Method for obtaining interspecific hybrid offspring of phreatia and vandopsis by embryo rescue

Abstract

The present application relates to the field of biotechnology breeding, and particularly relates to a method for obtaining interspecific hybrid offspring of Rhynchostylis retusa and Vanda teres by embryo rescue. The present application takes Vanda teres as the father and Rhynchostylis retusa as the mother, and combines with the embryo rescue technology. In the embryo rescue technology, the composition of the embryo rescue medium is as follows: 2g / L of Hua Bao No.1, 100g / L of potato homogenate, 30g / L of sucrose, 0.5g / L of activated carbon, 7.0g / L of agar, and the rest is water, and pH is 5.6-5.8. The method of the present application can significantly promote the germination of the hybrid embryo of Rhynchostylis retusa and Vanda teres, potentially introduce new genetic traits, enhance the genetic diversity of Phalaenopsis varieties, and provide a new germplasm resource for Phalaenopsis breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology breeding, and particularly relates to a method for obtaining interspecific hybrid offspring of Phalaenopsis amabilis and Oncidium by embryo rescue. BACKGROUND

[0002] Phalaenopsis amabilis and Oncidium are both in the genus Phalaenopsis, and are excellent parents of Phalaenopsis variety improvement. Chloroplast genome data shows that Phalaenopsis amabilis and Oncidium are relatively distant in genetic relationship.

[0003] Phalaenopsis is one of the most important horticultural flowers in the world commercial flower cultivation. Phalaenopsis breeding has also changed from only focusing on large flower types to small flower types, multi-color, easy to flower, drought resistance, and cold resistance. It is possible to breed new Phalaenopsis germplasm with small flowers and cold resistance through distant hybridization. Phalaenopsis amabilis is a type of Phalaenopsis with small flowers, unique flower shape, bright flower color, and good cold resistance, and has important breeding value.

[0004] Distant hybridization incompatibility and early embryo abortion are the main obstacles in plant hybrid breeding. There is no report on distant hybridization of Phalaenopsis amabilis. Embryo rescue is one of the main technical means to obtain distant hybridization seedlings. Early in vitro culture of early abortion, degeneration, and non-viable embryos can obtain seedlings, thereby overcoming distant hybridization incompatibility and improving interspecific hybrid seedling rate. At present, there is no case of using embryo rescue and in vitro culture technology to realize interspecific hybridization of Phalaenopsis amabilis and Oncidium with relatively distant genetic relationship and obtain hybrid offspring plants. In the process of embryo rescue, different embryo ages, disinfection methods, culture medium types, hormone types and ratios, and seedling rooting will affect the success rate of rescue. Establishing an embryo rescue system suitable for interspecific hybrid embryos of Phalaenopsis amabilis and Oncidium can lay a foundation for distant hybridization breeding and germplasm innovation and utilization. SUMMARY

[0005] In order to break through the natural interspecific hybridization barrier of Phalaenopsis and create new germplasm resources, the present application provides a method for obtaining interspecific hybrid offspring of Phalaenopsis amabilis and Oncidium by embryo rescue.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following invention:

[0007] A method for obtaining interspecific hybrid offspring of Phalaenopsis amabilis and Oncidium by embryo rescue, comprising the following steps:

[0008] Crossing Oncidium as the male parent and Phalaenopsis amabilis as the female parent, picking the hybrid young fruit after 40d-60d of pollination, disinfecting the young fruit, slicing, then inoculating in embryo rescue medium for culture, waiting for the young embryo to germinate into a rooted seedling and transferring to a strong seedling rooting medium for culture, and obtaining a rooted hybrid seedling;

[0009] The composition of each 1L of the embryo rescue medium is: Huabao No. 1 2g / L, potato homogenate 100g / L, sucrose 30g / L, activated carbon 0.5g / L, agar 7.0g / L, the balance is water, pH 5.6-5.8.

[0010] Although Phalaenopsis x Cypripedium has been successfully reported, Phalaenopsis x Cypripedium has not been able to obtain offspring. The present application uses the hybrid offspring of Cypripedium and Phalaenopsis, and combines with a specific embryo rescue medium to carry out embryo rescue, to obtain breeding intermediate materials, to provide genetic resources for subsequent hybrid offspring and Phalaenopsis hybridization, to provide genetic resources for Phalaenopsis variety improvement, and to provide technical reserves for cultivating new varieties with both ornamental value and stress resistance.

