In-vitro preservation method of water chestnut germplasm resource test-tube plantlets

Abstract

The invention discloses an in-vitro preservation method of a test-tube plantlet of a water chestnut germplasm resource, which comprises the following steps of: sterilizing a water chestnut explant, inoculating the water chestnut explant on a stem tip starting culture medium, culturing the water chestnut explant into a single bud or a clumpy bud, transferring the single bud or the clumpy bud to a subculture medium, and carrying out subculture multiplication culture to obtain a certain number of groups; transferring to a test-tube plantlet in-vitro preservation culture medium, storing for a period of time at low temperature, inducing the explant to germinate under the illumination condition to form a test-tube plantlet, and continuously culturing and preserving. According to the method disclosed by the invention, on the basis of keeping the characteristics of germplasm resources, the growth of the water chestnut tissue culture seedlings is delayed by adopting a low-temperature storage mode, one subculture time of 8-12 months or even longer can be realized, and the resource preservation time of each generation is 8-12 times longer than that of a tissue culture and rapid propagation technology; due to the fact that the number of subculture times is small, labor is correspondingly reduced, the possibility of variety mixing and genetic variation caused by manual operation is reduced, land is saved, and the influence of atrocious weather conditions and plant diseases and insect pests on safe preservation of germplasm resources is reduced.

Description

Technical Field

[0001] This invention relates to a method for preserving vegetable germplasm resources, and more particularly to an in vitro preservation method for water chestnut germplasm resources in the form of test-tube seedlings, belonging to the field of in vitro preservation of water chestnut germplasm resources. Background Technology

[0002] Crop germplasm resources are the material basis for crop breeding, biological science research and agricultural production. The richer the germplasm resources, the greater the potential for gene development, and the more conducive it is to original innovation in agricultural science and technology and the development of modern seed industry.

[0003] Water chestnut is a perennial shallow-water herbaceous plant belonging to the genus *Echinochloa* in the family Cyperaceae. It is also known as water caltrop, ground chestnut, and black taro. China is one of the origins of water chestnut, with a long history of cultivation and abundant germplasm resources. It is a major aquatic vegetable, with its underground corm as the edible organ, and is widely cultivated in the Yangtze River basin and areas south of it. Water chestnut is an asexually propagated crop, but it still possesses sexual reproductive capabilities. Water chestnut seeds are difficult to germinate, and the offspring of seedlings can exhibit variations in genetic characteristics. Therefore, water chestnut germplasm resources are generally preserved alive in resource nurseries. The asexual reproduction of water chestnut cultivation leads to various pathogen infections, genetic degeneration of seed tubers, and serious disease-carrying phenomena in the bulbs. This not only results in a huge workload for annual preservation and renewal but also makes it susceptible to adverse weather conditions, pests, and diseases, threatening the safety of water chestnut germplasm resource preservation.

[0004] Currently, water chestnut germplasm resources are mostly preserved in the field in a living state. For example, the National Aquatic Vegetable Germplasm Resource Nursery (Wuhan) preserves water chestnut germplasm resources by planting them in resource ponds, each with an area of ​​3 square meters. 2 Each pond contains one resource, and the ponds are separated from each other and at the bottom by cement walls to prevent different resources from mixing. To prevent resource loss, a backup set of resources is usually grown in a large ceramic container. All resources planted in the field need to be harvested and replanted every year, including a series of agricultural operations such as seedling cultivation, transplanting, fertilization, irrigation, drainage, weeding, pest and disease control, and harvesting. The construction and preservation of resource ponds require a large amount of land, manpower, materials, and funds for maintenance.

[0005] Conventional methods for preserving water chestnut germplasm resources often have the following drawbacks:

[0006] (1) Germplasm resources are prone to disease: The conventional method of preserving water chestnut germplasm resources is asexual reproduction and field planting. The seed water chestnuts, soil and resource cultivation management are prone to carrying pathogens and becoming susceptible to disease.

