A culture medium and method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds

Abstract

The present invention relates to a culture medium and method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds, and relates to the fields of plant biotechnology and plant cultivation technology. The culture medium includes a protocorm induction culture medium, a cluster bud differentiation culture medium, a cluster bud proliferation culture medium, and a rooting and seedling strengthening culture medium; the culture method includes the following specific steps: (1) sterile sowing and protocorm induction; (2) cluster bud differentiation culture; (3) cluster bud proliferation culture; and (4) cluster bud rooting and seedling strengthening culture. The present invention uses seeds of Tiangui Cymbidium orchid from different plants and flowers for cross-pollination as explants to construct a sterile sowing and culture system, which promotes rapid seed germination and a high germination rate, overcomes the disadvantages of using bulbs or tender shoots as explants, such as waste of materials and low induction rate, while improving production efficiency and reducing production costs.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology and plant cultivation technology, and in particular to a culture medium and a method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds. Background Art

[0002] Bulbophyllum tianguii is an epiphytic herbaceous plant of the genus Bulbophyllum (Orchiaceae) in the Orchidaceae family. It typically grows on karst canyon cliffs, understory rocks, mountaintop rocks, or on humus tree stems at altitudes of 700-1300 meters. It is currently found only in the Yachang Orchidaceae National Nature Reserve in Leye County, Guangxi, China, and in the Tiangui region of Wangmo, Xiuwen, Kaiyang, and Xifeng. Bulbophyllum tianguii boasts vibrant colors, unique shapes, and graceful, elegant blossoms, making it highly ornamental and medicinal. Due to its low natural reproduction rate and recent damage from human exploitation, Bulbophyllum tianguii is now listed as a Class II protected plant on the National List of Key Protected Wild Plants and as a Vulnerable (VU) species on the IUCN Red List of Threatened Species.

[0003] Most Bulbophyllum species remain in the wild and are in urgent need of development. Research on tissue culture and rapid propagation of Bulbophyllum species is relatively limited. Li Honglin et al. (2007) used stem segments to induce tissue culture and rapid propagation of Bulbophyllum kwangtungense, achieving a propagation coefficient of 2-3 times per 50 days. Huang Chongcheng et al. (2019) used pseudobulbs and young shoots as explants to obtain sterile material of wild Bulbophyllum sp., achieving a proliferation coefficient of 2.9 after 60 days of culture. Wang Xiaofeng et al. (2017) also established a tissue culture and rapid propagation method for Bulbophyllum delitescens using young shoots. Although these studies produced sterile material, the explants were sampled from the wild and contained endophytic fungi, resulting in high contamination rates and low induction rates, wasting significant amounts of wild resources. Sterile seed sowing has successfully established a factory-based rapid propagation system for various orchids, such as Bletilla striata, Dendrobium officinale, and Cymbidium chinense. This method, which produces large numbers of seedlings in a short period of time, is currently a cost-effective and rapid propagation method. However, the only report on sterile seeding of the genus Bletilla striata is by Tian Haijuan et al. (2022), who established a sterile seeding system using hybrid seeds of two Bletilla striata species. Tiangui Cymbidium truncatum, a new species reported by Lang Kaiyong and Luo Dun in 2007, uses division to propagate the plant, resulting in a slow propagation rate and a low reproduction coefficient, hindering large-scale and industrialized promotion and damaging resources. Therefore, establishing sterile seeding and rapid propagation technology for Tiangui Cymbidium truncatum and factory-based propagation of high-quality seedlings would not only effectively protect this precious resource but also provide seedlings and lay the foundation for its development and utilization. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a culture medium and method for promoting the rapid seedling formation of Tiangui Cymbidium orchid seeds. The purpose of the present invention is to construct a sterile sowing and culture system using seeds of Tiangui Cymbidium orchid from different plants and flowers as explants to quickly and efficiently obtain a large number of Tiangui Cymbidium orchid seedlings.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, a culture medium for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds, the culture medium comprising a protocorm induction medium, a cluster bud differentiation medium, a cluster bud proliferation medium, and a rooting and seedling strengthening medium;

[0007] The protocorm induction medium comprises the following components in the following amounts: MS basal medium, 1.0-2.0 mg / L 6-BA, 100-200 ml / L coconut milk, 0.1 mg / L NAA, 0-30 g / L sucrose and 0-7 g / L agar, with a pH value of 5.4-5.8;

