Method for realizing plant regeneration through hybrid embryo rescue of cymbidium faberi
By optimizing the embryo rescue technology for Cymbidium hybrids, the problem of embryo abortion in orchid hybrid seeds has been solved, achieving efficient plant regeneration and breeding progress, providing hybrid offspring with novel phenotypes, and addressing the shortcomings of traditional breeding methods.
Patent Information
- Application Number
- CN202511308567.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-15
- Publication Date
- 2025-11-14
AI Technical Summary
Embryo abortion and insufficient viability in orchid hybrid seeds lead to low survival rates from traditional sowing, limiting the number of novel trait varieties and breeding progress.
By employing the embryo rescue technology for Cymbidium hybrids, including steps such as disinfection, aseptic germination, rhizome proliferation, bud differentiation, and rooting induction, the in vitro embryo culture system is optimized to improve embryo survival rate and plant regeneration efficiency.
It significantly improved the embryo survival rate and plant regeneration efficiency of orchid hybrids, provided alternative hybrid offspring with novel phenotypes, and shortened the breeding cycle.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a method for regenerating plants through the rescue of hybrid Cymbidium goeringii embryos, specifically a method for significantly improving the survival rate of hybrid orchid embryos and the efficiency of plant regeneration by optimizing the embryo rescue and in vitro culture system, applicable to orchid breeding and germplasm resource protection. Background Technology
[0002] Hybrid breeding of Orchids in my country started relatively late. Domestically, the selection and breeding of Orchids mainly relies on the domestication of wild resources or screening from natural mutations, resulting in slow progress in hybrid breeding of Chinese Orchids. Due to the small size of orchid seeds, the lack of endosperm, and incomplete embryo development, hybrid orchids often suffer embryonic abortion under natural conditions due to abnormal endosperm development. The survival rate of traditional sowing is less than 5%, and seeds must coexist with specific fungi to germinate in small numbers. Currently, traditional breeding of premium varieties still relies on primitive methods such as natural variation and division propagation, leading to a scarcity of varieties with novel traits and increasing pressure on wild resource collection. To overcome the problems of insufficient seed viability and embryonic abortion in hybrid orchids, this invention proposes a method for obtaining regenerated plants through in vitro culture of Cymbidium hybrid embryos, providing a selection of hybrid offspring with novel phenotypes. This is of great significance for shortening the hybrid breeding cycle of premium orchids. Summary of the Invention
[0003] The purpose of this invention is to provide a method for regenerating plants by rescuing hybrid embryos of Cymbidium faberi. By using in vitro culture of hybrid embryos, the problem of seed embryo abortion and insufficient viability is solved, and regenerated plants of hybrid offspring with novel phenotypes can be obtained, providing a new technical path for the rapid propagation of premium orchids.
[0004] The objective of this invention is achieved through the following technical solutions.
[0005] A method for regenerating plants by rescuing hybrid embryos of Cymbidium faberi includes the following steps:
[0006] (1) Select a male parent with novel traits and a female parent for hybridization.
[0007] (2) Disinfection: Take mature, unopened Cymbidium hybrid capsules that have matured 90-110 days after pollination and disinfect the surface of the capsules.
[0008] (3) Non-symbiotic germination: The seeds are cultured under sterile conditions by alternating light and dark and synergistic effects of hormones until the embryo begins to swell.
[0009] (4) Rhizome proliferation and bud differentiation: The enlarged embryos were inoculated into the culture medium for proliferation until rhizomes were formed. Rhizomes with good growth were selected and transferred to the bud differentiation culture medium and cultured in a photoperiodic manner (16h light / 8h dark, light intensity 2000 Lux) to induce the formation of clustered buds.
[0010] (5) Rooting induction: Cut off buds longer than 2cm, transfer them to rooting medium, and culture for 40 days to obtain regenerated plants.
[0011] (6) Selection of F1 generation of hybrids: screening for hybrid offspring with novel phenotypes.
[0012] In step (1) above, individuals that exhibit excellent performance in both flower traits (such as flower color and flower shape) and leaf traits (such as leaf color and leaf shape) are selected as hybrid parents for the hybridization of Cymbidium.
[0013] After the Cymbidium goeringii pods in step (2) above are refrigerated at 4-6℃ for a period of time, they are taken out, the surface dust is washed off, and the surface is disinfected with 75% alcohol and 0.1% HgCl2 solution.
