Method for obtaining interspecific filial generation of elephant trunk orchid and calanthe calyx orchid by using embryo rescue

Abstract

The invention relates to the field of biotechnology breeding, in particular to a method for obtaining an interspecific filial generation of elephant trunk orchid and calanthe calyx orchid by using embryo rescue. According to the method, the calanthe ciliata is used as a male parent, the elephant trunk is used as a female parent, the embryo rescue technology is combined, in the embryo rescue technology, every 1 L of embryo rescue culture medium is composed of 2 g / L of Huabao No.1, 100 g / L of potato homogenate, 30 g / L of cane sugar, 0.5 g / L of activated carbon, 7.0 g / L of agar and the balance of water, and the pH is 5.6-5.8. According to the method, germination of the hybrid embryo of the calanthe ciliata and the elephant trunk can be remarkably promoted, new genetic characteristics are introduced potentially, and the hybrid embryo is obtained. The genetic diversity of the phalaenopsis variety is enhanced, and a new germplasm resource is provided for breeding of the phalaenopsis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology breeding, and in particular to a method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid by utilizing embryo rescue. Background Art

[0002] Elephant orchid and Calyx orchid belong to the same genus Phalaenopsis and are excellent parents for Phalaenopsis variety improvement. Chloroplast genome data show that Elephant orchid and Calyx orchid are distantly related.

[0003] Phalaenopsis orchids are one of the most important horticultural flowers in commercial floriculture worldwide. Phalaenopsis breeding has shifted from focusing solely on large flowers to smaller flowers with diverse colors, easy flowering, drought resistance, and cold tolerance. Breeding new Phalaenopsis germplasm with advantages such as small flowers and cold tolerance through distant hybridization is a possibility. The elephant trunk orchid, a smaller type of Phalaenopsis, boasts unique flower shapes, vibrant colors, and good cold tolerance, making it of great breeding value.

[0004] Incompatibility and early embryo abortion in distant hybridization are the main obstacles to plant hybridization breeding, and there have been no reports on distant hybridization of elephant orchids. Embryo rescue is one of the main technical means to obtain distant hybridization seedlings. Early in vitro culture of immature embryos that are aborted, degraded, or unable to seed is performed to obtain seedlings, thereby overcoming the incompatibility of distant hybridization and improving the seedling rate of interspecific hybrids. At present, there are no cases in China and abroad using immature embryo rescue in vitro culture technology to achieve interspecific hybridization of relatively distantly related elephant orchids and calyx orchids and obtain hybrid offspring plants. During the embryo rescue process, different embryo ages, disinfection methods, culture medium types, hormone types and ratios, seedling rooting and other factors will affect the rescue success rate. Establishing an embryo rescue system suitable for hybrid embryos of elephant orchids and calyx orchids can lay the foundation for distant hybridization breeding and innovative utilization of germplasm. Summary of the Invention

[0005] In order to overcome the natural hybridization barrier between species of Phalaenopsis and create new germplasm resources, the present invention provides a method for obtaining interspecific hybrid offspring of Phalaenopsis and Phalaenopsis by using embryo rescue.

[0006] To achieve the above object, the present invention adopts the following invention: A method for obtaining interspecific hybrid offspring of Elephant Orchid and Calyx Orchid by using embryo rescue comprises the following steps: Hybridization is carried out using Cymbidium calyx as the male parent and Cymbidium elegans as the female parent. Hybrid young fruits are picked 40 to 60 days after pollination, disinfected, sliced, and then inoculated into embryo rescue medium for cultivation. After the young embryos germinate into rooted seedlings, they are transferred to seedling rooting medium for cultivation to obtain rooted hybrid seedlings. When the rooted hybrid seedlings have 4 to 5 leaves and 5 to 8 roots, they are transplanted and planted for cultivation. The composition of the embryo rescue medium per 1 L is: 2 g / L Huabao No. 1, 100 g / L potato homogenate, 30 g / L sucrose, 0.5 g / L activated carbon, 7.0 g / L agar, and the balance is water, with a pH of 5.6-5.8.

[0007] Successful hybridization of calyx orchids with phalaenopsis has been reported, but hybridization of elephant trunk orchids with phalaenopsis has failed to produce offspring. The present invention utilizes hybrid progeny of elephant trunk orchids with calyx orchids, combined with a specific embryo rescue medium, to obtain breeding intermediate material. This provides genetic resources for subsequent hybridization of hybrid progeny with phalaenopsis, and offers technical reserves for the cultivation of new varieties with both ornamental value and stress resistance.

