Breeding method of high-cold-resistance gastrodia elata suitable for being planted in high-altitude area

Abstract

The invention provides a breeding method of high-cold-resistant gastrodia elata suitable for being planted in a high-altitude area, and relates to the technical field of gastrodia elata breeding. The method comprises the following steps: selecting wild gastrodia elata in a high altitude area of more than 2000 meters and gastrodia elata planted in a large scale in an altitude area of less than 1500 meters for cross breeding, treating the obtained seeds in a manner of combining chitosan oligosaccharide solution soaking and low-temperature treatment, and promoting germination and growth by using domesticated germination bacteria and armillaria mellea, so as to obtain the gastrodia elata seed with the high altitude area of more than 2000 meters and the high altitude area of less than 1500 meters. The seed germination rate of the cold-resistant gastrodia elata in the high-altitude area can be greatly increased, the yield and quality of the gastrodia elata are improved, the growth cycle of the cold-resistant gastrodia elata is shortened, and the breeding cycle is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of Gastrodia elata breeding, and particularly to a method for breeding highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas. Background Art

[0002] With the increasing attention of people to health and the booming development of the traditional Chinese medicine market, the market demand for Gastrodia elata in the fields of health products, functional foods, and traditional Chinese medicine decoction pieces has increased sharply. However, the wild Gastrodia elata resources are extremely limited. Relying solely on the collection of wild resources far from meets the growing market demand. In this context, artificial cultivation and variety breeding have become the key to solving the problem of Gastrodia elata supply. Through breeding, people hope to obtain new Gastrodia elata varieties with stronger medicinal effects, shorter growth cycles, and better stress resistance, so as to improve the yield and quality, and at the same time protect wild resources.

[0003] In the process of Gastrodia elata breeding, the breeding of Gastrodia elata at high altitudes (>2000 meters) has always been a research hotspot. The special environmental conditions in high-altitude areas provide unique natural advantages for the growth of Gastrodia elata. These areas usually have lower temperatures, larger day-night temperature differences, higher and stable humidity, good light conditions, and fewer pests and diseases. These conditions are conducive to obtaining more high-quality, high-yield, and stress-resistant Gastrodia elata varieties, and are conducive to Gastrodia elata accumulating more active ingredients, enhancing its medicinal value and market competitiveness.

[0004] However, at present, the cultivation and breeding of artificially cultivated Gastrodia elata mainly focus on areas below 1800 meters above sea level. For example, Gastrodia elata Jinhong No. 1 screened by people through unremitting efforts in recent years is suitable for planting at an altitude of 1000 - 1600 meters. For the breeding of Gastrodia elata for planting at higher altitudes (such as above 2000 meters), relatively few studies have been carried out at present. The reasons include: (1) As the altitude rises, the temperature drops significantly, and the day-night temperature difference further increases. The stress resistance of many Gastrodia elata varieties is weak, resulting in slow growth or even inability to grow normally. Even if some Gastrodia elata varieties can tolerate the cold temperature and temperature difference changes, their slow growth rate will lead to an extended breeding cycle; (2) Gastrodia elata breeding is a complex and delicate process that requires selecting single plants with excellent traits from a large number of germplasm resources. However, the germplasm resources in high-altitude areas (especially above 2000 meters) are relatively scarce. The germplasm resources collected from the wild often need to be cultured for several generations before a sufficient number of populations for screening can be obtained; (3) The germination rate of Gastrodia elata seeds is generally low, and its natural germination rate is less than 10%. In order to obtain enough single-plant materials, the breeding work has to be carried out by increasing the amount of seeds used and the number of generations in breeding, which further extends the breeding cycle and increases the breeding difficulty.

[0005] In view of this, the present application is proposed. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention proposes a method for breeding highly cold-tolerant Gastrodia elata suitable for planting in high-altitude areas, aiming to overcome at least one of the above-mentioned defects, in order to breed a new variety of Gastrodia elata that can be suitable for planting in higher-altitude areas (such as above 2000 meters), with high yield, good appearance, and strong cold tolerance in a short period.

[0007] To solve the above technical problems, the present invention provides the following technical solutions: A method for breeding highly cold-tolerant Gastrodia elata suitable for planting in high-altitude areas, the method includes, during the breeding process, using a combination of soaking with chitosan oligosaccharide solution and low-temperature treatment to treat each generation of Gastrodia elata seeds during the breeding process.

