Culture method for improving emergence rate of wheat haploid

Abstract

The invention discloses a culture method for increasing the emergence rate of wheat haploids, and belongs to the technical field of seed and seedling cultivation. The method comprises the following specific operation steps: disinfecting and sterilizing young embryos obtained through wheat and corn hybridization when the size of the young embryos is 0.5-1.0 mm, inoculating the young embryos into a breathable culture bottle containing a 1 / 2MS culture medium, culturing the young embryos on a culture shelf with the temperature of 23-25 DEG C and the LED light source of red-green (R: G = 4: 1, R4G1) for 7-10 days, turning off the LED light source after the young embryos germinate, continuing dark culture for 10-15 days, and then transferring the young embryos to a common light source to grow into seedlings. By combining the immature embryo rescue technology and the culture treatment of the LED light source after inoculation, the emergence rate of the wheat haploid can be remarkably increased, direct'one-step seedling formation 'is achieved, and the workload is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of seed and seedling cultivation, and specifically to a cultivation method for improving the emergence rate of wheat haploids. Background Art

[0002] As an efficient breeding method, wheat doubled haploid (DH) breeding technology can achieve gene homozygosity within 1-2 generations, quickly fix genetic variations, and provide important technical support for the rapid breeding of new wheat varieties. Among them, the method of inducing wheat haploids by wheat×maize hybridization is the most widely used wheat DH production technology at home and abroad.

[0003] The prerequisite for applying this technology to breeding is to obtain a large number of haploid seedlings. However, due to the serious endosperm abortion phenomenon in the hybrid caryopses produced by this technology, which cannot provide nutrition for the normal growth and development of haploid embryos, it is necessary to carry out embryo rescue in a timely manner to promote the germination of wheat haploid embryos into seedlings. Research has pointed out that the emergence rate of wheat haploid embryos (the percentage of wheat haploid seedlings to the number of inoculated embryos) and the emergence speed of embryos are affected by multiple factors such as embryo age, embryo size, medium type, and culture conditions. Currently, the most commonly used embryo rescue method is as follows: The hybrid caryopses are cultured for 14-16 days, the embryo size is 0.5-1.0 mm, the haploid embryos are taken out and inoculated into 1 / 2MS medium, and cultured in a dark environment at 23°C. After the coleoptile length of the embryo reaches 1-2 cm, the emerged embryos are taken out in turn and transferred to another culture bottle containing 1 / 2MS medium for light culture (the culture conditions are: 23°C, light intensity of 5000 lx, and photoperiod of 16 h), while the unemerged embryos continue to be cultured in the dark.

[0004] However, in large-scale production, the above method has the following problems: 1. The average embryo emergence rate is relatively low (30-60%) and the stability is poor; 2. There are significant differences in the emergence speed of embryos in the same culture bottle, and the emergence uniformity is poor. It is impossible to directly carry out light culture for the whole bottle, and the germinated seedlings need to be taken out gradually, which is laborious and increases the risk of contamination.

[0005] During the process of plant tissue culture, environmental factors (such as light, temperature, and humidity) directly regulate the morphogenesis, growth and development, and material synthesis of seedlings. Among them, light quality plays an extremely important role in the growth and differentiation of plant cells, tissues, and organs. Light quality refers to the light with different wavelengths (300 - 2600 nm) in sunlight, which is divided into visible light and invisible light. Visible light can be divided into seven colors of light: red, orange, yellow, green, cyan, blue, and violet according to wavelength. This part of light can directly affect the growth and development and material synthesis of tissue-cultured seedlings. However, there is a close relationship between the growth and development of tissue-cultured seedlings of different plants and the light quality composition. Therefore, in actual tissue-culture production, the responses of different plant growth and development stages to different light qualities still need to be analyzed and studied specifically. Under controllable environmental conditions, using light-emitting diodes (LEDs, which have the characteristics of low energy consumption, long service life, environmental protection, and low heat generation) can significantly reduce energy consumption, and can freely combine into various composite light qualities according to the growth needs of plants themselves, improving tissue-culture efficiency.

[0006] The emergence of wheat haploid young embryos is a necessary condition for obtaining wheat doubled haploids. Conventional research mainly focuses on the effects of the embryo age for embryo rescue, culture medium, and culture temperature, etc. on the emergence rate, and there are few reports on promoting the emergence of young embryos using different light quality ratios. Summary of the Invention

[0007] In view of the above problems, the present invention provides a culture method for improving the emergence rate of wheat haploids. After inoculation, the culture treatment with an LED light source (different light quality ratios) can significantly improve the emergence rate of wheat haploids.

[0008] One technical object of the present invention is to provide a culture method for improving the emergence rate of wheat haploids, including the following steps:

[0009] S1. Take out the wheat hybrid ears that have been in vitro cultured in an artificial climate incubator for 14 - 16 days, peel the hybrid caryopses, and pre-screen the caryopses with embryos.

