select wheat seeds that have grown in the wild for more than two years and are intact and good, rinse them with water, put them in a container after natural air drying, and then soak and clean them in a sterile environment. The obtained wheat seeds are reserved for use and used as wheat female parents. Select high-quality and high-yield varieties suitable for local planting, resistant to pests and diseases, weak tillering, resistant to lodging, large ears, and high fruiting rate as wheat male parents, and hybridize the wheat male parent and the wheat female parent to obtain hybrid combination first-generation wheat seeds; Step 3: placing the first generation wheat seeds in an induction medium, maintaining the culture temperature between 23° C. and 27° C., the relative humidity between 80% and 90%, the light intensity between 1700 and 3100 Lux, and the light duration at 7 hours per day for 55 to 60 days, thereby obtaining young wheat seedlings; Step 4: Plant the young wheat seedlings at a staggered spacing of 0.3-0.6m. When the wheat matures, harvest them uniformly to obtain the second-generation wheat hybrid seeds. Step 5: The second-generation wheat hybrid seeds are subjected to gene editing to edit the target DNA fragment in the genome of the recipient wheat, thereby obtaining gene-edited wheat in which the target DNA fragment is changed, and the target wheat having increased grain hardness compared with the recipient wheat is screened from the gene-edited wheat; Step six: Plant the wheat with increased hardness in step five, and harvest it uniformly after it matures to obtain the third-generation wheat hybrid seeds.

[0015] Wherein: the land described in step 1 is a test site, and the test site needs to be artificially divided into multiple areas of similar size, and a greenhouse is laid in the area of ​​the test site, and a detachable connecting frame is set between the two sides of the greenhouse and the ground; In step 4 and step 6, the plot is watered by spraying when the wheat is planted, and the topsoil is hoeed 3 to 5 days after the watering is completed, so as to break the compaction of the topsoil; Place the second generation seeds in a container, add alcohol with a concentration of 6%-18% in the container, and soak for 3-6 hours. After completion, take the second generation seeds out of the container and air dry them. Finally, soak them in a diethyl sulfate solution for 3-5 hours, and the concentration of the diethyl sulfate solution is 0.05-0.5 mol / L. The land in step 1 needs to be covered with a nutrient soil layer before planting wheat, and the nutrient soil layer includes manure, wood ash, and shredded grass; During the planting period, regular surveys and records are required to record the different traits of each test material at different stages of wheat growth, including pre-wintering, wintering, greening and seedling stages. The recorded traits include cold resistance, disease resistance and high yield. The depth of the furrows is 9-12 cm, the spacing between adjacent furrows is 15 cm, and the deep plowing depth is not less than 28 cm; The wheat described in step 4 and step 6 is fertilized with liquid fertilizer once on the 8th to 12th day after the wheat seedlings have fully emerged, and then fertilized with liquid fertilizer again after 6 to 10 days. The amount of fertilizer applied each time is 10 to 16 grams per square meter, and conventional seedling management is performed.

[0016] Example 2 A molecular breeding method for transgenic wheat comprises the following steps: Step 1: Selection and preparation of planting site: Select a flat area with sufficient sunlight during the day, turn the soil, remove straw, weeds, stones and other debris from the soil, apply base fertilizer, plough the land, grind the soil, make ridges on the leveled land, form drainage ditches between ridges, and dig ditches in the ridges; Step 2: Processing of wheat seeds: First, select wheat seeds that have grown in the wild for more than two years and are intact and good, rinse them with water, put them in a container after natural air drying, and then soak and clean them in a sterile environment. The obtained wheat seeds are reserved for use and used as wheat female parents. Select high-quality and high-yield varieties suitable for local planting, resistant to pests and diseases, weak tillering, resistant to lodging, large ears, and high fruiting rate as wheat male parents, and hybridize the wheat male parent and the wheat female parent to obtain hybrid combination first-generation wheat seeds; Step 3: placing the first generation wheat seeds in an induction medium, maintaining the culture temperature between 23° C. and 27° C., the relative humidity between 80% and 90%, the light intensity between 1700 and 3100 Lux, and the light duration at 7 hours per day for 55 to 60 days, thereby obtaining young wheat seedlings; Step 4: Plant the young wheat seedlings at a staggered spacing of 0.3-0.6m. When the wheat matures, harvest them uniformly to obtain the second-generation wheat hybrid seeds. Step 5: The second-generation wheat hybrid seeds are subjected to gene editing to edit the target DNA fragment in the genome of the recipient wheat, thereby obtaining gene-edited wheat in which the target DNA fragment is changed, and the target wheat having increased grain hardness compared with the recipient wheat is screened from the gene-edited wheat; Step 6: Plant the wheat with increased hardness in step 5, and harvest it uniformly after it matures, so as to obtain the third-generation wheat hybrid seeds; Step 7: Use the second-generation wheat hybrid seeds as the female parent and the third-generation wheat hybrid seeds as the male parent to crossbreed with the female parent to obtain hybrid wheat seeds. Then select high-quality selenium-rich wheat seeds for crossbreeding with them. After sowing and maturity, you will get selenium-rich wheat seeds with hard and high grains.