[0008] Compared with the prior art, the present invention has at least the following beneficial effects: In view of the problems of low germination rate of Gastrodia elata seeds and long breeding cycle in the prior art during the breeding process, the present invention proposes to use a combination of soaking with chitosan oligosaccharide solution and low-temperature treatment to treat each generation of Gastrodia elata seeds during the breeding process, thereby improving the seed germination rate, so that the selected variety can be obtained with as few breeding generations as possible; in addition, by domesticating Armillaria mellea and germination fungi and using them in the cultivation of Gastrodia elata, the single growth cycle of Gastrodia elata is shortened, thus shortening the breeding cycle. Description of the Drawings

[0009] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not constitute a limitation to the embodiments of the present invention. In the drawings: Figure 1 It is a physical comparison diagram of Gastrodia elata cv. Chuan Tianma Gao Hong No. 1 (TMGH-1), Gastrodia elata cv. Chuan Tianma Jin Hong No. 1 (JHCK), and the traditional red Gastrodia elata from the origin selected by the method of the present invention. Among them, (a) is Gastrodia elata cv. Chuan Tianma Jin Hong No. 1, (b) is Gastrodia elata cv. Chuan Tianma Gao Hong No. 1, and (c) is the traditional red Gastrodia elata from the origin; Figure 2 It is a comparison diagram of the yield per mu of Gastrodia elata cv. Chuan Tianma Gao Hong No. 1 (TMGH-1), Gastrodia elata cv. Chuan Tianma Jin Hong No. 1 (JHCK), and the traditional red Gastrodia elata from the origin selected by the method of the present invention; Figure 3 It is a comparison diagram of the detection results of the Chinese Pharmacopoeia indicators of Gastrodia elata cv. Chuan Tianma Gao Hong No. 1, Gastrodia elata cv. Chuan Tianma Jin Hong No. 1, and the traditional red Gastrodia elata from the origin selected by the method of the present invention. Detailed Embodiments

[0010] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments and drawings. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and do not limit the present invention.

[0011] Specifically, as Figures 1 to 3 shown, this embodiment provides a high-cold-resistant Gastrodia elata breeding method suitable for planting in high-altitude areas, which includes the following steps: Step S1, obtaining parents Based on the systematic breeding in southwestern Sichuan for many years and the approval of the new variety of Gastrodia elata Jinhong No. 1 (JHCK) in Sichuan, the Gastrodia elata varieties in high-altitude areas above 2000 meters in the Qinling-Bashan Mountains and Wumeng Mountains were screened. Select wild Gastrodia elata with strong growth and high gastrodin content (for example, greater than 0.5%) as the female parent. The female parent in this embodiment is selected from wild Gastrodia elata at an altitude of about 2500 meters in the Wumeng Mountains. It is required to select Gastrodia elata individuals with short but thick bodies, plump tubers, no disease spots, no miscellaneous bacteria infection, no black dot rot, and plump and intact bud mouths; the weight of a single Gastrodia elata tuber is more than 100 grams, with regular shape, smooth surface, and bright color; Correspondingly, in the areas around 1500 meters above sea level in the Qinling-Bashan Mountains and Wumeng Mountains (selected at about 1500 meters in this embodiment, and actually can be lower, such as 1300 meters, 1200 meters, etc.), the Gastrodia elata varieties that have been planted on a large scale are selected as the male parent. Specifically, Gastrodia elata Jinhong No. 1 (JHCK) is selected as the male parent in this embodiment. It is required that the tubers should be plump, full, with regular shape, smooth surface, bright color, no disease spots, no miscellaneous bacteria infection, no black dot rot, plump and intact bud mouths, and the weight of a single Gastrodia elata tuber is more than 200 grams, with uniform size.

