Breeding method for resisting stripe rust of wheat

Abstract

The invention belongs to the technical field of plant breeding, and particularly relates to a stripe rust resistant breeding method for wheat. The method comprises the following steps: (1) obtaining and planting wheat stripe rust high-resistance resources and breeding trunk parents; (2) screening a differential culture medium with an anther callus differentiation rate of 1-1.5%; (3) obtaining F1 hybrids; (4) obtaining haplobionts; (5) obtaining a wheat haplobiont root system; (6) obtaining wheat anther culture seedlings; and (7) planting wheat anther culture seedlings in a greenhouse, harvesting seeds from a single plant to obtain an improved stripe-rust-resistant wheat family, and sowing in a field to obtain stripe-rust-resistant wheat. The screening method and the breeding method are carried out at the same time, the wheat stripe rust disease-resistant breeding speed is greatly increased, the flower rust disease-resistant breeding period is remarkably shortened, and the important industrial value is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and specifically relates to a wheat breeding method for resistance to stripe rust. Background Art

[0002] Wheat stripe rust is a fungal disease caused by Puccinia striiformis West. f. sp. tritici Eriks, which is an important biological disaster affecting the safe production of wheat and is harmful to wheat production. Wheat stripe rust belongs to Puccinia striiformis f. sp. tritici of the genus Puccinia, and is an obligate biotrophic fungus.

[0003] In China, a comprehensive prevention and control strategy for wheat stripe rust is adopted, mainly by planting disease-resistant varieties, supplemented by cultivation management and chemical control measures. As one of the main measures for the integrated management of wheat stripe rust, in the years and regions where wheat stripe rust breaks out, the use of fungicides for unified prevention and control is an important means to control stripe rust. Even in regions where the resistance genes to stripe rust are rationally distributed, fungicides are also a necessary supplement to the prevention and control measures of wheat stripe rust. Since the 1970s, triazole fungicides have been widely used in the prevention and control of wheat stripe rust in China, and have played a huge role in controlling wheat diseases. However, due to the long-term single use in production, a series of problems have occurred, such as an increase in the amount of pesticide used, an increase in the number of uses, a decline in the control effect, the generation of resistance in the pathogen, and a decline in wheat quality.

[0004] Based on the fact that stripe rust is a serious disease that endangers wheat production. Although chemical agents can effectively control the economic losses caused by wheat diseases, due to considerations of environmental safety and human health, cultivating and promoting disease-resistant varieties is still considered the most fundamental, economical and safe way. Summary of the Invention

[0005] The purpose of the present invention is to provide a wheat breeding method for resistance to stripe rust, so as to realize the screening and breeding of disease-resistant wheat.

[0006] To this end, the present invention provides the following technical solutions.

[0007] One aspect of the present invention provides a wheat breeding method for resistance to stripe rust, and the method includes the following steps: (1) Obtain and plant high-resistance resources to wheat stripe rust and breeding backbone parents; (2) Inoculate the anthers of the backbone parents in step (1) into anther induction medium I for culture to obtain anther callus, and then transfer the obtained anther callus to a differentiation medium for differentiation culture, and screen a differentiation medium with an anther callus differentiation rate of 1-1.5%; (3) Cross the backbone parents described in step (1) to obtain an F1 hybrid; (4) Sow and grow the F1 hybrid, select the anthers of the main spike and inoculate them into anther induction medium II for culture to obtain anther callus, and then inoculate it into the differentiation medium screened in step (2) for differentiation culture to obtain haploid plants; (5) After the haploid plant seedlings grow 5 cm, transfer them to a rooting medium for rooting culture, and then harden off the seedlings to obtain the roots of wheat haploid plants; (6) Soak the roots of wheat haploid plants with a solution containing dimethyl sulfoxide and colchicine, then wash and transplant them to obtain wheat anther culture seedlings; (7) Plant the wheat anther culture seedlings, harvest the seeds individually to obtain improved wheat families resistant to stripe rust, and then sow them in the field to obtain wheat resistant to stripe rust.

[0008] The anther induction medium I includes plant polysaccharides. The plant polysaccharides are wolfberry polysaccharides and / or bletilla striata polysaccharides. The differentiation medium includes plant polypeptides. The plant polypeptides are phytosulfokine and / or theanine polypeptide.

[0009] The rooting medium includes ginsenoside Rb2 at 0.1 - 0.3 mg / L and paclobutrazol at 200 - 400 mg / L.

[0010] In a preferred embodiment of the present invention, step (1) includes: planting wheat resources in plots by the multi-year and multi-site method in a severely affected area of wheat stripe rust, investigating the incidence of stripe rust of each material in the wheat resource bank, and selecting wheat varieties or families with an incidence of less than 0.5% and stable resistance as highly resistant resources to wheat stripe rust; Select local main cultivated varieties with insufficient resistance to wheat stripe rust but excellent agronomic traits as breeding backbone parents; Plant the highly resistant resources to wheat stripe rust and breeding backbone parents in the normal season.

[0011] In a preferred embodiment of the present invention, in step (2), the composition of the anther induction medium I is as follows: NH4NO3 200 - 400 mg / L, KNO3 1200 - 1500 mg / L, KH2PO4 400 - 600 mg / L, CaCl2 100 - 200 mg / L, MgSO4·7H2O 100 - 200 mg / L, Na2-EDTA 35 - 36 mg / L, FeSO4·7H2O 25 - 28 mg / L, MnSO4·4H2O 3 - 5 mg / L, ZnSO4·7H2O 1 - 3 mg / L, H3BO3 1 - 2 mg / L, KI 0.5 - 1.5 mg / L, CaSO4·5H2O 0.1 - 0.3 mg / L, CoCl2·6H2O 0.1 - 0.3 mg / L, glycine 2 - 3 mg / L, vitamin B1 0.5 - 1.5 mg / L, vitamin B6 0.5 - 1.5 mg / L, nicotinic acid 0.5 - 1 mg / L, inositol 100 - 300 mg / L, KT 1 - 3 mg / L, IAA 0.5 - 1 mg / L, plant polysaccharide 80 - 100 g / L, agar 5 - 7 g / L, the balance is water, and the pH value is 5.8.