[0011] Further, the hybridization specifically comprises picking up pollen masses of Cypripedium at the flowering stage, removing stamens of Cypripedium, and sending the pollen masses of Cypripedium into pistils of Cypripedium for pollination.

[0012] Further, the disinfection specifically comprises: soaking young fruits with dishwashing liquid for 10-15 minutes, washing, soaking with 75% alcohol for 30-50 seconds, rinsing with sterile water, sterilizing with 0.1-0.3% HgCl2 for 15-20 minutes, and rinsing with sterile water for 4-5 times.

[0013] Further, the culture time in the embryo rescue medium is 40-60 days, and the conditions are: the temperature is 20-30℃, the LED white: LED red is 5-8:2-4, the light intensity is 30-40μmol·m-2·s-1, and the light time is 10-14 hours per day. -2 ·s -1 ~40μmol·m -2 ·s -1 , the light time is 10-14 hours per day.

[0014] Further, the composition of each 1L of the strong seedling rooting medium is: Huabao No. 1 1.5g / L, peptone 2.0g / L, NAA 1mg / L, myo-inositol 120mg / L, glycine 2.0mg / L, thiamine hydrochloride 0.2mg / L, pyridoxal hydrochloride 0.5mg / L, nicotinic acid 0.5mg / L, banana homogenate 75g / L, activated carbon 0.5g / L, agar 7g / L, the balance is water, and pH 5.6.

[0015] Further, the culture time in the strong seedling rooting medium is 100-120 days, and the culture conditions are the same as those in the embryo rescue medium.

[0016] Further, the substrate for planting culture is mixed by pine bark, pottery clay and coarse vermiculite in equal volume ratio.

[0017] Further, the conditions for the inoculation culture are as follows: the temperature is 20-25 DEG C during the day and 18-22 DEG C at night, the light time is 10-14 hours per day, the humidity is 75-85% during the day and 90-100% at night.

[0018] Compared with the prior art, the present application has the following advantages:

[0019] In the present application, the germination rate of the hybrid embryo of Phalaenopsis and Thrixspermum is effectively improved to 90% and the survival rate of transplanting is 95% through specific culture medium formula and culture conditions.

[0020] The present application is helpful to introduce new genetic characteristics and enhance the genetic diversity of the butterfly orchid variety through the distant hybridization of Phalaenopsis and Thrixspermum and the effective embryo rescue technology.

[0021] The specially designed hybrid embryo rescue culture medium formula, seedling rooting culture medium formula and culture conditions of Phalaenopsis and Thrixspermum in the present application are crucial to the germination of the hybrid embryo and the growth of the seedling. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced.

[0023] Figure 1The seed germination in different culture mediums is shown in the figure, A-F is M1-M6 in turn.

[0024] Figure 2 The seedling and rooting culture effect of different culture mediums is shown in the figure, 1: NAA 0 mg / L + potato homogenate 75 g / L; 2: NAA 0 mg / L + banana homogenate 75 g / L; 3: NAA 1.0 mg / L + potato homogenate 75 g / L; 4: NAA 1.0 mg / L + banana homogenate 75 g / L, 1-4 are marked with different components. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present application. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0026] Distant hybrid breeding refers to hybridization between species, genera or even higher taxonomic units, and artificially obtaining germplasm resources with excellent traits. Through hybridization, the segregation of offspring traits can be expanded, and genetic diversity can be enriched. Therefore, distant hybridization technology is an important way to expand genetic basis, increase genetic variation and create new germplasm resources. Distant hybridization is to exchange genes on the basis of overcoming reproductive isolation, so as to form hybrid offspring different from the parents. Although there are many allopolyploids in nature, it is difficult for humans to perform distant hybridization. Scholars speculate that the unstable characteristics of allopolyploids produced by natural hybridization, such as appearance and gene structure, may be eliminated due to evolutionary adaptation process, thereby forming stable species. However, artificial distant hybridization of different species has short process, large changes, and it is difficult to obtain fertile offspring. The development of hybrid embryo and endosperm tissue is not coordinated, and the endosperm is degraded too early, which cannot provide nutrition for the hybrid embryo, leading to degradation or degeneration in the later development stage, and showing hybrid sterility, degradation and non-survival, etc., which greatly limits the success rate of distant hybridization.

[0027] Embryo rescue is one of the key technologies to improve the survival rate of distant hybrid offspring. More than 95% of distant hybrid embryos cannot be sowed into seedlings, or the embryos are aborted or degenerated in the early development stage. Therefore, in vitro culture of immature embryos, especially early development embryos, is generally not easy to succeed. Suitable culture medium, hormone ratio and culture conditions are the basic conditions for successful in vitro culture.