[0008] The cluster bud differentiation medium comprises the following components in the following amounts: B5 basal medium, 0-0.2 mg / L IBA, 0.5-1.0 mg / L AgNO3, 0-30 g / L sucrose and 0-7 g / L agar, with a pH value of 5.4-5.8;

[0009] The cluster bud proliferation culture medium comprises the following components in the following amounts: MS basal culture medium, 1.0-3.0 mg / L 6-BA, 0.1-0.5 mg / L KT, 0.1-0.2 mg / L NAA, 50-100 g / L banana puree, 0-30 g / L sucrose, 0-1.0 g / L activated carbon and 0-7 g / L agar, with a pH of 5.4-5.8;

[0010] The rooting and seedling strengthening culture medium comprises the following components in the following amounts: MS basal culture medium, 0.2-1.0 mg / L 6-BA, 0.1-0.5 mg / L KT, 0.1-0.5 mg / L NAA, 100-200 ml / L coconut milk (banana puree may also be used), 0-30 g / L sucrose, 0-1.0 g / L activated carbon and 0-7 g / L agar, and has a pH of 5.4-5.8.

[0011] Furthermore, the protocorm induction medium comprises the following components in the following amounts: MS basal medium, 2.0 mg / L 6-BA, 200 ml / L coconut water, 0.1 mg / L NAA, 30 g / L sucrose and 7 g / L agar, with a pH value of 5.4-5.8;

[0012] The cluster bud differentiation medium includes the following components in the following amounts: B5 basal medium, 0.2 mg / L IBA, 1.0 mg / L AgNO3, 30 g / L sucrose and 7 g / L agar, with a pH value of 5.4-5.8;

[0013] The cluster bud proliferation culture medium comprises the following components in the following amounts: MS basal medium, 2.0 mg / L 6-BA, 0.5 mg / L KT, 0.2 mg / L NAA, 100 g / L banana puree, 30 g / L sucrose, 1.0 g / L activated carbon and 7 g / L agar, with a pH of 5.4-5.8;

[0014] The rooting and seedling strengthening culture medium comprises the following components in the following amounts: MS basic culture medium, 1.0 mg / L 6-BA, 0.2 mg / L KT, 0.5 mg / L NAA, 200 ml / L coconut milk, 30 g / L sucrose, 1.0 g / L activated carbon and 7 g / L agar, and has a pH of 5.4-5.8.

[0015] In a second aspect, a method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds comprises the following specific steps:

[0016] (1) Aseptic sowing and protocorm induction: After sterilizing unopened mature capsules of Tiangui volute, the seeds are taken out and inoculated into the protocorm induction medium for inducing seed germination, and cultured in the dark in a tissue culture room for 7-23 days (d), such as 7, 10, 15, 20, 23 days, etc., and then cultured in light for 40-70 days, such as 40, 45, 50, 55, 60, 65, 70 days, etc., to obtain germinated seeds;

[0017] (2) Cluster bud differentiation culture: transferring the germinated seeds to the cluster bud differentiation medium and culturing them under light for 120-150 days, for example, 120, 125, 130, 145, 150 days, etc., until cluster buds are induced to grow, specifically cluster buds with one leaf and a height of 0.5-1.5 cm;

[0018] (3) cluster bud proliferation culture: transferring the cluster buds to the cluster bud proliferation medium and culturing them under light for 90-120 days, for example, 90, 100, 110, 120 days, etc., to obtain proliferated cluster buds (cluster buds with a height of about 2-3 cm);

[0019] (4) Cultivation of rooted and strong seedlings of clustered buds: The proliferated clustered buds are transferred to the rooted and strong seedling culture medium and cultured under light for 90-120 days, for example, 90, 100, 110, 120 days, etc., to obtain rooted test tube seedlings.

