A high cold-resistant gordonii breeding method suitable for high altitude areas

Abstract

The application provides a high-cold-tolerance Gastrodia elata breeding method suitable for planting in high-altitude areas and relates to the technical field of Gastrodia elata breeding. Wild Gastrodia elata in high-altitude areas above 2000 m and Gastrodia elata planted in large scale in high-altitude areas below 1500 m are selected for hybridization breeding. The obtained seeds are treated by combining chitosan solution soaking with low-temperature treatment, and the germination and growth of the seeds are promoted by using domesticated germination bacteria and Armillaria mellea, so that the seed germination rate of the high-cold-tolerance Gastrodia elata in high-altitude areas is greatly improved, the yield and quality of the Gastrodia elata are improved, and the growth cycle and the breeding cycle of the high-cold-tolerance Gastrodia elata are shortened.

Description

Technical Field

[0001] This invention relates to the field of Gastrodia elata breeding technology, specifically to a method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas. Background Technology

[0002] With increasing public awareness of health and the booming development of the traditional Chinese medicine market, the market demand for Gastrodia elata in health products, functional foods, and processed medicinal herbs has grown rapidly. However, wild Gastrodia elata resources are extremely limited, and relying solely on harvesting wild resources is far from meeting the ever-increasing market demand. Against this backdrop, artificial cultivation and variety selection have become key to solving the supply problem of Gastrodia elata. Through selection and breeding, people hope to obtain new varieties of Gastrodia elata with stronger medicinal effects, shorter growth cycles, and better resistance to adverse conditions, in order to improve yield and quality while protecting wild resources.

[0003] In the breeding of Gastrodia elata, the selection of high-altitude (>2000 meters) Gastrodia elata has always been a research hotspot. The special environmental conditions of high-altitude areas provide unique natural advantages for the growth of Gastrodia elata. These areas typically have lower temperatures, larger diurnal temperature ranges, higher and more stable humidity, good light conditions, and fewer pests and diseases. These conditions are conducive to obtaining higher-quality, higher-yielding, and more resilient Gastrodia elata varieties, and also to the accumulation of more effective components in Gastrodia elata, thereby enhancing its medicinal value and market competitiveness.

[0004] However, the cultivation and breeding of artificially grown Gastrodia elata are currently mainly concentrated in areas below 1800 meters above sea level. For example, the Sichuan Gastrodia elata Jin Hong No. 1, selected through tireless efforts in recent years, is suitable for cultivation at an altitude of 1000-1600 meters. Breeding for cultivation at higher altitudes (e.g., above 2000 meters) is currently less common. The reasons include: (1) As altitude increases, temperature drops significantly and the diurnal temperature range increases further. Many Gastrodia elata varieties are less resistant to adverse conditions, resulting in slow growth or even failure to grow normally. Even if some Gastrodia elata varieties can withstand cold temperatures and temperature differences, their slow growth rate will lead to a longer breeding cycle; (2) Gastrodia elata breeding is a complex and delicate process that requires selecting individual plants with excellent traits from a large number of germplasm resources. However, germplasm resources in high-altitude areas (especially areas above 2000 meters) are relatively scarce. Germplasm resources collected from the wild often need to be cultured for multiple generations before a sufficient number of populations can be obtained for screening; (3) The germination rate of Gastrodia elata seeds is generally low, with a natural germination rate of less than 10%. In order to obtain enough individual plant materials, the breeding work has to be carried out by increasing the amount of seeds used and the number of generations, which further prolongs the breeding cycle and increases the difficulty of breeding.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas. The aim is to overcome at least one of the aforementioned defects, with the goal of breeding new Gastrodia elata varieties that are suitable for cultivation in higher altitude areas (e.g., above 2000 meters) with high yield, good quality, and strong cold resistance within a short period of time.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] A method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas includes treating each generation of Gastrodia elata seeds during the breeding process by combining soaking in chitosan oligosaccharide solution with low-temperature treatment.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects:

[0010] This invention addresses the problems of low seed germination rate and long breeding cycle in the existing technology of Gastrodia elata breeding process. It proposes a method that combines soaking in chitosan oligosaccharide solution with low-temperature treatment to treat each generation of Gastrodia elata seeds during the breeding process, thereby improving the seed germination rate. This allows for obtaining bred varieties with as few breeding generations as possible. In addition, by domesticating Armillaria mellea and germination bacteria and using them in the cultivation of Gastrodia elata, the individual growth cycle of Gastrodia elata is shortened, thereby shortening the breeding cycle. Attached Figure Description