[0012] Step S2, cultivation and domestication of strains Gastrodia elata is a perennial symbiotic herbaceous parasitic plant of the Orchidaceae family. It has no roots and green leaves and cannot carry out photosynthesis to synthesize organic substances. Its nutrients are mainly obtained from Armillaria mellea in wood, and in the germination process of Gastrodia elata seeds, the germination fungus plays a crucial role. Therefore, both of these strains are domesticated in this embodiment. Exemplarily, in order to ensure that Armillaria mellea can tolerate the environmental conditions in high-altitude areas during the subsequent forest land planting process and ensure the successful planting and growth of Gastrodia elata in high-altitude areas, the Armillaria mellea strain source is obtained from the original place of the female parent Gastrodia elata and a separate cultivation treatment is carried out. Specifically, the cultivation and domestication of Armillaria mellea include the following steps: S21. In the original habitat of the female parent Gastrodia elata, search for the Armillaria mellea symbiotic with it by observing the rhizomorphs or mycelia formed near the Gastrodia elata plants. Use sterile tools (such as sterile knives and sterile tweezers) to collect Armillaria mellea samples, ensure the integrity and pollution-free of the samples, and place the samples into sterile containers. Then, bring the collected Armillaria mellea samples back to the laboratory for mycelium isolation. Mycelium isolation can be carried out using the tissue isolation method or the spore isolation method; S22. Then, inoculate the isolated Armillaria mellea into the sterilized first medium for primary culture to obtain Armillaria mellea with high vitality and good growth. Among them, the first medium is a commercially available potato dextrose agar (PDA) medium. This type of medium contains rich carbon sources, nitrogen sources, vitamins, and inorganic salts, which can meet the nutritional requirements for the growth of Armillaria mellea. At the same time, its coagulability is good, which is convenient for observing the growth of mycelia. During the culture process, control the temperature of the incubator at 15°C to 25°C, keep the relative humidity at 60% to 70%, regularly observe the growth of Armillaria mellea, and ensure good mycelial growth. In addition, regularly detect the activity and quantity of Armillaria mellea to ensure the healthy growth of Armillaria mellea on the PDA medium and provide sufficient Armillaria mellea strains for the subsequent domestication of chitosan oligosaccharide; S23. Prepare the second medium with chitosan oligosaccharide solutions of different mass fractions, and domesticate the Armillaria mellea obtained from the primary culture in step S22 in ascending order of the mass fraction of chitosan oligosaccharide to obtain highly stress-resistant Armillaria mellea.

[0013] Specifically, the second medium is prepared by the following method: A. Weigh 50 parts of miscellaneous 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 by weight, stir and mix them evenly 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 medium; B. Put the prepared initial medium into culture bottles, and then place the culture bottles into an autoclave for sterilization. The sterilization conditions are: 121°C, 20 minutes; C. Prepare chitosan oligosaccharide solutions with mass fractions of 0.02%, 0.05%, and 0.15% using sterile water for standby. After the sterilized initial medium cools to room temperature, add the chitosan oligosaccharide solutions with corresponding mass fractions (0.02%, 0.05%, and 0.15%) to the sterilized initial medium respectively to prepare the second medium with different chitosan oligosaccharide contents. Among them, the addition amount of the chitosan oligosaccharide solution is preferably such that the water content of the finally obtained second medium reaches 60%. Preferably, when adding the chitosan oligosaccharide solution with the corresponding concentration to the sterilized initial medium, filter and sterilize the chitosan oligosaccharide solution using a 0.22 μm filter membrane.

[0014] Further, the three different second culture media corresponding to the chitosan oligosaccharide mass fractions of 0.02%, 0.05% and 0.15% are respectively denoted as the third culture medium (0.02%), the fourth culture medium (0.05%) and the fifth culture medium (0.15%). Then, the domestication of the Armillaria mellea obtained by the preliminary culture in step S22 is specifically as follows: First, inoculate the Armillaria mellea that has been preliminarily cultured and grows well onto the third culture medium, and maintain the same temperature (15°C to 25°C) and humidity (60% to 70%) conditions as in the preliminary culture, and culture for about 7 to 10 days to obtain the Armillaria mellea after the first domestication; then, inoculate the Armillaria mellea after the first domestication onto the fourth culture medium, and culture for about 7 to 10 days under the same conditions as the first domestication to obtain the Armillaria mellea after the second domestication; afterwards, inoculate the Armillaria mellea after the second domestication onto the fifth culture medium, and culture for about 10 to 15 days under the same conditions as the second domestication to obtain the Armillaria mellea after the third domestication, which is the highly stress-resistant Armillaria mellea, and then transfer it to an environment of 0°C to 5°C for storage.

[0015] It should be noted that the second culture medium of the present invention has a composition closer to the substrate conditions of the symbiosis between Gastrodia elata and Armillaria mellea in the natural environment. For example, its components such as sawdust and rice bran can provide richer natural components such as lignin and cellulose for Armillaria mellea, enabling it to grow in a more natural environment and helping to enhance the symbiotic adaptability between Armillaria mellea and Gastrodia elata; and by adding chitosan oligosaccharide solutions with different mass fractions and domesticating Armillaria mellea in the order of increasing mass fraction, physiological changes related to stress resistance can be gradually induced in Armillaria mellea, enabling it to better adapt to the harsh environment in high-altitude areas, such as low temperature.

