A method for rescuing distant hybrid embryos of peony into seedlings

Abstract

The present invention discloses a method for rescuing peony distant hybrid embryos and forming seedlings, belonging to the technical field of plant breeding. The present invention provides a culture medium specifically for rescuing peony distant hybrid embryos and forming seedlings, the culture medium comprising a germination medium, an embryo callus induction medium, a differentiation medium, and a rooting medium; the present invention also provides a method for rescuing peony distant hybrid embryos and forming seedlings using the dedicated culture medium. The present invention overcomes the common problem of embryo abortion in the process of distant hybridization between tree peonies and peony by performing an embryo rescue operation in a timely manner after pollination. By optimizing seed disinfection, embryo separation, culture medium formulation, and culture conditions, the normal development of embryos can be effectively promoted. The staged induction and differentiation culture strategy ensures the formation of embryo callus and its effective transformation into adult plants, significantly improving the success rate of embryo rescue in the process of distant hybridization between tree peonies and peony, and providing technical support for the cultivation of new varieties of tree peonies for both oil and ornamental purposes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a method for rescuing distant hybrid embryos of peony into seedlings. Background Art

[0002] Peony belongs to the genus Paeonia in the Ranunculaceae family and is known as the "King of Flowers" for its gorgeous flowers and rich cultural connotations. Among them, oil-producing peonies have important economic value due to their high seed yield and rich content of unsaturated fatty acids such as α-linolenic acid. However, the flower shape and color of oil-producing peonies are relatively simple, and their ornamental value is insufficient, which limits their diversified development in the ornamental and oil-producing fields. To overcome this limitation, existing breeding methods usually use peony varieties with high ornamental value to perform distant hybridization with oil-producing peonies, in order to cultivate new varieties with both oil-producing and ornamental value.

[0003] However, due to the severe incompatibility between tree peonies and herbaceous peonies, traditional distant hybridization often results in embryo abortion approximately 14 days after pollination, leading to low seed set. Therefore, an effective embryo rescue method is urgently needed to improve the seed set rate and subsequent breeding efficiency of distant hybridization. Summary of the Invention

[0004] To address the above technical issues, the present invention provides a method for rescuing distant hybrid peony embryos to seedlings. The present invention significantly improves the success rate of embryo rescue during distant hybridization between tree peonies and herbaceous peony by optimizing seed disinfection, embryo separation, culture medium formulation, and culture conditions.

[0005] The present invention provides the following technical solutions:

[0006] Technical solution 1: A culture medium specifically used for rescuing distant hybrid embryos of peony into seedlings, the culture medium comprising a germination medium, an embryonic callus induction medium, a differentiation medium, and a rooting medium;

[0007] The germination medium comprises: MS basal medium, 1.0 mg / L 6-BA, 0.1 mg / L IAA, 1.0 mg / L GA3, 30 g / L sucrose and 3.6 g / L agar;

[0008] The components of the embryonic callus induction medium are: MS basal medium, 0.1 mg / L NAA, 0.2 mg / L 2,4-D, 1.0 mg / L TDZ, 30 g / L sucrose and 3.6 g / L agar;

[0009] The components of the differentiation medium are: MS basal medium, 0.2 mg / L TDZ, 0.2 mg / L NAA, 30 g / L sucrose and 3.6 g / L agar;

[0010] The rooting medium comprises the following components: 1 / 2MS basal medium, 1.0 mg / L IBA, 1.0 mg / L GA3, 30 g / L sucrose and 3.6 g / L agar.

[0011] Technical Solution 2: A method for rescuing peony distant hybrid embryos into seedlings, comprising the following steps:

[0012] After cross-pollination of distant peony varieties, immature seeds are picked and disinfected; the disinfected seeds are placed in a germination medium for germination culture; embryos are separated from the germinated and cultured seeds, and embryo rescue is performed until the seeds are rescued into seedlings; the embryo rescue includes method 1 and method 2; method 1 is to transplant embryos separated from germinated seeds into the germination medium for 50 days of culture until the seeds are rescued into seedlings; method 2 is to place embryos separated from fully developed seeds into the embryo callus induction medium for induction culture, transplant the induced callus into the differentiation medium for differentiation culture to obtain differentiated buds, and place the buds into the rooting medium for rooting culture until the seeds are rescued into seedlings.

[0013] Furthermore, the disinfection treatment comprises soaking the immature seeds in 75% by volume anhydrous ethanol for 30 seconds, and then transferring them to a 5% sodium hypochlorite disinfectant and soaking them for 7 minutes. Before soaking the immature seeds in 75% by volume anhydrous ethanol, the immature seeds are first placed in an aqueous solution diluted 300 times with detergent and gently shaken for 5 minutes to remove surface impurities.