[0010] S2. Sterilize the caryopses with embryos using sodium hypochlorite solution for 30 minutes, take out the young embryos under a dissecting microscope, and inoculate them into a breathable culture bottle containing 1 / 2 MS medium.

[0011] S3. After inoculation, place the culture bottle on a culture rack with an LED light source of red - green for 7 - 10 days, where the light quality ratio of red - green is 4:1. After the young embryos germinate, turn off the LED light source and continue dark culture for 10 - 15 days, and then turn on the ordinary light source and continue culture for 7 - 10 days.

[0012] Further preferably, in step S1, the temperature of the artificial climate incubator is 23°C, the humidity is 80%, the light intensity is 10000 Lx, and the light duration is 14 h / darkness 10 h.

[0013] Further preferably, in step S2, the concentration of the sodium hypochlorite solution is 15%.

[0014] Further preferably, in step S3, the culture flask is cultured at a temperature of 23-25 °C.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] Firstly, the present invention effectively improves the production efficiency of wheat doubled haploids, can significantly shorten the emergence time of wheat haploid immature embryos, solves the problem of inconsistent germination of immature embryos, reduces the workload of seedling transfer, and at the same time increases the emergence rate of immature embryos by about 20%.

[0017] Secondly, the method adopted by the present invention is energy-saving and environment-friendly. The LED light source has low energy consumption and long service life, and is suitable for large-scale application.

[0018] Thirdly, the method of the present invention is easy to operate, the method is easy to standardize, is suitable for laboratory and industrial production, and has broad application prospects in wheat haploid breeding and other crop breeding. Detailed implementation mode

[0019] In order to enable those skilled in the art of the present technology 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.

[0020] Example 1

[0021] A culture method for improving the emergence rate of wheat haploids. After sterilizing wheat hybrid caryopses, they are inoculated onto a 1 / 2MS medium and cultured on a culture rack at 23-25 °C with an LED light source of red-green (R:G = 4:1, R4G1) for 7-10 days to promote the collective germination of immature embryos into seedlings. The rescue of immature embryos is changed from "transferring seedlings bottle by bottle operation" to "batch operation", reducing the workload, improving the emergence efficiency of immature embryos, and increasing the emergence rate of wheat haploids by 20%.

[0022] Specifically, the present invention includes the following steps:

[0023] 1) Take out wheat hybrid spikes (wheat-corn hybrids) that have been in vitro cultured for 14-16 days in an artificial climate incubator (temperature 23 °C, humidity 80%, light intensity 10000 Lx, light duration 14 h / dark 10 h), peel the hybrid caryopses, pre-inspect and screen out caryopses with embryos, and the size of the immature embryos is 0.5-1.0 mm.

[0024] 2) Sterilize the caryopses with embryos using sodium hypochlorite solution for 30 minutes. Under a dissecting microscope, take out the young embryos and inoculate them into a breathable culture flask containing 1 / 2 MS medium; its concentration is 15%; the preparation method is: take 150 ml of sodium hypochlorite solution and put it into 850 ml of sterile water.

[0025] 3) After inoculation, place the culture flask on a culture rack at 23 - 25°C with an LED light source of red - green (R:G = 4:1, R4G1) and culture for 7 - 10 days. After the young embryos germinate, turn off the LED light source and continue dark culture for 10 - 15 days, then turn on the ordinary light source and continue culture for 7 - 10 days until the wheat haploid plants grow to 3 leaves.

[0026] It should be understood that the temperature, humidity, and light intensity of the artificial climate incubator in step 1) of the present invention, and the temperature of the culture room in step 3) are not the core content of this method. However, to ensure the integrity and operability of this young embryo culture method, specific requirements are imposed on steps 1) and 3). The core content of this method is that after inoculation, batch LED light source treatment is carried out, which can significantly improve the emergence rate of all wheat materials.

[0027] Example 2

[0028] In May - June 2023, using Yunmai 112 DH line as the material, after inoculating the young embryos, study the effect of different ratios of LED light source treatment on the emergence rate of Yunmai 112 wheat haploids. The specific operation steps are as follows:

[0029] 1) Take out the wheat hybrid ears that have been in vitro cultured for 14 - 16 days in an artificial climate incubator (temperature 23°C, humidity 80%, light intensity 10000 Lx, light duration 14 h / darkness 10 h), peel the hybrid caryopses, pre - examine and screen out the caryopses with embryos, and the size of the young embryos is 0.5 - 1.0 mm.

[0030] 2) Sterilize the caryopses with embryos using 15% sodium hypochlorite solution for 30 minutes. Under a dissecting microscope, take out the young embryos and inoculate them into a breathable culture flask containing 1 / 2 MS medium, and evenly inoculate 20 haploid young embryos in each flask.

