Method for rescuing seedlings of peony distant hybrid embryos

Abstract

The invention discloses a peony far-source hybrid embryo rescue seedling method, and belongs to the technical field of plant breeding. The invention provides a culture medium special for rescue seedling of peony far-source hybrid embryos. The culture medium comprises a germination culture medium, an embryo callus induction culture medium, a differential culture medium and a rooting culture medium, the invention also provides a peony far-source hybrid embryo rescue seedling method obtained by applying the special culture medium. According to the method, embryo rescue operation is carried out at proper time after pollination, the common embryo abortion problem in the far-source hybridization process of the peony and the Chinese herbaceous peony is solved, normal development of embryos can be effectively promoted by optimizing seed disinfection, embryo separation, a culture medium formula and culture conditions, formation of embryo calluses is ensured through staged induction and differentiation culture strategies, and the survival rate of the embryos is increased. The embryo rescue success rate in the far-source hybridization process of the peony and the Chinese herbaceous peony is obviously improved, and a technical support is provided for the cultivation of a new variety of the oil-appreciation dual-purpose peony.

Description

Technical Field

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

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

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

[0004] In order to solve the above technical problems, the present invention proposes a method for rescuing distant hybrid embryos of peony to seedlings. The distant hybrid embryo rescue method of peony provided by the present invention significantly improves the success rate of embryo rescue in the process of distant hybridization between peony and herbaceous peony by optimizing seed disinfection, embryo separation, culture medium formula 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 embryo callus induction medium, a differentiation medium and a rooting medium;

[0007] The components of the germination medium are: 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 embryo 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: 1 / 2MS basic 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 the germination medium for germination culture; embryos are separated from the seeds for germination culture, and embryo rescue is performed until the seedlings are rescued; the embryo rescue includes mode 1 and mode 2; mode 1 is that the embryos separated from the germinated seeds are transplanted to the germination medium for culture for 50 days until the seedlings are rescued; mode 2 is that the embryos separated from the fully developed seeds are placed in the embryo callus induction medium for induction culture, the induced callus is transplanted to the differentiation medium for differentiation culture to obtain differentiated buds, and the buds are placed in the rooting medium for rooting culture until the seedlings are rescued.

[0013] Furthermore, the disinfection treatment is to soak the immature seeds in anhydrous ethanol with a volume concentration of 75% for 30 seconds, and then transfer to a 5% sodium hypochlorite disinfectant for 7 minutes. Before soaking the immature seeds in anhydrous ethanol with a volume concentration of 75%, the immature seeds are first placed in an aqueous solution diluted with detergent to 300 times, 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 embryo callus induction culture, the differentiation culture and the rooting culture are all cultured under the conditions of 23° C., 65% humidity, 16 h 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 extracts seeds 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, but 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, and the staged induction and differentiation culture strategy ensures the formation of embryo callus and its effective transformation into adult plants, thereby significantly improving the success rate of embryo rescue in the process of distant hybridization between peony and herbaceous peony.

[0022] Specific effects include: (1) improving the seedling rate: timely embryo rescue operations 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 that have both oil and ornamental value to meet the diversified market needs.

[0023] The present invention provides an efficient peony distant hybridization embryo rescue method, which overcomes the common embryo abortion problem in the distant hybridization process between peony and herbaceous peony by timely performing the embryo rescue operation after pollination, significantly improves the seedling rate, and provides technical support for the cultivation of new varieties of oil-viewing and ornamental peonies. 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 drawings required for use in the embodiments will be briefly introduced below. 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 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 taken out; D: plants grown into seedlings;

[0026] Figure 2 The figure shows the process of embryonic callus induction, bud differentiation and rooting culture, where A: embryo taken out from mature and plump seeds; B: induced callus; C: differentiated buds; 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 Gaochun International Slow City Kangzhiyuan Peony Garden, distant hybridization was carried out with peony as the female parent and peony as the male parent. The immature seeds were collected on the 14th day after pollination.