[0007] (2) Preservation of germplasm resources is easily affected by severe weather: conventional methods of preserving water chestnut germplasm resources involve planting and growing in the field, which is often affected by severe weather such as strong winds, hail, high temperatures, and frost damage.

[0008] (3) Requires substantial land resources: The conventional method for preserving water chestnut germplasm resources involves field planting. Each resource requires a separate resource pool of a certain area, and isolation is necessary between resource pools to prevent mixing. To prevent resource loss, a backup set of resources is usually grown in a large ceramic jar. Prolonged continuous cropping of water chestnuts in the same resource pool can exacerbate pests and diseases, so after several years of cultivation, crop rotation with other crops is necessary. Therefore, the conventional preservation method requires considerable land resources.

[0009] (4) Requires a lot of labor costs: Conventional methods require multiple agricultural operations such as seedling raising, transplanting, irrigation, drainage, weeding, spraying pesticides to prevent and control pests and diseases, and harvesting, which require a lot of manpower to continuously carry out field management.

[0010] By using tissue culture for in vitro preservation, water chestnut germplasm resources can be preserved indoors, reducing the threat of harsh weather and pests to these resources and providing an additional method for their preservation. However, the use of tissue culture for rapid propagation has drawbacks, such as rapid plant growth, multiple subcultures, high labor requirements, and short preservation time, which need further improvement.

[0011] Plant growth requires specific environmental conditions such as temperature, light, and water. When these conditions fail to meet these needs, plant growth and development are negatively impacted. Temperature is a crucial environmental factor. When the ambient temperature remains consistently below the temperature required for normal plant growth, it creates low-temperature stress, inhibiting growth and even causing death. Low-temperature stress is an abiotic stress, encompassing both cold stress (0–15°C) and freezing stress (<0°C). The phenomenon of growth retardation or reproductive impairment in crops occurring within the temperature range below their optimal growth temperature and above 0°C is called chilling injury. The degree of damage caused by chilling injury depends on the intensity and duration of the low temperature. Different crops have different sensitivities to low temperatures. Studies have shown that different rice varieties have different sensitivities to low temperatures. Low temperature stress has a significant inhibitory effect on the occurrence of rice tillers. The maximum number of tillers decreases significantly with the extension of low temperature treatment time. When the low temperature is 12℃, less than 3 days has little effect on the maximum number of rice tillers, but 3 to 6 days of 12℃ low temperature will seriously affect the occurrence of rice tillers, thereby reducing yield (Wang Lizhi, Wang Chunyan, Li Zhongjie, Li Rui, Li Yuyao, Meng Ying, Wang Lianmin. Effects of low temperature during tillering stage of cold injury on rice tillers in Heilongjiang. Heilongjiang Agricultural Sciences, 2009, (4): 18-20). Cotton growth and development will be seriously affected if the temperature is below 15℃ (Junjuan, Yin Zujun, Wang Delong, Wang Shuai, Fan Weili, Guo Lixue, Ye Wuwei. Research progress on cotton's resistance to low temperature. China Cotton, 2016, 43(12): 1-6). The minimum temperature for potato plant growth is 5-7℃. Seedlings suffer cold damage at -0.5-0.8℃ and frost damage at -2℃; mature plants die completely at -4℃ (Yang Jinhui, Lin Xuan, Song Yong. Research progress on potato resistance to low temperature stress. Chinese Horticulture Abstracts, 2014, (10):67-68,188). The growth limit temperature for cucumber is 10-30℃, with daytime temperatures of 25-30℃ and nighttime temperatures of 13-15℃. Below 10℃, physiological activities become disordered. Without training or with a sudden drop in temperature, plants will suffer cold damage at 5-10℃ and die at 2-3℃ (Wang Heli. Research progress on low temperature damage and cold tolerance of cucumber. Journal of Tarim Agricultural University, 1999, 11(4):58-60). Water chestnut seedlings tiller and divide slowly at temperatures of 15–20℃, and divide most rapidly at 25–30℃. Below 5℃, the above-ground stems of water chestnuts wither (Ke Weidong, Liu Yiman, Huang Xinfang. Technical Guidelines for Safe Production of Aquatic Vegetables (2nd Edition), Beijing: China Agriculture Press, 2014, 69). No literature reports on whether water chestnut tissue culture seedlings can tolerate 4℃ low temperatures and for how long. Summary of the Invention