[0017] Wherein: the land described in step 1 is a test site, and the test site needs to be artificially divided into multiple areas of similar size, and a greenhouse is laid in the area of ​​the test site, and a detachable connecting frame is set between the two sides of the greenhouse and the ground; In step 4 and step 6, the plot is watered by spraying when the wheat is planted, and the topsoil is hoeed 3 to 5 days after the watering is completed, so as to break the compaction of the topsoil; Place the second generation seeds in a container, add alcohol with a concentration of 6%-18% in the container, and soak for 3-6 hours. After completion, take the second generation seeds out of the container and air dry them. Finally, soak them in a diethyl sulfate solution for 3-5 hours, and the concentration of the diethyl sulfate solution is 0.05-0.5 mol / L. The land in step 1 needs to be covered with a nutrient soil layer before planting wheat, and the nutrient soil layer includes manure, wood ash, and shredded grass; During the planting period, regular surveys and records are required to record the different traits of each test material at different stages of wheat growth, including pre-wintering, wintering, greening and seedling stages. The recorded traits include cold resistance, disease resistance and high yield. The depth of the furrows is 9-12 cm, the spacing between adjacent furrows is 15 cm, and the deep plowing depth is not less than 28 cm; The wheat in step 4 and step 6 is fertilized with liquid fertilizer on the 8th to 12th day after the wheat seedlings are fully grown, and then fertilized with liquid fertilizer again after 6 to 10 days. The amount of fertilizer applied each time is 10 to 16 grams per square meter, and conventional seedling management is carried out. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A molecular breeding method for transgenic wheat, characterized in that: The steps include: Step 1: Selection and preparation of planting site: Select a flat area with sufficient sunlight during the day, turn the soil, remove straw, weeds, stones and other debris from the soil, apply base fertilizer, plough the land, grind the soil, make ridges on the leveled land, form drainage ditches between ridges, and dig ditches in the ridges; Step 2: Processing of wheat seeds: First, select wheat seeds that have grown in the wild for more than two years and are intact and good, rinse them with water, put them in a container after natural air drying, and then soak and clean them in a sterile environment. The obtained wheat seeds are reserved for use and used as wheat female parents. Select high-quality and high-yield varieties suitable for local planting, resistant to pests and diseases, weak tillering, resistant to lodging, large ears, and high fruiting rate as wheat male parents, and hybridize the wheat male parent and the wheat female parent to obtain hybrid combination first-generation wheat seeds; Step 3: placing the first generation wheat seeds in an induction medium, maintaining the culture temperature between 23° C. and 27° C., the relative humidity between 80% and 90%, the light intensity between 1700 and 3100 Lux, and the light duration at 7 hours per day for 55 to 60 days, thereby obtaining young wheat seedlings; Step 4: Plant the young wheat seedlings at a staggered spacing of 0.3-0.6m. When the wheat matures, harvest them uniformly to obtain the second-generation wheat hybrid seeds. Step 5: The second-generation wheat hybrid seeds are subjected to gene editing to edit the target DNA fragment in the genome of the recipient wheat, thereby obtaining gene-edited wheat in which the target DNA fragment is changed, and the target wheat having increased grain hardness compared with the recipient wheat is screened from the gene-edited wheat; Step 6: Plant the wheat with increased hardness in step 5, and harvest it uniformly after it matures, so as to obtain the third-generation wheat hybrid seeds; Step 7: Use the second-generation wheat hybrid seeds as the female parent and the third-generation wheat hybrid seeds as the male parent to crossbreed with the female parent to obtain hybrid wheat seeds. Then select high-quality selenium-rich wheat seeds for crossbreeding with them. After sowing and maturity, you will get selenium-rich wheat seeds with hard and high grains.

2. A transgenic wheat molecular breeding method according to claim 1, characterized in that: The land described in step 1 is a test site, and the test site needs to be artificially divided into multiple areas of similar size, and a greenhouse is laid in the area of ​​the test site, and a detachable connecting frame is set between the two sides of the greenhouse and the ground.

3. The method for molecular breeding of transgenic wheat according to claim 1, characterized in that: In step 4 and step 6, the land is watered by spraying when wheat is planted, and the topsoil is hoeed 3 to 5 days after watering to break up the compaction of the topsoil.

4. The method for molecular breeding of transgenic wheat according to claim 1, characterized in that: Place the second-generation seeds in a container, add alcohol with a concentration of 6%-18% to the container, and soak for 3-6 hours. After completion, take the second-generation seeds out of the container and air-dry them. Finally, place them in a diethyl sulfate solution and soak for 3-5 hours. The concentration of the diethyl sulfate solution is 0.05-0.5 mol / L.

5. The method for molecular breeding of transgenic wheat according to claim 1, characterized in that: The land described in step 1 needs to be covered with a nutrient soil layer before planting wheat, and the nutrient soil layer includes manure, wood ash, and chopped grass.

6. A transgenic wheat molecular breeding method according to claim 1, characterized in that: During the planting period, regular surveys and records are required to record the different characteristics of each test material at different stages of wheat growth. The recording periods include before wintering, wintering period, greening period and seedling stage. The recorded characteristics include cold resistance, disease resistance and yield.

7. A transgenic wheat molecular breeding method according to claim 1, characterized in that: The depth of the furrows is 9-12 cm, the spacing between adjacent furrows is 15 cm, and the deep plowing depth is not less than 28 cm.

8. The method for molecular breeding of transgenic wheat according to claim 1, characterized in that: The wheat described in step 4 and step 6 is fertilized with liquid fertilizer once on the 8th to 12th day after the wheat seedlings have fully emerged, and then fertilized with liquid fertilizer again after 6 to 10 days. The amount of fertilizer applied each time is 10 to 16 grams per square meter, and conventional seedling management is performed.