[0014] Furthermore, the pH of the germination medium, the embryo callus induction medium, the differentiation medium and the rooting medium are all 5.8.

[0015] Furthermore, the germination culture, the embryonic callus induction culture, the differentiation culture and the rooting culture are all cultured at 23° C., 65% humidity, 16 hours of light per day and a light intensity of 6000 lx.

[0016] Furthermore, the immature seeds are picked on the 14th day after pollination.

[0017] The present invention performs seed picking 14 days after pollination and timely rescues embryos, thereby preventing embryo abortion.

[0018] Furthermore, the distant hybridization includes hybridization with tree peony as the female parent and tree peony as the male parent.

[0019] The solution provided by the present invention is applicable to various distant hybrid combinations of tree peonies and herbaceous peonies, and is not limited to specific male and female parent varieties.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The method for rescuing peony distant hybrid embryos to seedlings provided by the present invention can effectively promote the normal development of embryos by optimizing seed disinfection, embryo separation, culture medium formula and culture conditions. The staged induction and differentiation culture strategy ensures the formation of embryo callus and its effective transformation into adult plants, significantly improving the success rate of embryo rescue in the process of distant hybridization between tree peonies and herbaceous peony.

[0022] Specific effects include: (1) improving the seedling rate: timely embryo rescue operation can reduce embryo abortion and significantly improve the seed seedling rate; (2) improving breeding efficiency: accelerating the breeding cycle of hybrid offspring and shortening the time for selecting new varieties; (3) expanding genetic diversity: effectively utilizing the genetic resources of distant hybridization to avoid gene homogeneity within the variety group, promoting the diversity of new varieties and the accumulation of excellent traits; (4) promoting the breeding of varieties with both oil and ornamental value: providing technical support for new peony varieties with both oil and ornamental value to meet the diversified market needs.

[0023] The present invention provides an efficient embryo rescue method for distant hybridization of tree peonies. By performing the embryo rescue operation in a timely manner after pollination, the method overcomes the common problem of embryo abortion in the process of distant hybridization between tree peonies and herbaceous peony, significantly improves the seedling rate of seeds, and provides technical support for the cultivation of new varieties of tree peonies for both oil and ornamental purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the development process of embryo culture, where A: sterilized immature seeds; B: seeds with developed radicles; C: embryos with radicles removed; D: plants grown into seedlings;

[0026] Figure 2 The diagram shows the callus induction, bud differentiation and rooting process of the embryo, where A: embryo taken from mature and plump seeds; B: induced callus; C: differentiated bud; D: plant with formed roots. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Seed collection

[0034] In the Gaochun International Slow City Kangzhiyuan Peony Garden, distant hybridization was carried out with tree peony as the female parent and tree peony as the male parent. The immature seeds were collected on the 14th day after pollination.

[0035] 2. Seed disinfection

[0036] (2) Place the collected immature seeds in a 300-fold dilution of detergent in water and gently shake for 5 minutes to remove surface impurities. Rinse thoroughly with sterile distilled water until no foam remains. Use sterile filter paper to absorb the moisture on the surface of the seeds and then disinfect them according to the disinfection scheme for each treatment group in Table 1. The contamination rate, mortality rate, and survival rate of the seeds in each treatment group are detailed in Table 1. Each group was set up with 30 materials and 3 biological replicates.

[0037] Table 1 Disinfection treatment

[0038]

[0039] According to Table 1, the disinfection method of treatment group 2 has the best disinfection effect ( Figure 1 A).

[0040] 3. Seed germination culture

[0041] The sterilized seeds were placed in the culture medium of each treatment group in Table 2, and the pH of the culture medium was adjusted to 5.8. The seeds were cultured at 23°C, 65% relative humidity, and 16 hours per day with a light intensity of 6000 lx. After one week, the seeds were fully developed; after two weeks, the seeds began to germinate and grow radicles ( Figure 1 B), the embryo germination rate and contamination rate were calculated (see Table 2 for details). Each group of experiments had 30 materials and 3 biological replicates.

[0042] Table 2 Effects of different plant growth regulators on immature seed germination

[0043]

[0044]

[0045] According to Table 2, after culture in the medium of treatment group 5, the embryo germination rate can reach 100%.

