Method for obtaining distant hybrid offspring of broccoli and kai-lan by embryo rescue

Abstract

The application discloses a method for obtaining a distant hybrid offspring of broccoli and Brassica rapa by embryo rescue, and relates to the technical field of plant breeding. The application takes diploid broccoli germplasm resource 'B-5' as a female parent, diploid Brassica rapa 'YW-8' as a male parent, and carries out artificial sexual hybridization breeding. By using repeated pollination and high-efficiency in-vitro embryo rescue technology, a new distant hybrid germplasm of broccoli and Brassica rapa is obtained, the survival rate and growth potential of hybrid zygote embryos are effectively improved, the genetic material of Brassica rapa is potentially introduced, the broccoli germplasm resource library is enriched and expanded, and new materials and new ideas are provided for the breeding of broccoli.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and more specifically to a method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue. Background Technology

[0002] Broccoli (Brassica oleracea L. var. italica Plenck) is a globally popular vegetable belonging to the Brassica genus of the Brassicaceae family. The florets of broccoli are rich in protein, vitamins, minerals, and the anti-cancer compound sulforaphane. This active ingredient can significantly reduce the incidence of various cancers, making it highly favored by consumers worldwide and earning it the reputation of "the crown jewel of vegetables."

[0003] *Brassica campestris* L. ssp. chinensis (L.) Makino var. rosularis Tsen et Lee is a variety of *Brassica campestris* subspecies of *Brassica rapa* in the Brassicaceae family. It is cold-resistant, high in vitamin C, and has significant breeding value. It is a major leafy vegetable cultivated in the middle and lower reaches of the Yangtze River in my country. Due to its fleshy and tender leaves, delicious flavor, and rich vitamins and minerals, it is highly popular with consumers, and market demand is continuously increasing.

[0004] Distant hybridization breeding refers to hybridization between different species, genera, and even species with more distant kinship. It is an important way to create high-quality new varieties. Through hybridization, the gene pool for breeding screening can be greatly expanded and enriched, and various excellent genes can be effectively promoted for recombination and exchange. This not only improves stress resistance, yield and quality, but also provides new materials and germplasm needed for breeding of superior varieties. At the same time, it lays an important genetic foundation for the analysis of related functional genes and the study of important traits.

[0005] Embryo rescue is one of the key technologies for obtaining offspring from distant hybridization. By culturing early-aborted embryos in vitro, hybrid seedlings can be obtained, effectively solving the problem of low breeding efficiency caused by embryo abortion due to incompatibility in distant hybridization.

[0006] Currently, there are no reports on the use of embryo rescue to obtain offspring plants of distant hybrids of broccoli and tatsoi, both domestically and internationally. Therefore, establishing an embryo rescue system suitable for hybrid embryos of broccoli and tatsoi is crucial for distant hybridization breeding, the creation and utilization of new germplasm, and improving breeding efficiency. Summary of the Invention

[0007] In view of this, the present invention provides a method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue, which breaks through the natural hybridization barrier between species of Brassica and creates new germplasm resources.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue includes the following steps: Broccoli was used as the female parent and tatsoi as the male parent for cross-pollination. After pollination, the hybrid ovary was removed, sterilized, and the ovules were taken out and inoculated into the embryo rescue medium. After the embryos grew into seedlings, they were transferred to the proliferation medium for rapid propagation. The seedlings obtained from rapid propagation were then transferred to the seedling rooting medium for rooting culture to obtain hybrid seedlings.

[0010] Preferred composition of embryo rescue medium per 1L: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L 6-BA, 0.05% activated carbon, with the remainder being sterile water, pH 5.6~5.8; The composition of each 1L of proliferation medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 1.5 mg / L 6-BA, 0.1 mg / L NAA, with the remainder being sterile water, pH 5.6~5.8; Each 1L of seedling rooting medium consists of: 1 / 2 MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L NAA, with the remainder being sterile water, and a pH of 5.6–5.8.

[0011] Preferred method: The specific method of hybrid pollination is as follows: collect fresh pollen from the day the cauliflower opens during the peak flowering period, remove the stamens from the broccoli, and evenly apply the pollen from the cauliflower to the pistil of the broccoli for pollination. Harvest the hybrid ovary 21 days after pollination.

