Malus xiaozhuzi and malus hallingani far-off hybridization embryo rescue and seedling raising method and application

Abstract

The present application relates to the field of plant biotechnology, in particular to a method for rescuing seedlings from Malus robusta and Malus baccata far-off hybridization zygotes and application, and the method is summarized as follows: a method for rescuing seedlings from Malus robusta and Malus baccata far-off hybridization zygotes is provided.The present application carries out interspecific far-off hybridization by taking Malus robusta as female parent and Malus baccata as male parent.According to the degeneration of far-off hybridization zygotes, the zygotes are rescued.The present application determines the suitable sampling period for rescuing zygotes of hybrid combinations, the concentration and ratio of exogenous hormones required for zygote germination, proliferation and expansion, rooting culture, and the suitable hardening and transplanting method.The zygote rescue system for interspecific hybridization of Malus robusta and Malus baccata established by the present application has a zygote germination rate of 74.3%, a proliferation coefficient of 5.1, a rooting rate of 95.5% in rooting culture, and a hardening and transplanting survival rate of 96.4%.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a method and application for rescuing seedlings from distant hybridization embryos of *Malus baccata* and *Malus hanfuensis*. Background Technology

[0002] Apple (Malus doumeri) is a plant belonging to the genus *Malus pumila* Mill. of the family Rosaceae. It is mainly distributed in Guangxi, Fujian, Yunnan, and Vietnam, in mixed mountain forests or valleys at altitudes of 700-2400 meters. It is a shrub or small tree, 4-10 meters tall, and is a tropical wild apple. Chloroplast sequence data indicates a distant relationship with cultivated apples. Apple (*Malus × domestica* Borkh) is one of the most widely cultivated fruits in the world. The fruit is sweet and juicy, rich in dietary fiber, vitamins, and other nutrients. Currently, Fuji, Gala, and Red Delicious are the world's main cultivated varieties. With the deepening of global apple breeding efforts, the channels for utilizing genetic resources are becoming increasingly narrow, and fruit flavor, appearance quality, and resistance are becoming increasingly homogeneous. With global warming and market demand for variety and quality diversity, conventional breeding methods are no longer sufficient. Developing new apple germplasm with superior quality, strong resistance, and low chilling requirements through distant hybridization is one possibility.

[0003] Incompatibility in distant hybridization and early embryo abortion are major obstacles in fruit tree hybridization breeding. The earliest report of distant hybridization between apple and pear was in 1952, using 'Fertility' pear as the female parent and 'Crawley Beauty' apple as the male parent, resulting in 16 hybrid seeds. In the 1980s, Japanese scholars hybridized Japanese pear (Pyrus setotina Rehd. var. Culta Rehd.) with apple (Malus × domesticica Borkh), but all hybrid seeds died within 6 months of germination under natural conditions (Liang Chunli, 2005; Sun Liang, 2008; Zhao Xiaodong, 2010; Yang Xuyuan, 2017). Embryo rescue is one of the main technical means to obtain seedlings from distant hybrids. Early in vitro culture of early aborted, degenerated, and seedless embryos to obtain seedlings plays an important role in overcoming incompatibility in distant hybridization and improving the seedling survival rate of interspecific hybrids. Currently, there are almost no cases, both domestically and internationally, of using embryo rescue in vitro culture technology to achieve interspecific hybridization between apple and juvenile crabapple with relatively distant kinship and obtain hybrid offspring. During the embryo rescue process, factors such as different fruit species, embryo age, low-temperature stratification method, embryo sterilization, culture medium type, hormone type and ratio, and seedling rooting can all affect the rescue success rate. Establishing a suitable hybrid embryo rescue system for 'apple' and 'juvenile crabapple' can lay the foundation for apple distant hybridization breeding and expanding germplasm resource diversity. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for rescuing seedlings from distant hybridization of *Malus baccata* and *Malus hanfuensis*. The established interspecific embryo rescue system for *Malus baccata* and *Malus hanfuensis* achieved an embryo germination rate of 74.3%, a proliferation coefficient of 5.1 in the proliferation culture, a rooting rate of 95.5% in the rooting culture, robust seedling growth, good vigor, and a survival rate of 96.4% after hardening and transplanting.

