A medium combination and embryo rescue method for broccoli and chinese cabbage distant hybrid embryo rescue

Abstract

This invention discloses a culture medium combination and embryo rescue method for distant hybridization of broccoli and Chinese cabbage, belonging to the field of hybridization breeding technology. Based on the growth characteristics of broccoli and Chinese cabbage, this invention establishes an embryo rescue technology system suitable for distant hybridization of broccoli and Chinese cabbage, including germination medium, proliferation medium, and rooting medium, as well as an embryo rescue method. The method of this invention can overcome reproductive barriers in distant hybridization and efficiently create new germplasm for cruciferous vegetables. Through in vitro culture, this method rescues immature hybrid embryos, overcomes interspecific reproductive isolation, and achieves gene aggregation of the disease resistance and high quality of broccoli with the early maturity and wide adaptability of Chinese cabbage. This provides a reference for improving distant hybridization technology in cruciferous vegetables, creating new germplasm that combines the advantages of both parents, and promoting vegetable germplasm innovation and industrial upgrading.

Description

Technical Field

[0001] This invention relates to the field of hybridization breeding technology, and in particular to a culture medium combination and embryo rescue method for rescuing embryos from distant hybridization of broccoli and Chinese cabbage. Background Technology

[0002] Embryo rescue technology, as an important means of obtaining distant hybrid offspring, occupies a pivotal position in the field of plant genetics and breeding. This technology, through the cultivation of distant hybrid offspring, can significantly increase the probability of obtaining interspecific hybrids, greatly accelerate the transfer of superior traits between species, and powerfully promote the innovative development of germplasm resources. In practical applications, Yu Hailong conducted related research in 2015. Ogura CMS kale was hybridized with Brassica napus restoration material, and embryo rescue technology was used to successfully cultivate interspecific restoration hybrids of kale. Zhang Shuangshuang et al. also conducted similar research in 2021, hybridizing Chinese cabbage and Ethiopian mustard, and using embryo rescue technology to cultivate interspecific hybrids with strong resistance to black rot. Researchers such as Lü Fengxian used Chinese cabbage and kale as parents to hybridize, and on the basis of the hybrid offspring, used embryo rescue technology to obtain interspecific hybrids, and successfully bred a new type of oilseed rape vegetable by chromosome doubling treatment. Peng Lisha et al. carried out related research in 2016. They selected parental materials with clubroot resistance characteristics from Brassica napus and Chinese cabbage, and successfully cultivated new interspecific germplasm by using interspecific hybridization as a key technical means, combined with embryo rescue technology.

[0003] Broccoli is a mainstream vegetable widely cultivated internationally, but broccoli breeding research in China started relatively late, resulting in a scarcity of superior resources. The "National Major Scientific Research Joint Project on Broccoli Varieties," launched in 2018, has yielded significant results, successfully cultivating 23 varieties with independent intellectual property rights. However, the widespread outbreak and spread of clubroot disease has become a major bottleneck restricting broccoli yield increases, severely impacting the high-quality development and economic benefits of the broccoli industry. Currently, due to the extreme scarcity of highly clubroot-resistant resources in cultivated broccoli, future research needs to focus on closely related and wild species. It is hoped that resistance genes contained in these resources can be transferred to cultivated crops through backcrossing or distant hybridization. This initiative will provide crucial foundational materials for disease-resistant breeding of broccoli, as well as the discovery and research of disease-resistant genes, helping to improve broccoli's resistance to clubroot and promoting the sustainable development of the broccoli industry. Chinese cabbage, as a cruciferous crop rich in clubroot resistance genes, has become a key gene donor for disease-resistant breeding. However, there are natural obstacles to the distant hybridization of broccoli and Chinese cabbage. Due to endosperm degeneration and insufficient nutrient supply, hybrid embryos are prone to abortion, making it difficult to successfully transmit disease-resistant genes. This problem has long restricted the progress of disease-resistant breeding in broccoli. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium combination and embryo rescue method for distant hybridization of broccoli and Chinese cabbage, thereby solving the problems existing in the prior art. Based on the growth characteristics of broccoli and Chinese cabbage, this invention establishes an embryo rescue technology system suitable for distant hybridization of broccoli and Chinese cabbage, including germination culture medium, proliferation culture medium, and rooting culture medium, as well as an embryo rescue method. The method of this invention can overcome reproductive barriers in distant hybridization and efficiently create new germplasm for cruciferous vegetables.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a culture medium combination for rescuing embryos from distant hybrids of broccoli and Chinese cabbage, comprising a germination medium, a proliferation medium, and a rooting medium; The germination medium is ① or ②: ①MS+0.5-1mg / L 6-BA+0.1mg / L NAA+1mg / L CH+1g / L AC; ②MS+0.5-1mg / L 6-BA+0.1-0.5 mg / L NAA+1mg / L KT+1mg / L CH+1g / L AC; The proliferation medium was: MS + 0.2-0.9 mg / L 6-BA + 0.1-0.5 mg / L NAA; The rooting medium was: MS + 0.5-0.9 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC.