[0011] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0012] Figure 1 The images show a comparison of the actual products of Sichuan Gastrodia elata Gao Hong No. 1 (TMGH-1), Sichuan Gastrodia elata Jin Hong No. 1 (JHCK), and traditional red Gastrodia elata (HCK) selected by the method of the present invention. Among them, (a) is Sichuan Gastrodia elata Jin Hong No. 1, (b) is Sichuan Gastrodia elata Gao Hong No. 1, and (c) is traditional red Gastrodia elata.

[0013] Figure 2 This is a comparison chart of the yield per mu of Sichuan Gastrodia elata Gaohong No. 1 (TMGH-1), Sichuan Gastrodia elata Jinhong No. 1 (JHCK), and traditional red Gastrodia elata (HCK) from the production area, bred using the method of this invention.

[0014] Figure 3 This is a comparison chart of the test results of the Chinese Pharmacopoeia for the Sichuan Gastrodia elata Gaohong No. 1, Sichuan Gastrodia elata Jinhong No. 1, and traditional red Gastrodia elata from the production area, selected by the method of this invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0016] Specifically, such as Figures 1 to 3 As shown in the figure, this embodiment provides a method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas, which includes the following steps:

[0017] Step S1: Obtaining Parental Samples

[0018] Based on years of systematic breeding and approval of the new variety Sichuan Gastrodia elata Jin Hong No. 1 (JHCK) in southwestern Sichuan, we focused on screening Sichuan Gastrodia elata varieties from high-altitude areas above 2000 meters in the Qinba Mountains and Wumeng Mountains. We selected robust wild Gastrodia elata with high gastrodin content (e.g., greater than 0.5%) as the parent plant. In this example, the parent plant was selected from wild Gastrodia elata from Wumeng Mountains at an altitude of about 2500 meters. The selection criteria were that the individual Gastrodia elata should be short but robust, with full tubers, free from disease spots, bacterial infection, black spots and rot, and with full and intact buds. The weight of a single Gastrodia elata tuber should be more than 100 grams, with a regular shape, smooth surface and bright color.

[0019] Accordingly, in areas with an altitude of around 1500 meters in the Qinba Mountains and Wumeng Mountains (this example is roughly at 1500 meters, but in practice it can be lower, such as 1300 meters or 1200 meters), a large-scale cultivated Gastrodia elata variety is selected as the male parent. In this example, the Sichuan Gastrodia elata Jin Hong No. 1 (JHCK) is specifically selected as the male parent. The tubers should be plump, full, regular in shape, smooth in surface, bright in color, free from disease spots, bacterial infection, black spots and rot, with plump and intact buds, and each Gastrodia elata tuber weighing more than 200 grams and of uniform size.

[0020] Step S2: Cultivation and domestication of microbial strains

[0021] Gastrodia elata is a perennial symbiotic herbaceous parasitic plant of the Orchidaceae family. It lacks roots and green leaves and cannot photosynthesize to synthesize organic matter. Its nutrition is mainly obtained from wood through Armillaria mellea, and the germination fungus plays a crucial role in the germination of Gastrodia elata seeds. Therefore, this embodiment acclimates both of these fungal species. For example, to ensure that Armillaria mellea can tolerate the environmental conditions of high-altitude areas during subsequent forest planting, and to ensure the successful cultivation and growth of Gastrodia elata in high-altitude areas, this embodiment obtains Armillaria mellea inoculum from the original habitat of the mother plant, Gastrodia elata, and performs separate cultivation treatment. Specifically, the cultivation and acclimation of Armillaria mellea includes the following steps:

[0022] S21. In the native habitat of the mother plant, *Gastrodia elata*, observe the mycelial cords or hyphae forming near the plants to identify the symbiotic *Armillaria mellea*. Collect *Armillaria mellea* samples using sterile tools (such as sterile scalpels and forceps), ensuring sample integrity and uncontaminated conditions, and place the samples in sterile containers. Then, bring the collected *Armillaria mellea* samples back to the laboratory for hyphal isolation. Hyphae isolation can be performed using tissue isolation or spore isolation methods.