[0020] Furthermore, in step (1), the capsule of the Tiangui orchid is a capsule obtained by artificial cross-pollination. Artificial cross-pollination is used to increase the seed germination rate. The capsule of the Tiangui orchid is obtained by: selecting a flower that has been flowering for 2-5 days between 9:00 and 11:00 in the morning on a sunny day, dipping anthers with a sterilized toothpick, and inoculating them onto the stigma of another flower to complete pollination, bagging, and marking. After growing for 7-10 months, the capsule pods are taken after turning light yellow, and placed at 4°C for later use. The specific steps for disinfecting the capsule of the Tiangui orchid are as follows: first, clean the surface of the capsule with detergent, rinse with tap water, place on a clean bench, and then soak in 75% v / v alcohol for 1 minute, then rinse with sterile water once, then soak in 1‰ HgCl2 for 10 minutes, and then rinse with sterile water 5 times. The disinfection method has a good disinfection and sterilization effect.

[0021] Furthermore, the conditions for dark culture in the tissue culture room in step (1) are: temperature of 25±2°C, shaded with shade cloth, and conditions for light culture are: temperature of 25±2°C, intensity of 1000-1500 lux, for example, 1000, 1100, 1200, 1300, 1400, 1500 lux, etc., and illumination time of 8-14 h / day, for example, 8, 9, 10, 11, 12, 13, 14 h / day;

[0022] In steps (2) to (4), the conditions for light culture are: temperature of 25±2°C, intensity of 1000-1500 lux, and light duration of 8-14 h / day, for example, 8, 9, 10, 11, 12, 13, 14 h / day.

[0023] Furthermore, the rooted test tube plantlets further comprise the following culturing steps:

[0024] (5) Transplantation and acclimation: The rooted test tube seedlings are removed from the culture room and acclimated for 7-23 days, for example, 7, 10, 15, 20, 23 days, etc., and then planted in a transplanting medium to obtain transplanted acclimated seedlings;

[0025] (6) Cultivation and management: Cultivate and manage the transplanted and domesticated seedlings.

[0026] Furthermore, the seedling hardening is mainly carried out with diffuse light, the light intensity is 1000-1500lx, the temperature is 15-25℃, and the humidity is 60-80%.

[0027] Furthermore, the planting time in step (5) is from March to May, or from September to November each year; the transplanting medium includes at least two of peat soil, bark, moss, perlite, and vermiculite. The transplanting medium needs to be disinfected with a 600-fold dilution of thiophanate-methyl powder.

[0028] Furthermore, the transplanting matrix includes any one of peat soil: bark: moss with a volume ratio of 1:1:1, bark: perlite: vermiculite with a volume ratio of 1:1:1, bark: moss with a volume ratio of 1:1, vermiculite: perlite with a volume ratio of 2:1, and peat soil: perlite: vermiculite with a volume ratio of 1:1.

[0029] Furthermore, the cultivation management in step (6) includes fertilization, water control and shading.

[0030] Furthermore, the cultivation management in step (6) specifically includes: spraying the transplanted and acclimated seedlings with a mixture of 0.02-0.05 mg / L BRs and 0.5-1 g / L Huabao No. 1 25-35 days after transplanting, for example, 25, 27, 30, 32, 35 days, etc., spraying once every 7-23 days, for example, 7, 10, 15, 20, 23 days, etc., and spraying at least 3 times in succession; spraying 100-200 times of Lanlingwang (microbial agent) once every 8-12 days during the growing season; the light intensity throughout the year is 1000-2000 lux; the temperature is maintained at 15°C-25°C in summer, and the seedlings are naturally overwintered in winter, and the transplanting medium is kept moist.

[0031] The principle of the present invention is described as follows:

[0032] The key features of MS basal medium are its high nitrate, potassium, and ammonium content, which ensures the mineral nutrients necessary for tissue growth and accelerates callus growth. Due to the high ion concentration in the formula, even slight variations in ingredients during preparation, storage, and sterilization will not affect the ionic balance. MS solid medium can be used for callus induction, seed germination, and the induction culture of embryos, stem segments, stem tips, and anthers.

[0033] The main feature of B5 basal culture medium is that it contains low ammonium content, which is not easy to inhibit the growth of the culture and is conducive to the differentiation culture of cluster buds.

[0034] 6-BA, also known as 6-(N-benzyl)aminopurine, is the first synthetic cytokinin. It has multiple benefits, including inhibiting the decomposition of chlorophyll, nucleic acids, and proteins in plant leaves, maintaining greenness and preventing aging; and directing amino acids, auxins, and inorganic salts to the treated area. It is widely used in tissue culture.