[0008] Furthermore, the hybridization specifically involves picking up the pollen mass of the calyx ridge orchid during the blooming period, removing the stamens of the elephant orchid, and sending the pollen mass of the calyx ridge orchid into the pistil of the elephant orchid for pollination.

[0009] Furthermore, the disinfection is specifically as follows: soak the young fruit in detergent for 10 min to 15 min, wash and soak in 75% alcohol for 30s to 50s, rinse with sterile water, sterilize with 0.1% to 0.3% HgCl2 for 15 min to 20 min, and rinse with sterile water 4 to 5 times.

[0010] Furthermore, the embryo rescue medium was cultured for 40 days to 60 days under the following conditions: temperature of 20°C to 30°C, LED white:LED red = 5-8:2-4, and light intensity of 30 μmol·m -2 ·s -1 ~40 μmol·m -2 ·s -1 , the lighting time is 10h~14h per day.

[0011] Furthermore, the composition of the seedling rooting culture medium per 1L is: 1.5g / L Huabao No. 1, 2.0g / L peptone, 1mg / L NAA, 120mg / L inositol, 2.0mg / L glycine, 0.2mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride, 0.5mg / L nicotinic acid, 75g / L banana homogenate, 0.5g / L activated carbon, 7g / L agar, and the balance is water, pH 5.6.

[0012] Furthermore, the culture time in the seedling rooting medium is 100 days to 120 days, and the conditions are the same as those in the embryo rescue medium.

[0013] Furthermore, the matrix for planting and culture is made by mixing pine bark, clay, and coarse vermiculite in equal volume ratios.

[0014] Furthermore, the conditions for planting and culture are as follows: daytime temperature of 20℃~25℃, nighttime temperature of 18℃~22℃, light duration of 10h~14h per day, daytime humidity of 75%~85%, and nighttime humidity of 90%~100%.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention, through a specific culture medium formulation and culture conditions, effectively increases the germination rate of young embryos from distant hybridization of elephant trunk orchids and calyx ridge orchids to 90%, and the transplant survival rate to 95%. The present invention can effectively improve the survival and growth potential of young embryos. In addition, through strict aseptic operation and optimized culture conditions, the risk of microbial contamination and poor growth of seedlings is greatly reduced. The culture medium ratio in the present invention helps to increase the germination rate of embryos and promote rooting of strong seedlings, overcoming the obstacle that distant hybridization embryos cannot survive under natural conditions.

[0016] The present invention, through distant hybridization of elephant trunk orchids and calyx orchids, combined with effective embryo rescue techniques, helps introduce new genetic traits and enhance the genetic diversity of Phalaenopsis varieties. In the present invention, distant hybridization of elephant trunk orchids and calyx orchids can potentially introduce genetic traits such as small flowers, a wider range of flower shapes and colors, and cold tolerance. This expansion of germplasm resources provides new materials for Phalaenopsis breeding to cultivate apple varieties with new characteristics, providing a new solution to the bottleneck problems currently encountered in Phalaenopsis breeding.

[0017] The specially designed formula of the rescue culture medium for the hybridization of elephant trunk orchid and calyx ridge orchid, the formula of the culture medium for rooting strong seedlings and the culture conditions are all crucial to the germination of the immature embryos and the growth of the seedlings. These ingredients play an important role in the continued development of the hybrid immature embryos until the germination process, helping the immature embryos to develop and germinate. At the same time, the present invention proposes a detailed distant hybridization embryo rescue process, including steps such as pollination, fruiting, seeding, disinfection, cultivation and transplanting. This complete operating process can not only be proven to be effective, but also can be standardized, meaning that it can be applied to large-scale breeding work. Large-scale operation can promote industrial development and provide more high-quality hybrid seedlings to the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the invention in the prior art, the drawings required for use in the embodiments or the description of the prior art 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 any creative work.

[0019] Figure 1 This shows the germination of seeds in different culture media. In the figure, AF are M1-M6.