[0012] In a preferred embodiment of the present invention, the anther induction culture conditions are as follows: the temperature is 35 - 37 °C, and the time is 24 - 30 h.

[0013] In a preferred embodiment of the present invention, in step (2), the composition of the differentiation medium is as follows: dextran 70 - 90 g / L, plant polypeptide 10 - 30 mg / L, carvacrol 10 - 30 mg / L, isorhamnetin 1 - 3 mg / L, crude toxin extract 1 - 2 mg / L, agar 5 - 6 g / L, the balance is water, and the pH value is 5.8.

[0014] In a preferred embodiment of the present invention, the differentiation culture conditions are as follows: the temperature is 25 - 27 °C, the light intensity is 1500 - 2000 Lux, and the light cycle is 12 h light / 12 h dark.

[0015] In a preferred embodiment of the present invention, in step (2), when performing differentiation culture, the concentration level of the crude toxin added to the differentiation medium with the anther callus differentiation rates of both the highly stripe rust-resistant resources and the breeding backbone parents being 1 - 1.5% is screened, and the crude toxin level of the average value of the two is determined as the stripe rust crude toxin screening pressure.

[0016] In a preferred embodiment of the present invention, in step (4), the composition of the anther induction medium II is as follows: NH4NO3 200 - 400 mg / L, KNO3 1200 - 1500 mg / L, KH2PO4 400 - 600 mg / L, CaCl2 100 - 200 mg / L, MgSO4·7H2O 100 - 200 mg / L, Na2-EDTA 35 - 36 mg / L, FeSO4·7H2O 25 - 28 mg / L, MnSO4·4H2O 3 - 5 mg / L, ZnSO4·7H2O 1 - 3 mg / L, H3BO3 1 - 2 mg / L, KI 0.5 - 1.5 mg / L, CaSO4·5H2O 0.1 - 0.3 mg / L, CoCl2·6H2O 0.1 - 0.3 mg / L, glycine 2 - 3 mg / L, vitamin B1 0.5 - 1.5 mg / L, vitamin B6 0.5 - 1.5 mg / L, nicotinic acid 0.5 - 1 mg / L, inositol 100 - 300 mg / L, KT 1 - 3 mg / L, IAA 0.5 - 1 mg / L, glutathione 180 - 200 mg / L, casein hydrolysate 0.3 - 0.5 g / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 100 - 120 mg / L, lactic acid 40 - 60 mg / L, quercitol 5 - 10 mg / L, psicose 70 - 80 mg / L, agar 5 - 7 g / L, with the balance being water, and the pH value being 5.8.

[0017] In a preferred embodiment of the present invention, the anther induction culture conditions are as follows: the temperature is 35 - 37 °C, and the time is 24 - 30 h.

[0018] In a preferred embodiment of the present invention, in step (4), the differentiation culture conditions are as follows: the temperature is 25 - 27 °C, the light intensity is 1500 - 2000 Lux, and the light cycle is 12 h light / 12 h dark.

[0019] In a preferred embodiment of the present invention, in step (5), the composition of the rooting medium is as follows: 1 / 2 MS basal medium, ginsenoside Rb2 0.1 - 0.3 mg / L, paclobutrazol 200 - 400 mg / L, with the balance being water, and the pH value being 5.8.

[0020] In a preferred embodiment of the present invention, the rooting culture conditions are as follows: the temperature is 27 - 29 °C, the light intensity is 2500 - 3000 Lux, and the light cycle is 12 h light / 12 h dark.

[0021] In a preferred embodiment of the present invention, in step (5), the seedling hardening process is as follows: Open the sealing film of the culture bottle for rooting culture, perform bottle-opening seedling hardening in the culture room for 4 - 6 days, and then transfer it to a place with a light intensity of 4000 - 6000 Lux, a temperature of 25 - 30 °C, and a humidity of 70 - 85%, and use natural light for seedling hardening for 7 - 10 days.

[0022] In a preferred embodiment of the present invention, in step (6), the soaking conditions are: room temperature, 24 - 28 h.

[0023] By means of the above technical solutions, the present invention has at least the following advantages: (1) The screening and breeding methods of the present invention are carried out simultaneously, greatly accelerating the breeding speed of wheat stripe rust resistance and significantly shortening the breeding cycle of wheat flower rust resistance, and having important industrial value.

[0024] (2) By regulating the wheat anther culture process, the present invention selects appropriate anther induction media I and II. In anther induction medium I, plant polysaccharides such as wolfberry polysaccharide and / or bletilla striata polysaccharide are added. It can not only replace the sugar component in the conventional medium, but also, due to the high biological activity of wolfberry polysaccharide and / or bletilla striata polysaccharide, significantly improve the induction rate of anther callus; in anther induction medium II, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, lactic acid, quercitol, and allulose are added. The combined action of these several components is particularly suitable for the callus induction of the main spike anthers of F1 hybrids and can improve the induction rate.

[0025] (3) During the differentiation culture process, the prepared differentiation medium contains dextran, plant polypeptide, carvacrol, and isorhamnetin. Compared with the conventional differentiation medium, the specially prepared differentiation medium of the present invention contains various differentiation-promoting active substances such as plant polypeptide phytosulfokine and / or theanine polypeptide, carvacrol, and isorhamnetin. It not only provides a sufficient nutrient source for the differentiation culture of anther callus, but also can promote the differentiation of callus and improve the differentiation rate. A small amount of ginsenoside Rb2 is added to the rooting medium, which, in synergistic action with paclobutrazol, can reduce the usage amount of paclobutrazol. The promoting effect of ginsenoside Rb2 on the synthesis of DNA and RNA can, to a certain extent, promote the rooting effect of paclobutrazol on plants.