[0005] To achieve the above-mentioned technical objectives and effects, the present invention is implemented through the following technical solution:

[0006] A method for rescuing seedlings from distant hybridization of *Malus baccata* and *Malus hanfuensis* includes the following steps:

[0007] S1: Using Hanfu apple as the male parent, pollen was collected, frozen, and stored for later use; using apple as the female parent, artificial emasculation and pollination were carried out during the bellflower flowering period the following year;

[0008] S2: Harvest hybrid young fruits about 55-60 days after hybridization of the mother plant, sterilize and aseptically peel out the whole seeds, and inoculate them on embryo rescue medium.

[0009] S3: Then transfer to a culture room with a temperature of 24±2℃, LED white red 7:3 lamp tubes, light intensity of 5000Lx, and light time of 12h / d. Culture for about 40 days. After the embryos sprout new shoots, cut off sterile new shoots with a length of 1.5-2.0cm and inoculate them into the proliferation medium. Culture under the same conditions, subculture once every 35-40 days, with a maximum of 8 subcultures.

[0010] S4: After the hybrid seedlings are propagated, healthy clump seedlings with a height of more than 2cm are cut into individual plants and inoculated into rooting substrate. Under the same culture conditions, rooted hybrid seedlings can be obtained in 25-30 days. When each plant has 3-4 roots, more than 6 true leaves, and a height of more than 2.0cm, it can be directly transplanted.

[0011] S5: Prepare the nutrient pots in advance and disinfect them with a 500-fold dilution of carbendazim solution. The substrate is coconut coir: perlite: organic fertilizer in a ratio of 7:3:1. Expose the plants to sunlight for more than 12 hours. After transplanting, water them thoroughly to help them settle. During the day, roll up the sides of the greenhouse and cover it with an 8-needle shade net (blocking about 50% of the sunlight). Keep the humidity at around 50% and pay attention to ventilation. After 2 weeks, you can obtain a distant hybrid of apple and Hanfu apple.

[0012] Furthermore, step S1 specifically includes: using 'Hanfu Apple' (Malus × domesticica Borkh) as the male parent, collecting natural flowers during the bell-shaped flowering period of the previous year, peeling off the anthers at room temperature, drying them under a 100-watt incandescent lamp, collecting them in 1.5ml or 2ml centrifuge tubes after the pollen has split open and dispersed, and storing them at -80℃ for use the following year; using 'Linqie' (Malus doumeri) as the female parent, performing artificial emasculation pollination during the bell-shaped flowering period of the following year, immediately covering the flowers with sulfuric acid paper bags after pollination, and removing the bags after 10-14 days.

[0013] Furthermore, step S2 specifically includes: taking hybrid young fruits that are about 55-60 days old, cleaning the young fruits with detergent, soaking them in 75% alcohol for 1 minute in a clean bench, rinsing them 3 times with sterile water, sterilizing them with 0.1% HgCl2 for 6-8 minutes, rinsing them 4-5 times with sterile water, peeling off the young embryos, inoculating them on embryo rescue medium, and placing them in the dark at 4℃ for 15-20 days.

[0014] Furthermore, the preparation of the culture medium specifically includes:

[0015] Basic culture medium and substrate: MS medium, with agar powder 5-7 g / L, sucrose 20-30 g / L, pH=5.8; 1 / 2 MS medium, coconut coir, perlite.

[0016] Cultivation conditions: temperature 24±2℃, LED light tube white to red ratio 7:3, light intensity 3000Lx, light duration 12 hours / day.

[0017] Embryo rescue medium: MS + 6-BA 0.5-1.0 mg / L + IAA 0.1-0.2 mg / L + PVA 0.1 mg / L, culture for 30-40 days.

[0018] Proliferation medium: MS + 6-BA 0.2-0.5 mg / L + NAA 0.1-0.2 mg / L + GA3 0.1-0.3 mg / L + PVA 0.1-0.3 mg / L. Germinated aseptic shoots with immature embryos are cut into 2.0-2.5 cm long stem segments and inoculated into the proliferation medium. Subculture once every 20 days or so, up to 8 subcultures.

[0019] Rooting substrate culture: 8g coconut coir + 2g perlite + 20ml rooting culture solution (1 / 2MS + NAA 0.5-1.0mg / L + IAA 1.0-1.5mg / L). Select seedlings with good growth and a height greater than 4cm for rooting culture. After about 40 days of culture, they can be directly transplanted into nutrient pots. The transplanting time is mid-February.

[0020] On the other hand, this invention proposes the application of the above method in apple distant hybridization breeding.