[0020] Figure 2 This shows the effect of seedling rooting culture on different culture media. In the figure, 1: NAA 0mg / L + potato homogenate 75g / L; 2: NAA 0mg / L + banana homogenate 75g / L; 3: NAA 1.0mg / L + potato homogenate 75g / L; 4: NAA 1.0mg / L + banana homogenate 75g / L. Only the different components are marked in 1 to 4. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0022] Distant hybridization involves interspecific, intergeneric, and even higher taxonomic units hybridization to artificially obtain germplasm with superior traits. This hybridization can enhance the segregation of traits in offspring and enrich genetic diversity. Therefore, distant hybridization is an important method for expanding the genetic base, increasing genetic variation, and creating new germplasm resources. Distant hybridization involves gene exchange while overcoming reproductive isolation, resulting in hybrid offspring that are distinct from their parents. Although allopolyploids are common in nature, artificial distant hybridization presents challenges. Scholars speculate that the unstable morphological and genetic characteristics of naturally occurring allopolyploids may be eliminated through evolutionary adaptation, leading to the formation of stable species. However, artificially hybridizing interspecific species is a short and highly variable process, making it difficult to obtain fertile offspring. Hybrid embryos and endosperm tissues develop incoherently, leading to premature degradation of the endosperm, which in turn prevents the hybrid embryo from nutrient supply, leading to late-stage failure or degeneration. This manifests as hybrid sterility, failure, and inviability, significantly limiting the success rate of distant hybridization.

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

[0024] The present invention intends to obtain breeding intermediate materials by obtaining hybrid offspring of elephant trunk orchid and calyx ridge orchid, so as to provide genetic resources for hybridization of subsequent hybrid offspring with Phalaenopsis, and provide technical reserves for cultivating new varieties with both ornamental value and stress resistance.

[0025] Example 1: A method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue.

[0026] S1, using Cymbidium selengensis as the male parent, use a toothpick to pick up the pollen mass during the flowering period for later use; using Elephant trunk orchid as the female parent, use a toothpick to remove the stamens during the flowering period, and put the Cymbidium selengensis pollen mass into the pistil. Put it into a sulfuric acid paper bag immediately after pollination. 3 days after pollination, the stigma of Elephant trunk orchid completely wraps the pollen mass, and the petals begin to shrink. Remove the bag after 12 days when the fruit begins to swell.

[0027] In S2, hybrid young fruits were picked 50 days after pollination, and after disinfection, they were cut into 4 mm thick slices under sterile conditions and inoculated on embryo rescue medium for culture. The temperature was 25 °C, LED white: red = 7:3, and the light intensity was 35 μmol·m -2 ·s -1 , and cultured for 50 days under the condition of 12 hours of light per day, the young embryos germinated into rooted seedlings.

[0028] The young fruit disinfection method is as follows: take hybrid young fruits, soak them in detergent for 13 minutes, clean them with running water, soak them in 75% alcohol by volume for 30 seconds in a clean bench, rinse them with sterile water for 3 times, sterilize them with 0.1% HgCl2 by mass for 17 minutes, and rinse them with sterile water for 5 times.

[0029] The embryo rescue medium includes: 2 g / L Huabao No. 1, 100 g / L potato homogenate, 30 g / L sucrose, 0.5 g / L activated carbon, 7.0 g / L agar, and a pH of 5.7.

[0030] S3, transfer the young embryos of the rooted seedlings to the seedling rooting medium, and culture them under the same culture conditions as the seed germination culture for 110 days to obtain rooted hybrid seedlings. When the seedlings grow into strong seedlings with 4 to 5 leaves and 5 to 8 roots, they can be transplanted.

[0031] The seedling rooting culture comprises: Huabao No. 1 1.5 g / L + peptone 2.0 g / L + NAA 1 mg / L + inositol 120 mg / L + glycine 2.0 mg / L + thiamine hydrochloride 0.2 mg / L + pyridoxine hydrochloride 0.5 mg / L + niacin 0.5 mg / L, banana homogenate 75 g / L, activated carbon 0.5 g / L, agar 7 g / L, and the balance is water, with a pH of 5.6.

[0032] S4. Select strong seedlings in bottles, open the bottles and harden them in the culture room for 4 days. Before transplanting, add 20-30 ml of water to the bottles and soak them for 30 minutes to soften the culture medium. Take out the bottle seedlings together with the culture medium, wash the root culture medium with running water, remove old roots and dead leaves, and place them in a plastic basket to drain the water.

[0033] S5. Prepare a cultivation medium by mixing pine bark, clay, and coarse vermiculite in a volume ratio of 1:1:1. Cover the old roots with the soil, leaving the rhizome exposed. Cultivate in a lighted incubator at 26°C during the day and 21°C at night, with 14 hours of light daily. Humidity should be 80% during the day and 95% at night. Water the medium every four days to keep it moist. Disinfect with carbendazim (900x diluted) every eight days. New roots should develop approximately 10 days after planting.

[0034] Example 2: A method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue.