[0023] S22. Next, the isolated Armillaria mellea was inoculated into a sterilized first culture medium for preliminary cultivation, resulting in highly viable and well-growing Armillaria mellea. The first culture medium was commercially available potato dextrose agar (PDA) medium. This type of medium is rich in carbon, nitrogen, vitamins, and inorganic salts, meeting the nutritional requirements for Armillaria mellea growth. Its good coagulation properties also facilitate observation of mycelial growth. During cultivation, the temperature of the incubator was controlled between 15°C and 25°C, and the relative humidity was maintained between 60% and 70%. The growth of Armillaria mellea was observed regularly to ensure good mycelial growth. Furthermore, the activity and quantity of Armillaria mellea were regularly monitored to ensure healthy growth on the PDA medium, providing sufficient Armillaria mellea strain for subsequent chitosan oligosaccharide domestication.

[0024] S23. Prepare a second culture medium using chitosan oligosaccharide solutions with different mass fractions, and acclimate the Armillaria mellea obtained in the initial culture in step S22 in order of increasing chitosan oligosaccharide mass fraction to obtain highly resistant Armillaria mellea.

[0025] Specifically, the second culture medium is prepared using the following method:

[0026] A. Weigh out 50 parts by weight of hardwood sawdust, 30 parts of rice bran, 15 parts of wheat bran, 1 part of gypsum, 5 parts of soybean meal, 2 parts of yeast extract, 0.5 parts of potassium dihydrogen phosphate, and 5 parts of glucose. Mix them thoroughly to obtain a mixture. Add an appropriate amount of water to make the water content of the mixture reach about 40% to obtain the initial culture medium.

[0027] B. Pour the prepared culture medium into culture flasks, then place the culture flasks in an autoclave for sterilization. Sterilization conditions: 121℃, 20 minutes;

[0028] C. Using sterile water, prepare chitosan oligosaccharide solutions with mass fractions of 0.02%, 0.05%, and 0.15% for later use. After the sterilized culture medium has cooled to room temperature, add the corresponding mass fractions (0.02%, 0.05%, and 0.15%) of chitosan oligosaccharide solution to the sterilized culture medium to prepare second culture media with different chitosan oligosaccharide contents. The amount of chitosan oligosaccharide solution added should be such that the final water content of the second culture medium reaches 60%. Preferably, when adding the chitosan oligosaccharide solution of the corresponding concentration to the sterilized culture medium, filter the chitosan oligosaccharide solution through a 0.22 μm filter membrane for sterilization.

[0029] Furthermore, the three different secondary culture media corresponding to chitosan oligosaccharide mass fractions of 0.02%, 0.05%, and 0.15% are respectively designated as the third culture medium (0.02%), the fourth culture medium (0.05%), and the fifth culture medium (0.15%). The acclimatization of the *Armillaria mellea* obtained from the initial culture in step S22 specifically involves: first, inoculating the well-grown *Armillaria mellea* from the initial culture onto the third culture medium, maintaining the same temperature (15°C to 25°C) and humidity (60% to 70%) as the initial culture. Under 0% conditions, Armillaria mellea was cultured for about 7 to 10 days to obtain the first domesticated Armillaria mellea. Then, the first domesticated Armillaria mellea was inoculated onto the fourth culture medium and cultured for about 7 to 10 days under the same conditions as the first domestication to obtain the second domesticated Armillaria mellea. After that, the second domesticated Armillaria mellea was inoculated onto the fifth culture medium and cultured for about 10 to 15 days under the same conditions as the second domestication to obtain the third domesticated Armillaria mellea, which is the highly resistant Armillaria mellea. Then, it was transferred to an environment of 0℃~5℃ for storage.

[0030] It should be noted that the second culture medium of the present invention has a composition that is closer to the substrate conditions under which Gastrodia elata coexists with Armillaria mellea in the natural environment. For example, the components it contains, such as hardwood sawdust and rice bran, can provide Armillaria mellea with richer natural components such as lignin and cellulose, enabling it to grow in a more natural environment and helping to enhance the symbiotic adaptability between Armillaria mellea and Gastrodia elata. Furthermore, by adding chitosan oligosaccharide solutions of different mass fractions and acclimating Armillaria mellea in order of increasing mass fraction, it is possible to gradually induce stress-related physiological changes in Armillaria mellea, enabling it to better adapt to the harsh environment of high-altitude areas, such as low temperature.