[0035] 2. Seed disinfection

[0036] (2) The collected immature seeds were placed in a 300-fold diluted aqueous solution of detergent, gently shaken for 5 minutes to remove surface impurities, and then rinsed thoroughly with sterile distilled water until no foam remained. After using sterile filter paper to absorb the moisture on the surface of the seeds, they were further disinfected according to the disinfection scheme of 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 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 full; after two weeks, the seeds began to germinate and grow radicles ( Figure 1 B), the embryo germination rate and contamination rate were statistically analyzed (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, GA 3 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 medium was adjusted to 5.8. After culturing for 50 days at 23°C, 65% humidity, 16h light per day, and 6000lx light intensity, all transplanted embryos successfully grew into complete plants, with a survival rate of 100% ( Figure 1 D).

[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 2A in the medium was adjusted to pH 5.8. The culture medium was placed at 23°C and 65% relative humidity for 35 days under the conditions of 16 hours per day and 6000 lx light intensity to induce callus tissue. Each group was set up with 30 materials and 3 biological replicates. The callus induction rate is shown in Table 3. The results show that the induction rate of cultured in the medium of treatment group 1 was 71.11% ( Figure 2 B).

[0051] Table 3 Embryonic callus induction

[0052]

[0053] Subsequently, the induced callus tissue was transplanted into the culture medium of each treatment group in Table 4, and the pH value of the culture medium was adjusted to 5.8. The culture medium was placed at 23°C and 65% relative humidity, with 16 hours of light per day and a light intensity of 6000 lx for 45 days to promote bud differentiation. Each group was set up with 30 materials and 3 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 in Table 5, and the pH value of the culture medium was adjusted to 5.8. The buds were cultured for 30 days at 23°C and 65% relative humidity with 16 hours of light per day and a light intensity of 6000 lx. The buds successfully grew into complete plants, and the rooting rate of the culture medium of each treatment group was counted. Each group was set with 30 materials and 3 biological replicates.

[0059] Table 5 Rooting situation

[0060]

[0061] According to Table 5, the rooting rate of the culture medium of treatment group 4 reached 100% ( Figure 2 D).

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design 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 all fall within the protection scope determined by the claims of the present invention.

Claims

1. A culture medium specifically used for rescuing distant hybrid embryos of peony into seedlings, characterized in that: The culture medium includes a germination medium, an embryo callus induction medium, a differentiation medium and a rooting medium; The components of the germination medium are: 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 embryo 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 basic medium, 1.0 mg / L IBA, 1.0 mg / L GA3, 30 g / L sucrose and 3.6 g / L agar.

2. 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 the germination medium described in claim 1 for germination culture; embryos are separated from the seeds for embryo rescue until they are rescued into seedlings; the embryo rescue includes method 1 and method 2; the method 1 is to separate embryos from germinated seeds and transplant them into the germination medium described in claim 1 for culture for 50 days until they are rescued into seedlings; the method 2 is to place embryos separated from fully developed seeds in the embryo callus induction medium described in claim 1 for induction culture, transplant the induced callus into the differentiation medium described in claim 1 for differentiation culture to obtain differentiated buds, and place the buds in the rooting medium described in claim 1 for rooting culture until they are rescued into seedlings.

3. The method according to claim 2, 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.

4. The method according to claim 2, characterized in that: The pH of the germination medium, the embryo callus induction medium, the differentiation medium and the rooting medium are all 5.

8.

5. The method according to claim 2, characterized in that: The germination culture, the embryo callus induction culture, the differentiation culture and the rooting culture are all cultured under the conditions of 23° C., 65% humidity, 16 hours of light per day and a light intensity of 6000 lx.

6. The method according to claim 2, characterized in that The immature seeds are picked on the 14th day after pollination.

7. The method according to claim 2, characterized in that The distant hybridization includes hybridization with tree peony as the female parent and tree peony as the male parent.

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