[0026] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following provides a detailed description of the preferred embodiments of the present invention as follows. Detailed implementation manners

[0027] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, the wolfberry polysaccharide (purity greater than 98%) used in the examples of the present invention was purchased from Lanzhou Waters Biotechnology Co., Ltd.; the bletilla striata polysaccharide (purity greater than 98%) was purchased from Lanzhou Waters Biotechnology Co., Ltd.; the plant sulfopeptide molecular formula is Tyr (SO3H)-Ile-Tyr (SO3H)-Thr-Gln; the theanine polypeptide sequence is CCYXXXXX, where C is cysteine, Y is tyrosine, and X is theanine, with a molecular weight of 1168.48. 1 / 2MS minimal medium: Product No.: M8526; purchased from Solarbio.

[0029] Unless otherwise specified, the preparation method of the crude toxin extract in the embodiment of the present invention is as follows: wheat ears infected with stripe rust are cut from the field, brought back to the laboratory and stored at -20°C, the wheat ears are taken out for extraction, broken with a blender, 10 g of the sample is weighed into a 150 mL conical flask, 50 mL of 84% acetonitrile aqueous solution is added, the mixture is mixed with a homogenizer for 3 minutes and then filtered, the filtrate is passed through a MycoSep 226 multifunctional purification column, 4 mL of the purified liquid is taken and blown with nitrogen in a 50°C water bath until almost dry, the liquid is dissolved with methanol and 10 mmol / L ammonium acetate solution in a volume ratio of 1:1, the mixture is mixed and the volume is adjusted to 1 mL, the liquid is passed through a 0.22 μm MICRO PES polyethersulfone membrane, and the liquid is stored at -20°C for use.

[0030] Example 1: The composition of anther induction medium I is as follows: NH4NO3 300 mg / L, KNO3 1300 mg / L, KH2PO4 500 mg / L, CaCl2 150 mg / L, MgSO4·7H2O 150 mg / L, Na2-EDTA 35.5 mg / L, FeSO4·7H2O 26.5 mg / L, MnSO4·4H2O 4 mg / L, ZnSO4·7H2O 2 mg / L, H3BO3 1.5 mg / L, KI 1.0 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.2 mg / L, glycine 2.5 mg / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, nicotinic acid 0.75 mg / L, inositol 200 mg / L, KT 2 mg / L, IAA 0.75 mg / L, wolfberry polysaccharide 90 g / L, agar 6 g / L, with the balance being water and the pH value being 5.8.

[0031] The composition of the differentiation medium is as follows: dextran 80 g / L, phytosulfokine 20 mg / L, carvacrol 20 mg / L, isorhamnetin 2 mg / L, crude toxin extract 1.5 mg / L, agar 5.5 g / L, with the balance being water and the pH value being 5.8.

[0032] The composition of anther induction medium II is as follows: NH4NO3 300 mg / L, KNO3 1300 mg / L, KH2PO4 500 mg / L, CaCl2 150 mg / L, MgSO4·7H2O 150 mg / L, Na2-EDTA 35.5 mg / L, FeSO4·7H2O 26.5 mg / L, MnSO4·4H2O 4 mg / L, ZnSO4·7H2O 2 mg / L, H3BO3 1.5 mg / L, KI 1.0 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.2 mg / L, glycine 2.5 mg / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, nicotinic acid 0.75 mg / L, inositol 200 mg / L, KT 2 mg / L, IAA 0.75 mg / L, glutathione 190 mg / L, hydrolyzed casein 0.4 g / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 110 mg / L, lactic acid 50 mg / L, quercitol 7.5 mg / L, psicose 75 mg / L, agar 6 g / L, with the balance being water and the pH value being 5.8.

[0033] The composition of the rooting medium is as follows: 1 / 2MS basal medium, ginsenoside Rb2 0.2 mg / L, paclobutrazol 300 mg / L, with the balance being water and the pH value being 5.8.

[0034] (1) First, wheat resources are planted in plots using the multi-year and multi-location method in severely affected areas of wheat stripe rust. The incidence of stripe rust in each material in the wheat resource bank is investigated, and wheat varieties with an incidence of stripe rust lower than 0.5% and stable resistance are selected as highly resistant resources to wheat stripe rust; local main cultivated varieties with insufficient resistance to wheat stripe rust but excellent agronomic traits are selected as the main breeding parents; they are planted in the normal season to obtain highly resistant resources to wheat stripe rust and the main breeding parents.

[0035] (2) The anthers of the obtained main breeding parents are inoculated into anther induction medium I and cultured at 36°C for 27 h to obtain anther callus. The obtained anther callus is transferred to a differentiation medium and cultured under the conditions of a temperature of 26°C, a light intensity of 2000 Lux, and a photoperiod of 12 h light / 12 h dark for differentiation culture. When performing the differentiation culture, the concentration level of the crude toxin added to the differentiation medium with a differentiation rate of 1.5% for both the highly resistant resources to stripe rust and the anther callus of the main breeding parents is screened, and the crude toxin level of the average value of the two is determined as the screening pressure of wheat stripe rust crude toxin.

[0036] (3) Cross between the obtained highly resistant resources to wheat stripe rust and the main breeding parents to obtain F1 hybrids.

[0037] (4) The obtained F1 hybrids are sown and planted in the normal season. Select the anthers of strong main spikes and inoculate them into anther induction medium II and culture at 36°C for 27 h to obtain anther callus, and select anther callus with a diameter of 0.6 cm and inoculate it into the differentiation medium with the screening pressure of wheat stripe rust crude toxin and culture under the conditions of a temperature of 26°C, a light intensity of 1500 Lux, and a photoperiod of 12 h light / 12 h dark to obtain haploid plants.