[0031] Furthermore, the germinating fungus used in this embodiment is *Osmunda japonica* germinating fungus, which also needs to undergo chitosan oligosaccharide stress acclimatization. The acclimatization method is the same as that for *Armillaria mellea*, which is achieved by adding chitosan oligosaccharide solutions with mass fractions of 0.02%, 0.05%, and 0.15%. The acclimatization is also carried out using the third, fourth, and fifth culture media. The resulting germinating fungus is a highly stress-resistant germinating fungus, which will not be described in detail here (unless otherwise specified, the germinating fungus and *Armillaria mellea* mentioned below refer to the acclimatized germinating fungus and *Armillaria mellea*).

[0032] Step S3: Obtaining Hybrid Seeds

[0033] S31. Flowering period adjustment

[0034] The collected samples of *Gastrodia elata* from high-altitude areas were placed in a greenhouse environment with controllable temperature and humidity. The temperature and humidity inside the greenhouse were gradually adjusted to simulate the relatively warm climate characteristics of low-altitude areas, thereby accelerating the growth rate of the mother *Gastrodia elata* and advancing its flowering period to match the flowering period of the father *Gastrodia elata*. Alternatively, the father *Gastrodia elata* was subjected to low-temperature dormancy treatment to slow its growth rate and match its flowering period with that of the mother *Gastrodia elata*. The flowering period of the father *Gastrodia elata* can be slightly earlier than that of the mother *Gastrodia elata*, for example, 1-2 days earlier. Flowering period adjustment is an existing technology and will not be elaborated upon here.

[0035] S32, pollination

[0036] During the flowering period of the male parent, *Gastrodia elata*, mature pollen is collected and applied to the stigma of the female parent. After pollination, the pollination date, variety information of the male and female parents, etc., are recorded on a tag.

[0037] S33. Fruit collection: After pollination, the capsules begin to swell. The capsules mature in about 20 to 30 days (when the six longitudinal sutures around the capsule are very obvious, the longitudinal sutures of the fruit are raised but not cracked, the capsules soften when gently squeezed by hand, the seeds are easily scattered when the shell is peeled off, and the seeds are light yellow, this is the sign that the capsules are mature). Harvest them in time and put them into sterile paper bags for later use.

[0038] Step S4, Seed Treatment and Sowing

[0039] S41. Place the harvested and unopened Gastrodia elata capsules in a low-temperature sterile environment of 0℃~2℃ for 5~7 days to induce the Gastrodia elata seeds into a short period of dormancy. During the low-temperature treatment, the ambient humidity should be maintained at 60~70%. Under this humidity range, the capsules will not crack on their own, which is beneficial for subsequent chitosan oligosaccharide soaking treatment.

[0040] S42. After the low-temperature treatment, transfer the capsules to an environment of 10℃~12℃ and place them for 2 hours. Then, use a sterile needle or a fine toothpick (diameter ≤0.5mm) to poke holes in the gastrodia capsules after the low-temperature treatment in step S41. When poking holes, avoid making the holes too large to prevent seed loss. Make holes evenly in the lower part of the capsule (avoiding the end of the stalk), with 3~5 holes per capsule. Disinfect the poking tools with 75% alcohol before poking to prevent pathogen contamination.

[0041] S43. Immerse the perforated Gastrodia elata capsules in a pre-prepared 0.1% (by mass) chitosan oligosaccharide solution (temperature controlled at 8-10°C). Then, place the chitosan oligosaccharide solution containing the Gastrodia elata capsules in a vacuum device and evacuate to approximately -0.06 MPa for 1 minute to allow the air inside the capsules to escape. Then, slowly release the pressure to allow the solution to penetrate into the capsule under atmospheric pressure. During this process, the capsules can be removed from the vacuum device and gently squeezed 1-2 times (preferably under sterile conditions). Then, place them back into the vacuum device to ensure the solution penetrates. Repeat the above steps of evacuation and gentle squeezing 2-3 times. Then, remove the capsules from the chitosan oligosaccharide solution (the total soaking time is approximately 15 minutes from start to finish) and place them on sterile gauze to drain for 1-1.5 hours. It should be noted that this step uses vacuum negative pressure combined with gentle squeezing, which allows the chitosan oligosaccharide solution to penetrate into the shell in a short time without damaging the seeds inside, even with a very small puncture hole. This improves processing efficiency and prevents soaking failure caused by uneven penetration. The smaller puncture hole also prevents the seeds inside the shell from leaking out.