[0035] S1, using Cymbidium selengensis as the male parent, use a toothpick to pick up the pollen mass during the flowering period for later use; using Elephant trunk orchid as the female parent, use a toothpick to remove the stamens during the flowering period, and put the Cymbidium selengensis pollen mass into the pistil. Put it into a sulfuric acid paper bag immediately after pollination. 3 days after pollination, the stigma of Elephant trunk orchid completely wraps the pollen mass, and the petals begin to shrink. Remove the bag after 10 days when the fruit begins to swell.

[0036] In S2, hybrid young fruits were picked 40 days after pollination, sterilized, and cut into 3 mm thick slices under sterile conditions. The slices were inoculated on embryo rescue medium for culture. The temperature was 25 °C, the LED white: red ratio was 7:3, and the light intensity was 30 μmol·m -2 ·s -1 , and cultured for 40 days under the condition of 12 hours of light per day, the young embryos germinated into rooted seedlings.

[0037] The young fruit disinfection method is as follows: take hybrid young fruits, soak them in detergent for 10 minutes, clean them with running water, soak them in 75% alcohol by volume for 30 seconds in a clean bench, rinse them with sterile water for 3 times, sterilize them with 0.1% HgCl2 by mass for 15 minutes, and rinse them with sterile water for 5 times.

[0038] The embryo rescue medium includes: 2 g / L Huabao No. 1, 100 g / L potato homogenate, 30 g / L sucrose, 0.5 g / L activated carbon, 7.0 g / L agar, and a pH of 5.6.

[0039] S3, transfer the young embryos of the rooted seedlings to the seedling rooting medium, and culture them under the same culture conditions as the seed germination culture for 100 days to obtain rooted hybrid seedlings. When the seedlings grow into strong seedlings with 4 to 5 leaves and 5 to 8 roots, they can be transplanted.

[0040] The seedling rooting culture medium is composed of Huabao No. 1 1.5g / L + peptone 2.0g / L + NAA 1mg / L + inositol 120mg / L + glycine 2.0mg / L + thiamine hydrochloride 0.2mg / L + pyridoxine hydrochloride 0.5mg / L + nicotinic acid 0.5mg / L, banana homogenate 75g / L, activated carbon 0.5g / L, agar 7g / L, and pH 5.6.

[0041] S4. Select strong seedlings in bottles, open the bottles and harden them in the culture room for 3 days. Before transplanting, add appropriate amount of water to the bottles and soak them for 30 minutes to soften the culture medium. Take out the bottle seedlings together with the culture medium, wash the root culture medium with running water, remove old roots and dead leaves, and place them in a plastic basket to drain the water.

[0042] S5. Prepare a cultivation medium by mixing pine bark, clay, and coarse vermiculite in a volume ratio of 1:1:1. Cover the old roots with the soil, leaving the rhizome exposed. Cultivate in a lighted incubator at 25°C during the day and 20°C at night, with 14 hours of light daily. Keep the humidity at 75% during the day and 90% at night. Water every three days to keep the medium moist. Disinfect with 800x carbendazim every six days. New roots should develop approximately 10 days after planting.

[0043] Example 3: A method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue.

[0044] S1, using Cymbidium selengensis as the male parent, use a toothpick to pick up the pollen mass during the flowering period for later use; using Elephanthus chinensis as the female parent, use a toothpick to remove the stamens during the flowering period, and put the Cymbidium selengensis pollen mass into the pistil. Put it into a sulfuric acid paper bag immediately after pollination. 3 days after pollination, the stigma of Elephanthus chinensis completely wraps the pollen mass, and the petals begin to shrink. Remove the bag after 114 days when the fruit begins to swell.

[0045] In S2, hybrid young fruits were picked 60 days after pollination, sterilized, and cut into 5 mm thick slices under sterile conditions. The slices were inoculated on embryo rescue medium for culture. The temperature was 25 °C, LED white: red = 7:3, and the light intensity was 40 μmol·m -2 ·s -1 , and cultured for 60 days under the condition of 12 hours of light per day, the young embryos germinated into rooted seedlings.

[0046] The young fruit disinfection method is as follows: take hybrid young fruits, soak them in detergent for 15 minutes, clean them with running water, soak them in 75% alcohol by volume for 30 seconds in a clean bench, rinse them with sterile water for 3 times, sterilize them with 0.1% HgCl2 by mass for 20 minutes, and rinse them with sterile water for 5 times.

[0047] The embryo rescue medium comprises: 2 g / L Huabao No. 1, 100 g / L potato homogenate, 30 g / L sucrose, 0.5 g / L activated carbon, 7.0 g / L agar, and the balance is water, with a pH of 5.8.