[0031] 3) Divide the culture flasks into 12 portions, with 6 flasks in each portion (a total of 120 wheat haploid young embryos), and place them under 12 different pre - prepared and installed LED light sources (the control is in the dark condition) for 7 - 10 days respectively. The temperature of the culture room is 23°C. Then turn off all light sources and conduct dark culture for 14 days, and then conduct statistics on the emergence rate.

[0032] The test results are summarized in Table 1. It can be seen from the results that under the illumination of the LED light source with red - green (R:G = 4:1, R4G1), the emergence rate can be significantly improved, which is 20.83% higher than that of the control (cultured only under dark conditions). In addition, the emergence rate of the light source with red - purple (R:P = 1:1, RP) is slightly higher than that of the control, while the emergence rates under the other 9 light source conditions are significantly lower than that of the control. Moreover, after the treatment with the light source R4G1, the emergence rate of wheat haploid immature embryos is relatively fast, the root system is developed and the growth is neat. There is no need to remove the overly tall seedlings for light culture during dark culture, and the whole batch can be directly subjected to light culture for seedling strengthening.

[0033] Table 1. Effects of different LED light sources on the emergence rate of wheat (Yunmai 112) haploid immature embryos in 2023

[0034]

[0035] Example 3

[0036] From July to August 2023, using the Yunmai 114 DH line as the material, after inoculating the immature embryos, the effects of different ratios of LED light source treatments on the emergence rate of wheat haploids of Yunmai 114 were studied. The specific operation steps are as follows:

[0037] 1) Take out the wheat hybrid ears that have been in vitro cultured for 14 days in an artificial climate incubator (temperature 23°C, humidity 80%, light intensity 10000 Lx, light duration 14 h / dark 10 h), peel the hybrid caryopses, and pre - screen the caryopses with embryos.

[0038] 2) Sterilize the caryopses with embryos with 15% sodium hypochlorite solution for 30 minutes, take out the immature embryos under a dissecting microscope, and inoculate them into a breathable culture bottle containing 1 / 2 MS medium. Uniformly inoculate 20 haploid immature embryos in each bottle.

[0039] 3) Divide the culture bottles into 12 portions, with 10 bottles in each portion (a total of 200 wheat haploid immature embryos), and place them under 12 different LED light sources (the control is dark conditions) that have been prepared and installed in advance for 7 - 10 days. The temperature in the culture room is 23°C. Then turn off all light sources for dark culture for 14 days, and then conduct the statistics of the emergence rate.

[0040] The test results are summarized in Table 2. It can be seen from the results that under the illumination of the LED light source with red - green (R:G = 4:1, R4G1), it is significantly 22% higher than that of the control (dark conditions). In addition, the emergence rate of the light source with red - blue (R:2B = 1:2, R2B) is slightly higher than that of the control, while the emergence rates under the other 9 light source conditions are significantly lower than that of the control. Similarly, after the treatment with the light source R4G1, the emergence rate of wheat haploid immature embryos is uniform and fast, and the root system is in good condition. The whole batch can be directly subjected to light culture for seedling strengthening.

[0041] Table 2. Effects of Different LED Light Sources on the Emergence Rate of Haploid Young Embryos of Wheat (Yunmai 114) in 2023

[0042]

[0043] The above embodiments are only explanations of the present invention, and they are not limitations of the present invention. Those skilled in the art can make modifications to these embodiments without creative contributions according to their 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 cultivation method for improving the haploid emergence rate of wheat, characterized in that: The following steps are involved: S1. Take out the wheat hybrid ears cultured in vitro for 14-16 days in an artificial climate incubator, peel off the hybrid caryopsis, and pre-screen and screen out the embryonic caryopsis; S2. Sterilize the embryo caryopsis with sodium hypochlorite solution for 30 minutes, remove the immature embryo under a dissecting microscope, and inoculate it into a breathable culture bottle containing 1 / 2MS culture medium; S3. After the inoculation is completed, the culture bottle is placed on a culture rack with a red-green LED light source and cultured for 7-10 days, wherein the red-green light quality ratio is 4:

1. After the young embryos germinate, the LED light source is turned off and dark culture is continued for 10-15 days, and then the ordinary light source is turned on and culture is continued for 7-10 days.

2. A cultivation method for improving the haploid emergence rate of wheat according to claim 1, characterized in that: In step S1, the temperature of the artificial climate incubator is 23°C, the humidity is 80%, the light intensity is 10000Lx, and the light duration is 14h / darkness 10h.

3. A cultivation method for improving the haploid emergence rate of wheat according to claim 1, characterized in that: In step S2, the concentration of the sodium hypochlorite solution is 15%.

4. A cultivation method for improving the haploid emergence rate of wheat according to claim 1, characterized in that: In step S3, the culture bottle is placed at a temperature of 23-25°C for cultivation.

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