[0042] S44. Spraying seeds onto leaves: Peel the capsules of Gastrodia elata after draining them in step S43, take out the seeds, and dry them; take the spawn of the germinating fungus of the genus Gastrodia elata from the culture bottle and place it in a basin; then gently sprinkle the Gastrodia elata seeds on the spawn leaves, mixing them evenly as you sprinkle.

[0043] S45. Indoor sowing: First, prepare the substrate, selecting substrates with short Armillaria mellea cultivation time, tender mycelia, vigorous growth, and mycelium that has penetrated the bark of the wood segment, free from contamination by other fungi (prepared through domesticated Armillaria mellea); and prepare the sowing bed in advance (for example, by using a hemp planting box with a depth of 30cm, a width of 60cm, and a length of 1m); after step S44, lay a layer of damp leaves at the bottom of the bed, divide the mixed Gastrodia elata seeds and mycelium leaves into two portions, scatter one portion at the bottom, place the substrate, spacing the substrate 3 to 4cm apart, cover with sand until the substrate is level, then lay damp leaves again, scatter the other half of the mixed mycelium leaves on the top layer, place the Armillaria mellea substrate, cover with soil to a thickness of 8 to 10cm, and then cover with agricultural film to complete indoor sowing;

[0044] S46. After sowing, adjust the room temperature to 15℃~25℃ and keep the soil moisture between 60%~70%. After 4~6 months of cultivation, harvest a large amount of rice hemp and white hemp, which will be used as hemp seeds for transplanting.

[0045] Step S5: Transplanting and cultivation in high-altitude areas:

[0046] In spring (March to April) or autumn (September to October), the hemp seeds are transplanted and cultivated in pre-selected forest land at an altitude of over 2000 meters (specifically, the forest land we selected has an altitude of 2500 meters). Specifically, first, the land is prepared, leaves and branches are spread, and phorate is applied for insect control. Then, tree sticks are placed at 5-7 cm intervals (for inoculation with Armillaria mellea), and Armillaria mellea inoculum is placed between the sticks, with inoculum placed at both ends of the sticks, and inoculum placed between sticks spaced 25 cm apart. Next, the rice hemp and white hemp (which need to be selected) obtained in step S46 are placed between the rows, covered with about 3 cm of soil, and a second layer is cultivated on top of the soil using a similar method. After the second layer is planted, it is covered with 10 cm of soil, and then covered with dry leaves; this completes the transplanting of the hemp seeds. After transplanting, manage the plants according to existing field management methods. Harvest the Gastrodia elata during its dormant growth period (late autumn). Select large arrow-shaped tubers with flower buds growing at the top. Choose arrow-shaped tubers with red and plump terminal buds, free from pests and diseases, without damage, and of suitable size as seed tubers for seed cultivation. After that, let the seed tubers dry for 1-2 days to remove some moisture, store them in moist sand, and maintain the storage temperature at 2-5℃. Then, cultivate the seed tubers in wooden or plastic boxes, add sand or sandy loam soil, maintain humidity, regulate temperature, and promote the bolting and flowering of Gastrodia elata.

[0047] Step S6, self-pollination: When the hemp plants grow to the budding stage, self-pollination is carried out to obtain self-homozygous capsules and seeds, which are then screened and preserved;

[0048] Step S7: To improve seed homozygosity, repeat operations S4 to S6 on the preserved seeds. After multiple generations of self-pollination and screening, obtain *Gastrodia elata* seeds with stable genetic traits and high homozygosity for large-scale cultivation or further breeding of new varieties. The above are the main steps of *Gastrodia elata* breeding according to this invention. It should be understood that this method may also include other steps, all of which should fall within the scope of protection of this invention without departing from the inventive concept.