[0006] Furthermore, the germination medium is ① or ②: ①MS+1mg / L 6-BA+0.1mg / L NAA+1mg / L CH+1g / L AC; ②MS+1mg / L 6-BA+0.1mg / L NAA+1mg / L KT+1mg / L CH+1g / L AC.

[0007] Furthermore, the germination medium is: MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 1 mg / L CH + 1 g / L AC.

[0008] Furthermore, the proliferation medium is: MS + 0.9 mg / L 6-BA + 0.1 mg / L NAA.

[0009] The present invention also provides the application of the above-mentioned culture medium combination in the rescue of embryos from distant hybrids of broccoli and Chinese cabbage.

[0010] This invention also provides a method for rescuing embryos from distant hybrids of broccoli and Chinese cabbage, comprising the following steps: The hybrid ovules were extracted 12-14 days after the cross-pollination of broccoli and Chinese cabbage. The hybrid ovules were inoculated into the above germination medium and cultured for differentiation to obtain adventitious buds; The adventitious buds were inoculated into the above-mentioned proliferation medium and cultured to obtain clustered buds; The clustered buds were inoculated in 1 / 2 MS medium. After 3 pairs of true leaves sprouted, they were transferred to the above-mentioned rooting medium for rooting culture and hardening off to obtain hybrid plants.

[0011] Furthermore, the differentiation culture temperature is 23±2℃, the light exposure time is 12h, the light intensity is 1500 lx, and the differentiation culture time is 7-15d.

[0012] Furthermore, the temperature for the propagation culture was 23±2℃, the light duration was 12h, the light intensity was 1500 lx, and the culture was carried out until at least two true leaves were fully expanded.

[0013] Furthermore, the rooting culture temperature is 23±2℃, the light duration is 12h, the light intensity is 1500 lx, and the culture is carried out until there are more than 3 main roots with a root length of more than 5mm.

[0014] The present invention discloses the following technical effects: Based on the growth characteristics of broccoli and Chinese cabbage, this invention establishes an embryo rescue technology system suitable for distant hybridization between broccoli and Chinese cabbage, including optimal germination medium, proliferation medium, and rooting medium, as well as embryo rescue methods. The method of this invention can overcome reproductive barriers in distant hybridization and efficiently create new germplasm for cruciferous vegetables.

[0015] Distant hybridization between broccoli and Chinese cabbage often fails to yield hybrid plants due to abnormal endosperm development and early zygotic embryo abortion. This invention utilizes in vitro culture to rescue immature hybrid embryos, overcoming interspecific reproductive isolation and achieving gene aggregation of the superior traits of broccoli (disease resistance and high quality) and Chinese cabbage (early maturity and wide adaptability). Its advantages are: 1. It can obtain stable and fertile interspecific hybrids, providing basic materials for chromosome engineering and gene introgression; 2. It can rapidly create new germplasm possessing the advantages of both parents, improving vegetable resistance, quality, and yield, significantly shortening the breeding cycle, increasing seed setting and seedling rates, obtaining virus-free healthy seedlings, improving seedling quality, and supporting green breeding; 3. It can improve the distant hybridization technology of cruciferous plants, providing methodological references for interspecific hybridization and wild resource utilization, and promoting vegetable germplasm innovation and industrial upgrading.