[0028] The application aims to obtain breeding intermediate materials by obtaining hybrid offspring of Phalaenopsis amabilis and Oncidiopsis glauca, cross the hybrid offspring with Phalaenopsis, provide genetic resources for Phalaenopsis variety improvement, and provide technical reserves for cultivating new varieties with ornamental value and stress resistance.

[0029] Embodiment 1: A method for obtaining interspecific hybrid offspring of Phalaenopsis amabilis and Oncidiopsis glauca by embryo rescue.

[0030] S1, using Oncidiopsis glauca as the father, using toothpicks to pick pollen at the flowering stage; using Phalaenopsis amabilis as the mother, using toothpicks to take away the stamens, and sending the Oncidiopsis glauca pollen into the pistil, immediately after pollination, wrapping the sulfuric acid paper bag, 3 days after pollination of Phalaenopsis amabilis, the stigma is completely wrapped with pollen blocks, and the petals begin to wilt, and the bag is removed after 12 days when the fruit begins to swell.

[0031] S2, picking hybrid young fruits 50 days after pollination, disinfecting the young fruits, and cutting the young fruits into 4mm-thick slices under sterile conditions, and inoculating the slices on the embryo rescue medium for culture. The culture is carried out under the condition that the temperature is 25℃, the LED white: red = 7:3, the light intensity is 35μmol·m -2 ·s -1 , and the light time is 12h per day for 50 days, and the embryo germinates into a small plant with roots.

[0032] The disinfection method of the young fruit is as follows: taking the hybrid young fruit, soaking it in detergent for 13min, washing it clean with running water, placing it in a clean bench, soaking it in alcohol with a volume fraction of 75% for 30s, rinsing it with sterile water for 3 times, sterilizing it with HgCl2 with a mass fraction of 0.1% for 17min, and rinsing it with sterile water for 5 times.

[0033] The embryo rescue medium: flower treasure No.1 2g / L, potato homogenate 100g / L, sucrose 30g / L, activated carbon 0.5g / L, agar 7.0g / L, pH 5.7.

[0034] S3, the embryo with roots is transferred to the strong seedling rooting medium, and cultured under the same culture condition as the seed germination culture for 110 days, and the rooted hybrid seedling is obtained, and when the seedling grows to a strong seedling with 4-5 leaves and 5-8 roots, it can be transplanted.

[0035] The strong seedling rooting culture: flower treasure No.1 1.5g / L+peptone 2.0g / L+NAA 1mg / L+inositol 120mg / L+glycine 2.0mg / L+thiamine hydrochloride 0.2mg / L+pyridoxal hydrochloride 0.5mg / L+nicotinic acid 0.5mg / L, banana homogenate 75g / L, activated carbon 0.5g / L, agar 7g / L, and the rest is water, pH 5.6.

[0036] S4. Select strong seedlings in bottles and harden them off in the culture room for 4 days. Before transplanting, add 20-30ml of water to the bottle and soak for 30 minutes to soften the culture medium. Take out the seedlings along with the culture medium, wash the culture medium off the roots with running water, remove old roots and withered leaves, and place them in a plastic basket to drain.

[0037] S5: Prepare a cultivation substrate by mixing pine bark, clay, and coarse vermiculite in a 1:1:1 volume ratio. When planting, cover the old roots of the plant with the substrate, leaving the rhizome exposed. Place the plant in a light-controlled incubator with a daytime temperature of 26℃ and a nighttime temperature of 21℃, providing 14 hours of light per day. Maintain a humidity level of 80% during the day and 95% at night. Water every 4 days to keep the substrate moist, and spray with a 900-fold dilution of carbendazim for disinfection every 8 days. New roots will emerge approximately 10 days after transplanting.

[0038] Example 2: A method for obtaining interspecific hybrid offspring of Cymbidium faberi and Cymbidium calycifolium using embryo rescue.

[0039] S1 uses *Cymbidium goeringii* as the male parent, and collects pollen masses with a toothpick during the peak flowering period for later use; uses *Cymbidium faberi* as the female parent, and removes the stamens with a toothpick during the flowering period, then transfers the pollen masses from *Cymbidium goeringii* into the pistil. Immediately after pollination, cover the pistil with a sulfuric acid paper bag. Three days after pollination, the stigma of *Cymbidium faberi* completely encloses the pollen masses, and the petals begin to wither. Remove the bag after 10 days when the fruit begins to swell.

