Method for obtaining distant intergeneric hybrids of Ampelopsis latifolia

Abstract

The invention discloses a method for obtaining distant intergeneric hybrids of orchid. The method provided by the invention comprises the following steps: carrying out intergeneric distant hybridization on cymbidium sinense 'Jianxiaoji', cymbidium ensifolium 'Qingshan Yuquan', cymbidium ensifolium 'Xianshan Red' and winter impatiens of cymbidium as male parents, and culturing hybrid seeds into seedlings at the stages of sterile sowing, germination, differentiation and rooting and seedling strengthening, so as to obtain hybrid seedlings with complete plants. According to the method, the distant intergeneric hybrid seedlings obtained through specific hybridization conditions are high in hybridization fruit setting rate, high in germination success rate, short in germination starting time and good in seedling forming effect. According to the method provided by the invention, the success rate of distant hybridization of the orchid can be increased, the affinity obstacle of distant hybridization of the orchid genus and the orchid genus is overcome, the limitation that the genetic relationship between the orchid genus and the orchid genus is extremely far and natural geographical isolation exists is broken through, new germplasm is provided for planting and appreciating the orchid, and the method has important significance.

Description

Technical Field

[0001] This invention relates to the field of orchid hybridization breeding technology, and more specifically, to a method for obtaining intergeneric hybrids of distant relatives of Orchididae. Background Technology

[0002] *Cymbidium axillarifolium*, also known as the mandible orchid, gets its name from the shape of its flowers, which resemble the open mandibles of an insect. It belongs to the genus *Cymbidium* in the family Orchidaceae. Maxillaria The genus *Cymbidium* is a general term for plants in the Orchidaceae family, and is one of the largest genera within this family. *Cymbidium* is widely distributed in the tropical and subtropical regions of Central and South America. It thrives in hot, humid, and sunny environments, but also exhibits some cold and drought tolerance. Currently, some native species of *Cymbidium* have been introduced to China and are being developed into new commercial orchids, with large-scale artificial propagation and commercial production, such as *Cymbidium slender-leavedii*. Maxillaria tenuifolia The fine-leaved axillary lip orchid has advantages such as bright flower color, strong fragrance (coconut scent), long flowering period (1-2 months), and heat and drought resistance. However, it also has disadvantages such as solitary flowers and generally small and inconspicuous flowers.

[0003] And Orchid ( Cymbidium The plant possesses excellent ornamental traits such as long inflorescences, numerous flowers, delicate fragrance, and upright plant shape. If hybrid vigor can be fully utilized by conducting distant hybridization between *Cymbidium stenoptera* and other orchid species to combine the advantages of both, the plant shape, flower type, inflorescence, flower quantity, and fragrance of *Cymbidium stenoptera* will be significantly improved, greatly enhancing its ornamental value and promotion potential.

[0004] However, orchid hybridization, especially between distantly related genera, often faces significant challenges such as incompatibility (inability to produce seeds), embryo abortion (seeds failing to develop normally), or sterile offspring. When using the genus *Maxillariinae* for distant intergeneric hybridization, reproductive obstacles and breeding challenges arise, including incompatibility before fertilization, incompatibility after fertilization (post-zygotic barrier), and seed non-germination. *Maxillariinae* is a genus within the subtribe of the tribe Cymbidieae. Currently, existing technologies disclose hybridization between *Maxillariinae* and the genus *Maxillariinae*, which also belongs to the subtribe. Lycaste ), Angoulême Anguloa ), genus Diplosperma ( Bifrenaria They are capable of distant hybridization.