[0012] Preferred method: The disinfection process is as follows: Remove the hybrid ovary from the mother plant, rinse it with sterile water, and then, under sterile conditions, treat it with 75% ethanol for 30 seconds, followed by treatment with 2.5% sodium hypochlorite for 5 minutes. After that, rinse it with sterile water 3 to 5 times, 2 minutes each time.

[0013] Preferred culture time: embryo rescue medium for 40-50 days. After ovule inoculation, culture in the dark for 7 days, then transfer to light culture. Culture conditions: temperature 25℃, light intensity 30 μmol·m⁻². -2 ·s -1 The light exposure time is 14 hours per day. -1 The relative humidity is 50%~70%.

[0014] Preferred: The culture time in the proliferation medium is 30-35 days, and the culture conditions are the same as those for embryo rescue.

[0015] Preferred culture medium for seedling rooting is used for 35-45 days, with culture conditions identical to those for embryo rescue.

[0016] Preferred method: After obtaining hybrid seedlings, transplant them into the field when they have 4-5 leaves and 5-6 roots. The transplanting substrate is a mixture of garden soil, coconut coir, vermiculite and perlite in a volume ratio of 2:2:1:2.

[0017] Preferred conditions for field transplanting are: daytime temperature of 20℃~25℃, nighttime temperature of 15℃~18℃, and light duration of 14 h·d. -1 The relative humidity is 50%~70%.

[0018] The present invention also provides the application of any of the above methods, or the distant hybrid offspring of broccoli and tatsoi obtained by any of the above methods, in breeding.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue, and the technical effects achieved are as follows: In this invention, by screening to obtain the optimal culture medium formula and culture conditions, the germination rate of embryos from distant hybrids of broccoli and tatsoi was increased from 28.33% to 86.33%, the proliferation coefficient from 2.76 to 5.13, the root length from 1.98 cm to 3.08 cm, and the number of roots from 4.33 to 7.03. The field transplant survival rate reached 95.00%. This invention can effectively improve the survival rate and growth potential of hybrid embryos by adjusting the culture medium ratio to increase the germination rate of embryos and promote proliferation and rooting, effectively overcoming the obstacle that distant hybrid embryos cannot survive normally under natural conditions.

[0020] This invention utilizes distant hybridization between broccoli and tatsoi, combined with efficient embryo rescue technology, to facilitate the exchange of genetic material and introduce new genetic characteristics, thereby enhancing the genetic diversity of broccoli germplasm. In this invention, distant hybridization between broccoli and tatsoi can potentially introduce genetic material such as cold resistance and high vitamin C content from tatsoi. Experimental results show that the vitamin C content of the hybrid offspring is 12.62% higher than that of the maternal parent 'B-5', enriching and expanding the broccoli germplasm resource bank and providing new materials and ideas for broccoli breeding.

[0021] The embryo rescue culture medium, proliferation culture medium, and rooting culture medium formulations for broccoli and tatsoi in this invention are crucial for embryo germination, seedling proliferation, and rooting. Furthermore, the complete set of procedures for embryo rescue from distant hybridization proposed in this invention is highly efficient and easy to operate, which is of positive significance for large-scale production applications and can provide the market with more high-quality hybrid seedlings. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The attached figure is a schematic diagram of the finishing process provided by the present invention.

[0024] Figure 2 The attached figure shows a diagram of sterile hybrid offspring obtained by embryo rescue according to the present invention.

[0025] Figure 3 The attached figure shows a diagram of the transplanted hybrid offspring provided by the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This invention discloses a method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue. In this embodiment, raw materials not mentioned are all conventional commercially available materials, and operational steps not mentioned are all conventional processes, which will not be described in detail here. The overall process of this invention can be found in [link to invention]. Figure 1 .