[0016] Further, the selected germinating fungus in this embodiment is Osmunda japonica Thunb. germinating fungus, which also needs to be stressed and domesticated with chitosan oligosaccharide. The domestication method refers to that of Armillaria mellea, and it is also achieved by adding chitosan oligosaccharide solutions with mass fractions of 0.02%, 0.05% and 0.15%. The third culture medium, the fourth culture medium and the fifth culture medium are also used for domestication, and the obtained germinating fungus is the highly stress-resistant germinating fungus, which will not be elaborated here (unless otherwise specified, the germinating fungus and Armillaria mellea mentioned later are both domesticated germinating fungus and Armillaria mellea).

[0017] Step S3, Obtaining Hybrid Seeds S31, Flowering Period Adjustment Place the collected female Gastrodia elata samples from high-altitude areas in a greenhouse environment with adjustable temperature and humidity. Gradually adjust the temperature and humidity in the greenhouse to simulate the relatively warm climate characteristics of low-altitude areas, so as to accelerate the growth rate of the female Gastrodia elata and advance the flowering period to coincide with that of the male Gastrodia elata; and / or, conduct a low-temperature dormancy treatment on the male Gastrodia elata to slow down its growth rate to coincide with the flowering period of the female Gastrodia elata. The flowering period of the male Gastrodia elata can be slightly earlier than that of the female Gastrodia elata, for example, 1 - 2 days earlier; the adjustment of the flowering period belongs to the prior art and will not be elaborated here. S32. Pollination During the flowering period of the male Gastrodia elata, collect mature pollen and smear it on the stigma of the female. After pollination, record the pollination date, variety information of the male and female, etc. with a marker. S33. Fruit collection: After pollination, the capsule begins to swell, and the Gastrodia elata capsule matures in about 20 - 30 days (when the 6 longitudinal sutures around the capsule are very obvious, the longitudinal sutures of the fruit protrude but do not crack, gently squeeze the capsule with your hand and it becomes soft, peel the fruit shell and the seeds are easy to scatter, and the seeds are light yellow, which is the sign of capsule maturity). Pick it in time and put it into a sterile paper bag for standby. Step S4. Seed treatment and sowing S41. Place the unpollinated Gastrodia elata capsule picked and placed in a low-temperature sterile environment of 0℃ - 2℃ for 5 - 7 days to promote the Gastrodia elata seeds to enter a short-term dormancy. During the low-temperature treatment, the environmental humidity is maintained at 60 - 70%. Conducting the low-temperature treatment within this humidity range, the fruit shell will not crack by itself, which is beneficial for the subsequent soaking treatment with chitosan oligosaccharide. S42. After the low-temperature treatment is completed, transfer the capsule to an environment of 10℃ - 12℃ and place it for 2 hours. Then use a puncturing tool such as a sterile needle or a thin toothpick (diameter ≤ 0.5mm) to puncture the Gastrodia elata capsule after the low-temperature treatment in step S41. When puncturing, avoid the holes being too large to cause seed loss, and evenly puncture the middle and lower part of the capsule (avoiding the fruit stalk end), with 3 - 5 holes per fruit; disinfect the puncturing tool with 75% alcohol before puncturing to prevent pathogen contamination. S43. Place the punctured Gastrodia elata capsules into a pre-prepared chitosan oligosaccharide solution with a mass fraction of 0.1% (temperature controlled at 8 - 10 °C) for soaking. Then, place the chitosan oligosaccharide solution with the soaked Gastrodia elata capsules in a vacuum device, evacuate to about -0.06 MPa, and maintain 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 fruit shell under atmospheric pressure. During this period, the capsules can be taken out of the vacuum device and gently squeezed 1 - 2 times (preferably carried out in a sterile environment), and then put back into the vacuum device to ensure the solution penetrates. Repeat the above steps of evacuation and gentle squeezing 2 - 3 times. Then, take out the capsules from the chitosan oligosaccharide solution (the total soaking time from the start to the end is about 15 minutes), and place them on a sterile gauze to drain for 1 - 1.5 hours. It should be noted that in this step, by combining the method of vacuum negative pressure with gentle squeezing, even when the puncture holes are