[0005] According to the China Biographies Database, *Cymbidium* belongs to the subtribe of the tribe Cymbidieae, while *Cymbidium* belongs to a different subtribe and a different branch of evolutionary phylogeny. The two genera are very distantly related, representing a significant genetic distance in taxonomy. *Cymbidium* is mainly distributed in southern North America and South America, including Brazil, Ecuador, and Guatemala, with no natural distribution in Asia. *Cymbidium*, on the other hand, is mainly distributed in Asia and Oceania, including China, Malaysia, and Western Australia, with no natural distribution in the Americas. Therefore, *Cymbidium* and *Cymbidium* are not only distantly related but also naturally geographically isolated, making natural hybridization impossible under natural conditions. Furthermore, there are currently no documented records of successful hybridization between *Cymbidium* and *Cymbidium*. Current technology has no successful precedent for hybridization between *Cymbidium* and *Cymbidium*, and it is theoretically almost impossible, considered a virtually insurmountable barrier in orchid hybridization. In order to overcome the reproductive obstacles in hybridization breeding of the genus *Cymbidium* and to break the limitations of the distant kinship and natural geographical isolation between *Cymbidium* and *Orchid*, this invention application is hereby proposed. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the obstacles of existing distant hybridization between the genera *Cymbidium* and *Cymbidium*, to create new germplasm resources of *Cymbidium*, and to provide a method for obtaining intergeneric hybrids of *Cymbidium* with advantages such as high hybridization fruit setting rate, successful germination, and good quality.

[0007] The first objective of this invention is to provide a method for obtaining intergeneric hybrids of the distant relatives of Orychomycium argenteum.

[0008] A second objective of this invention is to provide an application of the method.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for obtaining intergeneric hybrids of *Cymbidium axillariae*, comprising the following steps: (1) Using *Cymbidium axillaryum* as the female parent, pollen from *Cymbidium* species was used to pollinate *Cymbidium axillaryum*; the *Cymbidium* species were selected from *Cymbidium sinense* 'Qijian Baimo' ( Cymbidium sinense 'Qi Jian Bai Mo', Jianlan 'Qingshan Yuquan' ( Cymbidium ensifolium 'Qing Shan Yu Quan', Jianlan 'Xianshan Hong' ( Cymbidium ensifolium 'Xian ShanHong' or Winter Phoenix Orchid ( Cymbidium dayanum ); (2) After the hybrid pods that have reached a development time of 110-160 days are sterilized, they are aseptically sown and cultured in an aseptic germination medium to obtain germinating hybrid seeds; the formula of the germination medium is: 1 / 2MS + Flower Treasure No. 1 1.0-2.0 g / L + Inositol 0.1-0.2 g / L + 6-BA 1.0-1.5 mg / L + NAA 0.2-0.5 mg / L + Activated Carbon 0.5-1.0 g / L + Sucrose 25-30 g / L + Carrageenan 6.0-7.0 g / L, pH=5.8±0.2; (3) The germinated seeds were transferred to the differentiation medium for further culture; the formulation of the differentiation medium was: 1 / 2 MS + inositol 0.1~0.2 g / L + 6-BA 1.0~1.5 mg / L + NAA 0.2~0.5 mg / L + activated carbon 0.2~0.5 g / L + sucrose 25~30 g / L + carrageenan 6.0~7.0 g / L; (4) The differentiated buds are transferred to a rooting and seedling strengthening medium for culture to obtain hybrid seedlings with complete plants; the formula of the rooting and seedling strengthening medium is: 1 / 2MS + inositol 0.1~0.2 g / L + 6-BA 0.1~0.3 mg / L + NAA 0.2~0.5 mg / L + activated carbon 0.2~0.5 g / L + 10.0%~15.0% coconut juice + sucrose 20~30 g / L + carrageenan 6.0~7.0 g / L.