[0012] The main objective of this invention is to provide a method for in vitro preservation of water chestnut germplasm seedlings;

[0013] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0014] A method for in vitro preservation of water chestnut germplasm seedlings, comprising:

[0015] (1) After disinfecting the explants of water chestnuts, they were inoculated onto water chestnut shoot tip initiation culture medium for shoot tip initiation culture until they grew into single shoots or clusters of shoots;

[0016] (2) Transfer the single bud or cluster buds obtained in step (1) to the subculture medium for subculture proliferation culture;

[0017] (3) The shoot clusters obtained from subculture were divided into single shoots and transferred to the in vitro preservation medium of test-tube seedlings and stored at a low temperature of 4-6℃; wherein the in vitro preservation medium of test-tube seedlings is MS + 6-BA 0.1-0.5 mg / L + AC 0.0-1.0 g / L + sucrose 30-80 g / L + agar 5-6 g / L, and the pH value of the medium is 5.8-6.0;

[0018] (4) After the low-temperature storage is completed, the explants are cultured under normal temperature and light conditions to induce regrowth.

[0019] (5) The germinated explants were cultured under light at 13-18℃ to induce the formation of test-tube seedlings, and the seedlings were cultured and preserved under the same conditions.

[0020] In a preferred embodiment of the present invention, the shoot tip initiation culture medium in step (1) is: LS + 6-BA 0.5-1.0 mg / L + NAA 0.0-0.5 mg / L + sucrose 30 g / L + agar 5-6 g / L; the pH value of the culture medium is 5.8-6.0; the conditions for shoot tip initiation culture include: culture temperature of 24-26℃, light intensity of 2000-2500 lx, and light exposure of 10-12 h per day; preferably, the shoot tip initiation culture medium is: LS + 6-BA 0.8 mg / L + sucrose 30 g / L + agar 6 g / L.

[0021] In a preferred embodiment of the present invention, the subculture medium in step (2) is: LS + 6-BA 0.5-1.0 mg / L + sucrose 30 g / L + agar 5-6 g / L; the pH value of the medium is 5.8-6.0; the conditions for the subculture proliferation culture include: culture temperature of 24-26℃, light intensity of 2000-2500 lx, and light exposure of 10-12 h per day.

[0022] In a preferred embodiment of the present invention, the explants obtained from subculture proliferation culture are first transferred to a culture medium for in vitro preservation of test-tube seedlings and then stored at low temperature. After the low-temperature storage, the culture container is removed and placed under normal temperature culture conditions for culture until the explants germinate. The germinated explants are then cultured under light conditions to form test-tube seedlings, and the test-tube seedlings are then cultured and preserved in vitro under light conditions. The low-temperature storage includes: transferring the explants obtained from subculture proliferation culture in step (2) to the culture medium for in vitro preservation of test-tube seedlings. On the culture medium, it is then placed at a temperature of 4-6℃ for 1-5 months. During low-temperature storage, no light is required. The explants obtained from the subculture in step (2) are transferred to the in vitro preservation medium of the test-tube seedlings. The transferred explants stop growing at 4-6℃. If they are placed in a suitable cultivation environment, they can continue to grow. Therefore, the in vitro preservation time of water chestnut germplasm resources can be further extended. As the low-temperature storage time is extended, the germination survival rate of the transferred explants after being placed in suitable growth conditions will decrease.

[0023] In a preferred embodiment of the present invention, the low-temperature storage time in step (3) is 4 to 24 weeks, more preferably 12 to 19 weeks, and most preferably 12 weeks.

[0024] In a preferred embodiment of the present invention, the low-temperature storage in step (3) does not require light exposure.