[0028] Example 1 A method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue includes the following steps: Hybridization was carried out using broccoli 'B-5' (introduced from the National Vegetable Germplasm Resource Bank) as the female parent and tatsoi 'YW-8' (trade name: Huangxinwu, bred by the Institute of Horticulture, Anhui Academy of Agricultural Sciences, distributed by Anhui Kele Horticulture Technology Co., Ltd., brand / trademark: Aidi) as the male parent. Twenty-one days after pollination, the hybrid ovaries were harvested, sterilized, and the ovules were removed and inoculated into embryo rescue medium. After the embryos developed into seedlings, they were transferred to a proliferation medium for rapid propagation. The seedlings obtained from rapid propagation were then transferred to a strong seedling rooting medium for rooting culture to obtain hybrid seedlings. When the seedlings grew to 4-5 leaves and 5-6 roots, they were transplanted into the field.

[0029] To further optimize the technical solution: the composition of each 1L embryo rescue medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L 6-BA, 0.05% activated carbon, and the balance is sterile water with a pH of 5.6~5.8; The composition of each 1L of proliferation medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 1.5 mg / L 6-BA, 0.1 mg / L NAA, with the remainder being sterile water, pH 5.6~5.8; Each 1L of seedling rooting medium consists of: 1 / 2 MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L NAA, with the remainder being sterile water, and a pH of 5.6–5.8. To further optimize the technical scheme: the hybridization process involves collecting fresh pollen from newly opened flowers of *Tatsoi* during its peak flowering period, removing the stamens from *Brassica oleracea*, and then evenly applying the pollen from *Tatsoi* to the pistils of *Brassica oleracea* for pollination.

[0030] To further optimize the technical solution: the disinfection process is as follows: 21 days after distant hybridization and pollination, the hybrid ovary is removed from the mother plant, rinsed with sterile water, placed on a clean bench, treated with 75% ethanol for 30 seconds, then treated with 2.5% sodium hypochlorite for 5 minutes, and then rinsed with sterile water 3 times, 2 minutes each time.

[0031] To further optimize the technical solution: the embryo rescue medium was cultured for 40-50 days. After inoculation of the ovules, they were first cultured in the dark for 7 days, and then transferred to light culture. The culture conditions were: culture temperature of 25℃ and light intensity of 30 μmol·m⁻¹. -2 ·s -1 The illumination time is 14 h·d -1 The relative humidity is 50%.

[0032] To further optimize the technical solution: the culture time in the proliferation medium is 30 days, and the culture conditions are the same as those for embryo rescue.

[0033] To further optimize the technical solution: the seedling rooting medium was cultured for 35 days, and the culture conditions were the same as those for embryo rescue.

[0034] To further optimize the technical solution: the transplanting substrate is made of garden soil, coconut coir, vermiculite and perlite in a volume ratio of 2:2:1:2.

[0035] To further optimize the technical solution: the field transplanting conditions are to maintain a daytime temperature of 20℃, a nighttime temperature of 15℃, and a light duration of 14 h·d. -1 The relative humidity is 50%.

[0036] Example 2 A method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue includes the following steps: Hybridization was carried out using broccoli 'B-5' (introduced from the National Vegetable Germplasm Resource Bank) as the female parent and tatsoi 'YW-8' (trade name: Huangxinwu, bred by the Institute of Horticulture, Anhui Academy of Agricultural Sciences, distributed by Anhui Kele Horticulture Technology Co., Ltd., brand / trademark: Aidi) as the male parent. Twenty-one days after pollination, the hybrid ovaries were harvested, sterilized, and the ovules were removed and inoculated into embryo rescue medium. After the embryos developed into seedlings, they were transferred to a proliferation medium for rapid propagation. The seedlings obtained from rapid propagation were then transferred to a seedling rooting medium for rooting culture to obtain hybrid seedlings. When the seedlings grew to 4-5 leaves and 5-6 roots, they were transplanted into the field.

[0037] To further optimize the technical solution: the composition of each 1L embryo rescue medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L 6-BA, 0.05% activated carbon, and the balance is sterile water with a pH of 5.6~5.8; The composition of each 1L of proliferation medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 1.5 mg / L 6-BA, 0.1 mg / L NAA, with the remainder being sterile water, pH 5.6~5.8; Each 1L of seedling rooting medium consists of: 1 / 2 MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L NAA, with the remainder being sterile water, and a pH of 5.6–5.8. To further optimize the technical scheme: the hybridization process involves collecting fresh pollen from newly opened flowers of *Tatsoi* during its peak flowering period, removing the stamens from *Brassica oleracea*, and then evenly applying the pollen from *Tatsoi* to the pistils of *Brassica oleracea* for pollination.