[0010] This invention marks the first successful hybridization between *Cymbidium* and *Orchid* species using a hybridization breeding method, creating a distant intergeneric hybrid of *Cymbidium*. The invention employs suitable parent plants: *Cymbidium stenoptera* as the female parent and *Cymbidium sinense* 'Qijian Baimo', *Cymbidium ensifolium* 'Qingshan Yuquan', *Cymbidium ensifolium* 'Xianshan Hong', and *Cymbidium faberi* as the male parents. Artificial pollination is performed at a time when pollen and stigma activity are high. Aseptic sowing is carried out using a germination medium with activated carbon concentration of 0.5–1.0 g / L. Subsequent cultivation in a suitable differentiation and rooting medium yields hybrid seedlings with complete leaves and roots. This method features a short germination initiation time, high germination success rate, high hybrid fruit set rate, and excellent seedling formation. The method provided by this invention can improve the success rate of distant hybridization of Cymbidium axillariae, overcome the affinity barrier between distant hybridization of Cymbidium axillariae and Cymbidium, and break the limitation that Cymbidium axillariae and Cymbidium are extremely distantly related and have natural geographical isolation. It provides new germplasm for the cultivation and ornamental use of Cymbidium axillariae, which is of great significance.

[0011] Preferably, in step (1), the *Cymbidium axillaria* is *Cymbidium slender-leaved* (…). Maxillaria tenuifolia ).

[0012] Preferably, the pollen in step (1) is fresh fresh pollen collected from flowers that have been in full bloom for 7 days.

[0013] More preferably, the pollination method in step (1) is artificial pollination.

[0014] More preferably, the pollination timing in step (1) includes sunny days with high pollen activity and stigma activity during the peak flowering period.

[0015] Preferably, the development time of the hybrid pods in step (2) is 120 to 150 days.

[0016] Preferably, the aseptic sowing method in step (2) is as follows: clean the hybrid pods, then soak them in a 70-75% ethanol solution for 1-3 minutes to sterilize them, then cut the pods longitudinally to take out the seeds, and sow the seeds on a sterile germination medium.

[0017] More preferably, the method of aseptic sowing is as follows: washing the hybrid pods with tap water for 2 minutes; and sterilizing the pods by immersing them in a 75% ethanol solution for 2 minutes on a clean bench.

[0018] Preferably, the germination conditions in step (2) are: to cultivate the seeds in the dark until they germinate, at a temperature of 24±1℃, for a cultivation time of 90~120 days.

[0019] More preferably, the germination medium is formulated as follows: 1 / 2 MS medium, Flower Treasure No. 1 1.0~2.0 g / L, 6-benzylaminopurine (6-BA) 1.0~1.5 mg / L, naphthaleneacetic acid (NAA) 0.2~0.5 mg / L, inositol 0.1 g / L, sucrose 30 g / L, activated carbon 0.5~1.0 g / L and carrageenan 7 g / L.

[0020] Furthermore, the components of the 1 / 2 MS medium include: KSO4 950 mg / L, (NH4)2SO4 825 mg / L, MgSO4•7H2O 185 mg / L, KH2PO4 85 mg / L, CaCl2 166 mg / L, MnSO4•4H2O 22.3 mg / L, ZnSO4•7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4•2H2O 0.25 mg / L, CuSO4•5H2O 0.025 mg / L, CoCl2•6H2O 0.025 mg / L, nicotinic acid 0.5 mg / L, pyridoxine hydrochloride 0.5 mg / L, thiamine hydrochloride 0.1 mg / L, glycine 2.0 mg / L, FeSO4•7H2O 27.8 mg / L. mg / L, Na·EDTA·H2O 37.25 mg / L.

[0021] Preferably, the culture conditions for differentiation culture in step (3) are: light intensity of 1500±100 lx, light duration of 12 h / d, temperature of 24±1 ℃, and culture for 120~150 days.

[0022] More preferably, the differentiation medium is formulated as follows: 1 / 2 MS medium, 6-benzylaminopurine 0.5~1.0 mg / L, naphthaleneacetic acid 0.2~0.5 mg / L, inositol 0.1 g / L, sucrose 30 g / L, activated charcoal 0.1~0.2 g / L and carrageenan 7 g / L.