very small, the chitosan oligosaccharide solution can penetrate into the fruit shell within a short time without damaging the internal seeds, which can improve the processing efficiency while preventing soaking failure caused by uneven penetration. The smaller puncture holes can prevent the internal seeds of the fruit shell from flowing out; S44. Mixing seeds with fungal leaves: Shell the Gastrodia elata capsules drained in step S43, take out the seeds, and let them dry; Take out the leaf fungal strains of the germinated fungus of the genus Mycena that have been cultured from the culture bottle and place them in a container; Then, gently sprinkle the Gastrodia elata seeds on the fungal leaves and mix them evenly while sprinkling; S45. Indoor sowing: First, prepare the fungus sticks, select the fungus sticks with short Armillaria mellea culture time, tender mycelial cords, strong growth, and the mycelium has invaded the cortex of the wood segment without contamination by miscellaneous bacteria (prepared from domesticated Armillaria mellea); And prepare a sowing bed in advance (for example, by using a hemp-planting box with a depth of 30 cm, a width of 60 cm, and a length of 1 m); After step S44 is completed, spread a layer of wet leaves at the bottom of the bed. Divide the fungal leaves with the mixed Gastrodia elata seeds into two parts. Sprinkle one part on the bottom layer, place the fungus sticks with a spacing of 3 - 4 cm between the tree sticks, cover with sand until the sticks are level, then spread wet leaves, sprinkle the other half of the seed-mixed fungal leaves on the upper layer, place the Armillaria mellea fungus sticks, cover with soil to a thickness of 8 - 10 cm, and then cover with agricultural film to complete indoor sowing; S46. After sowing, adjust the room temperature to 15 °C - 25 °C, keep the soil humidity between 60% - 70%, cultivate for 4 - 6 months to harvest a large amount of rice-shaped and white-shaped Gastrodia elata, and use them as the hemp seeds for transplantation; Step S5. Transplantation and cultivation in high-altitude areas: In March to April in spring or September to October in autumn, in a pre-selected forest land with an altitude of over 2,000 meters (specifically, the altitude of the forest land we selected is 2,500 meters), transplant and cultivate the said cannabis seeds. Specifically, first, prepare the land, spread leaves and branches, use phoxim to prevent pests, then place tree rods (for inoculating Armillaria mellea) at intervals of 5 - 7 cm, cut and place Armillaria mellea strains between the rods, place Armillaria mellea strains at both ends of the rods, and place Armillaria mellea strains between tree rods 25 cm apart; then, put the obtained rice cannabis and white cannabis (which need to be selected) into the rows between the trees, cover the tree rods with about 3 cm of soil, and then cultivate the second layer on the covered soil in a similar way. After the second layer is planted, cover the soil with 10 cm, and cover it with dry leaves on top; complete the transplanting of the cannabis seeds. After transplantation, manage according to the existing field management methods, and harvest the gastrodia elata during its growth dormancy period (late autumn). The arrow gastrodia is large in size and has a flower stalk bud growing at the top; select arrow gastrodia with a rosy, plump top bud, no pests or diseases, no damage, and of appropriate size as the seed tubers for cultivating seeds; then, let the cultivated seed tubers dry for 1 - 2 days to remove some moisture, and store them in moist sand, with the storage temperature maintained at 2 - 5°C; then cultivate the seed tubers in wooden boxes or plastic boxes, add sand or sandy loam, keep the humidity, adjust the temperature, and promote the gastrodia elata to bolt and flower; Step S6, self-crossing: When the gastrodia elata plant grows to the budding stage, conduct self-crossing pollination to obtain capsules and seeds after self-crossing and homozygosity, and conduct screening and preservation; Step S7, to improve the homozygosity of the seeds, repeat the operations of S4 to S6 on the preserved seeds. After multiple generations of self-crossing and screening, until obtaining gastrodia elata seeds with stable genetic traits and high homozygosity for large-scale planting or further new variety breeding. The above are the main steps for the gastrodia elata breeding of the present invention. It should be understood that this method may also include other steps, and without departing from the inventive concept of the present invention, they should all fall within the protection scope of the present invention.