[0035] NAA, or 1-naphthylacetic acid, is readily soluble in organic solvents and is an auxin analogue found in plant growth regulators. It promotes cell division and expansion, inducing the formation of adventitious roots. It is commonly referred to as commercial root-promoting powder or root-promoting agent and is widely used in plant cutting propagation.

[0036] KT, or 6-furfurylaminopurine, is a natural plant endogenous hormone and the first cytokinin discovered. It primarily promotes cell division, increases yield, and stimulates the division of medullary and plant cells. It is not only essential for the growth and development of young embryos, but also has significant effects on parthenogenesis in certain plants, stimulates fruit and grain growth, and prevents aging.

[0037] IBA, or indolebutyric acid, is a broad-spectrum indole plant growth regulator with wide adaptability and safety, making it an excellent rooting agent. IBA and NAA are two commonly used plant growth regulators for rooting cuttings. Of these, indolebutyric acid is the most effective because it circulates less within the plant and tends to remain near the site of application. While IBA has a strong rooting effect, it produces thin and long adventitious roots, while NAA produces fewer, thicker roots. Therefore, combining the two often yields better results.

[0038] Brassinosteroid (BR) is a novel endogenous plant hormone and the first active brassinosteroid (BR) to be isolated. It is internationally recognized as the most active, highly effective, broad-spectrum, and non-toxic plant growth hormone. Plant physiologists believe that it can fully stimulate the inherent potential of plants, promote crop growth and increase yields, improve cold tolerance, enhance disease and salt resistance, enhance stress tolerance, and reduce herbicide damage to crops.

[0039] Hyponex No. 1, a balanced nitrogen, phosphorus, and potassium formula with a nitrogen, phosphorus, and potassium ratio of 7:6:19, provides all plant nutrients from seedling cultivation through growth to flowering and fruiting. Hyponex No. 5, a high-nitrogen formula with a nitrogen, phosphorus, and potassium ratio of 30:10:10, promotes rapid growth of various seedlings and is widely used to accelerate the growth of new shoots of various Chinese and Western orchids.

[0040] AgNO3. In plant in vitro culture, adding AgNO3 to the differentiation medium can significantly promote callus organogenesis and somatic embryogenesis, promote the production of some seed buds that are difficult to regenerate, increase the number of adventitious buds produced by explants and improve the frequency of plant regeneration.

[0041] Coconut water is an organic additive that contains a variety of amino acids, such as lysine, dapoxetine, vitamins, etc. It is used to provide nutrition during plant tissue culture to promote tissue culture activity.

[0042] Banana puree: An organic additive that stabilizes pH and helps strengthen seedlings. It contains a large amount of sugars, which provide energy for cell division and differentiation in plant tissues. Bananas also contain plant hormones such as cytokinins, auxins, ethylene, and gibberellins.

[0043] Lanlingwang: A microbial agent, its main ingredients include Bacillus subtilis, Bacillus licheniformis, Trichoderma harzianum, lactic acid bacteria, nitrogen-fixing bacteria, actinomycetes and other beneficial microorganisms and their various secreted extraspore enzymes. It has the effects of improving disease resistance and accelerating germination and flowering.

[0044] The protocorm induction medium for inducing seed germination of the present invention comprises MS as a basal medium, 6-BA and NAA as plant hormones that promote seed germination and cell division, and coconut milk as a source of trace elements. This significantly promotes seed germination and protocorm induction. Sterilized Tiangui volute capsules are inoculated into the protocorm induction medium and cultured in the dark, allowing the seeds to gradually turn green. The seeds are then cultured in the light, swelling to form protocorms.

[0045] Among the cluster bud differentiation culture medium of the present invention, B5 is screened out as the best basic culture medium. The culture medium formula has the characteristic of low salt, which is conducive to the differentiation culture of cluster buds. IBA is conducive to the differentiation and development of cluster buds, and AgNO3 has the effect of stimulating differentiation.

[0046] The present invention provides a bud cluster proliferation culture medium comprising MS as the basal medium, 6-BA, KT, and NAA to promote cell division and bud cluster growth. Bananas are also beneficial for bud cluster growth and development, while activated carbon absorbs harmful substances produced during growth. After culture, the bud cluster proliferation coefficient reaches 6.67; the test tube seedlings exhibit dark green leaves, indicating good bud cluster growth.