[0048] S3, transfer the young embryos of the rooted seedlings to the seedling rooting medium, and culture them under the same culture conditions as the seed germination culture for 120 days to obtain rooted hybrid seedlings. When the seedlings grow into strong seedlings with 4 to 5 leaves and 5 to 8 roots, they can be transplanted.

[0049] The seedling rooting culture medium comprises: Huabao No. 1 1.5 g / L + peptone 2.0 g / L + NAA 1 mg / L + inositol 120 mg / L + glycine 2.0 mg / L + thiamine hydrochloride 0.2 mg / L + pyridoxine hydrochloride 0.5 mg / L + nicotinic acid 0.5 mg / L, banana homogenate 75 g / L, activated carbon 0.5 g / L, agar 7 g / L, and a pH of 5.6.

[0050] S4. Select strong seedlings in bottles, open the bottles and harden them in the culture room for 5 days. Before transplanting, add 20-30 ml of water to the bottles and soak them for 30 minutes to soften the culture medium. Take out the bottle seedlings together with the culture medium, wash the root culture medium with running water, remove old roots and dead leaves, and place them in a plastic basket to drain the water.

[0051] S5. Prepare a cultivation medium by mixing pine bark, clay, and coarse vermiculite in a volume ratio of 1:1:1. Cover the old roots with the soil, leaving the rhizome exposed. Cultivate in a lighted incubator at 27°C during the day and 22°C at night, with 14 hours of light daily. Humidity should be 85% during the day and 100% at night. Water every 5 days to keep the medium moist. Disinfect with 1000x carbendazim every 10 days. New roots should develop approximately 10 days after planting.

[0052] Verification Example 1: Effects of culture medium components on non-symbiotic seed germination.

[0053] Taking Example 1 as an example, gather the fruit of embryo age 50d, adopt single factor experimental design, study minimal medium MS, Huabao No. 1, the NAA of 0mg / L, 1mg / L, 2mg / L, no organic additives add, add potato 100g / L, add coconut milk 100g / L on the impact of seed germination.The fruit collected is disinfected with 75% alcohol for 30s, 0.1% mercuric chloride is disinfected for 18min, and sterile water rinses 5 times. In a clean bench, cut the fruit longitudinally with a scalpel. The seeds in the fruit are clamped with tweezers on the culture medium, and then 0.5ml sterile water is drawn in the bottle with a pipette. The culture bottle is shaken so that the seeds are evenly dispersed on the culture medium surface. 10 bottles are often organized, and 3 repetitions are repeated. After inoculation 60d, the seeds in the tissue culture bottle are taken out, and observation is taken with 10 times of stereomicroscopes. The seeds in every bottle of tissue culture bottle are randomly photographed and 3 visual fields are taken. According to the process of seed germination and seedling morphology development of elephant trunk orchid, it can be divided into S1 stage: the seed swells and breaks the seed coat; S2 stage: the embryo breaks the seed coat to form a green protocorm; S3 stage: the oblate spherical protocorm is formed and buds and rhizoids are produced; S4 stage: 3-5 young leaves and young roots are grown, and the seed germination status in the field of view is counted.

[0054] There were significant differences in the germination process of seeds in different basic media (P<0.05). Figure 1 As shown in Table 1, seeds germinated faster in M1 medium. After 60 days of incubation, the proportion of protocorms in the S3 stage in M1 medium was 48.48%, significantly higher than the 14.67% in M6 medium. In contrast, the protocorms in M6 medium were primarily concentrated in the S2 stage, accounting for 70.92%, significantly higher than the 42.51% in M6 medium. Later observations revealed that protocorms in MS medium exhibited higher mortality rates with increasing incubation time. Significant differences in seed germination progress were observed in media containing different organic additives (P < 0.05). The addition of organic additives significantly increased seed germination rates, with the most significant effect being achieved in M1 supplemented with 100 g / L potato. After 60 days of cultivation, 87.07% of the protocorms in the medium without organic additives were still concentrated in the S2 stage, while those in the S3 and S4 stages accounted for only 0.63%. The proportion of protocorms in these stages reached over 30%. Adding 100g / L of potato increased the proportion of protocorms in the S3 stage from 0.63% in the unsupplemented medium to 48%, a 47.37% improvement. Coconut milk did not significantly promote seed germination in the elephant orchid. Therefore, Huabao No. 1 is more suitable as a basic medium for asymbiotic germination culture of the elephant orchid. See Table 1 for a comparison of culture data obtained in different media.