[0021] The beneficial effects of this invention are:

[0022] Distant hybridization breeding refers to hybridization between species, genera, or even higher taxonomic units to artificially and directionally obtain germplasm resources with superior traits. Through hybridization, phenotypic segregation in offspring can be expanded, enriching genetic diversity. Therefore, distant hybridization technology is an important way to expand the genetic base, increase genetic variation, and create new germplasm resources. Distant hybridization involves gene exchange based on overcoming reproductive isolation, thereby forming hybrid offspring distinct from the parents. Although allopolyploids exist abundantly in nature, artificial distant hybridization presents certain difficulties. Scholars speculate that the unstable characteristics of allopolyploids produced by natural hybridization, such as phenotypic morphology and gene structure, may be eliminated through evolutionary adaptation (natural selection), thus forming stable species. However, artificial distant hybridization is a short and highly variable process, making it difficult to obtain fertile offspring. Incoordination between the hybrid embryo and endosperm development, premature endosperm degradation, and failure to provide nutrition to the hybrid embryo lead to late-stage developmental decline or degeneration, manifesting as hybrid sterility, decline, and inability to survive, greatly limiting the success rate of distant hybridization.

[0023] Embryo rescue is one of the key technologies for improving the survival rate of distant hybrid offspring. Since more than 95% of distant hybrid embryos cannot be sown into seedlings, or the embryos fail or degenerate in the early stages of development, in vitro culture is carried out in the early and middle stages of embryonic development. Artificial cultivation can improve the germination rate of hybrid embryos and thus form seedlings. Immature embryos, especially those in the early stages of development, are generally not easy to succeed in in vitro culture. Appropriate culture medium, hormone ratio, and culture conditions are the basic conditions for the success of in vitro culture.

[0024] In this invention, through specific culture medium formulations and cultivation conditions, the germination rate of embryos from distant hybrids of *Malus baccata* and *Malus hanfuensis* was effectively increased to 74.3%, and the rooting rate reached as high as 95.5%. These data show that this invention can effectively improve the survival and growth potential of embryos. Furthermore, through strict aseptic operation and optimized nutritional conditions, the risk of seedling contamination and poor growth was significantly reduced. The hormone ratio in this technical solution helps to improve the germination rate of embryos, promote tissue proliferation, and accelerate the rooting process, overcoming the obstacle that distant hybrid embryos cannot survive under natural conditions.

[0025] This technical solution, through distant hybridization of *Apple* and *Kanfu* apples combined with effective embryo rescue techniques, helps introduce new genetic traits and enhance the genetic diversity of apple varieties. The distant hybridization of *Apple* and *Kanfu* apples in this solution can potentially introduce genetic characteristics of tropical apples, such as better resistance to pests and diseases or better adaptability to tropical climates. This expansion of germplasm resources provides new materials for apple breeding to cultivate apple varieties with new characteristics, offering a new solution to current bottlenecks in apple breeding (such as limited resistance and homogenization of flavor and appearance).

[0026] The specially designed culture medium formula (such as MS + 6-BA, NAA, GA3, and other hormone ratios) and culture conditions (such as light intensity and temperature control) in this invention are crucial for embryo germination and seedling proliferation. These hormones promote cell division, prolong the cell differentiation stage, and promote root development and nutrient absorption within the plant, contributing to the rapid and healthy growth of seedlings. This invention also proposes a detailed process for rescuing distant hybrid embryos, including pollination, fruit harvesting, seed collection, sterilization, culture, proliferation, and transplanting. This complete operational process is not only proven effective but also standardized, meaning it can be applied to large-scale breeding operations. Large-scale operation can drive industrial development and provide the market with more high-quality fruit tree seedlings.

[0027] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of 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.

[0029] Figure 1 This is a schematic diagram of the overall process of the present invention;

[0030] Figure 2 A schematic diagram of aborted seeds from mature fruits of distant hybridization;

[0031] Figure 3 Schematic diagram of embryo rescue from distant hybrid seeds at different developmental stages;

[0032] Figure 4 A schematic diagram of a distant hybrid seedling of *Crataegus pinnatifida* × *Hanfu* obtained through embryo rescue;

[0033] Figure 5 A schematic diagram of transplanting seedlings of a distant hybrid of *Crataegus pinnatifida* and *Cynodon dactylon*.

[0034] Figure 6 A schematic diagram of the 55th day after the cross between *Rhododendron simsii* and *Hanfu*.

[0035] Figure 7 A schematic diagram of embryonic development on day 55 after the cross between *Impatiens balsamina* and *Hanfu*.