[0023] Preferably, the conditions for rooting and seedling cultivation in step (4) are: light intensity of 2000±100 lx, light duration of 12 h / d, temperature of 24±1 ℃, and cultivation for 120~150 days.

[0024] More preferably, the formula of the rooting and seedling strengthening culture medium is: 1 / 2 MS medium, 6-benzylaminopurine 0.2~0.5 mg / L, naphthaleneacetic acid 0.5~1.0 mg / L, inositol 0.1 g / L, sucrose 30 g / L, 10.0%~15.0% coconut juice, activated carbon 0.2~0.3 g / L and carrageenan 7 g / L.

[0025] The present invention also provides the application of the above method in the cultivation of seedlings of distant hybrids between the genera *Cymbidium* and *Cymbidium*.

[0026] The present invention has the following beneficial effects: This invention overcomes the limitations of the distant kinship and natural geographical isolation between the genera *Cymbidium* and *Orchid*, solving the problem of compatibility barriers in distant hybridization between these genera. It enables successful intergeneric hybridization breeding and the acquisition of hybrids within the *Cymbidium* genus. The method provided by this invention, through extensive experimental verification, demonstrates that selecting suitable male parents, such as *Cymbidium sinense* 'Qijian Baimo', *Cymbidium ensifolium* 'Qingshan Yuquan', *Cymbidium ensifolium* 'Xianshan Hong', and *Cymbidium faberi*, and performing distant intergeneric hybridization with *Cymbidium sclerotiorum* under specific conditions, can successfully produce fruit and obtain hybrid seeds and seedlings. This method utilizes aseptic sowing of hybrid pods with a development time of 120-150 days, resulting in a high hybridization fruit set rate. Using a suitable germination medium for aseptic sowing improves the germination success rate of the hybrids and shortens the initial germination time. Furthermore, selecting suitable differentiation and rooting media for subsequent cultivation leads to excellent seedling formation. The method provided by this invention can improve the success rate of distant hybridization of Cymbidium axillariae, overcome the affinity barrier between distant hybridization of Cymbidium axillariae and Cymbidium, and break the limitation that Cymbidium axillariae and Cymbidium are extremely distantly related and have natural geographical isolation. It provides new germplasm for the cultivation and ornamental use of Cymbidium axillariae, which is of great significance. Attached Figure Description

[0027] Figure 1A diagram showing the phylogenetic relationships between the genera *Cymbidium* and *Orchid*.

[0028] Figure 2 Image of a small-leaved axillary lip orchid.

[0029] Figure 3 Images of flowers from *Cymbidium goeringii* and *Cymbidium ensifolium* 'Qingshan Yuquan'.

[0030] Figure 4 The image shows the hybrid pods from Example 1.

[0031] Figure 5 This is a diagram showing the budding process during differentiation culture in Example 1.

[0032] Figure 6 This is a diagram showing the rooting and seedling cultivation process in Example 1.

[0033] Figure 7 This is a diagram showing the rooting and seedling cultivation process in Example 3.

[0034] Figure 8 Images showing the seed germination in Examples 1, 2, and 1 (Comparative Example 1).

[0035] Figure 9 Images showing the seed germination in Examples 3, 4, and Comparative Example 2.

[0036] Figure 10 Images showing seed germination in Examples 5, 6, and Comparative Example 3.

[0037] Figure 11 Images showing the seed germination in Examples 7, 8, and Comparative Example 4. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] Example 1 To fully utilize hybrid vigor, this application previously conducted distant hybridization between *Cymbidium* and a large number of *Orchid* species. The phylogenetic relationships between the two are as follows: Figure 1 As shown, these two genera are naturally geographically isolated, making natural hybridization impossible under natural conditions. The results indicate that most hybrid combinations fail to produce hybrid pods or result in seed germination. This example demonstrates a method that successfully induces hybridization.