[0038] (5) After the haploid plant seedlings grow to 5 cm, transfer them to a rooting medium and culture under the conditions of a temperature of 28°C, a light intensity of 2500 Lux, and a photoperiod of 12 h light / 12 h dark for rooting culture. After 15 days, open the sealing film for acclimatization: Open the sealing film of the culture bottle for rooting culture and perform acclimatization in the culture room for 5 days, then transfer it to a place with a light intensity of 5000 Lux, a temperature of 28°C, and a humidity of 70%, and use natural light for acclimatization for 10 days to obtain the roots of wheat haploid plants.

[0039] (6) Soak the roots of wheat haploid plants in a solution of 2% dimethyl sulfoxide and 0.5‰ colchicine at room temperature for 24 h, then rinse the medicinal liquid with clean water, and then transplant the plants to the greenhouse, shade them for 7 days and then perform routine cultivation management to obtain wheat anther culture seedlings.

[0040] (7) Plant the wheat anther culture seedlings in the greenhouse, harvest seeds from individual plants to obtain improved wheat families resistant to stripe rust, and then sow them in the field to obtain wheat resistant to stripe rust.

[0041] Example 2: The composition of anther induction medium I is: NH4NO3 200 mg / L, KNO3 1500 mg / L, KH2PO4 400 mg / L, CaCl2 200 mg / L, MgSO4·7H2O 100 mg / L, Na2-EDTA 36 mg / L, FeSO4·7H2O 25 mg / L, MnSO4·4H2O 5 mg / L, ZnSO4·7H2O 1 mg / L, H3BO3 2 mg / L, KI 0.5 mg / L, CaSO4·5H2O 0.3 mg / L, CoCl2·6H2O 0.3 mg / L, glycine 2 mg / L, vitamin B1 1.5 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 1 mg / L, inositol 100 mg / L, KT 3 mg / L, IAA 0.5 mg / L, Bletilla striata polysaccharide 80 g / L, agar 7 g / L, the balance is water, and the pH value is 5.8.

[0042] The composition of the differentiation medium is: dextran 70 g / L, theanine polypeptide 30 mg / L, carvacrol 10 mg / L, isorhamnetin 3 mg / L, crude toxin extract 1 mg / L, agar 6 g / L, the balance is water, and the pH value is 5.8.

[0043] The composition of anther induction medium II is: NH4NO3 200 mg / L, KNO3 1500 mg / L, KH2PO4 400 mg / L, CaCl2 200 mg / L, MgSO4·7H2O 100 mg / L, Na2-EDTA 36 mg / L, FeSO4·7H2O 25 mg / L, MnSO4·4H2O 5 mg / L, ZnSO4·7H2O 1 mg / L, H3BO3 2 mg / L, KI 0.5 mg / L, CaSO4·5H2O 0.3 mg / L, CoCl2·6H2O 0.3 mg / L, glycine 2 mg / L, vitamin B1 1.5 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 1 mg / L, inositol 100 mg / L, KT 3 mg / L, IAA 0.5 mg / L, glutathione 200 mg / L, casein hydrolysate 0.3 g / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 120 mg / L, lactic acid 40 mg / L, quercitol 10 mg / L, allulose 70 mg / L, agar 7 g / L, the balance is water, and the pH value is 5.8.

[0044] The composition of the rooting medium is: 1 / 2 MS basal medium, ginsenoside Rb2 0.3 mg / L, paclobutrazol 400 mg / L, the balance is water, and the pH value is 5.8.

[0045] (1) First, wheat resources are planted in small plots using the multi-year and multi-location method in severely affected areas of wheat stripe rust. The incidence of stripe rust in each material in the wheat resource bank is investigated, and wheat varieties with an incidence of stripe rust lower than 0.5% and stable resistance are selected as highly resistant resources to wheat stripe rust; local main cultivated varieties with insufficient resistance to wheat stripe rust but excellent agronomic traits are selected as the main breeding parents; they are planted in the normal season to obtain highly resistant resources to wheat stripe rust and the main breeding parents.

[0046] (2) The anthers of the obtained main breeding parents are inoculated into anther induction medium I and cultured at 35°C for 30 h to obtain anther callus. The obtained anther callus is transferred to a differentiation medium and cultured under the conditions of a temperature of 27°C, a light intensity of 1500 Lux, and a photoperiod of 12 h light / 12 h dark for differentiation culture. When performing differentiation culture, the concentration level of crude toxin added to the differentiation medium with a differentiation rate of 1.5% for both the highly resistant resources to stripe rust and the anther callus of the main breeding parents is screened, and the crude toxin level of the average value of the two is determined as the screening pressure of stripe rust crude toxin.

[0047] (3) Cross between the obtained highly resistant resources to wheat stripe rust and the main breeding parents to obtain F1 hybrids.

[0048] (4) The obtained F1 hybrids are sown and planted in the normal season. Select the anthers of strong main spikes and inoculate them into anther induction medium II and culture at 37°C for 24 h to obtain anther callus, and select anther callus with a diameter of 0.6 cm and inoculate it into the differentiation medium with the screening pressure of stripe rust crude toxin and culture under the conditions of a temperature of 25°C, a light intensity of 2000 Lux, and a photoperiod of 12 h light / 12 h dark to obtain haploid plants.

[0049] (5) After the haploid plant seedlings grow to 5 cm, transfer them to a rooting medium and culture them under the conditions of a temperature of 29°C, a light intensity of 2500 Lux, and a photoperiod of 12 h light / 12 h dark for rooting culture. After 15 days, open the sealing film for acclimatization: Open the sealing film of the culture bottle for rooting culture, perform bottle-opening acclimatization in the culture room for 5 days, and then transfer it to a light intensity of 6000 Lux, a temperature of 25°C, and a humidity of 85%, and acclimatize it with natural light for 10 days to obtain the roots of wheat haploid plants.