[0047] The rooting and seedling strengthening culture medium of the present invention comprises MS as the base medium, 6-BA and KT to promote the growth of clustered buds, NAA to promote the rooting of clustered buds, coconut milk to promote strong seedlings, and activated carbon to absorb harmful substances produced during growth and strengthen the rooting of seedlings. The culture medium can greatly promote the rooting and seedling strengthening of test tube seedlings. After cultivation, the leaves of the test tube seedlings are dark green, the rooting rate is 100%, the number of roots reaches 6.91, the root length reaches 3.15 cm, the root system is strong, the stems are thick, the leaves are dark green, and the seedlings are tall.

[0048] During the transplanting and acclimation process, the optimal transplanting substrate is a mixture of vermiculite and perlite in a volume ratio of 2:1, achieving a 100% transplant survival rate and high-quality, uniform seedlings. During the transplanting and acclimation process, a mixture of 0.02 mg / L BRs and 1.0 g / L Huabao No. 1 was sprayed three times, followed by a 200-fold concentration of Lanlingwang (a microbial agent) every 10 days. This achieved the best results and the highest seedling quality.

[0049] Beneficial effects of the present invention:

[0050] (1) The present invention is the first to use seeds of Tiangui Cymbidium that are cross-pollinated with different plants and flowers as explants, and after disinfection, they are sterile sown to construct a sterile sowing system, inducing the formation of protocorms, promoting rapid seed germination and high germination rate, overcoming the disadvantages of using bulbs or young shoots as explants, wasting materials and having a low induction rate, while improving production efficiency and reducing production costs.

[0051] (2) The present invention establishes a method for rapidly propagating seedlings of Tiangui Cymbidium by using seeds and its culture medium, including the steps of obtaining seeds, pre-sowing treatment of seeds, sowing and protocorm induction, cluster bud induction culture, cluster bud proliferation culture, cluster bud rooting and seedling cultivation, and transplanting and domestication; the method of the present invention is used to propagate Tiangui Cymbidium seedlings, with high seed germination rate and fast germination; fast cluster bud differentiation and high uniformity; cluster bud proliferation coefficient reaches 6.67 / 120d; rooting rate is 100%, the number of roots is 6.91, the root length is 3.15cm, the root system is strong, the stem is thick, the leaves are dark green, and the seedlings are tall; the transplanting survival rate is 100%, and the growth is vigorous; the method has the characteristics of fast propagation speed, large yield, excellent seedling quality, short time, strong practical operability, etc., and can obtain a large number of Tiangui Cymbidium seedlings in a relatively short time, which is of great significance to the protection and sustainable utilization of Tiangui Cymbidium germplasm resources and has a very broad application prospect.

[0052] (3) The present invention establishes a transplanting and domestication technology system for Tiangui Cymbidium, with a transplanting survival rate of up to 100%; establishes an application method and application concentration for combining the application of plant growth regulators brassinolide BRs, Huabao No. 1, and the microbial agent Lanlingwang in transplanting and domestication management, thereby promoting the growth and development of domesticated seedlings and improving the stress resistance of plants.

[0053] (4) The method of the present invention is simple, easy to operate, has broad market prospects, and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The growth of the clustered buds in Example 1 after 120 days of inoculation into the rooting and seedling strengthening medium.

[0055] Figure 2 The results of Example 1 are as follows: the cluster buds were cultured in the cluster bud differentiation medium for 120 days.

[0056] Figure 3 This is the growth condition of the clustered buds after 120 days of proliferation culture in Example 1.

[0057] Figure 4 The growth conditions of the rooted seedlings in Example 1 after 3 months of transplanting and acclimatization. DETAILED DESCRIPTION

[0058] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0059] Example 1

[0060] A method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds comprises the following specific steps:

[0061] Step 1: Obtaining seeds

[0062] Tiangui orchid capsules are collected from the Guangxi Yachang Orchidaceae National Nature Reserve. They are mature, unopened capsules that have been artificially pollinated from different plants and flowers. The specific operation method is: on a sunny day between 9:00 and 11:00 am, select a flower that has been blooming for 2-5 days. After dipping the anther with a sterilized toothpick, inoculate it onto the stigma of another flower. Complete pollination, bag, and mark. After 7-10 months of growth, wait until the capsule pods turn light yellow and harvest.