[0014] The culture medium composition described in step (3) above is: Flower Treasure No. 1 + agar + sucrose + AC + iron salt + organic matter, with additional NAA and 6-BA. It is cultured in the dark at 25℃ for 20 days, and then transferred to light culture.
[0015] In step (3) above, the seed coat needs to be punctured with a dissecting needle before aseptic seed culture.
[0016] The proliferation medium composition described in step (4) above is: Flower Treasure No. 1 + agar + sucrose + peptone + iron salt + organic matter, with the addition of NAA, 6-BA and AC. After inoculation, the medium is first cultured in the dark for 7 days, and then transferred to a light intensity of 2000 Lux and a temperature of 25℃ for further culture.
[0017] The bud differentiation medium described in step (4) above is MS + agar + sucrose, with additional NAA and 6-BA. Rhizomes are cut into 0.5-1.0 cm long segments before inoculation.
[0018] In step (5) above, select adventitious buds with good growth and a length of 2 cm or more and transfer them to rooting medium: MS + agar + sucrose + NAA + 6-BA. Induce rooting at (25±2)℃, with a light intensity of 2000 Lux for 12 h / d.
[0019] In step (6) above, select individual plants from the F1 generation of hybrids that show outstanding performance in both floral traits (such as flower color and flower shape) and leaf traits (such as leaf color and leaf shape).
[0020] The hybrid capsules of Cymbidium goeringii used in the method of this invention were obtained from Southwest University of Science and Technology.
[0021] The advantages of using the technology of this invention are:
[0022] This invention proposes a method for plant regeneration through embryo rescue in Cymbidium faberi hybrids. By using embryo rescue technology, regenerated plants with hybrid embryos are obtained, providing novel phenotypic hybrid offspring for selection. This shortens the breeding cycle through vegetative breeding. This method innovates upon the traditional breeding methods of premium orchids, which rely on natural variation and division propagation, providing a new technological option for the breeding of premium orchids. Attached Figure Description
[0023] Figure 1 The image shows a disinfected Cymbidium goeringii pod, as described in Example 1 of this invention.
[0024] Figure 2 This is an example of aseptic germination culture of seeds in Example 1 of the present invention.
[0025] Figure 3 Example 1 of the present invention is a rhizome proliferation culture.
[0026] Figure 4 This is a cluster of shoots induced in Example 1 of the present invention.
[0027] Figure 5 This is an example of seedling cultivation in Example 1 of the present invention. Detailed Implementation
[0028] The present invention will be described in detail below with reference to specific embodiments.
[0029] The hybrid capsules of Cymbidium used in the examples were obtained from Southwest University of Science and Technology.
[0030] Example 1:
[0031] The female parent was a hybrid of the butterfly flower and the male parent, the edge grass leaf variegation.
[0032] Disinfection: 90-110 days after pollination, harvest mature, unopened Cymbidium hybrid capsules and refrigerate them. After a period of time, take them out and rinse the surface of the capsules with running water. Then place them on a pre-sterilized laminar flow hood and disinfect the surface of the capsules with 75% alcohol and 0.1% mercuric chloride. After that, put them into sterile culture bottles for later use.
[0033] Non-symbiotic germination: After sterilization, blot the surface moisture of the pods with sterile filter paper, place them on a pre-sterilized work board, cut open the pods with a clean, sterile scalpel, remove the seeds, gently puncture the seed coat with a dissecting needle, and then evenly sprinkle the seeds onto the culture medium with sterile forceps, ensuring the surface of each bottle of medium is evenly covered. The embryo rescue medium uses Flower Treasure No. 1 as the basic medium, supplemented with agar, sucrose, activated carbon, and additional plant hormones NAA and 6-BA. The inoculated medium is then incubated in the dark at 25℃ for 20 days, followed by incubation under light.
[0034] Proliferation Culture: After embryo germination, the enlarged embryos are inoculated into a culture medium for proliferation until rhizomes are formed. The proliferation culture medium consists of: Flower Treasure No. 1 + agar + sucrose + peptone + FeNaEDTA + organic matter, with the addition of NAA, 6-BA and AC. After inoculation, the embryos are first cultured in the dark for 7 days, and then transferred to a light intensity of 2000 Lux and a temperature of 25℃ for further culture.