[0050] (6) Soak the roots of wheat haploid plants in a solution of 2% dimethyl sulfoxide and 0.5‰ colchicine at room temperature for 24 h, then rinse the medicinal solution with clean water, and then transplant the plants into the greenhouse, shade for 7 days and then perform routine cultivation management to obtain wheat anther culture seedlings.

[0051] (7) Plant the wheat anther culture seedlings in the greenhouse, harvest seeds from each individual plant to obtain improved wheat families resistant to stripe rust, and then sow them in the field to obtain wheat resistant to stripe rust.

[0052] Example 3: The composition of anther induction medium I is as follows: NH4NO3 400 mg / L, KNO3 1200 mg / L, KH2PO4 600 mg / L, CaCl2 100 mg / L, MgSO4·7H2O 200 mg / L, Na2-EDTA 35 mg / L, FeSO4·7H2O 28 mg / L, MnSO4·4H2O 3 mg / L, ZnSO4·7H2O 3 mg / L, H3BO3 1 mg / L, KI 1.5 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.1 mg / L, glycine 3 mg / L, vitamin B1 0.5 mg / L, vitamin B6 1.5 mg / L, nicotinic acid 0.5 mg / L, inositol 300 mg / L, KT 1 mg / L, IAA 1 mg / L, Bletilla striata polysaccharide 100 g / L, agar 5 g / L, with the balance being water and the pH value being 5.8.

[0053] The composition of the differentiation medium is as follows: dextran 90 g / L, phytosulfokine 10 mg / L, carvacrol 30 mg / L, isorhamnetin 1 mg / L, crude toxin extract 2 mg / L, agar 5 g / L, with the balance being water and the pH value being 5.8.

[0054] The composition of anther induction medium II is as follows: NH4NO3 400 mg / L, KNO3 1200 mg / L, KH2PO4 600 mg / L, CaCl2 100 mg / L, MgSO4·7H2O 200 mg / L, Na2-EDTA 35 mg / L, FeSO4·7H2O 28 mg / L, MnSO4·4H2O 3 mg / L, ZnSO4·7H2O 3 mg / L, H3BO3 1 mg / L, KI 1.5 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.1 mg / L, glycine 3 mg / L, vitamin B1 0.5 mg / L, vitamin B6 1.5 mg / L, nicotinic acid 0.5 mg / L, inositol 300 mg / L, KT 1 mg / L, IAA 1 mg / L, glutathione 180 mg / L, hydrolyzed casein 0.5 g / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 100 mg / L, lactic acid 60 mg / L, quercitol 5 mg / L, psicose 80 mg / L, agar 5 g / L, with the balance being water and the pH value being 5.8.

[0055] The composition of the rooting medium is as follows: 1 / 2MS basal medium, ginsenoside Rb2 0.1 mg / L, paclobutrazol 200 mg / L, with the balance being water and the pH value being 5.8.

[0056] (1) First, wheat resources were planted in plots in areas severely affected by wheat stripe rust using the multi-site method for many years. The incidence of stripe rust in various materials in the wheat resource library was investigated, and wheat varieties with an incidence rate lower than 0.5% and stable resistance were selected as high-resistance resources for wheat stripe rust. Local main cultivated varieties with insufficient resistance to wheat stripe rust but excellent agronomic traits were selected as main breeding parents. High-resistance resources for wheat stripe rust and main breeding parents were obtained by planting in the main season.

[0057] (2) The anthers of the obtained main trunk parent were inoculated into anther induction medium I and cultured at 37°C for 24 h to obtain anther callus. The obtained anther callus was transferred to differentiation medium and cultured at a temperature of 25°C, a light intensity of 2000 Lux, and a photoperiod of 12 h light / 12 h dark. During the differentiation culture, the crude toxin concentration level added to the differentiation medium with a differentiation rate of 1.5% for the anther callus of the high-resistance resources and the breeding main trunk parent was screened. The crude toxin level of the average value of the two was determined as the stripe rust crude toxin screening pressure.

[0058] (3) The obtained wheat stripe rust-resistant resources are hybridized with the main breeding parent to obtain F1 hybrids.

[0059] (4) The obtained F1 hybrid was sown and planted in the main season, and the healthy main ear anthers were selected and inoculated into anther induction medium II and cultured at 35°C for 30 hours to obtain anther callus. The anther callus with a diameter of 0.6 cm was selected and inoculated into the differentiation medium of the stripe rust crude toxin screening pressure and cultured at a temperature of 27°C, a light intensity of 1500 Lux, and a photoperiod of 12 hours of light / 12 hours of dark to obtain haploid plants.

[0060] (5) After the haploid seedlings grow to 5 cm, they are transferred to the rooting medium and cultured under the conditions of a temperature of 27 ° C, a light intensity of 3000 Lux, and a photoperiod of 12 h light / 12 h dark. After 15 days, the sealing film is opened to harden the seedlings: the sealing film of the culture bottle for rooting culture is opened, and the seedlings are hardened in the culture room for 5 days. Then, the seedlings are transferred to a light intensity of 4000 Lux, a temperature of 30 ° C, and a humidity of 70%, and hardened under natural light for 10 days to obtain the root system of wheat haploid plants.

[0061] (6) The roots of wheat haploid plants were soaked in a solution of 2% dimethyl sulfoxide and 0.5‰ colchicine at room temperature for 24 h, and then the solution was rinsed with clean water. The plants were then transplanted into a greenhouse and shaded for 7 days before conventional cultivation and management to obtain wheat flower seedlings.

[0062] (7) Plant wheat seedlings in greenhouses and harvest individual plants to obtain improved wheat lines resistant to stripe rust. Then, plant them in the field to obtain wheat resistant to stripe rust.