[0055] Table 1: The percentage of S1-S4 protocorms cultured for 60 days in different culture media.

[0056]

[0057] Note: The above culture medium is supplemented with activated carbon 0.5g / L and agar 7.0g / L, with a pH of 5.6-5.8.

[0058] Verification Example 2 The hybrid rooted seedlings of Example 1 were used as materials and Huabao No. 1 was used as the basic culture medium to study the effects of 0 mg / L and 1 mg / L NAA, 75 g / L potato and 75 g / L banana homogenates on seedling growth. The above culture media were supplemented with 2.0 g / L peptone, 120 mg / L inositol + 2.5 mg / L glycine + 0.2 mg / L thiamine hydrochloride + 0.8 mg / L pyridoxine hydrochloride + 0.8 mg / L nicotinic acid, 0.5 g / L activated carbon, 7.0 g / L agar, and the pH was 5.6-5.8. Figure 2 The results showed that the culture medium containing Huabao No. 1 1.5g / L + peptone 2.0g / L + NAA 1.0mg / L + inositol 120mg / L + glycine 2.5mg / L + thiamine hydrochloride 0.2mg / L + pyridoxine hydrochloride 0.8mg / L + niacin 0.8mg / L + banana homogenate 75g / L, activated carbon 0.5g / L, agar 7.0g / L, pH 5.6~5.8 had the best effect on seedling growth and rooting.

[0059] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A method for obtaining interspecific hybrid offspring of Elephant Orchid and Calyx Orchid by using embryo rescue, characterized in that: The steps include: Hybridization is carried out using Cymbidium calyx as the male parent and Cymbidium elegans as the female parent. Hybrid young fruits are picked 40 to 60 days after pollination, disinfected, sliced, and then inoculated into embryo rescue medium for cultivation. After the young embryos germinate into rooted seedlings, they are transferred to seedling rooting medium for cultivation to obtain rooted hybrid seedlings. When the rooted hybrid seedlings have 4 to 5 leaves and 5 to 8 roots, they are transplanted and planted for cultivation. The composition of the embryo rescue medium per 1 L is: 2 g / L Huabao No. 1, 100 g / L potato homogenate, 30 g / L sucrose, 0.5 g / L activated carbon, 7.0 g / L agar, and the balance is water, with a pH of 5.6-5.

8.

2. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue according to claim 1, characterized in that: The hybridization specifically comprises the following steps: picking up the pollen mass of the calyx ridge orchid during the blooming period, taking away the stamens of the elephant orchid, and sending the pollen mass of the calyx ridge orchid into the pistil of the elephant orchid for pollination.

3. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid by using embryo rescue according to claim 1, characterized in that: The disinfection is specifically as follows: soak the young fruit in detergent for 10 to 15 minutes, wash and soak in 75% alcohol for 30 to 50 seconds, rinse with sterile water, sterilize with 0.1 to 0.3% HgCl2 for 15 to 20 minutes, and rinse with sterile water 4 to 5 times.

4. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue according to claim 1, characterized in that: The embryo rescue medium was cultured for 40 to 60 days under the following conditions: temperature of 20°C to 30°C, LED white:LED red = 5 to 8:2 to 4, and light intensity of 30 μmol·m -2 ·s -1 ~40 μmol·m -2 ·s -1 , the lighting time is 10h~14h per day.

5. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid by using embryo rescue according to claim 1, characterized in that: The composition of the seedling rooting culture medium per 1L is: 1.5g / L Bao No. 1, 2.0g / L peptone, 1mg / L NAA, 120mg / L inositol, 2.0mg / L glycine, 0.2mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride, 0.5mg / L nicotinic acid, 75g / L banana homogenate, 0.5g / L activated carbon, 7g / L agar, and the balance is water, pH 5.

6.

6. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue according to claim 4, characterized in that: The culture time in the seedling rooting medium is 100 days to 120 days, and the conditions are the same as those in the embryo rescue medium.

7. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue according to claim 1, characterized in that: The matrix for planting culture is a mixture of pine bark, clay and coarse vermiculite in equal volume ratios.

8. The method for obtaining interspecific hybrid offspring of Cymbidium orchid and Cymbidium orchid using embryo rescue according to claim 1, characterized in that: The conditions for planting and cultivation are daytime temperature of 20℃~25℃, nighttime temperature of 18℃~22℃, light duration of 10h~14h per day, daytime humidity of 75%~85%, and nighttime humidity of 90%~100%.

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