[0018] In addition, to verify the effects of low-temperature treatment and soaking in chitosan oligosaccharide solution on the germination rate of Gastrodia elata seeds in the present invention, we conducted different comparative experiments on the seeds obtained after self-crossing and homozygosity in step S6. We took 60 mature Gastrodia elata capsules picked in the same batch and divided them evenly into twelve groups (numbered in the order of 1, 2, 3... 11, 12). Among them, different low-temperature treatments and / or soaking treatments in chitosan oligosaccharide solution were carried out on the Gastrodia elata capsules between groups, and the same low-temperature treatment and / or soaking treatment in chitosan oligosaccharide solution were used for 5 Gastrodia elata capsules within each group (during the subsequent seed dressing and sowing processes, the treatment methods for the 5 Gastrodia elata capsules within the same group were the same and were all treated separately, aiming to conduct individual comparisons within the group to ensure the accuracy of the data). Then, seed dressing and sowing were carried out in the same way as in S44 and S45 (sowing indoors 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 samples, 5 fields of view were randomly selected under the microscope, and the total number of seeds, the total number of dead seeds, and the total number of germinated seeds in each field of view were calculated respectively, and the germination rate was calculated; among them, the germination rate = (total number of germinated seeds / total number of counted seeds) × 100%; after statistics, the germination rate results of each group are as follows in the table: It should be noted that for each group of Gastrodia elata capsules in the above table, the treatment and sowing of their seeds were carried out with reference to (or similar to) the method in step S4. In the above table, the Gastrodia elata seeds in groups 1 to 4 were not soaked in chitosan oligosaccharide solution. Among them, the Gastrodia elata seeds in groups 1 to 3 were all subjected to low-temperature treatment (from 4°C to 0°C), and the Gastrodia elata seeds in group 4 were sown immediately in the form of fresh seeds after picking; the Gastrodia elata seeds in groups 5 to 12 were all soaked in chitosan oligosaccharide solution, and low-temperature treatment was carried out for all except group 9. It can be seen from the microscopic examination results in the above table that compared with the Gastrodia elata seeds in group 4 without low-temperature treatment, the average microscopic germination rate of the Gastrodia elata seeds in groups 1 to 3 with low-temperature treatment was significantly higher, indicating that the selection of low-temperature treatment helps to improve the seed germination rate. This may be because low temperature helps the seeds enter a short dormant period, which may help to improve their seed germination rate; by comparing the results of groups 1 to 3, it can be seen that the Gastrodia elata seeds stored at 4°C for 5 days in group 1 had the highest average microscopic germination rate, and as the temperature decreased, the average microscopic germination rates of groups 2 and 3 gradually decreased. This may be because within this temperature range, the decrease in temperature had an inhibitory effect on the germination of Gastrodia elata seeds, such as a decrease in the enzyme activity inside the seeds.

[0019] Referring further to the above table, in Groups 5 to 7, the seeds were soaked in a chitosan oligosaccharide solution with a mass fraction of 0.1% and were respectively treated with low temperatures of 4°C, 2°C, and 0°C. The results showed that the average germination rates detected by microscopy in Groups 5 to 7 were significantly higher than those in Groups 1 to 3 that were not soaked in the chitosan oligosaccharide solution. This indicates that the treatment method of soaking in the chitosan oligosaccharide solution combined with low-temperature treatment can further increase the germination rate of Gastrodia elata seeds. In addition, it can be seen from Groups 5 to 7 that when the treatment method of soaking in the chitosan oligosaccharide solution combined with low-temperature treatment is adopted, the average germination rates detected by microscopy in Groups 6 and 7 with stress temperatures of 2°C and 0°C are actually higher than that in Group 5 with a stress temperature of 4°C. This shows that although low-temperature conditions have an adverse effect on seed germination, the soaking treatment with the chitosan oligosaccharide solution can alleviate this adverse effect to a certain extent. Especially under low-temperature conditions of 2°C and 0°C, the germination rate of the seeds still remains at a relatively high level. In addition, according to the results of Groups 8 and 9 in the above table, when the temperature of the low-temperature treatment is -2°C, even if the seeds are soaked in the chitosan oligosaccharide solution, the average germination rate detected by microscopy drops significantly to 10.2%, indicating that the temperature has become the main factor affecting Gastrodia elata seeds at this time. In Group 9, the seeds were not treated with low temperature but only soaked in a chitosan oligosaccharide solution with a mass fraction of 0.1%. At this time, the germination rate of the seeds was only 15.4% (significantly higher than that in Group 4), indicating that soaking in the chitosan oligosaccharide solution can help improve the germination rate of the seeds, but the improvement effect is limited when using only the soaking method alone.