[0063] Example 4: The composition of anther induction medium I is as follows: NH4NO3 200 mg / L, KNO3 1500 mg / L, KH2PO4 500 mg / L, CaCl2 150 mg / L, MgSO4·7H2O 200 mg / L, Na2-EDTA 36 mg / L, FeSO4·7H2O 27 mg / L, MnSO4·4H2O 4 mg / L, ZnSO4·7H2O 3 mg / L, H3BO3 1.5 mg / L, KI 1.0 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.3 mg / L, glycine 2 mg / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, nicotinic acid 0.75 mg / L, inositol 200 mg / L, KT 2 mg / L, IAA 0.75 mg / L, wolfberry polysaccharide 80 g / L, agar 6 g / L, with the balance being water and the pH value being 5.8.

[0064] The composition of the differentiation medium is as follows: dextran 80 g / L, theanine polypeptide 30 mg / L, carvacrol 20 mg / L, isorhamnetin 1 mg / L, crude toxin extract 1.5 mg / L, agar 5.5 g / L, with the balance being water and the pH value being 5.8.

[0065] The composition of anther induction medium II is as follows: NH4NO3 400 mg / L, KNO3 1200 mg / L, KH2PO4 600 mg / L, CaCl2 200 mg / L, MgSO4·7H2O 100 mg / L, Na2-EDTA 35.5 mg / L, FeSO4·7H2O 26.5 mg / L, MnSO4·4H2O 4 mg / L, ZnSO4·7H2O 2 mg / L, H3BO3 1.5 mg / L, KI 1.0 mg / L, CaSO4·5H2O 0.1 mg / L, CoCl2·6H2O 0.3 mg / L, glycine 2.5 mg / L, vitamin B1 1 mg / L, vitamin B6 1 mg / L, nicotinic acid 0.75 mg / L, inositol 200 mg / L, KT 3 mg / L, IAA 0.75 mg / L, glutathione 200 mg / L, hydrolyzed casein 0.4 g / L, 4-hydroxyethylpiperazineethanesulfonic acid 110 mg / L, lactic acid 50 mg / L, quercitol 8 mg / L, allulose 70 mg / L, agar 6 g / L, with the balance being water and the pH value being 5.8.

[0066] The composition of the rooting medium is as follows: 1 / 2 MS basal medium, ginsenoside Rb2 0.3 mg / L, paclobutrazol 300 mg / L, with the balance being water and the pH value being 5.8.

[0067] (1) First, wheat resources were planted in plots using the multi-year and multi-location method in severely wheat stripe rust disease areas. The incidence of stripe rust disease of each material in the wheat resource bank was investigated, and wheat varieties with an incidence of less than 0.5% and stable resistance were selected as highly resistant resources to wheat stripe rust disease; local main cultivated varieties with insufficient resistance to wheat stripe rust disease but excellent agronomic traits were selected as the main breeding parents; they were planted in the normal season to obtain highly resistant resources to wheat stripe rust disease and the main breeding parents.

[0068] (2) The anthers of the obtained main breeding parents were inoculated into anther induction medium I and cultured at 36°C for 27 h to obtain anther callus. The obtained anther callus was transferred to a differentiation medium and cultured under the conditions of a temperature of 26°C, a light intensity of 2000 Lux, and a photoperiod of 12 h light / 12 h dark. When performing the differentiation culture, the concentration level of the crude toxin added to the differentiation medium with a differentiation rate of 1.5% for both the highly resistant resources to stripe rust disease and the anther callus of the main breeding parents was screened, and the crude toxin level of the average value of the two was determined as the screening pressure of the stripe rust disease crude toxin.

[0069] (3) Cross between the obtained highly resistant resources to wheat stripe rust disease and the main breeding parents to obtain F1 hybrids.

[0070] (4) The obtained F1 hybrids were sown and planted in the normal season. The anthers of the strong main ears were selected and inoculated into anther induction medium II and cultured at 36°C for 27 h to obtain anther callus. The anther callus with a diameter of 0.6 cm was selected and inoculated into the differentiation medium with the screening pressure of the stripe rust disease crude toxin and cultured under the conditions of a temperature of 26°C, a light intensity of 1500 Lux, and a photoperiod of 12 h light / 12 h dark to obtain haploid plants.

[0071] (5) After the haploid plant seedlings grew to 5 cm, they were transferred to a rooting medium and cultured under the conditions of a temperature of 28°C, a light intensity of 2500 Lux, and a photoperiod of 12 h light / 12 h dark for rooting culture. After 15 days, the sealing film was opened for acclimatization: the sealing film of the culture bottle for rooting culture was opened, and acclimatization was carried out in the culture room for 5 days, and then transferred to a light intensity of 5000 Lux, a temperature of 28°C, and a humidity of 70%, and acclimatized with natural light for 10 days to obtain the roots of wheat haploid plants.

[0072] (6) The roots of wheat haploid plants were soaked in a solution of 2% dimethyl sulfoxide and 0.5‰ colchicine at room temperature for 24 h, and then the medicinal solution was rinsed off with clean water. Then the plants were transplanted into the greenhouse, shaded for 7 days, and then managed by conventional cultivation to obtain wheat anther culture seedlings.

[0073] (7) The wheat anther culture seedlings were planted in the greenhouse, and seeds were harvested from individual plants to obtain improved wheat families resistant to stripe rust disease. Then they were sown in the field to obtain wheat resistant to stripe rust disease.

[0074] Comparative Example 1: The only difference between this Comparative Example 1 and Example 1 is that: in the anther induction medium I, the wolfberry polysaccharide is replaced with malt polysaccharide, and the rest is the same as in Example 1.

[0075] Comparative Example 2: The only difference between this Comparative Example 2 and Example 1 is that: phytosulfokine is not added to the differentiation medium, and the rest is the same as in Example 1.

[0076] Comparative Example 3: The only difference between this Comparative Example 3 and Example 1 is that: carvacrol is not added to the differentiation medium, and the rest is the same as in Example 1.