[0016] For endangered plants, embryo rescue can help preserve and propagate germplasm resources, and a mature embryo rescue system can form a standardized process. Therefore, the method of this invention provides a reference for hybridization breeding of other plants, and significantly improves breeding efficiency and innovation capabilities. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A diagram showing the state of hybrid ovules inoculated into germination medium; Figure 2 The embryo emergence of hybrid ovules on M6 germination medium; Figure 3 The diagram shows the growth status of adventitious shoots on the proliferation medium; where A represents the state after 15 days of proliferation culture; and B represents the state after 30 days of proliferation culture. Figure 4 The images show the seedling rooting culture and hardening-off stages; where A represents the seedlings after 15 days of cultivation on the rooting medium; B represents the seedlings after 20 days of hardening-off; and C represents the seedlings after 40 days of hardening-off. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0025] The broccoli and Chinese cabbage mentioned in the following examples were provided by the Vegetable Genomics and Genetic Improvement Laboratory of the College of Horticulture, Shenyang Agricultural University.

[0026] In the following embodiments, the abbreviations used have the following meanings: Table 1. List of Abbreviations Example 1 1. Interspecific hybridization Experimental materials: Qinghuacai Tailv 6 was used as the female parent, and Chinese cabbage CR510 (carrying resistance to clubroot disease) was used. CRA3.7 (Genes) as the father.

[0027] To ensure synchronized flowering of Chinese cabbage and broccoli, the sowing time was adjusted accordingly. Chinese cabbage underwent a second germination and sowing process in January of the following year. The first batch of treated Chinese cabbage began bolting on February 5, 2023, while broccoli began producing small heads and branching from January 30, 2023, reaching pollination standards by February 15, 2023. Hybridization and pollination between broccoli and Chinese cabbage were carried out during peak flowering. Two days before hybridization began, all fully bloomed Chinese cabbage inflorescences were removed, and isolation measures were implemented.

[0028] On the day of hybridization, all open flowers and some buds that, while not fully open, already have stigmas on the broccoli inflorescences are removed, leaving only the unopened buds for emasculation. Pollen from the flowers that opened that day on the bagged branches of the Chinese cabbage is then used to pollinate these treated buds, thus constructing a hybrid embryo from broccoli and Chinese cabbage. After pollination, the Chinese cabbage inflorescences are isolated in bags to avoid interference from non-parental pollen.

[0029] 2. Embryo rescue After interspecific hybridization and pollination of broccoli and Chinese cabbage, observe the condition of the seed pods and select plump individuals. After disinfecting the scissors with alcohol, cut off the hybrid seed pods from the base. Then, wrap the cut seed pods with a slightly damp paper towel, place them in a pre-prepared sealed bag, and put the sealed bag in an insulated container.

[0030] Before performing embryo rescue operations, the clean bench should be disinfected with ultraviolet light in advance. Hands and arms should be thoroughly sprayed with alcohol before entering the bench. The seed pods to be used should be washed with tap water at least three to five times to remove external dust and impurities. After absorbing excess water with filter paper, they should be placed on a filter screen and then placed in a small beaker for subsequent operations.

[0031] Place the prepared small beaker on a UV-sterilized clean bench and add enough 75% alcohol to completely cover the contents. Immerse for 50 seconds, then remove the alcohol. Add a 10% sodium hypochlorite solution to the beaker and let it soak for 15 minutes. After this step, pour out the sodium hypochlorite and rinse the seed pods four to five times with sterile water. After the final rinse, soak the seed pods in sterile water for 5 minutes.

[0032] After completing the disinfection process, the seed pod is quickly placed on sterile filter paper, and any residual moisture on its surface is carefully absorbed to ensure that no excess water droplets adhere. Then, a strictly sterilized scalpel is picked up, and with extremely gentle movements, the ovary is slowly cut along the ventral and dorsal sutures.

[0033] During the procedure, it is necessary to control the force at all times to avoid causing unnecessary damage to the ovules inside. The goal is to open the ovary precisely and gently, and then remove the ovules with the help of tweezers.

[0034] 2.1 Optimization of embryo rescue time Hybrid pods were harvested at 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 days post-hybridization. They were sterilized using the method described above. Plump, green hybrid ovules were selected and inoculated onto a culture medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 1 mg / L CH + 1 g / L AC). Each sampling time point was repeated three times, with 90-350 ovules inoculated each time. All inoculated ovules were cultured at (23±2) °C under 12 hours of light per day and a light intensity of 1500 lx.