[0040] S2: Hybrid young fruits harvested 40 days after pollination were sterilized and then aseptically sliced ​​into 3mm thick slices, which were then inoculated onto embryo rescue medium for culture. The culture was conducted at 25℃ with an LED white:red ratio of 7:3 and a light intensity of 30 μmol·m⁻¹. -2 ·s -1 When cultured for 40 days under a light exposure of 12 hours per day, the embryos germinated into seedlings with roots.

[0041] The method for disinfecting young fruits is as follows: Take hybrid young fruits, soak them in detergent for 10 minutes, rinse them with running water, place them in a clean bench and soak them in 75% alcohol for 30 seconds, rinse them three times with sterile water, sterilize them with 0.1% HgCl2 for 15 minutes, and rinse them five times with sterile water.

[0042] The embryo rescue culture medium consisted of: Flower Treasure No. 1 2 g / L, potato homogenate 100 g / L, sucrose 30 g / L, activated carbon 0.5 g / L, agar 7.0 g / L, and pH 5.6.

[0043] S3. Transfer the embryos of the rooted seedlings to the seedling rooting medium and culture them under the same conditions as seed germination culture for 100 days to obtain rooted hybrid seedlings. When the seedlings grow to the point of having 4 to 5 leaves and 5 to 8 roots, they can be transplanted.

[0044] The strong seedling rooting medium is Hua Bao 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 + pyridoxal 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, pH 5.6.

[0045] S4, selecting strong seedling bottles, opening the bottle in the culture room for 3 days, adding an appropriate amount of water in the bottle before transplanting for 30 min to soften the medium, taking out the seedling together with the medium, washing the root medium with running water, removing old roots and dead leaves, and placing in a plastic basket to drain water.

[0046] S5, preparing a cultivation matrix by mixing pine bark, pottery clay and coarse vermiculite at a volume ratio of 1:1:1, covering the old roots of the plant with the cultivation soil, exposing the rhizome part, and placing in a light culture box for culture, with a culture temperature of 25℃ in the daytime and 20℃ at night, 14h light per day, a culture box humidity of 75% in the daytime and 90% at night. Watering once every 3 days to keep the matrix moist, and spraying with carbendazim 800 times for sterilization once every 6 days. New roots can be extracted after about 10 days of planting.

[0047] Example 3: A method for obtaining interspecific hybrid offspring of Phalaenopsis amabilis and Oncidium by embryo rescue.

[0048] S1, using Oncidium as the female parent, taking the pollen mass with a toothpick at the flowering stage for standby; using Oncidium as the male parent, taking away the stamens with a toothpick at the flowering stage, and sending the Oncidium pollen mass into the pistil. Immediately after pollination, a sulfuric acid paper bag is used to cover the flower. The stigma is completely wrapped in the pollen mass 3 days after pollination of the female parent, and the petals begin to wilt. The bag is removed when the fruit begins to swell 114 days after pollination.

[0049] S2, picking hybrid young fruits 60 days after pollination, disinfecting the young fruits, and cutting the young fruits into 5mm-thick slices under sterile conditions, and inoculating the slices on the embryo rescue medium for culture. The culture is carried out at a temperature of 25℃, LED white: red = 7:3, light intensity of 40 μmol·m -2 ·s -1 , and light time of 12h per day for 60 days, and the young embryos germinate into small seedlings with roots.

[0050] The disinfection method of the young fruits is as follows: taking the hybrid young fruits, soaking them in dishwashing liquid for 15 min, washing them clean with running water, immersing them in alcohol with a volume fraction of 75% for 30 s in a super-clean bench, rinsing them with sterile water for 3 times, sterilizing them with HgCl2 with a mass fraction of 0.1% for 20 min, and rinsing them with sterile water for 5 times.

[0051] The embryo rescue medium: Huaba No.1 2g / L, potato homogenate 100g / L, sucrose 30g / L, activated carbon 0.5g / L, agar 7.0g / L, the rest is water, pH 5.8.

[0052] S3, the seedling with root embryo is transferred to the strong seedling rooting medium, and is cultured under the same culture condition as the seed germination culture for 120d, so that the hybrid seedling with roots is obtained, and when the seedling grows to the strong seedling with 4-5 leaves and 5-8 roots, the seedling is transplanted.