[0077] Comparative Example 4: The only difference between this Comparative Example 3 and Example 1 is that: neither phytosulfokine nor carvacrol is added to the differentiation medium, and the rest is the same as in Example 1.

[0078] Comparative Example 5: The only difference between this Comparative Example 5 and Example 1 is that: quercitol is not added to the anther induction medium II, and the rest is the same as in Example 1.

[0079] Comparative Example 6: The only difference between this Comparative Example 6 and Example 1 is that: psicose is not added to the anther induction medium II, and the rest is the same as in Example 1.

[0080] Comparative Example 7: The only difference between this Comparative Example 6 and Example 1 is that: neither quercitol nor psicose is added to the anther induction medium II, and the rest is the same as in Example 1.

[0081] Comparative Example 8: The only difference between this Comparative Example 8 and Example 1 is that: ginsenoside Rb2 is not added to the rooting medium, and the rest is the same as in Example 1.

[0082] Experiment 1: Evaluation of the resistance of different wheat varieties to stripe rust 6000 wheat seedlings obtained in Examples 1 - 4 and Comparative Examples 1 - 8 were respectively selected as transplanted seedlings. The wheat planting base in Tianshui, Gansu was selected and evenly divided into 36 test fields according to the area size, and numbered 1 - 12 (three test fields in parallel for each group).

[0083] The seedlings of Examples 1 - 4 and Comparative Examples 1 - 8 were respectively transplanted into test fields No. 1 - 12. Then, the combination of the five - point sampling method and the reconnaissance method was used to record the disease (stripe rust) situation of the wheat. Data such as the diseased leaf rate, diseased spot rate, and diseased field rate of wheat stripe rust were recorded.

[0084] Diseased leaf rate (%): It represents the proportion of wheat plants infected with stripe rust, and is used to indicate the percentage of diseased leaves among the total number of surveyed leaves.

[0085] Diseased spot rate (%): It represents the proportion of diseased plants at the surveyed locations, and is used to indicate the percentage of surveyed locations among the total number of surveyed locations.

[0086] Diseased field rate (%): It represents the proportion of diseased fields, and is used to indicate the percentage of surveyed fields among the total number of surveyed fields.

[0087] Severity (%): It refers to the percentage of the area occupied by uredinia of Puccinia striiformis on diseased wheat leaves to the total leaf area, and is expressed by an 8-grade method, corresponding to 1%, 5%, 10%, 20%, 40%, 60%, 80% and 100% in sequence; When no stripe rust symptoms appear on the leaves, the severity is recorded as "0": When some leaves are diseased but the severity is less than 1%, it is recorded as trace amount and can be represented by "t". During the survey, the severity of each surveyed leaf is estimated visually, and finally the average severity is calculated (NY / T 1443.1-2007, Technical Specification for Evaluation of Wheat Resistance to Pests and Diseases: Part 1 Technical Specification for Evaluation of Wheat Resistance to Stripe Rust).

[0088] The statistical results are shown in Table 1: Table 1 Incidence of Wheat in Each Experimental Field ; It can be seen from the results in Table 1 that compared with Comparative Examples 1-8, the diseased leaf rate, diseased spot rate, etc. of wheat seedlings obtained by the methods of Examples 1-4 of the present invention are significantly reduced, indicating that the method of the present invention can obtain wheat resistant to stripe rust and has high disease resistance. Compared with Example 1, in Comparative Example 1, by replacing lycium barbarum polysaccharide with maltose, the diseased leaf rate and diseased spot rate of the obtained wheat are significantly increased, indicating that in addition to the functions of ordinary sugars, lycium barbarum polysaccharide also has the efficacy of improving wheat disease resistance. Compared with Comparative Example 2 where phytosulfokine was not added to the differentiation medium, Comparative Example 3 where carvacrol was not added to the differentiation medium, and Comparative Example 4 where neither phytosulfokine nor carvacrol was added to the differentiation medium, the diseased leaf rate, diseased spot rate, etc. of the obtained wheat are significantly decreased, indicating that there is a synergistic effect between phytosulfokine and carvacrol, which can improve the disease resistance of wheat. Similarly, compared with Comparative Example 5 where quercitol was not added to the anther induction medium II, Comparative Example 6 where psicose was not added to the anther induction medium II, and Comparative Example 7 where neither quercitol nor psicose was added to the anther induction medium II, the diseased leaf rate, diseased spot rate, etc. of the obtained wheat are significantly decreased, indicating that there is a synergistic effect between quercitol and psicose, which can improve the disease resistance of wheat. And in Comparative Example 8 where ginsenoside Rb2 was not added to the rooting medium, the diseased leaf rate, diseased spot rate, etc. of the obtained wheat decreased to a certain extent compared with Example 1.

[0089] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed methods and technical content within the scope of the technical solution of the present invention to obtain equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A wheat breeding method for resistance to stripe rust, characterized in that, It includes the following steps: (1) Obtain and plant high-resistant resources against wheat stripe rust and breeding backbone parents; (2) Inoculate the anthers of the backbone parents described in step (1) into anther induction medium I for culture to obtain anther callus, and then transfer the obtained anther callus to differentiation medium for differentiation culture, and screen the differentiation medium with an anther callus differentiation rate of 1-1.5%; (3) Cross the backbone parents described in step (1) to obtain F1 hybrids; (4) Sow and plant the F1 hybrids, select the main ear anthers and inoculate them into anther induction medium II for culture to obtain anther callus, and then inoculate them into the differentiation medium screened in step (2) for differentiation culture to obtain haploid plants; (5) After the haploid plant seedlings grow 5 cm, transfer them to rooting medium for rooting culture, and then harden off the seedlings to obtain the roots of wheat haploid plants; (6) Immerse the roots of wheat haploid plants in a solution containing dimethyl sulfoxide and colchicine, then wash and transplant them to obtain wheat anther culture seedlings; (7) Plant the wheat anther culture seedlings, harvest seeds from individual plants to obtain improved wheat families resistant to stripe rust, and then sow them in the field to obtain wheat resistant to stripe rust; The anther induction medium I includes plant polysaccharides, and the plant polysaccharides are wolfberry polysaccharides and / or bletilla striata polysaccharides; the differentiation medium includes plant polypeptides, and the plant polypeptides are phytosulfokine and / or theanine polypeptide; The rooting medium includes ginsenoside Rb2 at 0.1-0.3 mg / L and paclobutrazol at 200-400 mg / L.