[0035] Seven days after inoculation, the ovules were closely monitored for contamination. When the culture period reached 15 days, the embryo emergence rate (embryo emergence rate = number of embryos / number of ovules cultured) was statistically analyzed at different sampling time points. Through in-depth comparison and analysis of the embryo emergence rate data at each sampling time point, the optimal sampling time with the highest embryo emergence rate was precisely selected. This is the optimal sampling time for rescuing embryos from interspecific hybrids of broccoli and Chinese cabbage.

[0036] Embryo rescue was performed on ovules from broccoli and Chinese cabbage hybrids 7-16 days after the cross. The timing of the rescue resulted in different outcomes (see Table 2). The highest embryo emergence rate (5.92%) was achieved when rescue was performed on day 13. Ovules retrieved for rescue on days 12 and 14 after hybridization showed no significant difference compared to those retrieved on day 13 (5.11% and 5.08% respectively), but both were higher than at other times. This indicates that days 12, 13, and 14 are the optimal time for embryo rescue, representing a stable and efficient period.

[0037] When the ovules were removed on day 7 for rescue, the embryo emergence rate was only 0.59%, indicating that the ovules were small and the embryos were in the spherical embryo stage. The free nuclei of the endosperm cells had not yet been cellularized. Premature separation would lead to the collapse of the energy metabolism system. Therefore, the ovules should not be separated from the mother for embryo rescue on day 7 after hybridization pollination.

[0038] As the ovules grew larger with each passing day after hybridization pollination, the embryo emergence rate gradually increased to 1.23%, 2.11%, 3.95%, and 4.56% at 8, 9, 10, and 11 days, respectively. The embryo emergence rate began to decline significantly from 15 days after hybridization pollination, indicating that ovules in distant hybrids will suffer sterility if not rescued promptly in the later stages of fertilization. A comparison of the embryo emergence rates in Table 2 determined that the optimal time for rescuing ovules from broccoli and Chinese cabbage was 13 days after pollination.

[0039] Table 2. Embryo emergence rate of hybrid cauliflower and Chinese cabbage at different harvesting times. 2.2 Optimization of Germination Medium Composition On day 13 post-pollination, ovules were removed and grouped into 11 different culture media numbered M1-M11 (see Table 3). Each culture medium was used in triplicate, with 20-100 ovules inoculated into each medium (see Table 3). Figure 1 The culture medium inoculated with ovules was placed at (23±2) °C, with 12 hours of light per day and a light intensity of 1500 lx.

[0040] Table 3. Germination rescue medium for interspecific hybrid embryos of broccoli and Chinese cabbage Seven days after inoculation, the inoculation process was carefully observed. At 15 days post-inoculation, a comprehensive statistical analysis of the embryo emergence rate was conducted. Through comparative analysis of embryo emergence rate data from different culture media, the medium with the highest embryo emergence rate was selected and determined as the optimal germination medium for embryo rescue.

[0041] Different germination media formulations have varying effects on embryo development, leading to differences in embryo emergence rates (see Table 4). The optimal embryo rescue germination medium is M6 medium (see Table 4). Figure 2 The embryo emergence rate was as high as 5.92% on medium M1, and as low as 0% on medium M1. Medium M1 contained only basic MS medium without any added plant hormones, indicating that plant hormones have a strong influence on ovule germination. Starting with medium M2, gradually adding AC, 6-BA, CH, NAA, and KT increased the embryo emergence rate from 1.07% to a maximum of 5.92%. When KT was added to medium M7 for the first time, the medium contained five growth regulators, and the embryo emergence rate decreased from M7 onwards. This indicates that a combination of high doses of growth regulators does not necessarily improve ovule germination. Therefore, medium M6 (MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 1 mg / L CH + 1 g / L AC) had the highest embryo emergence rate, making it the optimal medium for embryo rescue and germination.

[0042] Table 4. Embryo emergence rate of broccoli and Chinese cabbage interspecific hybrids on different germination media. Note: Different lowercase letters indicate a significance level of 0.05, and the same applies below.

[0043] 2.3 Optimization of proliferation medium composition Successfully differentiated ovules (adventitious buds) were sequentially transferred to N1, N2, N3, N4, N5, and N6 proliferation media with different compositions (see Table 5), and placed under conditions of 23±2°C, 12 hours of light per day, and 1500 lx light intensity to promote their subsequent proliferation and differentiation process.