[0049] In addition, to verify the effects of low-temperature treatment and chitosan oligosaccharide solution soaking on the germination rate of Gastrodia elata seeds, we conducted different comparative experiments on the self-pollinated homozygous seeds obtained in step S6. Sixty mature Gastrodia elata capsules harvested from the same batch were divided into twelve groups (numbered sequentially as 1, 2, 3...11, 12). Different low-temperature treatments and / or chitosan oligosaccharide solution soaking treatments were applied to the Gastrodia elata capsules between groups, while the same low-temperature treatment and / or chitosan oligosaccharide solution soaking treatment was applied to the five Gastrodia elata capsules within each group (in the subsequent seed dressing and sowing process, the five Gastrodia elata capsules within the same group were treated in the same way and were treated individually to ensure the accuracy of the data through individual comparison within the group). Then, seed dressing and sowing were carried out according to the methods in S44 and S45 (indoor sowing, which has higher controllability and helps to ensure the accuracy of the experiment). Sampling and statistics were carried out 3 months after sowing. For the sampled specimens, five fields of view were randomly selected under a microscope. The total number of seeds, the total number of dead seeds, and the total number of germinating seeds were calculated for each field of view, and the germination rate was calculated. The germination rate was calculated as (total number of germinating seeds / total number of seeds counted) × 100%. The germination rate results for each group are shown in the table below.

[0050]

[0051] It should be noted that the seed treatment and sowing of each group of Gastrodia elata capsules in the table above are carried out in accordance with (or similar to) the method in step S4. In the table above, the Gastrodia elata seeds in groups 1 to 4 were not soaked in chitosan oligosaccharide solution. Among them, groups 1 to 3 were subjected to low-temperature treatment (from 4℃ to 0℃), and the Gastrodia elata seeds in group 4 were sown immediately after harvesting as fresh seeds. Groups 5 to 12 were soaked in chitosan oligosaccharide solution, and except for group 9, they were subjected to low-temperature treatment. The microscopic examination results in the table above show that the average germination rate of Gastrodia elata seeds in groups 1 to 3, which underwent low-temperature treatment, was significantly higher than that in group 4, which did not undergo low-temperature treatment. This indicates that low-temperature treatment helps improve the seed germination rate, possibly because low temperature helps the seeds enter a short dormancy period, which may help improve their germination rate. Comparing the results of groups 1 to 3, it can be seen that the average germination rate of Gastrodia elata seeds in group 1, which were stored at 4℃ for 5 days, was the highest. As the temperature decreased, the average germination rate of groups 2 and 3 gradually decreased. This may be because within this temperature range, the decrease in temperature inhibited the germination of Gastrodia elata seeds, for example, by reducing the enzyme activity inside the seeds.

[0052] Referring further to the table above, groups 5 through 7 were soaked in a 0.1% chitosan oligosaccharide solution and subjected to low-temperature treatments at 4℃, 2℃, and 0℃, respectively. The results showed that the average germination rate of groups 5 through 7 was significantly higher than that of groups 1 through 3, which did not undergo chitosan oligosaccharide solution soaking. This indicates that the treatment method of chitosan oligosaccharide solution soaking combined with low-temperature treatment can further increase the germination rate of Gastrodia elata seeds. Furthermore, as shown in groups 5 through 7, when the treatment method of chitosan oligosaccharide solution soaking combined with low-temperature treatment was used, the average germination rate of groups 6 and 7 (stressed at 2℃ and 0℃) was actually higher than that of group 5 (stressed at 4℃). This indicates that although low-temperature conditions have an adverse effect on seed germination, the chitosan oligosaccharide solution soaking treatment can alleviate this adverse effect to some extent, especially under the low-temperature conditions of 2℃ and 0℃, the seed germination rate still remains at a high level. Furthermore, the results from groups 8 and 9 in the table above show that when the low-temperature treatment temperature was -2℃, even with soaking in chitosan oligosaccharide solution, the average germination rate under microscopic examination decreased significantly to 10.2%, indicating that temperature became the main factor affecting the Gastrodia elata seeds at this point. Group 9, which did not undergo low-temperature treatment but only used a 0.1% (w / w) chitosan oligosaccharide solution for soaking, had a germination rate of only 15.4% (significantly higher than group 4), indicating that soaking in chitosan oligosaccharide solution can help improve the germination rate, but the effect of soaking alone is limited.