[0038] To further optimize the technical solution: the disinfection process is as follows: 21 days after distant hybridization and pollination, the hybrid ovary is removed from the mother plant, rinsed with sterile water, placed on a clean bench, treated with 75% ethanol for 30 seconds, then treated with 2.5% sodium hypochlorite for 5 minutes, and then rinsed with sterile water 4 times, 2 minutes each time.

[0039] To further optimize the technical solution: the embryo rescue medium was cultured for 45 days. After inoculation of ovules, they were first cultured in the dark for 7 days, and then transferred to light culture. The culture conditions were: culture temperature of 25℃ and light intensity of 30 μmol·m⁻¹. -2 ·s -1 The illumination time is 14 h·d -1 The relative humidity is 60%.

[0040] To further optimize the technical solution: the culture time in the proliferation medium was 33 days, and the culture conditions were the same as those for embryo rescue.

[0041] To further optimize the technical solution: the seedling rooting medium was cultured for 40 days, and the culture conditions were the same as those for embryo rescue.

[0042] To further optimize the technical solution: the transplanting substrate is made of garden soil, coconut coir, vermiculite and perlite in a volume ratio of 2:2:1:2.

[0043] To further optimize the technical plan: the field transplanting conditions are to maintain a daytime temperature of 22℃, a nighttime temperature of 17℃, and a light duration of 14 h·d. -1 The relative humidity is 60%.

[0044] Aseptic seedlings of hybrid offspring obtained by embryo rescue are shown Figure 2 Transplanted hybrid offspring Figure 3 .

[0045] Example 3 A method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue includes the following steps: Hybridization was carried out using broccoli 'B-5' (introduced from the National Vegetable Germplasm Resource Bank) as the female parent and tatsoi 'YW-8' (trade name: Huangxinwu, bred by the Institute of Horticulture, Anhui Academy of Agricultural Sciences, distributed by Anhui Kele Horticulture Technology Co., Ltd., brand / trademark: Aidi) as the male parent. Twenty-one days after pollination, the hybrid ovaries were harvested, sterilized, and the ovules were removed and inoculated into embryo rescue medium. After the embryos developed into seedlings, they were transferred to a proliferation medium for rapid propagation. The seedlings obtained from rapid propagation were then transferred to a seedling rooting medium for rooting culture to obtain hybrid seedlings. When the seedlings grew to 4-5 leaves and 5-6 roots, they were transplanted into the field.

[0046] To further optimize the technical solution: the composition of each 1L embryo rescue medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L 6-BA, 0.05% activated carbon, and the balance is sterile water with a pH of 5.6~5.8; The composition of each 1L of proliferation medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 1.5 mg / L 6-BA, 0.1 mg / L NAA, with the remainder being sterile water, pH 5.6~5.8; Each 1L of seedling rooting medium consists of: 1 / 2 MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L NAA, with the remainder being sterile water, and a pH of 5.6–5.8. To further optimize the technical scheme: the hybridization process involves collecting fresh pollen from newly opened flowers of *Tatsoi* during its peak flowering period, removing the stamens from *Brassica oleracea*, and then evenly applying the pollen from *Tatsoi* to the pistils of *Brassica oleracea* for pollination.

[0047] To further optimize the technical solution: the disinfection process is as follows: 21 days after distant hybridization and pollination, the hybrid ovary is removed from the mother plant, rinsed with sterile water, placed on a clean bench, treated with 75% ethanol for 30 seconds, then treated with 2.5% sodium hypochlorite for 5 minutes, and then rinsed with sterile water 5 times, 2 minutes each time.

[0048] To further optimize the technical solution: the embryo rescue medium was cultured for 40-50 days. After inoculation of the ovules, they were first cultured in the dark for 7 days, and then transferred to light culture. The culture conditions were: culture temperature of 25℃ and light intensity of 30 μmol·m⁻¹. -2 ·s -1 The illumination time is 14 h·d -1 The relative humidity is 70%.