[0053] The strong seedling rooting medium: Huaba No.1 1.5g / L+peptone 2.0g / L+NAA 1mg / L+inositol 120mg / L+glycine 2.0mg / L+thiamine hydrochloride 0.2mg / L+pyridoxal hydrochloride 0.5mg / L+nicotinic acid 0.5mg / L, banana homogenate 75g / L, activated carbon 0.5g / L, agar 7g / L, pH 5.6.

[0054] S4, the strong seedling bottle seedling is selected, the bottle is opened and the seedling is acclimated for 5d in the culture room, 20-30ml water is added in the bottle before transplanting to soften the culture medium for 30min, the bottle seedling is taken out together with the culture medium, the root culture medium is washed with running water, the old roots and withered leaves are removed, and the seedling is placed in a plastic basket to drain water.

[0055] S5, pine bark, pottery clay and coarse vermiculite are mixed according to the volume ratio of 1:1:1 to prepare the cultivation matrix, the old roots of the plant are covered with the cultivation soil, and the rhizome part is exposed. The seedling is placed in a light culture box for culture, the culture temperature is 27℃ in the daytime and 22℃ at night, the light illumination time is 14h per day, the humidity of the culture box is 85% in the daytime and 100% at night. Water is poured once every 5d to keep the matrix moist, and carbendazim 1000 times is sprayed once every 10d for sterilization. New roots are extracted after about 10d of planting.

[0056] Verification example 1: influence of medium components on seed nonsymbiotic germination.

[0057] With Example 1 as an example, the fruits of 50 d embryo age were collected, and the effects of basic medium MS, Hua Bao No. 1, 0 mg / L, 1 mg / L, 2 mg / L NAA, no organic additive, adding potato 100 g / L, and adding coconut juice 100 g / L on seed germination were studied by single factor experiment design. The collected fruits were sterilized with 75% alcohol for 30 s, 0.1% mercury for 18 min, and rinsed with sterile water for 5 times. The fruits were longitudinally cut with a surgical knife in the clean bench, and the seeds in the fruits were picked up with tweezers and placed on the culture medium. Then 0.5 ml sterile water was taken with a pipette gun into the bottle, and the bottle was shaken to make the seeds evenly dispersed on the surface of the culture medium. Each group had 10 bottles, and there were 3 repeats. After 60 d of inoculation, the seeds in the culture bottle were taken out, and the visual microscope was used to take pictures at 10 times. Three fields of view were randomly taken for each seed in the culture bottle. According to the seed germination and the morphological development process of the seedling of Pecteilis spicata, it was divided into S1 stage: seed swelling and breaking the seed coat; S2 stage: seed embryo breaking the seed coat to form green protocorm; S3 stage: forming flat spherical protocorm and producing bud points and false roots; S4 stage: sprouting 3-5 young leaves and young roots, and the seed germination status in the field of view was counted.

[0058] There were significant differences in the seed germination process in different basic media, P<0.05, as shown in Figure 1 and Table 1, the seeds germinated faster in M1 medium. After 60 d of culture, the S3 protocorms in M1 medium accounted for 48.48%, which was significantly higher than 14.67% in M6 medium; while the protocorms in M6 medium were mainly concentrated in S2 stage, accounting for 70.92%, which was significantly higher than 42.51% in M6 medium. Later observation found that the protocorms in MS medium showed a higher mortality rate with the extension of culture time. There were significant differences in the seed germination process in different organic additive media, P<0.05. The addition of organic additives significantly improved the seed germination rate, and the addition of potato 100 g / L in M1 medium had the most significant effect on promoting seed germination. After 60 d of culture, 87.07% of the protocorms in the medium without organic additives were concentrated in S2 stage, and the protocorms in S3 and S4 stages accounted for only 0.63%. The proportion of protocorms in S3 and S4 stages reached more than 30%, and the addition of potato 100 g / L increased the proportion of S3 stage protocorms from 0.63% without addition to 48%, an increase of 47.37%. Coconut juice could not significantly promote the seed germination of Pecteilis spicata. Therefore, Hua Bao No. 1 is more suitable as the basic medium for the non-symbiotic germination culture of Pecteilis spicata. The culture data obtained in different media are shown in Table 1 below.

[0059] Table 1: Proportion of S1-S4 protocorms in different culture media after 60 d of culture.

[0060]

[0061] Note: the above medium is added with activated carbon 0.5g / L, agar 7.0g / L, pH 5.6-5.8.

