Method for improving caryopsis rate of highland barley haploid induced by highland barley and corn hybridization

Abstract

The invention belongs to the field of plant biotechnology and highland barley breeding, and particularly relates to a method for improving the caryopsis rate of highland barley haploid induced by highland barley and corn hybridization. Highland barley ears are cut, pollinated and then taken back to a laboratory, the highland barley ears are inserted into a nutrient solution and placed in a growth room for in-vitro culture, a 50 mg / L 2, 4-D solution is sprayed in a concentrated mode after 24 hours, a 50 mg / L gibberellin solution is sprayed in a concentrated mode after 5 hours, and the highland barley ears are placed in the growth room for culture for 12 days after being dried. By spraying the 2, 4-D and GA3 solutions before and after, the thousand seed weight and plumpness of the caryopses are greatly improved, the caryopses rate can reach 90% or above, the embryo formation rate is effectively improved, and large-scale production of highland barley double haploids induced through highland barley and corn hybridization is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical fields of plant biotechnology and hulless barley breeding technology, and particularly relates to a method for improving the caryopsis rate of haploid hulless barley induced by hulless barley - maize hybridization. Background Art

[0002] Hulless barley ( Hordeum vul gare L. var. nudum HooK. f. ) belongs to the Gramineae family of plants, also known as naked barley, which is a special type of barley. With the completion of the hulless barley genome sequencing and the application of modern molecular breeding techniques, combined with off - site generation addition in Yuanmou, Yunnan, it still takes 5 - 8 years to obtain a homozygous and stable strain. Moreover, as the number of target traits aggregated in breeding increases, the stabilization time of hybrid offspring also lengthens. How to quickly fix excellent variations has become one of the key factors restricting the efficiency of hulless barley breeding. The doubled haploid (DH) technology can make any first - generation hybrid homozygous and stable, and combined with molecular breeding techniques, it can construct an accurate, rapid and efficient breeding technology system.

[0003] Regarding haploid breeding of barley, as early as 1934, Johansen discovered haploid barley plants. However, the frequency of spontaneous or induced haploids is too low to be used for breeding. Currently, the three main methods widely used in barley genetic breeding are the Hordeum bulbosum method, anther culture method, and microspore culture method, etc. The Hordeum bulbosum method was first proposed by Kasha and Kao (1970). Its principle is that after crossing Hordeum bulbosum with cultivated barley, the chromosomes of Hordeum bulbosum are lost during the development of the zygotic embryo, and a haploid embryo containing only cultivated barley chromosomes is obtained. After in vitro embryo rescue and chromosome doubling, a doubled haploid plant of cultivated barley is obtained. Some varieties and DH lines have been developed using this method at home and abroad. However, the Hordeum bulbosum method is affected by many factors such as the genotype of the parents, the growth conditions of Hordeum bulbosum, pollination techniques and conditions, and in vitro embryo culture conditions, making it difficult to be widely applied to barley haploid breeding on a large scale.

[0004] Anther culture and microspore culture can also produce barley DH. As early as the 1970s, Clapham reported the research on obtaining barley anther callus. After more than 20 years of improvement research, a mature anther culture technology method has been formed. The research by Lu Ruiju (2014) et al. first reported the technology of microspore regeneration into seedlings of hulless barley. Some barley and hulless barley varieties have also been developed using these two methods at home and abroad. However, both of these two methods have the problem that the DH production efficiency is limited by the barley genotype, and some genotypes cannot produce DH, which restricts the popularization and application of these two methods in breeding.

[0005] The method of inducing wheat haploid by distant hybridization between wheat and maize reported by Laurie et al. (1986) has no restriction on wheat genotypes, and wheat haploid can be obtained from all wheat genotypes. This method has the same principle of haploid production as Hordeum bulbosum × Hordeum vulgare, which may help to solve the problem of genotype restriction in hulless barley haploid production, but there is no such research report at home and abroad. At present, there are few studies on inducing hulless barley haploid by hulless barley-maize hybridization at home and abroad. The number of DHs produced by this method is small, the efficiency is low, and it is difficult to apply to the breeding of hulless barley.

[0006] In view of the above analysis, the technical problems urgently to be solved in the prior art are as follows: At present, the breeding of hulless barley is still traditional cross-breeding. The technical method of inducing double haploid of hulless barley by hulless barley-maize hybridization is not mature. The main problems include poor quality of induced caryopses, low caryopsis rate and embryo rate, and it is difficult to produce hulless barley double haploid on a large scale. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for improving the caryopsis rate of inducing hulless barley haploid by hulless barley-maize hybridization, which can effectively improve the embryo formation rate and is beneficial to the large-scale production of hulless barley double haploid by hulless barley-maize hybridization.

[0008] The above technical purpose of the present invention is achieved by the following technical solutions, including the following steps: Step 1: After emasculating the hulless barley, pollinate with maize pollen 3-4 days later. After 24 hours of pollination, spray 25-100 mg / L 2,4-D solution on the florets of the wheat ear, and place the wheat ear to dry at the ventilation opening. Step 2: 5 hours after spraying the 2,4-D solution, spray 25-100 mg / L GA3 solution on the florets of the wheat ear. After drying at the ventilation opening, place it in a growth chamber for 12 d.