[0044] Table 5. Composition of the medium for the proliferation of interspecific hybrids of broccoli and Chinese cabbage. For each culture medium, three replicate experiments were conducted, with 5-7 adventitious buds inoculated in each replicate. After ovule transfer, their proliferation dynamics were closely monitored, and the proliferation coefficient of ovules on each culture medium (proliferation coefficient = number of buds / original number of buds) was recorded in detail. Through systematic statistical analysis of the proliferation coefficients of each culture medium, the culture medium with the highest proliferation coefficient was identified as the most suitable proliferation medium.

[0045] Successfully differentiated ovules were cultured in a proliferation medium. To better observe the effect of the medium on the proliferation capacity of adventitious buds and obtain a large number of vigorous buds, the proliferation coefficient of the medium was calculated when the adventitious buds in the medium had grown to two fully expanded true leaves, using the bud growth status as a node (see [link to relevant documentation]). Figure 3 The effects of the six proliferation media varied (see Table 6). N1 showed the best proliferation effect, with a proliferation coefficient of 6.08%, significantly higher than the other five media. The effects of N3 and N5 media were not significantly different, but the data showed that N3 was slightly better than N5, with N3 containing 0.1 mg / L more NAA than N5. This indicates that adding a small amount of growth regulator can improve the proliferation effect of the media.

[0046] There was no significant difference between the N2 and N6 media, but both were lower than other media. The 6-BA added to N2 and N6 was only 0.1 mg / L, indicating that 6-BA had a significant effect on the proliferation of broccoli-Chinese cabbage hybrid embryos. A higher proliferation coefficient indicates better differentiation. Therefore, N1 (MS + 0.9 mg / L 6-BA + 0.1 mg / L NAA) is the optimal proliferation medium.

[0047] Table 6. Proliferation coefficients of ovules from broccoli and Chinese cabbage hybrids on different culture media. 2.4 Optimization of rooting medium composition Ovules undergo proliferation and differentiation on proliferation medium. When the clustered shoots grow to 2-3 fully expanded true leaves, they are transferred to 1 / 2 MS medium to promote their continued growth.

[0048] When the seedlings developed three pairs of true leaves, they were transferred to rooting media containing different types and concentrations of auxins (K1, K2, K3, K4, K5, K6, K7) (see Table 7). Each medium was tested in triplicate, with 13-30 seedlings inoculated in each replicate and placed at 23±2°C, 12 hours of light per day, and a light intensity of 1500 lx. During this stage, the rooting rate and rooting time of the seedlings on each medium were closely observed and recorded in detail. From the time of transfer until day 5, the rooting rate of the seedlings on each medium was recorded (rooting rate = number of rooted seedlings / total number of inoculated seedlings). Rooting was defined as the emergence of three strong white taproots, reaching a length of 5 mm and bearing obvious root hairs. The rooting time was recorded, and the average rooting time required for seedlings on each medium was calculated. Statistics were compiled up to day 15; rooting was no longer recorded after day 15.

[0049] Table 7. Components of rooting culture medium for interspecific hybrids of broccoli and Chinese cabbage Once the seedlings have developed 3-5 strong white taproots, reaching 5-8 mm in length and bearing noticeable root hairs, hardening-off can begin. Carefully remove the seedlings from the culture medium, thoroughly rinse off any remaining medium from the roots with clean water, then add tap water for hydroponics to help them adapt to the external environment. After the seedlings have developed new roots, prepare the seedling substrate by stirring it with water until it reaches a consistency where it can be formed into a ball without crumbling. Then, transplant the seedlings into seedling pots (see...). Figure 4 ).

[0050] Next, place the seedling pots in an artificial climate chamber with a temperature controlled at 21-25℃ and a humidity maintained at 80% for hardening-off. After 3 days, increase the water supply appropriately according to the growth status of the seedlings to meet their growth needs.

[0051] After 15 days of hardening-off, the seedlings' growth stabilized, and they could then be transplanted into large flowerpots for conventional fertilization and watering. The survival rate of the transplanted hybrid plants was recorded. Considering all factors, after 15 days of hardening-off on various culture media, the seedlings' growth stabilized, and they could then be transplanted into large flowerpots for conventional fertilization and watering. The survival rate of the transplanted hybrid plants was recorded (see Table 8).