[0041] The slender-leaved axillary orchid (of the genus *Axillary*) Maxillaria tenuifolia ) is the parent plant, such as Figure 2 As shown; Cymbidium ensifolium 'Qingshan Yuquan' ( Cymbidium ensifolium 'Qing Shan Yu Quan' is the parent gene, such as Figure 3 As shown, the specific steps for intergeneric hybridization and the cultivation of hybrid seeds into seedlings through aseptic sowing, germination, differentiation, and rooting and seedling strengthening are as follows: (1) Mother plant: In April and May, the mother plant, Orychophragmus simonii, is in full bloom and the stigma is highly active; (2) Male parent: In April and May, the male parent Cymbidium ensifolium 'Qingshan Yuquan' is in full bloom and has strong pollen activity; (3) Artificial pollination: In a greenhouse where both parents are in full bloom, choose a sunny day from 08:30 to 10:30 or from 14:30 to 16:30 to carry out intergeneric hybridization; select healthy flowering plants, select flowers that have been in bloom for 7 days, are normal and without deformities, and have not been pollinated, use a toothpick to remove the pollen clusters of the male parent Cymbidium ensifolium 'Qingshan Yuquan', and then place them into the stigma of the female parent Cymbidium simonii, and remove the lip of the pollinated flower to prevent insect pollination. Each pollinated flower is labeled with the hybridization combination and the pollination date, and a total of 3 flowers are pollinated; (4) Hybrid pods: Hybrid pods that have reached a development time of 120 days are harvested (e.g., Figure 4 (as shown) and aseptic sowing is carried out on the day of harvest; (5) Aseptic sowing: After washing the harvested capsules with detergent, rinse them with tap water for 2 minutes, dry them, soak them in 75% ethanol solution for 2 minutes on a clean bench, then use tweezers to pick out the fruits and place them on sterilized newspaper to dry. Cut off a small portion of both ends of the pod with a scalpel, then cut the pod longitudinally, pick out the seeds with tweezers, and sow the seeds evenly in a germination medium with activated carbon concentration of 1.0 g / L (formulation: 1 / 2MS + Flower Treasure No. 1 1.0 g / L + Inositol 0.1 g / L + 6-BA 1.0 mg / L + NAA 0.2 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 7.0 g / L, pH=5.8±0.2) for aseptic germination culture; place the culture bottle after sowing in the dark and culture until the seeds germinate at a temperature of 24±1℃; (6) Bud differentiation: Germinated seeds were transferred to differentiation medium (formulation: 1 / 2 MS + inositol 0.1 g / L + 6-BA 1.0 mg / L + NAA 0.2 mg / L + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 7.0 g / L) for culture; the culture flasks were placed under light, with a light intensity of 1500±100 lx, a light duration of 12 h / d, and a temperature of 24±1℃; after 120 days of culture, leaf buds were differentiated from the intermediate propagules. Figure 5 As shown; (7) Rooting and seedling strengthening: Rootless leaf buds were transferred to a rooting and seedling strengthening medium (formulation: 1 / 2 MS + inositol 0.1 g / L + 6-BA 0.2 mg / L + NAA 0.5 mg / L + activated carbon 0.3 g / L + 10.0%~15.0% coconut juice + sucrose 30 g / L + carrageenan 7.0 g / L); the culture bottles were placed under light for cultivation, with a light intensity of 2000±100 lx, a light duration of 12 h / d, and a temperature of 24±1℃; after 120 days of cultivation, the leaf buds grew into test-tube seedlings with complete leaves and roots, such as Figure 6 As shown.

[0042] Example 2 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 1. The only difference was that the activated carbon concentration in the germination medium was 0.5 g / L.

[0043] Example 3 Using *Cymbidium slender-leaved* as the female parent, *Cymbidium ensifolium* 'Xianshan Hong' (*Cymbidium faberi*) was developed. Cym. ensifolium 'XianShan Hong' was used as the male parent for intergeneric hybridization. The hybridization method, aseptic sowing method, and subsequent culture method were the same as in Example 1. The test-tube plantlets obtained after rooting and seedling strengthening were as follows: Figure 7 As shown.