[0062] Verification example 2

[0063] With hybridization of the root seedlings of example 1 as material, with Huaba No.1 as basic medium, the effect of 0mg / L, 1mg / L of NAA, organic additives homogenate potato 75g / L, banana 75g / L on seedling strengthening was studied, the above medium is added with peptone 2.0g / L, myo-inositol 120mg / L+glycine 2.5mg / L+thiamine hydrochloride 0.2mg / L+pyridoxal hydrochloride 0.8mg / L+nicotinic acid 0.8mg / L, activated carbon 0.5g / L, agar 7.0g / L, pH 5.6-5.8. As Figure 2 The results show that Huaba No.1 1.5g / L+peptone 2.0g / L+NAA 1.0mg / L+myo-inositol 120mg / L+glycine 2.5mg / L+thiamine hydrochloride 0.2mg / L+pyridoxal hydrochloride 0.8mg / L+nicotinic acid 0.8mg / L+banana homogenate 75g / L, activated carbon 0.5g / L, agar 7.0g / L, pH 5.6-5.8, the medium seedling rooting effect is best.

[0064] Although the preferred embodiments of the present application have been described, those skilled in the art, once knowing the basic creative concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. A method for obtaining interspecific hybrid offspring of Paphiopedilum and Cypripedium by embryo rescue, characterized in that, Comprising the following steps: Crossing with the male parent of Calanthe trullata and the female parent of Rhynchostylis retusa, picking the hybrid young fruits after pollination for 40-60 days, disinfecting the young fruits, slicing, then inoculating in the embryo rescue medium for culture, waiting for the young embryo to germinate into the rooted seedlings, transferring to the strong seedling rooting medium for culture, and obtaining the rooted hybrid seedlings; The composition of the embryo rescue medium is as follows: 2 g / L of Hua Bao No. 1, 100 g / L of potato homogenate, 30 g / L of sucrose, 0.5 g / L of activated carbon, 7.0 g / L of agar, and the balance of water, with pH being 5.6-5.

8. The composition of the strong seedling rooting medium is as follows: 1.5 g / L of Hua Bao No. 1, 2.0 g / L of peptone, 1 mg / L of NAA, 120 mg / L of inositol, 2.0 mg / L of glycine, 0.2 mg / L of thiamine hydrochloride, 0.5 mg / L of pyridoxine hydrochloride, 0.5 mg / L of nicotinic acid, 75 g / L of banana homogenate, 0.5 g / L of activated carbon, 7 g / L of agar, and the balance of water, with pH being 5.6-5.

8.

2. The method of claim 1, wherein the hybridization between Paphiopedilum and Peristylus is obtained by embryo rescue, characterized in that, The crossing is specifically as follows: picking the pollen mass of Calanthe trullata at the flowering stage, removing the stamens of Rhynchostylis retusa, and sending the pollen mass of Calanthe trullata into the pistil of Rhynchostylis retusa for pollination.

3. The method of claim 1, wherein the hybridization between Paphiopedilum and Cypripedium is obtained by embryo rescue. The disinfection is specifically as follows: soaking the young fruits in detergent for 10-15 min, washing, soaking in 75% alcohol for 30-50 s, rinsing with sterile water, sterilizing with 0.1-0.3% HgCl2 for 15-20 min, and rinsing with sterile water for 4-5 times.

4. The method of claim 1, wherein the hybridization between Paphiopedilum and Cypripedium is obtained by embryo rescue. The culture time in the embryo rescue medium is 40d-60d, the temperature is 20℃-30℃, the LED white:LED red is 5-8:2-4, the light intensity is 30μmol·m -2 ·s -1 ~40μmol·m -2 ·s -1 , and the light time is 10h-14h per day.

5. The method of claim 1, wherein the hybridization between Paphiopedilum and Cypripedium is obtained by embryo rescue. The culture time in the strong seedling rooting medium is 100-120 days, and the culture conditions are the same as those in the embryo rescue medium.

6. The method of claim 1, wherein the hybridization between Paphiopedilum and Cypripedium is obtained by embryo rescue. The substrate for the planting culture is a mixture of pine bark, pottery clay and coarse vermiculite in equal volume.

7. The method of claim 1, wherein the hybridization of the species between Paphiopedilum and Peristylus is obtained by embryo rescue. The conditions for the planting culture are as follows: the temperature is 20-25 ℃ during the day and 18-22 ℃ at night, the illumination time is 10-14 h per day, the humidity is 75-85% during the day and 90-100% at night.

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