[0063] Step 2: Sterile sowing and germination of seeds

[0064] The capsules were disinfected by first cleaning the surface with detergent, rinsing with tap water, placing them on a clean bench, and then soaking them in 75% v / v alcohol for 1 minute. The seeds were then rinsed once with sterile water, then soaked in 1‰ HgCl2 for 10 minutes, and then rinsed five times with sterile water. After disinfection, the seeds were removed and inoculated on protocorm induction medium. The seeds were cultured in the dark in a tissue culture room and then transferred to light for culture to obtain germinated seeds.

[0065] Protocorm induction medium: MS basal medium, 2.0 mg / L 6-BA, 0.1 mg / L NAA, 200 ml / L coconut water, 30 g / L sucrose, 7 g / L agar, pH 5.8; culture in the dark for 15 days and in the light for 60 days; ambient temperature is 25 ± 2°C, light intensity is 1500-2000 lux, and time is 12 hours / day;

[0066] Step 3: Cluster bud differentiation culture

[0067] The germinated seeds obtained in step 2 were transferred to the cluster bud differentiation medium and cultured for another 120 days to induce the growth of cluster buds with one leaf and a height of 0.5-1.5 cm ( Figure 2 );

[0068] Cluster bud differentiation medium: B5 basal medium, 0.2 mg / L IBA, 1.0 mg / L AgNO3, 30 g / L sucrose, and 7 g / L agar, pH 5.4-5.8; light culture temperature 25±2°C, intensity 1500-2000 lux, time 12 hours / day;

[0069] Step 4: Cluster bud proliferation culture

[0070] When the clustered buds obtained in step 3 are transferred to the clustered bud proliferation medium and cultured for 120 days, the proliferated clustered buds ( Figure 3 );

[0071] Cluster shoot proliferation medium: MS basal medium, 2.0 mg / L 6-BA, 0.5 mg / L KT, 0.2 mg / L NAA, 100 g / L banana puree, 30 g / L sucrose, 7 g / L agar, and 1.0 g / L activated carbon, pH 5.4-5.8; light culture temperature was 25 ± 2°C, intensity was 1500-2000 lux, and time was 12 h / d;

[0072] Step 5: Rooting and cultivation of clustered buds and seedlings

[0073] The proliferated buds obtained in step 4 are transferred to a rooting and seedling-strengthening medium and cultured for 120 days to obtain rooted test tube plantlets;

[0074] Rooting and seedling growth medium: MS, 1.0 mg / L 6-BA, 0.2 mg / L KT, 0.5 mg / L NAA, 200 ml L -1 coconut water, 30g / L sucrose, 1.0g / L activated carbon and 7g·L -1 Agar, pH 5.4-5.8 ( Figure 1 );

[0075] Step 6: Transplanting and Acclimation

[0076] The rooted test-tube seedlings obtained in step 5 are removed from the culture room in March-May or September-November, and after 15 days of hardening in bottles, the roots are washed, dried, and planted in a transplanting medium disinfected with a 600-fold dilution of thiophanate methyl powder in a plastic greenhouse. The transplanting medium is a mixture of vermiculite and perlite in a volume ratio of 2:1; the hardening is mainly carried out under scattered light, with a light intensity of 1500-3000 lx, a temperature of 15°C-25°C, and a humidity of 60-80%;

[0077] Step 7: Cultivation and management of transplanted and acclimated seedlings

[0078] During the transplanting and acclimation process described in step 6, 30 days after transplanting, spray 0.02 mg / L BRs and 1.0 g / L Huabao No. 1 mixture three times; spray 200 times Lanlingwang (microbial agent) every 10 days; the light intensity is 1000-2000 lux; the temperature is maintained at 15℃-25℃ in summer, and it is naturally overwintered in winter, and the substrate is kept moist ( Figure 4 ).

[0079] Example 2

[0080] Compared with Example 1, this Example 2 uses self-pollinated, cross-pollinated or naturally pollinated seeds to carry out the sterile sowing and seed germination experiments described in Step 2 of Example 1. The culture medium used is the same as that described in Step 2 of Example 1. The results are shown in Table 1.