[0009] Further preferably, in Step 1, the concentration of the 2,4-D solution is 50 mg / L.

[0010] Further preferably, in Step 2, the concentration of the GA3 solution is 50 mg / L.

[0011] Further preferably, in Step 3, the temperature of the growth chamber is 23 °C, the humidity is 85%, the light intensity is 10000 Lx, and the light duration is 14 h / dark 10 h.

[0012] In summary, the present invention has the following beneficial effects: First, the expected benefits and commercial values after the transformation of the technical solution of the present invention are: The height of haploid embryo rate is the premise basis that highland barley and corn hybridization induce to produce double haploid, and high-quality caryopsis is the key guarantee of haploid embryo normal growth and development. At present, domestic highland barley double haploid breeding demand is larger, and the demand of annual double haploid is about 5000-10000 strain, but the production efficiency of highland barley double haploid is lower at present, and market price is 500 yuan / strain, and production cost and use cost are higher, and breeding unit can not afford high amount breeding cost, greatly hinders the fast development of highland barley double haploid breeding. At present, only this team is carried out in domestic highland barley corn hybridization induction to produce double haploid commercial service, hormone treatment method provided by the present invention can improve haploid caryopsis rate and caryopsis quality, makes caryopsis rate reach more than 90%, and embryo rate improves 10%, and this technical method can improve the production efficiency of highland barley double haploid, thus reduces production cost. By using this technical method, the production price of double haploid highland barley can be reduced to 300 yuan per plant. By producing at least 5,000 DH strains per year, a commercial profit of about 1.5 million yuan can be obtained.

[0013] Second, the technical solution of the present invention fills the technical gaps in the industry at home and abroad: Currently, there are no reports on the production of doubled haploids using highland barley and corn hybridization. The present invention technically addresses the problems of low 1000-grain weight, underdeveloped grains, and low caryopsis rate in highland barley haploids. Through the hormone treatment method of the present invention, the embryo yield, caryopsis rate, and caryopsis quality of highland barley haploids are significantly improved. This provides strong technical support for the production of doubled haploids using highland barley and corn hybridization.

[0014] Third, the technical solution of the present invention overcomes technical prejudice: Currently, wheat doubled haploid technology is the most mature among wheat crops and has been widely used in wheat breeding. A common hormone treatment method after wheat-corn hybridization is to spray the entire wheat ear with a 100 mg / L 2,4-D solution 24 hours after pollination. This method can later produce high-quality caryopsis and a high embryo yield. Foreign countries have used a method of spraying the entire wheat ear with a 75 ppm GA3 solution 24 hours after pollination for bulbous barley-barley hybridization, which can later produce haploid embryos. The present invention, which sprays 50 mg / L of 2,4-D and GA3 separately 24 hours after pollination for highland barley-corn hybridization, produces caryopsis of poor quality, with a maximum caryopsis yield of only 16.88%. Using the hormone spraying method of the present invention, spraying 50 mg / L of 2,4-D followed by 50 mg / L of GA3 after pollination, can produce high-quality caryopsis with a caryopsis yield exceeding 90%.

[0015] In terms of industrial applications, the use of the hormone treatment method of the present invention significantly improves the production efficiency of hulless barley doubled haploids, reduces manpower and resource consumption, greatly shortens the breeding cycle of hulless barley varieties, enables breeding institutions to quickly homozygously stabilize a large number of materials in a short period of time, speeds up the hulless barley variety breeding process, and significantly improves the efficiency and success rate of hulless barley breeding. At the same time, it provides a stable and reliable technical guarantee for the industrial production of hulless barley doubled haploids. Description of the Drawings

[0016] Figure 1 Observation of caryopsis development after 12 days of in vitro culture of hulless barley ears. Figure A shows the caryopsis development status of hulless barley variety Zangqing 3000 after spraying different hormones, and Figure B shows the comparison of caryopsis status between spraying 2,4-D alone and spraying 50 mg / L of GA3 after spraying 50 mg / L of 2,4-D first. Detailed Embodiments

[0017] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0018] Embodiment 1 A hormone treatment method for increasing the caryopsis rate of hulless barley haploids. After 24 hours of culture in a growth chamber after pollination of hulless barley, first spray 50 mg / L of 2,4-D, and then spray 50 mg / L of GA3 after 5 hours. Compared with the traditional single hormone spraying method, this technical method significantly increases the caryopsis rate and caryopsis quality of hulless barley haploids, and increases the embryo rate of wheat haploids by 10%.