[0046] 4. Two Culture Methods for Embryo Rescue

[0047] (1) Method 1

[0048] The embryos separated from the germinated seeds were transplanted into the optimal medium for seed germination (treatment group 5, MS medium supplemented with 6-BA 1.0 mg / L, IAA 0.1 mg / L, GA3 1.0 mg / L, sucrose 30 g / L and agar 3.6 g / L) ( Figure 1 C in the medium). The pH value of the culture medium was adjusted to 5.8. After culturing for 50 days at 23°C, 65% humidity, 16 hours of light per day, and a light intensity of 6000 lx, all transplanted embryos successfully grew into complete plants, with a survival rate of 100% ( Figure 1 D) in.

[0049] (2) Method 2

[0050] The embryos separated from the fully developed seeds were transplanted into the culture medium of each treatment group in Table 3 ( Figure 2The culture medium was adjusted to pH 5.8 and cultured for 35 days at 23°C, 65% relative humidity, 16 hours per day, and 6000 lx light intensity to induce callus. Each group had 30 materials and 3 biological replicates. The callus induction rate is detailed in Table 3. The results showed that the induction rate of cultured in the medium of treatment group 1 was 71.11% ( Figure 2 B) in.

[0051] Table 3 Embryonic callus induction

[0052]

[0053] Subsequently, the induced calli were transplanted into the culture medium of each treatment group listed in Table 4. The pH of the culture medium was adjusted to 5.8. The calli were cultured for 45 days at 23°C and 65% relative humidity under conditions of 16 h of light per day and 6000 lx to promote bud differentiation. Each group had 30 samples and three biological replicates.

[0054] Table 4 Differentiation of embryonic callus

[0055]

[0056]

[0057] The callus differentiation rate and browning rate are shown in Table 4. The results show that the differentiation rate reached 55% and the browning rate reached 48% under the medium of treatment group 5, which has the best comprehensive effect ( Figure 2 C).

[0058] Finally, the differentiated buds were transplanted into the culture medium of each treatment group listed in Table 5, and the pH of the culture medium was adjusted to 5.8. The buds were cultured for 30 days at 23°C, 65% relative humidity, and a light intensity of 6000 lx with 16 h of light per day. The buds successfully grew into complete plants, and the rooting rate of the culture medium of each treatment group was calculated. Each group was set up with 30 materials and 3 biological replicates.

[0059] Table 5 Rooting situation

[0060]

[0061] According to Table 5, the rooting rate of the treated group 4 was 100% ( Figure 2 D) in.

[0062] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for rescuing distant hybrid embryos of peony into seedlings, characterized in that: The following steps are involved: After cross pollination of distant peony varieties, the immature seeds are picked and disinfected; The sterilized seeds are placed in a germination medium for germination culture; embryos are separated from the germination cultured seeds and embryo rescue is performed until the rescued seedlings are formed; the embryo rescue comprises method 1 and method 2; the method 1 is to separate embryos from germinated seeds and transplant them into the germination medium for 50 days for culturing until the rescued seedlings are formed; the method 2 is to place embryos separated from fully developed seeds in an embryo callus induction medium for induction culture, transplant the induced callus into a differentiation medium for differentiation culture to obtain differentiated buds, and place the buds in a rooting medium for rooting culture until the rescued seedlings are formed; The germination medium comprises: MS basal medium, 1.0 mg / L 6-BA, 0.1 mg / L IAA, 1.0 mg / L GA3, 30 g / L sucrose and 3.6 g / L agar; The components of the embryonic callus induction medium are: MS basal medium, 0.1 mg / L NAA, 0.2 mg / L 2,4-D, 1.0 mg / L TDZ, 30 g / L sucrose and 3.6 g / L agar; The components of the differentiation medium are: MS basal medium, 0.2 mg / L TDZ, 0.2 mg / L NAA, 30 g / L sucrose and 3.6 g / L agar; The rooting medium comprises: 1 / 2MS basal medium, 1.0 mg / L IBA, 1.0 mg / L GA3, 30 g / L sucrose and 3.6 g / L agar; The immature seeds are picked on the 14th day after pollination; The distant hybridization is a hybridization performed with tree peony as the female parent and tree peony as the male parent.

2. The method according to claim 1, characterized in that The disinfection treatment is to soak the immature seeds in anhydrous ethanol with a volume concentration of 75% for 30 seconds, and then transfer them to a 5% sodium hypochlorite disinfectant and soak them for 7 minutes.

3. The method according to claim 1, characterized in that The pH values ​​of the germination medium, the embryonic callus induction medium, the differentiation medium and the rooting medium are all 5.

8.

4. The method according to claim 1, wherein The germination culture, the embryonic callus induction culture, the differentiation culture and the rooting culture are all cultured at 23° C., 65% humidity, 16 h of light per day and a light intensity of 6000 lx.

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