[0025] In a preferred embodiment of the present invention, the in vitro preservation culture medium in step (3) is: MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L.

[0026] In a preferred embodiment of the present invention, the ambient temperature in step (4) is 24-26°C.

[0027] In a preferred embodiment of the present invention, the light cultivation conditions in step (4) include: light intensity of 2000-2500 lx, and light exposure of 10-12 hours per day.

[0028] In a preferred embodiment of the present invention, in step (5), the germinating explants are cultured under light at 15°C to form test-tube seedlings.

[0029] In a preferred embodiment of the present invention, the light cultivation conditions in step (5) include: light intensity of 2000-2500 lx and light exposure of 10-12 h per day.

[0030] Compared with conventional methods for preserving water chestnut germplasm resources, the in vitro preservation method of the water chestnut germplasm resources of this invention has the following main advantages: (1) The germplasm resources are disease-free: This invention preserves water chestnut germplasm resources by obtaining tissue culture seedlings through water chestnut stem tip culture. The entire process is aseptic, and the germplasm resources are disease-free. In contrast, conventional methods for preserving water chestnut germplasm resources involve asexual reproduction and field planting, and the seedlings, soil, and resource cultivation management are all susceptible to pathogens. This invention solves the problem of water chestnut seedlings being susceptible to disease in conventional methods. (2) The preservation of germplasm resources is not affected by adverse weather conditions: This invention belongs to an agricultural biotechnology and is carried out indoors, with artificial control of growth conditions such as temperature and light. The preservation of germplasm resources is not affected by adverse weather conditions. In contrast, conventional methods for preserving water chestnut germplasm resources involve planting and growing in the field, which is often affected by adverse weather conditions such as strong winds, hail, high temperatures, and frost damage. This invention solves the problem that conventional methods of preserving germplasm resources are easily threatened by severe weather. (3) Land saving: The water chestnut germplasm resources of this invention are grown in culture bottles and can be stored and cultivated indoors in a three-dimensional manner, saving space; while conventional methods of preserving water chestnut germplasm resources involve field planting, each resource requires a certain area of ​​resource pool for separate planting, and to ensure that the resources do not mix, the resource pools need to be isolated. To prevent the loss of resources, a set of resources is usually planted in a large ceramic jar as a backup. If water chestnuts are continuously planted in the same resource pool for too long, it will aggravate the occurrence of pests and diseases, so after several years of planting, they need to be rotated with other crops. Therefore, conventional preservation methods require more land resources. This invention saves more land compared with conventional methods. (4) Reduced labor costs: This invention, while preserving the inherent characteristics of the germplasm resources, slows down plant growth as much as possible, allowing for a single subculture that can be stored for six months to a year or more, requiring less manual labor. In contrast, conventional methods require multiple agricultural operations such as seedling raising, transplanting, irrigation, drainage, weeding, pest and disease control, and harvesting, necessitating a large amount of manual labor for continuous field management. This invention saves more labor costs compared to conventional methods.

[0031] The in vitro preservation method for water chestnut germplasm seedlings of the present invention differs from conventional water chestnut tissue culture rapid propagation technology in the following main ways:

[0032] ① Different purposes: The purpose of rapid propagation technology of water chestnut tissue culture is to make the tissue culture seedlings grow quickly and to carry out as many subcultures as possible within a unit of time to obtain a larger number of water chestnut tissue culture seedlings; while the purpose of the in vitro preservation method of water chestnuts is to make the tissue culture seedlings grow slowly, to ensure that the germplasm resources remain unchanged and can survive indefinitely, without pursuing an increase in the number of propagations, and to carry out as few subcultures as possible within a unit of time to reduce labor and the probability of mutation.

[0033] ②Different culture medium formulations: The culture medium formulation for rapid propagation of water chestnuts through tissue culture is conducive to the rapid propagation and growth of tissue culture seedlings; the culture medium formulation for in vitro preservation of water chestnuts causes tissue culture seedlings to grow slowly.