[0036] Figure 8 A schematic diagram showing different stages of cultivation of the maternal parent crabapple and its hybrid offspring;

[0037] Figure 9 A schematic diagram of rooting culture of tissue culture seedlings of the hybrid offspring of *Crassula ovata* and *Hymenopause chinensis*. Detailed Implementation

[0038] 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.

[0039] Example 1

[0040] The method for rescuing seedlings from distant hybridization of *Malus baccata* and *Malus hanfuensis* described in this embodiment includes the following steps:

[0041] S1: Pollen collection and pollination: Using Hanfu apple as the male parent, pollen was collected, frozen, and stored for later use; using apple as the female parent, artificial emasculation and pollination were carried out during the bellflower flowering period the following year.

[0042] S2: Harvesting young fruits for seed: Harvest young hybrid fruits from the mother plant about 55-60 days after hybridization, disinfect them, and aseptically extract the whole seeds, then inoculate them onto the embryo rescue medium.

[0043] S3: Then transfer to a culture room with a temperature of 24±2℃, LED white red 7:3 lamp tubes, light intensity of 5000Lx, and light time of 12h / d. Culture for about 40 days. After the embryos sprout new shoots, cut off sterile new shoots with a length of 1.5-2.0cm and inoculate them into the proliferation medium. Culture under the same conditions, subculture once every 35-40 days, with a maximum of 8 subcultures.

[0044] S4: After the hybrid seedlings are propagated, healthy clump seedlings with a height of more than 2cm are cut into individual plants and inoculated into rooting substrate. Under the same culture conditions, rooted hybrid seedlings can be obtained in 25-30 days. When each plant has 3-4 roots, more than 6 true leaves, and a height of more than 2.0cm, it can be directly transplanted.

[0045] S5: Prepare the nutrient pots in advance and disinfect them with a 500-fold dilution of carbendazim solution. The substrate is coconut coir: perlite: organic fertilizer in a ratio of 7:3:1. Expose the plants to sunlight for more than 12 hours. After transplanting, water them thoroughly to help them settle. During the day, roll up the sides of the greenhouse and cover it with an 8-needle shade net (blocking about 50% of the sunlight). Keep the humidity at around 50% and pay attention to ventilation. After 2 weeks, you can obtain a distant hybrid of apple and Hanfu apple.

[0046] In this embodiment, step S1 specifically includes: using 'Hanfu Apple' (Malus × domesticica Borkh) as the male parent, collecting natural flowers during the bell-shaped flowering period of the previous year, peeling off the anthers at room temperature, drying them under a 100-watt incandescent lamp, collecting the pollen in 1.5ml or 2ml centrifuge tubes after the pollen has split open and dispersed, and storing them at -80℃ for use the following year; using 'Linqie' (Malus doumeri) as the female parent, performing artificial emasculation pollination during the bell-shaped flowering period of the following year, immediately covering the flowers with sulfuric acid paper bags after pollination, and removing the bags after 10-14 days.

[0047] In this embodiment, step S2 specifically includes: taking hybrid young fruits that are about 55-60 days old, cleaning the young fruits with detergent, soaking them in 75% alcohol for 1 minute in a clean bench, rinsing them 3 times with sterile water, sterilizing them with 0.1% HgCl2 for 6-8 minutes, rinsing them 4-5 times with sterile water, peeling off the young embryos, inoculating them on embryo rescue medium, and placing them in the dark at 4℃ for 15-20 days.

[0048] In this embodiment, the preparation of the culture medium specifically includes:

[0049] Basic culture medium and substrate: MS medium, with agar powder 5-7 g / L, sucrose 20-30 g / L, pH=5.8; 1 / 2 MS medium, coconut coir, perlite.

[0050] Cultivation conditions: temperature 24±2℃, LED light tube white to red ratio 7:3, light intensity 3000Lx, light duration 12 hours / day.

[0051] Embryo rescue culture: MS + 6-BA 0.5-1.0 mg / L + IAA 0.1-0.2 mg / L + PVA 0.1 mg / L, culture for 30-40 days.

[0052] Proliferation medium: MS + 6-BA 0.2-0.5 mg / L + NAA 0.1-0.2 mg / L + GA3 0.1-0.3 mg / L + PVA 0.1-0.3 mg / L. Germinated aseptic shoots with immature embryos are cut into 2.0-2.5 cm long stem segments and inoculated into the proliferation medium. Subculture once every 20 days or so, up to 8 subcultures.