[0081] Table 1 Effects of different pollination methods on seed germination of Tiangui Cymbidium (n=3)

[0082]

[0083] Since orchid seeds are numerous and small, the germination status was counted 90 days after sowing, and the germination rate was preliminarily estimated (germinated seeds / total number of seeds sown). The germination status was divided into 5 levels, 1 "+" represents germination rate ≤ 20%, "++" represents germination rate ≤ 40% for 20%, "+++" represents germination rate ≤ 60% for 40%, "++++" represents germination rate ≤ 60% for 40%, "++++" represents germination rate ≤ 80% for 60%, and "+++++" represents germination rate ≤ 80% for 60%.

[0084] Example 3

[0085] Compared with Example 1, in Example 3, the amounts of 6-BA, NAA, and coconut milk in the protocorm induction medium and the culture time were changed. The remaining components were the same as in Example 1. The sterile sowing and seed germination experiments described in step 2 of Example 1 were carried out. The results are shown in Table 2.

[0086] Table 2 Culture of protocorm induction medium with different hormone ratios (n=3)

[0087]

[0088]

[0089] Example 4

[0090] Compared with Example 1, in Example 4, the amounts of IBA and AgNO3 in the bud cluster differentiation medium and the culture time were changed. The other components were the same as those in Example 1. The bud cluster differentiation culture described in step 3 of Example 1 was carried out. The results are shown in Table 3.

[0091] Table 3 Effects of different hormone ratios on the culture of cluster bud differentiation of Tiangui Cymbidium (n=3)

[0092]

[0093] Example 5

[0094] Compared to Example 1, Example 5 varied the amounts of 6-BA, KT, NAA, and banana puree in the bud cluster proliferation culture medium, as well as the incubation time. The remaining components remained the same as in Example 1. The bud cluster proliferation culture described in step 4 of Example 1 was performed. The results are shown in Table 4. The proliferation coefficient is the percentage of the number of proliferated individuals to the original number of individuals. Proliferation coefficient = growth index / original number of individuals.

[0095] Table 4 Effects of different hormone ratios on the proliferation of clustered buds of Tiangui Cymbidium (n=3)

[0096]

[0097] Example 6

[0098] Compared with Example 1, this Example 6 changes the amount of 6-BA, KT, NAA, and other additives in the seedling rooting culture and the culture time. The other components are the same as those in Example 1. The clustered bud seedling rooting culture described in step 5 of Example 1 is carried out. The results are shown in Table 5.

[0099] Rooting rate = number of rooted buds / number of rooted buds × 100%; number of roots = total number of roots / number of inoculations; root length = total length of root system / total number of roots.

[0100] Table 5 Effect of different hormone ratios on the rooting of clustered buds and seedlings of Tiangui Cymbidium (n=3)

[0101]

[0102] Example 7

[0103] Compared with Example 1, Example 7 changed the cultivation medium and the ratio. The rest was the same as Example 1. The transplanting and acclimation described in step 6 of Example 1 were carried out. The results are shown in Table 6. Wherein, the survival rate = the number of survivals / the total number of transplants × 100%.

[0104] Table 6 Transplant survival rate of tissue culture seedlings of Tiangui Cymbidium in different cultivation media (n=3)

[0105]

[0106]

[0107] Example 8

[0108] Compared with Example 1, Example 8 changed the fertilization management. The rest was the same as Example 1. The cultivation management of transplanted and acclimated seedlings recorded in step 7 of Example 1 was carried out. The results are shown in Table 7.

[0109] Table 7 Fertilization management after transplanting and acclimation of Tiangui Cymbidium tissue culture seedlings

[0110]

[0111] In summary, the present invention has established a method for rapidly propagating seedlings of Tiangui Cymbidium by utilizing seeds thereof and a culture medium thereof. The method of the present invention is used to propagate Tiangui Cymbidium seedlings, and the seed germination rate is high and the germination is fast; the cluster buds differentiate quickly and have high uniformity; the cluster bud proliferation coefficient reaches 6.67 / 120d; the rooting rate is 100%, the number of roots is 6.91, the root length is 3.15cm, the root system is strong, the stem is thick, the leaves are dark green, and the seedlings are tall; the transplanting survival rate is 100%, and the growth is vigorous; it has the characteristics of fast propagation speed, large yield, excellent seedling quality, short time requirement, strong practical operability, etc., and a large number of Tiangui Cymbidium seedlings can be obtained in a relatively short time, which is of great significance to the protection and sustainable utilization of Tiangui Cymbidium seed resources, and has a very broad application prospect.