[0049] To further optimize the technical solution: the culture time in the proliferation medium is 35 days, and the culture conditions are the same as those for embryo rescue.

[0050] To further optimize the technical solution: the seedling rooting medium was cultured for 45 days, and the culture conditions were the same as those for embryo rescue.

[0051] To further optimize the technical solution: the transplanting substrate is made of garden soil, coconut coir, vermiculite and perlite in a volume ratio of 2:2:1:2.

[0052] To further optimize the technical solution: the field transplanting conditions are to maintain a daytime temperature of 25℃, a nighttime temperature of 18℃, and a light duration of 14 h·d. -1 The relative humidity is 70%.

[0053] Technical effectiveness verification: Experimental design and grouping (see Tables 1-4): Embryo rescue medium screening: Using hybrid ovaries 21 days after pollination as material, after rinsing with sterile water, under sterile conditions, first treat with 75% ethanol for 30 seconds, then treat with 2.5% sodium hypochlorite for 5 minutes, take out and rinse with sterile water 3-5 times, 2 minutes each time.

[0054] Ovules were removed from a clean bench and inoculated into embryo rescue medium supplemented with MS as the basal medium, with sucrose (20, 30, and 40 g / L), 6-BA (0.2, 0.4, and 0.8 mg / L), and activated charcoal (0.01, 0.05, and 0.10%) added respectively. A total of nine experimental treatment combinations were performed, with three replicates. After 40–50 days of culture, the germination rate of immature embryos was calculated, and the optimal embryo rescue medium was selected. Immature embryo germination rate (%) = (number of germinating ovules / total number of inoculated ovules) × 100.

[0055] Table 1. Orthogonal experimental design for embryo rescue culture medium screening.

[0056] Selection of proliferation media: After 40-50 days of embryo culture, vigorous seedlings with uniform growth were selected as materials and inoculated into proliferation media with MS as the basal medium, supplemented with sucrose (20, 30, and 40 g / L), 6-BA (1.0, 1.5, and 2.0 mg / L), and NAA (0.01, 0.05, and 0.10 mg / L), respectively. A total of 9 experimental treatment combinations were performed, with 3 replicates. After 30-35 days of culture, the proliferation coefficient was calculated, and the optimal proliferation media was selected. Proliferation coefficient = total number of shoots after proliferation / total number of shoots at inoculation.

[0057] Table 2. Orthogonal experimental design for screening proliferation culture media

[0058] Screening of rooting medium for robust seedlings: After 30-35 days of proliferation culture, vigorous seedlings with uniform growth were selected as materials and inoculated into 1 / 2 MS as the basic medium, with sucrose 30 g / L and NAA (0.10, 0.20, and 0.30 mg / L) added respectively, for a total of 3 experimental treatment combinations, with 3 replicates. After 35-45 days of culture, root length and root number were counted to screen the optimal rooting medium for robust seedlings.

[0059] Table 3. Experimental Design for Screening Seedling Rooting Culture Medium

[0060] Field transplanting substrate screening: After the seedlings have rooted, the hybrid seedlings are placed indoors for 3 days to harden off. They are then removed from the culture bottles, the culture medium is washed off with clean water, and they are transplanted into the cultivation substrate. The experimental design for screening the cultivation substrate formula is shown in the table below.

[0061] Table 4. Experimental Design for Screening Cultivation Substrate Formulas

[0062] Vitamin C content determination: Flower heads of hybrid offspring and broccoli were collected at the harvest time, and rosette leaves were collected at the harvest time of tatsoi. The content was determined by 2,6-dichlorophenolindophenol titration.

[0063] Experimental results: By screening to obtain the optimal culture medium formula and culture conditions, the germination rate of the embryos from the distant hybridization of broccoli and tatsoi was increased from 28.33% to 86.33% (see Table 5, Effect of different treatments on the germination rate of the hybrid embryos of broccoli and tatsoi), the proliferation coefficient was increased from 2.76 to 5.13 (see Table 6, Effect of different treatments on the proliferation culture of the hybrid offspring), the root length was increased from 1.98 cm to 3.08 cm, and the number of roots increased from 4.33 to 7.03 (see Table 7, Effect of different treatments on the rooting culture of the hybrid offspring). The survival rate of transplanted seedlings in the field reached 95.00% (see Table 8, Effect of different substrate ratios on the transplant survival rate of the hybrid offspring of broccoli and tatsoi).