[0052] Table 8. Rooting of interspecific hybrids of broccoli and Chinese cabbage on different culture media The results showed that the average rooting time in K7 medium was longer than that in other media, while the rooting rate was lower. The amount of CH added in K7 medium was significantly less than in the other six media, indicating that CH has a certain impact on the rooting of hybrids. K2 medium had the shortest average rooting time at 8 days, but its rooting rate was lower than that of K1 medium (43.73%). The difference between K1 and K2 media was the presence of 6-BA, suggesting that 6-BA affects the rooting time of hybrids.

[0053] In summary, the survival rate of seedlings transplanted when they were in good rooting condition was 100%. The rooting rates of K1 (MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC) and K2 (MS + 0.9 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC) media were significantly higher than those of other media, and the seedling growth quality was also higher. Therefore, they can be identified as the optimal rooting media.

[0054] In summary, this invention successfully established a technology system for rescuing embryos from distant hybridizations of broccoli and Chinese cabbage. Using M6 germination medium (MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 1 mg / L CH + 1 g / L AC), the embryo emergence rate was 5.92%. Using N1 proliferation medium (MS + 0.9 mg / L 6-BA + 0.1 mg / L NAA), the proliferation coefficient was 6.08%. Using K1 (MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC) or K2 rooting medium (MS + 0.9 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC), the average rooting time was 8-11 days, and the rooting rate was 35.82%-43.73%.

[0055] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A culture medium composition for rescuing embryos from distant hybrids of broccoli and Chinese cabbage, characterized in that, Including germination medium, proliferation medium and rooting medium; The germination medium is ① or ②: ①MS+0.5-1mg / L 6-BA+0.1mg / L NAA+1mg / L CH+1g / L AC; ②MS+0.5-1mg / L 6-BA+0.1-0.5 mg / L NAA+1mg / L KT+1mg / L CH+1g / L AC; The proliferation medium was: MS + 0.2-0.9 mg / L 6-BA + 0.1-0.5 mg / L NAA; The rooting medium was: MS + 0.5-0.9 mg / L 6-BA + 0.2 mg / L NAA + 3 g / L CH + 1 g / L AC.

2. The culture medium combination according to claim 1, characterized in that, The germination medium is ① or ②: ①MS+1mg / L 6-BA+0.1mg / L NAA+1mg / L CH+1g / L AC; ②MS+1mg / L 6-BA+0.1mg / L NAA+1mg / L KT+1mg / L CH+1g / L AC.

3. The culture medium combination according to claim 2, characterized in that, The germination medium was: MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 1 mg / L CH + 1 g / L AC.

4. The culture medium combination according to claim 1, characterized in that, The proliferation medium was: MS + 0.9 mg / L 6-BA + 0.1 mg / L NAA.

5. The application of the culture medium combination according to any one of claims 1-4 in the rescue of embryos from distant hybridization of broccoli and Chinese cabbage.

6. A method for rescuing embryos from distant hybrids of broccoli and Chinese cabbage, characterized in that, Includes the following steps: The hybrid ovules were extracted 12-14 days after the cross-pollination of broccoli and Chinese cabbage. The hybrid ovules are inoculated into the germination medium described in any one of claims 1-4 and cultured for differentiation to obtain adventitious buds; The adventitious buds are inoculated into the proliferation medium described in any one of claims 1-4 and cultured to obtain clustered buds; The clustered buds were inoculated in 1 / 2 MS medium. After 3 pairs of true leaves emerged, they were transferred to the rooting medium described in any one of claims 1-4 for rooting culture and hardening off to obtain hybrid plants.

7. The method according to claim 6, characterized in that, The differentiation culture was conducted at a temperature of 23±2℃, with a light exposure time of 12h and a light intensity of 1500 lx, for a period of 7-15 days.

8. The method according to claim 6, characterized in that, The propagation culture was conducted at a temperature of 23±2℃, with a light duration of 12h and a light intensity of 1500 lx, until at least two true leaves were fully expanded.

9. The method according to claim 6, characterized in that, The rooting culture was conducted at a temperature of 23±2℃, with a light duration of 12h and a light intensity of 1500 lx, until the rooting reached at least 3 main roots with a length of at least 5mm.

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