[0053] Referring further to the table above, compared to group 7, groups 10 to 12 changed the mass fraction of chitosan oligosaccharides in the chitosan oligosaccharide solution. When soaking in a 0.15% chitosan oligosaccharide solution, the average germination rate observed under microscopy remained high, exceeding 30%. However, when soaking in a 0.05% chitosan oligosaccharide solution, the average germination rate was only 21.9%, indicating that the promoting effect of chitosan oligosaccharide on the germination of Gastrodia elata seeds decreased with decreasing chitosan oligosaccharide concentration. When soaking in a 0.25% chitosan oligosaccharide solution, the average germination rate was only 4.9%, indicating that the chitosan oligosaccharide concentration was too high at this point, producing adverse effects. Therefore, the combined treatment of chitosan oligosaccharide solution soaking and low-temperature treatment is a key factor in improving the germination rate of highly cold-resistant Gastrodia elata seeds, and both the mass fraction of the chitosan oligosaccharide solution and the temperature of the low-temperature treatment need to be optimized to achieve the best germination effect.

[0054] Furthermore, the length of the breeding cycle is mainly determined by the number of generations cultivated and the length of the growth cycle of each generation (a complete growth cycle is from seed → protocorm → rice hemp → white hemp → arrow hemp → seed). This invention, by combining chitosan oligosaccharide solution soaking with low-temperature treatment, can significantly improve the germination rate of highly cold-resistant Gastrodia elata seeds. This allows for obtaining more breeding samples in a short period, thereby reducing the number of cultivation generations. Furthermore, the growth cycle of Gastrodia elata is affected not only by its own genetic factors but also by the symbiotic fungus Armillaria mellea. Since the above method uses domesticated, highly resistant germinating bacteria and Armillaria mellea for breeding and cultivation, to verify their impact on the growth cycle of Gastrodia elata, we also conducted the following comparative experiment: comparing undomesticated germinating bacteria, Armillaria mellea (undomesticated) initially cultured in step S22, and domesticated germinating bacteria and Armillaria mellea (the culture methods were identical except for the fungal species). The seeds used were also from the same batch of self-crossed homozygous seeds obtained in step S6. The statistical results are as follows:

[0055]

[0056] As shown in the table above, although the self-pollinated homozygous seeds obtained in step S6 possess high cold resistance characteristics derived from the parent plant *Gastrodia elata*, their growth cycle remains very long (up to 1130 days) when planted at high altitudes without acclimation of *Armillaria mellea* and its germinating fungi. However, when using the acclimated germinating fungi and *Armillaria mellea* of this invention, the growth cycle of the highly cold-resistant *Gastrodia elata* can be significantly shortened. This may be because acclimation not only improves the stress resistance of the germinating fungi and *Armillaria mellea*, reducing the impact of low temperatures at high altitudes, but also introduces some chitosan oligosaccharide components during the subsequent introduction of fragments of highly stress-resistant *Armillaria mellea* mycelium. This component also promotes the growth of *Gastrodia elata*, thereby shortening the growth cycle.

[0057] Furthermore, using the aforementioned breeding methods, we crossbred wild Gastrodia elata (female parent) found at an altitude of 2500 meters in the Wumeng Mountains with Sichuan Gastrodia elata Jin Hong 1 (male parent), and in a short period of time, we cultivated a new variety—Sichuan Gastrodia elata Gao Hong 1 (TMGH-1)—that can be cultivated on a large scale in areas with altitudes of 1800-3200 meters. Figure 1 It can be seen that TMGH-1 exhibits excellent characteristics in terms of size and shape. Its individual specimens are even larger than JHCK red Gastrodia elata grown at an altitude of 1500 meters, and its appearance is remarkably similar to the expensive wild Gastrodia elata on the market. Figure 2 and Figure 3It can be seen that TMGH-1 has higher yield and better agronomic characteristics. Its performance in terms of dilute ethanol extract, total amount of gastrodin and p-hydroxybenzyl alcohol, ash content, moisture content and sulfur dioxide residue is excellent. Not only does its dilute ethanol extract, total amount of gastrodin and p-hydroxybenzyl alcohol meet the requirements of the Chinese Pharmacopoeia and are significantly better than JHCK and HCK, but its ash content is also lower. This not only indicates its excellent quality, but also shows that its growth environment and processing have been better controlled and managed. This makes TMGH-1 superior to other varieties in terms of safety, efficacy and quality stability, making it a high-quality and high-yield gastrodia variety.