[0044] Example 4 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 3. The only difference was that the activated carbon concentration in the germination medium was 0.5 g / L.

[0045] Example 5 Using *Cymbidium slender-leaved* as the female parent, *Cymbidium lancifolium* (also known as *Cymbidium*) is used as the female parent. Cymbidium dayanum Intergeneric hybridization was performed using the male parent as the male parent. The hybridization method, aseptic sowing method, and subsequent culture method were the same as in Example 1.

[0046] Example 6 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 5. The only difference was that the activated carbon concentration in the germination medium was 0.5 g / L.

[0047] Example 7 Using *Cymbidium slender-leaved* as the female parent, *Cymbidium sinense* 'Qijian Baimo' (*Cymbidium moniliforme*) was developed. Cymbidium sinense 'Qi Jian Bai Mo' was used as the male parent for intergeneric distant hybridization. Pods that had developed for 150 days were harvested and aseptically sown. The aseptic sowing method and subsequent culture method were the same as in Example 1.

[0048] Example 8 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 7, with the only difference being that the pod development time was 120 days.

[0049] Comparative Example 1 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 1. The only difference was that the activated carbon concentration in the germination medium was 0 g / L, that is, no activated carbon was added to the germination medium, but no germinating seeds were obtained in the end.

[0050] Comparative Example 2 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 3. The only difference was that the activated carbon concentration in the germination medium was 0 g / L, that is, no activated carbon was added to the germination medium, but no germinating seeds were obtained in the end.

[0051] Comparative Example 3 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 5. The only difference was that the activated carbon concentration in the germination medium was 0 g / L, that is, no activated carbon was added to the germination medium, and germinating seeds were obtained. However, the germination time was longer at 85 days and the germination rate was lower at 8.32%.

[0052] Comparative Example 4 The hybrid parental combination, hybridization time, hybridization method, and aseptic sowing method were the same as in Example 7. The only difference was that the pod development time was 90 days, resulting in hybrid pods, but no germinating seeds were ultimately obtained.

[0053] Comparative Example 5 Using *Cymbidium slender-leaved* as the female parent, the large-flowered Cymbidium 'Xianglan' (*Cymbidium faberi*) of the Orchid genus... Cym 'Xiang Lan' was used as the male parent for intergeneric distant hybridization. The hybridization method was the same as in Example 1, but no hybrid pods were obtained.

[0054] Comparative Example 6 Using *Cymbidium slender-leaved* as the parent plant, the Cymbidium 'Cherry Beauty' (*Cymbidium 'Cherry Beauty') is a large-flowered orchid from the genus *Cymbidium*. Cym 'Ying TaoMei Ren' was used as the male parent for intergeneric distant hybridization. The hybridization method was the same as in Example 1, but no hybrid pods were obtained.

[0055] Test Example 1 The number of pollinated flowers in the hybrid combinations of the above examples and comparative examples was counted. One month after pollination, the number of fruits set and the fruit set rate were calculated. After aseptic sowing, the number of days to germination and the germination rate were counted. The calculation formula is as follows: Germination rate = (Number of germinated seeds / Total number of seeds sown) * 100%; Fruit set rate = number of fruits / number of pollinated flowers * 100%.

[0056] The results are shown in Table 1 below. Examples 1-8, which used Cymbidium goeringii as the female parent and selected Cymbidium sinense 'Qijian Baimo', Cymbidium ensifolium 'Qingshan Yuquan', Cymbidium ensifolium 'Xianshan Hong', and Cymbidium faberi as the male parents for distant hybridization, all had a fruit set rate of 100%. However, the comparative examples 5 and 6, which used Cymbidium faberi 'Xianglan' and 'Yingtao Meiren' as male parents for hybridization with Cymbidium goeringii, both had a fruit set rate of 0% and could not produce hybrids. This indicates that selecting suitable male parents such as Cymbidium sinense, Cymbidium ensifolium, and Cymbidium faberi can avoid reproductive isolation problems between Cymbidium and Orchid species and improve the fruit set rate.