[0019] Specifically, the present invention includes the following steps: (1) After emasculating hulless barley, wait for 3 - 4 days until the stigmas at the base of the ear are feathery and spread out, cut along the roots on the ground, and take fresh corn pollen for pollination; ( ) Insert the pollinated ears into the nutrient solution and place them in a growth chamber for culture (temperature 23 °C, humidity 85%, light intensity 10000 Lx, light duration 14 h / dark 10 h); (3) After 24 hours of pollination, spray the florets of the ears with 2,4-D solution using a sprayer, and place the ears to dry at the ventilation opening; (4) After 5 hours of spraying the 2,4-D solution, spray the GA3 solution using the same operation method, dry at the ventilation opening and then place it in the growth chamber for culture for 12 d. During the culture period, change the culture solution every 4 days, and cut off the yellowed part from the bottom when changing the culture solution to ensure smooth nutrient absorption of the ears (temperature 23 °C, humidity 80%, light intensity 10000 Lx, light duration 14 h / dark 10 h).

[0020] It should be understood that the temperature, humidity and light intensity in the growth chamber in steps (2) and (4) of the present invention are not the core content of this method. However, to ensure the integrity and operability of this hormone treatment method, specific requirements are imposed on steps (2) and (4). The core content of this method is that after pollination, the wheat ears are first sprayed with 2,4-D treatment at 50 mg / L, and after the wheat ears are dried, they are sprayed with GA3 at 50 mg / L, which can significantly improve the caryopsis rate and caryopsis quality of all hulless barley materials.

[0021] Example 2 Effects of different concentrations of 2,4-D and GA3 on caryopsis rate and caryopsis quality.

[0022] Using the hulless barley variety Zangqing 3000 as the material, 24 h after pollination, the effects of spraying different concentrations of 2,4-D and GA3 on caryopsis rate and embryogenesis rate were studied. The specific operation steps are as follows: 1) Before the hulless barley variety heads, remove the wrapped flag leaves, use forceps to remove the stamens in each floret, put on a hybridization bag, and check whether the stigmas are feathery and spread out on the 3rd - 4th day; 2) Select wheat ears in the same state and cut them at the roots near the ground. Roll the wheat ears in the collected corn pollen 1 - 2 times to ensure that each floret is covered with corn pollen; 3) Insert the pollinated wheat ears into the nutrient solution and place them in the growth chamber for cultivation; 4) Spray different concentrations of GA3 and 2,4-D solutions 24 h after pollination. After spraying, place the wheat ears in a ventilated place for natural drying; 5) After completion, continue to cultivate in the growth chamber for 12 d and change the nutrient solution every 4 d.

[0023] The test results are summarized in Table 1. It can be seen from the results that when different concentrations of 2,4-D were sprayed alone 24 h after pollination, the caryopsis rate of the treatment with 50 mg / L 2,4-D was the highest (16.88%), but the overall caryopsis development was incomplete, the caryopsis quality was poor, shrunken and yellowish, the 1000-grain weight was low, and the number of effective caryopses was small ( Figure 1 ). When different concentrations of GA3 were sprayed alone after pollination, it could not effectively induce the development of hulless barley caryopses. The highest caryopsis rate of spraying 50 mg / L GA3 was only 3.41%.

[0024] Table 1 Effects of different concentrations of 2,4-D and GA3 on caryopsis rate Example 3 Using the hulless barley varieties Zangqing 3000, Zangqing 2000, Yunqing 604 and Yunqing 602 as materials, the specific operation steps are the same as those in Example 1. The test results are summarized in Table 2. It can be seen from the results that after pollination for 24 h, 2,4-D at a concentration of 50 mg / L was sprayed first, and then GA3 at the same concentration was sprayed 5 h later. Compared with the separate spraying of the two hormones in Example 2, the caryopsis rate increased by about 80%, the caryopses were plump, the effective caryopsis rate reached over 90%, and the high embryo rate reached over 11%, effectively improving the production efficiency of haploid embryos of hulless barley.

[0025] Table 2 Effects of spraying 2,4-D first and then GA3 after pollination on caryopsis rate The above embodiments are only explanations of the present invention, and they do not limit the present invention. Those skilled in the art can make modifications to the embodiments without creative contributions according to needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for improving the caryopsis rate of haploid hulless barley induced by hulless barley-corn hybridization, characterized in that, It includes the following steps: Step 1: After emasculating the hulless barley, pollinate it with corn pollen 3 - 4 days later. 24 hours after pollination, spray a 25 - 100 mg / L 2,4-D solution on the florets of the wheat ear, and place it at the ventilation opening to dry the wheat ear. Step 2: 5 hours after spraying the 2,4-D solution, spray a 25 - 100 mg / L GA3 solution on the florets of the wheat ear. After drying at the ventilation opening, place it in a growth chamber and culture for 12 days.

2. The method for improving the caryopsis rate of haploid hulless barley induced by hulless barley and corn hybridization according to claim 1, characterized in that In Step 1, the concentration of the 2,4-D solution is 50 mg / L.

3. A method for increasing the caryopsis rate of haploid hulless barley induced by hulless barley-corn hybridization according to claim 1, characterized in that, In Step 2, the concentration of the GA3 solution is 50 mg / L.

4. A method for improving the caryopsis rate of haploid hulless barley induced by hulless barley and corn hybridization according to claim 1, characterized in that, In Step 3, the temperature in the growth chamber is 23 °C, the humidity is 85%, the light intensity is 10000 Lx, and the light duration is 14 h / darkness 10 h.

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