[0034] ③ Different cultivation conditions: The cultivation conditions for rapid propagation of water chestnuts through tissue culture are conducive to the rapid growth of tissue culture seedlings; the cultivation conditions for in vitro preservation of water chestnuts are conducive to the slow growth of tissue culture seedlings.

[0035] ④ Different process flow: The process flow for rapid propagation of water chestnut tissue culture is: shoot tip initiation → subculture proliferation → formation of test tube seedlings (test tube bulbs). The process flow for in vitro preservation of water chestnut is: shoot tip initiation → subculture proliferation → low temperature preservation → formation of test tube seedlings. The process flow for in vitro preservation of water chestnut has the additional step of "low temperature preservation".

[0036] This invention utilizes the principle of low-temperature stress for in vitro preservation of water chestnut germplasm resources. During low-temperature storage, the plants cease growth. After a period of low-temperature storage, growth resumes under suitable environmental conditions such as temperature and light, thereby extending the in vitro preservation time of germplasm resources. However, as the low-temperature storage time increases, the germination rate gradually decreases, and the losses gradually increase, which also gradually reduces the safety of in vitro preservation of water chestnut germplasm resources. Therefore, it is more suitable to store water chestnut tissue culture seedlings at a low temperature of 4℃ for 12 to 19 weeks.

[0037] ⑤ Different germplasm resource preservation effects: When using water chestnut tissue culture for rapid propagation, subculture is generally required about once a month; when using in vitro water chestnut preservation methods, subculture can be performed every 8-12 months or even longer. In comparison, the in vitro preservation method of water chestnut germplasm resources in test-tube seedling form provided by this invention has advantages such as slow plant growth, fewer subcultures, less manual labor required, and longer preservation time.

[0038] Compared with conventional water chestnut tissue culture propagation technology, the in vitro preservation method of water chestnut germplasm resources of the present invention has the following main advantages: (1) Longer in vitro preservation time and fewer subcultures: The in vitro preservation method of the present invention, while maintaining the characteristics of the germplasm resources themselves, delays plant growth as much as possible, and can achieve subculture once every 8 to 12 months or even longer. In contrast, the water chestnut tissue culture propagation technology generally requires subculture once every month. The in vitro preservation method of the present invention has a preservation time of 8 to 12 times longer per generation than the tissue culture propagation technology. (2) Reduced labor costs: Due to fewer subcultures, the amount of labor required is also reduced. Taking a preservation time of 1 year as an example, the in vitro preservation method of the present invention requires about 1 / 6 of the labor required by the tissue culture propagation technology. (3) Safer: Due to fewer subcultures, the possibility of variety mixing caused by human operation is reduced, and the possibility of genetic variation is also reduced. Therefore, the in vitro preservation method of the present invention is safer than the existing water chestnut tissue culture propagation technology for preserving germplasm resources.

[0039] This invention overcomes the difficulties of conventional methods for preserving water chestnut germplasm resources, such as the susceptibility of seed tubers to disease and the vulnerability of preserved resources to harsh weather conditions. Utilizing plant tissue culture technology, it proposes a safe, convenient, and efficient method for in vitro preservation of water chestnut germplasm resources, while minimizing plant growth, extending the preservation time for each subculture, and reducing labor intensity. This invention solves the problems of conventional asexual reproduction preservation of water chestnut germplasm resources in the field, which easily leads to the degradation of seed tubers, severe disease in the bulbs, and an enormous workload for annual preservation and renewal. Furthermore, it addresses the susceptibility to harsh weather, pests, and diseases. By using in vitro tissue culture preservation, water chestnut germplasm resources are preserved indoors, reducing the threats posed by harsh weather and pests. The in vitro preservation method of this invention for preserving water chestnut germplasm resources allows for one subculture every 8-12 months or even longer, ensuring that the tissue culture seedlings maintain the germplasm characteristics and grow normally. This in vitro preservation method is also more efficient in saving labor costs, requiring approximately 1 / 6 of the labor required for rapid tissue culture propagation. Furthermore, this in vitro preservation method for water chestnut germplasm resources in the form of test-tube seedlings is safer than existing rapid tissue culture propagation techniques for preserving germplasm resources. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0041] Example 1: In vitro preservation of water chestnut germplasm seedlings