[0035] Inducing shoot clusters: Select healthy new rhizomes, cut them to 0.5-1.0 cm, and transfer them to shoot differentiation medium. Culture them under photoperiod (16 h light / 8 h dark, light intensity 2000 Lux) to induce shoot clusters.
[0036] Seedling strengthening and rooting: Use sterile tweezers and a scalpel to divide the cluster of seedlings into small seedlings and inoculate them into a culture medium to strengthen the seedlings. When the seedlings grow to 2-3cm in height, transfer them to a rooting culture medium and culture for 40 days to obtain regenerated plants.
[0037] Screening for single plants with novel traits among the F1 generation of hybrid regenerated plants.
Claims
1. A method for regenerating plants by rescuing hybrid embryos of Cymbidium faberi, comprising the following steps: (1) Select a male parent with novel traits and a female parent for hybridization. (2) Disinfection: Take mature, unopened Cymbidium hybrid capsules that have matured 90-110 days after pollination and disinfect the surface of the capsules. (3) Non-symbiotic germination: The seeds are cultured under sterile conditions by alternating light and dark and synergistic effects of hormones until the embryo begins to swell. (4) Rhizome proliferation and bud differentiation: The enlarged embryos were inoculated into the culture medium for proliferation until rhizomes were formed. Rhizomes with good growth were selected and transferred to the bud differentiation culture medium and cultured in a photoperiodic manner (16h light / 8h dark, light intensity 2000 Lux) to induce the formation of clustered buds. (5) Rooting induction: Cut off buds longer than 2cm, transfer them to rooting medium, and culture for 40 days to obtain regenerated plants. (6) Selection of F1 generation of hybrids: screening for hybrid offspring with novel phenotypes.
2. The method for plant regeneration through embryo rescue in Cymbidium faberi according to claim 1, characterized in that: In step (1), individuals that exhibit excellent performance in both flower traits (such as flower color and flower shape) and leaf traits (such as leaf color and leaf shape) are selected as hybrid parents for the Cymbidium hybridization.
3. The method for plant regeneration through embryo rescue in Cymbidium faberi according to claim 1, characterized in that: After the Cymbidium goeringii pods in step (2) are harvested, they are placed in a refrigerator at 4-6℃ for a period of time, then taken out and the surface dust is washed off. The surface is then disinfected with 75% alcohol and 0.1% HgCl2 solution.
4. The method for plant regeneration through embryo rescue in Cymbidium faberi according to claim 1, characterized in that: The culture medium described in step (3) is: Flower Treasure No. 1 + agar + sucrose + AC + iron salt + organic matter, with additional NAA and 6-BA. It is cultured in the dark for 20 days at a temperature of 25℃, and then transferred to light culture.
5. The method for plant regeneration through embryo rescue in Cymbidium faberi according to claim 1, characterized in that: Before the aseptic culture of the seeds described in step (3), the seed coat needs to be punctured with a dissecting needle.
6. The method for plant regeneration through embryo rescue in Cymbidium faberi according to claim 1, characterized in that: The proliferation medium described in step (4) is: Flower Treasure No. 1 + agar + sucrose + peptone + iron salt + organic matter, with the addition of NAA, 6-BA and AC. After inoculation, the medium is first cultured in the dark for 7 days, and then transferred to a light intensity of 2000 Lux and a temperature of 25℃ for further culture.
7. The method for regenerating plants by rescuing hybrid embryos of Cymbidium goeringii according to claim 1, characterized in that: The bud differentiation medium described in step (4) is MS + agar + sucrose, with additional NAA and 6-BA. Rhizomes are cut into 0.5-1.0 cm long segments before inoculation.
8. The method for regenerating plants by rescuing hybrid embryos of Cymbidium goeringii according to claim 1, characterized in that: Step (5): Select adventitious buds with good growth and a length of 2 cm or more and transfer them to rooting medium: MS + agar + sucrose + NAA + 6-BA. Induce rooting at (25±2)℃, light for 12 h / d, and light intensity of 2000 Lux.
9. The method for regenerating plants by rescuing hybrid embryos of Cymbidium goeringii according to claim 1, characterized in that: Step (6) describes selecting individual plants from the F1 generation of hybrids that exhibit outstanding floral traits (such as flower color and flower shape) and leaf traits (such as leaf color and leaf shape).