[0053] Rooting substrate culture: 8g coconut coir + 2g perlite + 20ml rooting culture solution (1 / 2MS + NAA 0.5-1.0mg / L + IAA 1.0-1.5mg / L). Select seedlings with good growth and a height greater than 4cm for rooting culture. After about 40 days of culture, they can be directly transplanted into nutrient pots. The transplanting time is mid-February.

[0054] On the other hand, this invention proposes the application of the above method in apple distant hybridization breeding.

[0055] Example 2

[0056] Hybrid seedlings were harvested 30 and 55 days post-pollination. Fruits were thoroughly cleaned with detergent, and seeds were disinfected. The seeds were then divided into two treatments: those harvested 30 and 60 days post-pollination were directly inoculated with intact embryos, while those with the seed coat removed and endosperm retained were treated separately. Inoculated seedlings with intact embryos were placed in the dark at 4°C for 20 days before being returned to normal culture conditions. There were six treatment groups, each with four different culture medium formulations. The results showed that inoculating intact embryos at 60 days post-pollination... - The culture medium was first incubated in the dark at 4°C for 15 days, then resumed normal culture. Tissue culture seedlings were obtained after a total culture period of 40 days and could then be used for proliferation culture. (Table 1)

[0057] Table 1. Germination culture methods and effects of crabapple × Hanfu hybrid seeds

[0058]

[0059] Note: Culture medium - MS+6-BA0.5mg / L+IAA0.1mg / L+PVA0.1mg / L;

[0060] - MS+6-BA0.5mg / L+IAA0.1mg / L+PVA0.1mg / L;

[0061] - MS+6-BA1.0mg / L+IAA0.1mg / L+PVA0.1mg / ;

[0062] - MS+6-BA1.0mg / L+IAA0.2mg / L+PVA0.1mg / L;

[0063] Seedlings that germinated after embryo rescue were inoculated into different culture media at the proliferation and differentiation stage. The temperature was 24±2℃, the LED light ratio was 7:3 (white to red), the light intensity was 3000 Lx, and the photoperiod was 12 hours / day. Subculture was performed every 20 days, and seedling height was measured every 10 days. The proliferation coefficient was calculated on day 40. It was found that II- Under the culture medium, the proliferation coefficient was 5.1, and the seedling height was 3.68 cm, indicating the best culture effect. (Table 2)

[0064] Table 2. Proliferation and differentiation of *Crassula ovata* × *Hanfu* hybrid seedlings

[0065]

[0066] Note: Culture medium

[0067] II- MS+6-BA0.2mg / L+NAA0.1mg / L+GA3 0.1mg / L+PVA0.1mg / L; Ⅱ- MS+6-BA0.5mg / L+NAA0.1mg / L+GA3 0.3mg / L+PVA0.3mg / L; Ⅱ- MS+6-BA0.2mg / L+NAA0.2mg / L+GA3 0.3mg / L+PVA0.3mg / L; Ⅱ- MS+6-BA0.5mg / L+NAA0.2mg / L+GA3 0.1mg / L+PVA0.1mg / L;

[0068] After propagation culture, once the seedlings exceed 2cm in height, they can be cut into individual seedlings, inoculated into rooting medium, and cultured for about 40 days before transplanting. Using agar-based media requires washing and hardening off the seedlings, both of which can lead to root loss and damage. - After cultivation, the seedlings can be directly transplanted after 40 days, with good root growth and a seedling height of over 6cm. This saves time, does not damage the root system, and achieves a seedling survival rate of over 96%. (Table 3)

[0069] Table 3. Rooting culture of *Crassula ovata* × *Hanfu* hybrid seedlings

[0070]

[0071] Note: Culture medium - 1 / 2 MS + NAA 0.5 mg / L + IAA 1.5 mg / L + sucrose 30 g / L + agar 7 g / L; - 1 / 2 MS + NAA 1.0 mg / L + IAA 1.0 mg / L + sucrose 30 g / L + agar 7 g / L; - 8g coconut coir + 2g perlite + 20ml rooting culture medium (1 / 2MS + NAA 0.5mg / L + IAA 1.5mg / L); - 8g coconut coir + 2g perlite + 20ml rooting culture medium (1 / 2MS + NAA 1.0mg / L + IAA 1.0mg / L)

[0072] In summary, this invention proposes a method and application for rescuing seedlings from distant hybridization of *Malus baccata* and *Malus hanfuensis*. *Malus baccata* was used as the female parent and *Malus hanfuensis* as the male parent for interspecific distant hybridization. Embryo rescue was performed to address embryo abortion in the distant hybridization. The suitable time for embryo collection, the concentration and ratio of exogenous hormones required for embryo germination, propagation, and rooting culture, and the appropriate hardening-off transplanting method were determined. The interspecific embryo rescue system for *Malus baccata* and *Malus hanfuensis* established using this technology achieved an embryo germination rate of 74.3%, a propagation coefficient of 5.1 in propagation culture, a rooting rate of 95.5% in rooting culture, robust seedling growth, and a survival rate of 96.4% after hardening-off transplanting.