[0057] Because inter-genus *Cymbidium* hybrid seeds are prone to browning during cultivation, leading to germination failure or extremely low germination rates, adding 0.5–1.0 g / L activated carbon to the germination medium can improve the germination success rate and germination rate of hybrid seeds, and shorten the initial germination time. However, if the activated carbon dosage exceeds 1 g, the excessively high concentration will adsorb both browning substances and nutrients and hormones, severely affecting the cultivation effect and causing seeds to fail to germinate. In the hybrid combinations that achieved fruit setting, the hybrid seeds of Examples 1 and 2 both germinated successfully, with germination rates exceeding 90%, while the seeds of Comparative Example 1 failed to germinate successfully. Figure 8 As shown; the seeds of Examples 3 and 4 germinated successfully, while the seeds of Comparative Example 2 failed to germinate. Figure 9 As shown; the seeds of Examples 5 and 6 both germinated successfully, with an initiation time of no more than 60 days and a germination rate of over 92%. The seeds of Comparative Example 3 also germinated successfully, but the initiation time was longer at 85 days and the germination rate was lower at 8.32%. Figure 10 As shown in the figure. In Comparative Example 3, the germination medium used without activated carbon also allowed seeds to germinate, but the probability was low, which, from another perspective, illustrates that the success of hybridization using this method is related to the selection of the male parent.

[0058] In the hybrid combinations of Examples 7 and 8, the seeds of both successfully germinated, while the seeds of Comparative Example 4 failed to germinate. Figure 11As shown, this may be related to the short development time of the pods, leading to immature embryos. Aseptic sowing of hybrid pods older than 150 days significantly reduces germination success and rate. Aseptic sowing of hybrid pods with a development time of 120-150 days improves the germination success and rate. Therefore, optimizing the methods and conditions for distant hybridization between *Cymbidium* and *Orchid* genera by selecting suitable male parents, harvesting hybrid pods with a development time of 120-150 days, and using a germination medium with activated carbon concentration of 0.5-1.0 g / L can improve the success rate of distant hybridization of *Cymbidium* and overcome the compatibility barrier in distant hybridization between *Cymbidium* and *Orchid*.

[0059] Table 1. Effects of different hybrid parent combinations, different fruit ages, and different activated carbon concentrations on intergeneric hybridization of *Cymbidium*.