[0020] Referring further to the above table, compared with Group 7, in Groups 10 to 12, the mass fraction of chitosan oligosaccharide in the chitosan oligosaccharide solution was changed. When soaked in a chitosan oligosaccharide solution with a mass fraction of 0.15%, the average germination rate detected by microscopy was still relatively high, above 30%; while when soaked in a chitosan oligosaccharide solution with a mass fraction of 0.05%, the average germination rate detected by microscopy was only 21.9%, indicating that as the concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution decreases, its promoting effect on the germination of Gastrodia elata seeds decreases; when soaked in a chitosan oligosaccharide solution with a mass fraction of 0.25%, the average germination rate detected by microscopy was only 4.9%, indicating that the concentration of chitosan oligosaccharide in the chitosan oligosaccharide solution is too high at this time and has produced an adverse effect. It can be seen from this that the combined treatment of soaking in the chitosan oligosaccharide solution and low-temperature treatment is the key factor to improve the germination rate of highly cold-tolerant 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.

[0021] Furthermore, the length of the breeding cycle is mainly determined by the number of generations of cultivation and the length of the growth cycle of each generation (a complete growth cycle is from seed → protocorm → millet corm → white corm → arrow corm → seed). By combining the soaking of the seeds in chitosan oligosaccharide solution with low-temperature treatment, the present invention can significantly improve the germination rate of high-cold-resistant Gastrodia elata seeds. In this way, more breeding samples can be obtained in a short period, thereby reducing the number of generations of cultivation. Further, the growth cycle of Gastrodia elata is not only affected by its own genetic factors but also by the symbiotic fungus Armillaria mellea. Since domesticated high-stress-resistant germination fungi and Armillaria mellea are used for breeding and cultivation in the above method, in order to verify their influence on the growth cycle of Gastrodia elata, the following comparative experiments were also carried out, that is, a comparison was made between non-domesticated germination fungi, the Armillaria mellea (non-domesticated) initially cultivated in step S22, and domesticated germination fungi and Armillaria mellea (the cultivation methods are the same except for the different strains of fungi), and the seeds used were also the same batch of seeds obtained after self-crossing and homozygosity in step S6. The statistical results are as follows: As can be seen from the above table, although the seeds obtained after self-crossing and homozygosity in step S6 have the high-cold-resistant characteristics from the female parent Gastrodia elata, when planted in high-altitude areas, if their Armillaria mellea and germination fungi are not domesticated, their growth cycle is still very long, reaching 1130 days. However, when using the domesticated germination fungi and Armillaria mellea of the present invention, it can significantly shorten the growth cycle of high-cold-resistant Gastrodia elata. This may be because through domestication, not only the stress resistance of the germination fungi and Armillaria mellea is improved, reducing the impact of low temperature in high-altitude areas on them, but also during the subsequent introduction of fragmented high-stress-resistant Armillaria mellea materials, some chitosan oligosaccharide components are introduced, and this component also has a growth-promoting effect on Gastrodia elata itself, thereby shortening the growth cycle.

[0022] Furthermore, through the above breeding method, we crossed and selected a wild Gastrodia elata female parent found at an altitude of 2500 meters in the Wumeng Mountains with Gastrodia elata cv. Jinhong No. 1 from Sichuan as the male parent, and quickly cultivated a new variety - Gastrodia elata cv. Gaohong No. 1 (TMGH-1) that can be planted on a large scale in the area with an altitude of 1800 - 3200 meters. From Figure 1 it can be seen that TMGH-1 exhibits excellent characteristics in terms of size, shape, etc. Its individual is even larger than that of Gastrodia elata cv. JHCK planted at an altitude of 1500 meters, and moreover, its appearance is even very similar to that of the expensive wild Gastrodia elata on the market. From Figure 2 and Figure 3It can be seen that TMGH-1 has higher yields and better agronomic properties, and performs excellently in terms of indicators such as the total amount of dilute ethanol extract, gastrodin, and p-hydroxybenzyl alcohol, ash content, moisture content, and sulfur dioxide residue. Not only does the total amount of its dilute ethanol extract, gastrodin, and p-hydroxybenzyl alcohol meet the requirements of the Chinese Pharmacopoeia and is significantly better than JHCK and HCK, but its ash content is lower. This not only demonstrates its excellent quality but also indicates that it has been better controlled and managed during the growth environment and processing. This makes TMGH-1 superior to other varieties in terms of safety, effectiveness, and quality stability, and it is a high-quality and high-yield gastrodia elata variety.

[0023] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above description is only for the specific embodiments of the present invention and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for breeding highly cold-tolerant Gastrodia elata suitable for planting in high-altitude areas, characterized in that, The method includes, during the breeding process, treating the Gastrodia elata seeds of each generation in the breeding process by combining the soaking with a chitosan oligosaccharide solution and low-temperature treatment.