[0112] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0113] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for promoting rapid seedling formation of Tiangui Cymbidium orchid seeds, comprising the following specific steps: (1) Aseptic sowing and protocorm induction: After sterilizing the unopened mature capsules of Tiangui volute, remove the seeds and inoculate them into a protocorm induction medium for seed germination. Place them in a tissue culture room for dark culture for 7-23 days, and then culture them in light for 40-70 days to obtain germinated seeds. (2) Cluster bud differentiation culture: the germinated seeds are transferred to a cluster bud differentiation medium and cultured under light for 120-150 days until cluster buds are induced to grow; (3) Cluster bud proliferation culture: transferring the cluster buds to a cluster bud proliferation culture medium and culturing them under light for 90-120 days to obtain proliferated cluster buds; (4) Rooting and seedling cultivation of clustered buds: transferring the proliferated clustered buds to a rooting and seedling cultivation medium and culturing them under light for 90-120 days to obtain rooted test tube seedlings; The protocorm induction medium includes the following components in the following amounts: MS basal medium, 2.0 mg / L 6-BA, 200 ml / L coconut milk, 0.1 mg / L NAA, 30 g / L sucrose and 7 g / L agar, with a pH value of 5.4-5.8; The cluster bud differentiation medium includes the following components in the following amounts: B5 basal medium, 0.2 mg / L IBA, 1.0 mg / L AgNO3, 30 g / L sucrose and 7 g / L agar, with a pH value of 5.4-5.8; The cluster bud proliferation culture medium comprises the following components in the following amounts: MS basal medium, 2.0 mg / L 6-BA, 0.5 mg / L KT, 0.2 mg / L NAA, 100 g / L banana puree, 30 g / L sucrose, 1.0 g / L activated carbon and 7 g / L agar, with a pH of 5.4-5.8; The rooting and seedling strengthening medium comprises the following components in the following amounts: MS basal medium, 1.0 mg / L 6-BA, 0.2 mg / L KT, 0.5 mg / L NAA, 200 ml / L coconut water, 30 g / L sucrose, 1.0 g / L activated carbon and 7 g / L agar, with a pH of 5.4-5.8; In step (1), the capsule of the Tiangui orchid is a capsule obtained by artificial cross-pollination.

2. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 1, characterized in that: In step (1), the dark culture conditions in the tissue culture room are: temperature of 25±2°C, shaded with shade cloth, and the light culture conditions are: temperature of 25±2°C, intensity of 1000-1500 lux, and illumination time of 8-14 h / day; In steps (2) to (4), the conditions for light culture are: temperature of 25±2°C, intensity of 1000-1500 lux, and light duration of 8-14 h / day.

3. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 1, characterized in that: The rooted test-tube plantlet further comprises the following culturing steps: (5) Transplantation and acclimation: The rooted test tube seedlings are removed from the culture room, hardened for 7-23 days, and then planted in a transplanting medium to obtain transplanted acclimated seedlings; (6) Cultivation and management: Cultivation and management of the transplanted and domesticated seedlings.

4. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 3, characterized in that: The time for planting in the transplanting medium in step (5) is from March to May, or from September to November each year; the transplanting medium includes at least two of peat soil, bark, moss, perlite, and vermiculite.

5. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 4, characterized in that: The transplanting matrix comprises any one of peat soil: bark: moss in a volume ratio of 1:1:1, bark: perlite: vermiculite in a volume ratio of 1:1:1, bark: moss in a volume ratio of 1:1, and vermiculite: perlite in a volume ratio of 2:

1.

6. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 3, characterized in that: The cultivation management in step (6) includes fertilization, water control and shading.

7. A method for promoting rapid seedling formation of Tiangui Cymbidium seeds according to claim 3, characterized in that: The cultivation management in step (6) specifically includes: spraying the transplanted and acclimated seedlings with a mixture of 0.02-0.05 mg / L BRs and 0.5-1 g / L Huabao No. 1 25-35 days after transplanting, spraying once every 7-23 days, and spraying at least 3 times in succession; spraying 100-200 times of Lanlingwang once every 8-12 days during the growing season; the light intensity throughout the year is 1000-2000 lux; the temperature is maintained at 15℃-25℃ in summer, and it overwinters naturally in winter, and the transplanting medium is kept moist.

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