[0060] In summary, this invention, through extensive experimentation, selected suitable male parents from the *Cymbidium* genera, such as *Cymbidium goeringii* 'Qijian Baimo', *Cymbidium ensifolium* 'Qingshan Yuquan', *Cymbidium ensifolium* 'Xianshan Hong', and *Cymbidium faberi*, and conducted distant hybridization with *Cymbidium sclerotiorum*, overcoming the compatibility barrier in distant hybridization between *Cymbidium* and *Cymbidium* genera, and successfully obtaining intergeneric hybrids of *Cymbidium*. The method provided by this invention has the advantage of high hybridization fruit set rate. Aseptic sowing is performed on hybrid pods that have developed for 120-150 days using a suitable germination medium (activated carbon concentration of 0.5-1.0 g / L), which improves the germination success rate of hybrids and shortens the initial germination time. Furthermore, selecting suitable differentiation and rooting media for subsequent cultivation results in good seedling formation. The method provided by this invention can improve the success rate of distant hybridization of Cymbidium axillariae, overcome the affinity barrier between distant hybridization of Cymbidium axillariae and Cymbidium, and break the limitation that Cymbidium axillariae and Cymbidium are extremely distantly related and have natural geographical isolation. It provides new germplasm for the cultivation and ornamental use of Cymbidium axillariae, which is of great significance.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for obtaining intergeneric hybrids of distant relatives of *Cymbidium axillaria*, characterized in that, Includes the following steps: (1) With the axillary lip orchid ( Maxillaria ) as the mother plant, using the genus Orchid ( Cymbidium The pollen from the material was used to pollinate the axillary lip orchid; the orchid material was selected from Cymbidium sinense 'Qijian Baimo' ( Cym. sinense 'Qi Jian Bai Mo', Jianlan 'Qingshan Yuquan' ( Cym. ensifolium 'Qing Shan Yu Quan', Jianlan 'Xianshan Hong' ( Cym. ensifolium 'XianShan Hong' or Winter Phoenix Orchid ( Cym. dayanum ); (2) Harvest hybrid pods that have reached a development time of 110 to 160 days, sterilize them, and then sow them under sterile conditions. They are then cultured in a sterile germination medium to obtain germinating hybrid seeds. The germination medium formula is as follows: 1 / 2 MS + Flower Treasure No. 1 1.0~2.0 g / L + Inositol 0.1~0.2 g / L + 6-BA 1.0~1.5 mg / L + NAA 0.2~0.5 mg / L + Activated Carbon 0.5~1.0 g / L + Sucrose 25~30 g / L + Carrageenan 6.0~7.0 g / L, pH=5.8±0.2; (3) Transfer the germinated seeds to the differentiation medium for further culture; The differentiation medium formulation is as follows: 1 / 2 MS + inositol 0.1~0.2 g / L + 6-BA 1.0~1.5 mg / L + NAA 0.2~0.5 mg / L + activated charcoal 0.2~0.5 g / L + sucrose 25~30 g / L + carrageenan 6.0~7.0 g / L; (4) The differentiated buds were transferred to a rooting and seedling culture medium to obtain hybrid seedlings with complete plants; The formula for the rooting and seedling strengthening culture medium is as follows: 1 / 2 MS + inositol 0.1~0.2 g / L + 6-BA 0.1~0.3 mg / L + NAA 0.2~0.5 mg / L + activated carbon 0.2~0.5 g / L + 10.0%~15.0% coconut juice + sucrose 20~30 g / L + carrageenan 6.0~7.0 g / L.

2. The method according to claim 1, characterized in that, In step (1), the *Cymbidium axillaria* is a narrow-leaved *Cymbidium axillaria* ( Maxillaria tenuifolia ).

3. The method according to claim 2, characterized in that, In step (1), the pollen is fresh pollen collected from flowers that have been in full bloom for 7 days.

4. The method according to claim 3, characterized in that, In step (1), the pollination method is artificial pollination.

5. The method according to claim 4, characterized in that, The development time of the hybrid pods in step (2) is 120-150 days.

6. The method according to claim 1, characterized in that, The aseptic sowing method in step (2) is as follows: clean the hybrid pods, then soak them in a 70%~75% ethanol solution for 1-3 minutes to sterilize them, then cut the pods lengthwise to take out the seeds, and sow the seeds on a sterile germination medium.

7. The method according to claim 1, characterized in that, The germination conditions in step (2) are: to cultivate the seeds in the dark until they germinate, at a temperature of 24±1 ℃, for a period of 90~120 days.

8. The method according to claim 1, characterized in that, The culture conditions for differentiation culture in step (3) are: light intensity of 1500±100 lx, light duration of 12 h / d, temperature of 24±1 ℃, and culture for 120~150 days.

9. The method according to claim 1, characterized in that, The conditions for rooting and seedling cultivation in step (4) are: light intensity of 2000±100 lx, light duration of 12 h / d, temperature of 24±1 ℃, and cultivation for 120~150 days.

10. The application of the method of claim 1 in the cultivation of seedlings of distant hybrids between the genera *Cymbidium* and *Cymbidium*.

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