2. The breeding method of highly cold-resistant Gastrodia elata suitable for high-altitude areas according to claim 1, characterized in that, It includes the following steps: Step S1, obtaining parents; Step S2, culturing and domesticating the strains; Step S3, obtaining hybrid seeds; Step S4, seed treatment and sowing; Step S5, transplanting in high-altitude areas; Step S6, obtaining capsules and seeds after self-crossing and homozygosity, screening and preserving them; Step S7, repeating the operations of S4 to S6 on the preserved seeds, and through multiple generations of self-crossing and screening until Gastrodia elata seeds with stable genetic traits and high homozygosity are obtained.

3. The breeding method of highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas according to claim 2, characterized in that, In step S1, obtaining parents includes obtaining the male parent and the female parent. The female parent is selected from wild Gastrodia elata above 2000 meters above sea level; the male parent is selected from cultivated Gastrodia elata at a scale below 1500 meters above sea level.

4. A method for breeding highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas according to claim 1 or 2 or 3, characterized in that, Step S2 includes culturing and domesticating Armillaria mellea, which includes the following steps: S21, in the original habitat of the female parent Gastrodia elata, searching for Armillaria mellea symbiotic with Gastrodia elata and conducting hyphal isolation; S22, inoculating the isolated Armillaria mellea into the sterilized first culture medium for preliminary culture to obtain Armillaria mellea with high vitality and good growth; S23, preparing the second culture medium with chitosan oligosaccharide solutions of different mass fractions, and domestication of the Armillaria mellea obtained by preliminary culture in step S22 is carried out in sequence according to the increasing order of the chitosan oligosaccharide mass fraction to obtain Armillaria mellea with high stress resistance.

5. A method for breeding high-cold-resistant Gastrodia elata suitable for planting in high-altitude areas as described in claim 4, characterized in that, The second culture medium is prepared by the following method: A. By weight, weigh 50 parts of miscellaneous 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, stir and mix evenly 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; B. Fill the prepared initial culture medium into culture bottles, and then put the culture bottles into an autoclave for sterilization treatment; C. Prepare chitosan oligosaccharide solutions with mass fractions of 0.02%, 0.05% and 0.1% with sterile water for standby. After the sterilized initial culture medium cools to room temperature, add the corresponding mass fraction of chitosan oligosaccharide solution to the sterilized initial culture medium respectively to prepare the second culture medium with different chitosan oligosaccharide contents.

6. The breeding method of highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas according to claim 5, characterized in that Using the second culture medium with different chitosan oligosaccharide contents, the Armillaria mellea obtained by preliminary culture in step S22 is domesticated in the above order according to the increasing mass fraction.

7. A method for breeding highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas as claimed in claim 1 or 2 or 3, characterized in that, Step S4 includes: S41, placing the picked and unopened Gastrodia elata capsules in a low-temperature sterile environment of 0°C to 2°C for 5 to 7 days to prompt the Gastrodia elata seeds to enter a short-term dormancy.

8. A method for breeding highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas according to claim 7, characterized in that, Step S4 also includes: S42, using a puncturing tool to puncture the Gastrodia elata capsules after low-temperature treatment in step S41; S43, putting the punctured Gastrodia elata capsules into a pre-prepared chitosan oligosaccharide solution with a mass fraction of 0.1 to 0.15% for soaking, and during the soaking process, ensure that the chitosan oligosaccharide solution can penetrate into the Gastrodia elata capsules; S44, breaking open the Gastrodia elata capsules treated in S43, taking out the seeds, drying them; then mixing them with the inoculated germination fungus material; S45, carrying out indoor sowing; S46. Manage and obtain the hemp seeds for transplantation.

9. A method for breeding highly cold-resistant Gastrodia elata suitable for planting in high-altitude areas as claimed in claim 8, characterized in that, In step S43, by means of vacuum pumping and gentle extrusion, ensure that the chitosan oligosaccharide solution can penetrate into the inside of the gastrodia elata capsule.

10. A method for breeding high-cold-resistant Gastrodia elata suitable for planting in high-altitude areas according to claim 9, characterized in that, Step S43 is specifically to place the chitosan oligosaccharide solution soaked with gastrodia elata capsules in a vacuum device, pump the vacuum to -0.06 MPa, maintain for 1 minute to allow the air inside the capsules to escape, and then slowly release the pressure to enable the chitosan oligosaccharide solution to penetrate into the inside of the fruit shells under atmospheric pressure. During this period, gently squeeze the capsules 1 - 2 times to ensure that the solution penetrates.

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