[0058] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high cold-tolerance Gastrodia elata breeding method suitable for high-altitude areas, the method comprising, during the breeding process, using a chitosan oligosaccharide solution to soak and combine low-temperature treatment to process each generation of Gastrodia elata seeds during the breeding process; characterized in that, The high cold-resistant Gastrodia elata breeding method comprises the following steps: Step S1, parent obtaining; Step S2, culture and domestication of the strain; Step S3, obtaining of hybrid seeds; Step S4, seed treatment and sowing; Step S5, transplanting in high-altitude areas; Step S6, obtaining of self-crossed and pure hybrid pericarp and seeds, screening and preservation; Step S7, repeating the operations of S4 to S6 on the preserved seeds, until the seeds with stable genetic traits and high purity are obtained through multiple generations of self-crossing and screening; In the step S1, the parent obtaining comprises father obtaining and mother obtaining, the mother is selected from wild Gastrodia elata in an altitude above 2000 meters, and the father is selected from red Gastrodia elata planted in an altitude below 1500 meters; Step S4 comprises: S41, placing the picked and uncracked Gastrodia elata pericarp in a low-temperature sterile environment at 0°C-2°C for 5-7 days to promote the Gastrodia elata seeds to enter a short-term dormancy; S42, using a puncture tool to puncture the Gastrodia elata pericarp after the low-temperature treatment in step S41; S43, placing the punctured Gastrodia elata pericarp into a prepared chitosan oligosaccharide solution with a mass fraction of 0.1-0.15% for soaking, and ensuring that the chitosan oligosaccharide solution can penetrate into the Gastrodia elata pericarp, wherein the chitosan oligosaccharide solution is ensured to penetrate into the Gastrodia elata pericarp by means of vacuumizing and gently pressing, specifically, placing the chitosan oligosaccharide solution with the Gastrodia elata pericarp in a vacuum device, vacuumizing to-0.06 MPa and maintaining for 1 minute to make the air in the Gastrodia elata pericarp escape, then slowly releasing the pressure to make the chitosan oligosaccharide solution penetrate into the pericarp under the atmospheric pressure, and gently pressing the Gastrodia elata pericarp 1-2 times during the process to ensure the solution to penetrate; S44, breaking the Gastrodia elata pericarp treated in S43, taking out the seeds and drying, and then mixing with the inoculation germination material; S45, indoor sowing; S46, management, and obtaining of the Gastrodia elata seeds for transplanting. Step S2 comprises culture and domestication of Armillaria mellea, which comprises the following steps:

2. The method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas as described in claim 1, characterized in that, S21, searching for Armillaria mellea symbiotic with Gastrodia elata in the original habitat of the mother Gastrodia elata, and separating the mycelium; S22, inoculating the separated Armillaria mellea into the sterilized first culture medium for preliminary culture to obtain Armillaria mellea with high activity and good growth; S23, preparing a second culture medium by using chitosan oligosaccharide solutions with different mass fractions, and domesticating the Armillaria mellea obtained in step S22 in order of the mass fraction of chitosan oligosaccharide from low to high to obtain Armillaria mellea with high stress resistance. The second culture medium is prepared by the following method:

3. The method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas as described in claim 2, characterized in that, According to weight parts, 50 parts of mixed wood chips, 30 parts of rice bran, 15 parts of wheat bran, 1 part of gypsum, 5 parts of soybean meal, 2 parts of yeast extract, 0.5 part of potassium dihydrogen phosphate and 5 parts of glucose are weighed and uniformly mixed to obtain a mixture, and an appropriate amount of water is added to make the water content of the mixture reach about 40% to obtain a culture medium primary body; The prepared culture medium primary body is loaded into a culture bottle, and then the culture bottle is placed in an autoclave for sterilization treatment; ​ A chitooligosaccharide solution with a mass fraction of 0.02%, 0.05% and 0.1% is prepared with sterile water, and after the initial medium is cooled to room temperature, the chitooligosaccharide solution with a corresponding mass fraction is added to the sterilized initial medium, thereby preparing the second medium with different chitooligosaccharide contents.

4. The method for breeding highly cold-resistant Gastrodia elata suitable for cultivation in high-altitude areas as described in claim 3, characterized in that, The Armillaria mellea preliminarily cultured in step S22 is domesticated in the order from low to high of the mass fraction of the second medium with different chitooligosaccharide contents.

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