[0073] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for rescuing seedlings from distant hybridization embryos of *Malus baccata* and *Malus hanfuensis*, characterized in that, Includes the following steps: S1: Using Hanfu apple as the male parent, pollen was collected, frozen, and stored for later use; using apple as the female parent, artificial emasculation and pollination were carried out during the bellflower flowering period the following year; S2: Harvest hybrid young fruits 55-60 days after hybridization of the mother plant, disinfect and aseptically peel out the whole seeds, inoculate them on embryo rescue medium, and place them in the dark at 4℃ for 15-20 days. S3: Then transfer to a culture room with a temperature of 24±2℃, LED white red 7:3 lamp tubes, light intensity of 5000Lx, and light time of 12h / d, and culture for 40 days. After the embryos sprout new shoots, cut off sterile new shoots with a length of 1.5-2.0cm and inoculate them into proliferation medium. Culture under the same culture conditions, subculture once every 35-40 days, with a maximum of 8 subcultures. S4: After the hybrid seedlings are propagated, healthy clump seedlings with a height of more than 2cm are cut into individual plants and inoculated into rooting substrate. Under the same culture conditions, rooted hybrid seedlings can be obtained in 25-30 days. When each plant has 3-4 roots, more than 6 true leaves, and a height of more than 2.0cm, it can be directly transplanted. S5: Prepare the nutrient pots in advance and disinfect them with a 500-fold carbendazim solution. The substrate is coconut coir: perlite: organic fertilizer 7:3:

1. Expose to sunlight for more than 12 hours. After transplanting, water thoroughly to settle the roots. During the day, roll up the sides of the greenhouse and cover it with an 8-needle shade net. Keep the humidity at 50% and pay attention to ventilation. After 2 weeks, you can obtain the distant hybrid plants of crabapple and Hanfu apple. The preparation of the culture medium specifically includes: Embryo rescue medium: MS + 6-BA 0.5-1.0 mg / L + IAA 0.1-0.2 mg / L + PVA 0.1 mg / L; Proliferation medium: MS + 6-BA 0.2-0.5 mg / L + NAA 0.1-0.2 mg / L + GA3 0.1-0.3 mg / L + PVA 0.1-0.3 mg / L; Rooting substrate: 8g coconut coir + 2g perlite + 20ml rooting culture solution; rooting culture solution: 1 / 2MS + NAA 0.5-1.0mg / L + IAA 1.0-1.5mg / L.

2. The method for rescuing seedlings from distant hybridization embryos of *Malus baccata* and *Malus hanfuensis* as described in claim 1, characterized in that: Step S1 specifically includes: using 'Hanfu Apple' as the male parent, collecting natural flowers during the bell-shaped flowering period of the previous year, peeling off the anthers at room temperature, drying them under a 100-watt incandescent lamp, collecting the pollen in 1.5ml or 2ml centrifuge tubes after the pollen has split open and dispersed, and storing them at -80℃ for use the following year; using 'Linqin' as the female parent, artificial emasculation and pollination are performed during the bell-shaped flowering period of the following year, and the pollination is immediately covered with a sulfuric acid paper bag, which is removed after 10-14 days.

3. The method for rescuing seedlings from distant hybridization embryos of *Malus baccata* and *Malus hanfuensis* as described in claim 1, characterized in that: Step S2 specifically includes: taking hybrid young fruits 55-60 days after hybridization, cleaning the young fruits with detergent, soaking them in 75% alcohol for 1 minute in a clean bench, rinsing them 3 times with sterile water, sterilizing them with 0.1% HgCl2 solution for 6-8 minutes, rinsing them 4-5 times with sterile water, peeling off the young embryos, and inoculating them onto embryo rescue culture medium.

4. The application of the method as described in any one of claims 1-3 in apple distant hybridization breeding.

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