2. The method according to claim 1, wherein Step (1) includes: planting wheat resources in plots by the multi-year and multi-point method in the severely affected areas of wheat stripe rust, investigating the incidence of stripe rust of each material in the wheat resource bank, and selecting wheat varieties or families with an incidence rate lower than 0.5% and stable resistance as high-resistant resources against wheat stripe rust; Select local main cultivated varieties with insufficient resistance to wheat stripe rust but excellent agronomic traits as breeding backbone parents; Plant high-resistant resources against wheat stripe rust and breeding backbone parents in the normal season.

3. The method according to claim 1, wherein In step (2), the composition of the anther induction medium I is as follows: NH4NO3 200 - 400 mg / L, KNO3 1200 - 1500 mg / L, KH2PO4 400 - 600 mg / L, CaCl2 100 - 200 mg / L, MgSO4·7H2O 100 - 200 mg / L, Na2-EDTA 35 - 36 mg / L, FeSO4·7H2O 25 - 28 mg / L, MnSO4·4H2O 3 - 5 mg / L, ZnSO4·7H2O 1 - 3 mg / L, H3BO3 1 - 2 mg / L, KI 0.5 - 1.5 mg / L, CaSO4·5H2O 0.1 - 0.3 mg / L, CoCl2·6H2O 0.1 - 0.3 mg / L, glycine 2 - 3 mg / L, vitamin B1 0.5 - 1.5 mg / L, vitamin B6 0.5 - 1.5 mg / L, nicotinic acid 0.5 - 1 mg / L, inositol 100 - 300 mg / L, KT 1 - 3 mg / L, IAA 0.5 - 1 mg / L, plant polysaccharide 80 - 100 g / L, agar 5 - 7 g / L, with the balance being water and the pH value being 5.8; The anther induction culture conditions are as follows: the temperature is 35 - 37 °C and the time is 24 - 30 h.

4. The method according to claim 1, wherein In step (2), the composition of the differentiation medium is as follows: dextran 70 - 90 g / L, plant polypeptide 10 - 30 mg / L, carvacrol 10 - 30 mg / L, isorhamnetin 1 - 3 mg / L, crude toxin extract 1 - 2 mg / L, agar 5 - 6 g / L, with the balance being water and the pH value being 5.8; The differentiation culture conditions are as follows: the temperature is 25 - 27 °C, the light intensity is 1500 - 2000 Lux, and the photoperiod is 12 h light / 12 h dark.

5. The method according to claim 1, characterized in that, In step (2), when performing differentiation culture, the crude toxin concentration level added to the differentiation medium with the anther callus differentiation rate of 1 - 1.5% for both the high resistance resources to stripe rust and the main breeding parents is screened, and the crude toxin level of the average of the two is determined as the screening pressure of the stripe rust crude toxin.

6. The method according to claim 1, wherein In step (4), the composition of the anther induction medium II is as follows: NH4NO3 200 - 400 mg / L, KNO3 1200 - 1500 mg / L, KH2PO4 400 - 600 mg / L, CaCl2 100 - 200 mg / L, MgSO4·7H2O 100 - 200 mg / L, Na2-EDTA 35 - 36 mg / L, FeSO4·7H2O 25 - 28 mg / L, MnSO4·4H2O 3 - 5 mg / L, ZnSO4·7H2O 1 - 3 mg / L, H3BO3 1 - 2 mg / L, KI 0.5 - 1.5 mg / L, CaSO4·5H2O 0.1 - 0.3 mg / L, CoCl2·6H2O 0.1 - 0.3 mg / L, glycine 2 - 3 mg / L, vitamin B1 0.5 - 1.5 mg / L, vitamin B6 0.5 - 1.5 mg / L, nicotinic acid 0.5 - 1 mg / L, inositol 100 - 300 mg / L, KT 1 - 3 mg / L, IAA 0.5 - 1 mg / L, glutathione 180 - 200 mg / L, casein hydrolysate 0.3 - 0.5 g / L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 100 - 120 mg / L, lactic acid 40 - 60 mg / L, quercitol 5 - 10 mg / L, psicose 70 - 80 mg / L, agar 5 - 7 g / L, with the balance being water and the pH value being 5.8; The anther induction culture conditions are: temperature 35 - 37°C, time 24 - 30 h.

7. The method according to claim 1, characterized in that In step (4), the differentiation culture conditions are: temperature 25 - 27°C, light intensity 1500 - 2000 Lux, light cycle 12 h light / 12 h dark.

8. The method according to claim 1, wherein In step (5), the rooting culture conditions are: temperature 27 - 29°C, light intensity 2500 - 3000 Lux, light cycle 12 h light / 12 h dark.

9. The method according to claim 1, characterized in that In step (5), the acclimatization process is as follows: Open the sealing film of the culture bottle for rooting culture, carry out bottle-opening acclimatization in the culture room for 4 - 6 days, and then transfer it to a place with a light intensity of 4000 - 6000 Lux, a temperature of 25 - 30°C, and a humidity of 70 - 85%, and acclimatize with natural light for 7 - 10 days.

10. The method according to claim 1, characterized in that, In step (6), the soaking conditions are: room temperature, 24 - 28 h.