[0064] The vitamin C content of the hybrid offspring was 12.62% higher than that of the parent 'B-5' (see Table 9, comparison of vitamin C content of broccoli, tatsoi and their hybrid offspring).

[0065] In summary, this invention enriches and expands the broccoli germplasm resource bank, providing new materials and ideas for broccoli breeding.

[0066] Table 5 Note: The data in the table are the mean ± standard deviation of three replicates. Different lowercase letters in the same column indicate significant differences between data (P<0.05), and different uppercase letters in the same column indicate extremely significant differences between data (P<0.01). The same applies to the following tables.

[0067] Table 6

[0068] Table 7

[0069] Table 8

[0070] Table 9

[0071] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0072] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue, characterized in that, Includes the following steps: Broccoli was used as the female parent and tatsoi as the male parent for cross-pollination. After pollination, the hybrid ovary was removed, sterilized, and the ovules were taken out and inoculated into the embryo rescue medium. After the embryos grew into seedlings, they were transferred to the proliferation medium for rapid propagation. The seedlings obtained from rapid propagation were then transferred to the seedling rooting medium for rooting culture to obtain hybrid seedlings.

2. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 1, characterized in that, The embryo rescue medium per 1L consists of: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L 6-BA, 0.05% activated carbon, with the remainder being sterile water, and a pH of 5.6–5.

8. The composition of each 1L of the proliferation medium is: MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 1.5 mg / L 6-BA, 0.1 mg / L NAA, with the remainder being sterile water, pH 5.6~5.8; The composition of each 1L of the seedling rooting medium is: 1 / 2 MS, 6.0 g / L agar powder, 30.0 g / L sucrose, 0.2 mg / L NAA, with the remainder being sterile water, and a pH of 5.6-5.

8.

3. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 2, characterized in that, The hybridization pollination process is as follows: during the peak flowering period, fresh pollen from the newly opened flowers of the tatsoi is collected, the stamens of the broccoli are removed, and the pollen from the tatsoi is evenly applied to the pistils of the broccoli for pollination. The hybrid ovary is harvested 21 days after pollination.

4. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 3, characterized in that, The disinfection process is as follows: the hybrid ovary is removed from the mother plant, rinsed with sterile water, and then treated with 75% ethanol for 30 seconds under sterile conditions, followed by treatment with 2.5% sodium hypochlorite for 5 minutes. The ovary is then rinsed with sterile water 3 to 5 times, 2 minutes each time.

5. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 4, characterized in that, The embryo rescue medium was cultured for 40-50 days. After inoculation, the embryos were first cultured in the dark for 7 days, then transferred to light culture. The culture conditions were: a temperature of 25℃ and a light intensity of 30 μmol·m⁻¹. -2 ·s -1 The illumination time is 14 h·d -1 The relative humidity is 50%~70%.

6. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 5, characterized in that, The culture time in the proliferation medium is 30-35 days, and the culture conditions are the same as those for embryo rescue.

7. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 6, characterized in that, The seedlings were cultured in the rooting medium for 35 to 45 days under the same conditions as the embryo rescue conditions.

8. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 7, characterized in that, After obtaining hybrid seedlings, transplant them into the field when they have grown to 4-5 leaves and 5-6 roots. The transplanting substrate is a mixture of garden soil, coconut coir, vermiculite and perlite in a volume ratio of 2:2:1:

2.

9. The method for obtaining distant hybrid offspring of broccoli and tatsoi using embryo rescue according to claim 8, characterized in that, The conditions for field transplanting are: daytime temperature of 20℃~25℃, nighttime temperature of 15℃~18℃, light duration of 14 h·d⁻¹, and relative humidity of 50%~70%.

10. The application of the method according to any one of claims 1 to 9, or the distant hybrid offspring of broccoli and tatsoi obtained by the method according to any one of claims 1 to 9, in breeding.

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