[0042] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants were in vitro preserved through shoot tip initiation, subculture, and induction to form test-tube plantlets. The steps included:

[0043] 1. Stem tip initiation: Using Tuanfeng water chestnut as the experimental material, clean, disease-free apical or lateral buds of Tuanfeng water chestnuts were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto water chestnut stem tip initiation medium (LS + 6-BA 1.0 mg / L + sucrose 30 g / L + agar 6 g / L) with a pH of 5.8. The culture temperature was 26℃, the light intensity was 2500 lx, and the light intensity was 10 hours per day. Buds or clustered buds appeared after 30 days.

[0044] 2. Subculture: Explants were transferred to a subculture medium containing LS + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L for subculture. The pH of the subculture medium was 5.8. Under a culture temperature of 26℃ and a light intensity of 2500 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0045] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the explants are transferred to MS medium for in vitro preservation of test-tube seedlings, which is composed of MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L, with a pH of 5.8.

[0046] 4. Low-temperature storage: Place the culture container containing the explants in a refrigerator at 4°C and store for 5 weeks (35 days).

[0047] 5. Room temperature culture: The culture container that had been stored at low temperature for 12 weeks was taken out of the refrigerator and placed at a temperature of 25℃ and a light intensity of 2500 lx, with 10 hours of light per day. After 4 weeks at 25℃, about 97.78% of the explants germinated.

[0048] 6. In vitro preservation: After the explants germinate again, place the culture container in a light incubator at a temperature of 15℃ and a light intensity of 2500 lx for 10 hours a day. The explants will grow into test-tube seedlings and can be preserved under these culture conditions for 10 months without transfer.

[0049] Example 2: In vitro preservation of water chestnut germplasm seedlings

[0050] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants were in vitro preserved through shoot tip initiation, subculture, and induction to form test-tube plantlets. The steps included:

[0051] 1. Stem tip initiation: Using Tuanfeng water chestnut as the experimental material, clean, disease-free apical or lateral buds of Tuanfeng water chestnuts were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto water chestnut stem tip initiation medium (LS + 6-BA 1.0 mg / L + sucrose 30 g / L + agar 6 g / L) with a pH of 5.8. The culture temperature was 26℃, the light intensity was 2500 lx, and the light intensity was 10 hours per day. Buds or clustered buds appeared after 30 days.

[0052] 2. Subculture: Explants were transferred to a subculture medium containing LS + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L for subculture. The pH of the subculture medium was 5.8. Under a culture temperature of 26℃ and a light intensity of 2500 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0053] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the buds are transferred to MS medium for in vitro preservation of test-tube seedlings, which is composed of MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L, with a pH of 5.8.

[0054] 4. Low-temperature storage: Place the culture container containing the transplanted explants in a refrigerator at 4°C for 19 weeks (i.e., 133 days).

[0055] 5. Room temperature culture: The culture container that had been stored at low temperature for 19 weeks was taken out of the refrigerator and placed at a temperature of 25℃ and a light intensity of 2500 lx, with 10 hours of light per day. After 4 weeks at 25℃, about 86.67% of the explants germinated.

[0056] 6. In vitro preservation: After the explants germinate again, place the culture container in a light incubator at a temperature of 15℃ and a light intensity of 2500 lx for 10 hours a day. The explants will grow into test-tube seedlings and can be preserved under these culture conditions for 10 months without transfer.

[0057] Example 3: In vitro preservation of water chestnut germplasm seedlings

[0058] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants were in vitro preserved through shoot tip initiation, subculture, and induction to form test-tube plantlets. The steps included:

[0059] 1. Stem tip initiation: Using Tuanfeng water chestnut as the experimental material, clean, disease-free apical or lateral buds of Tuanfeng water chestnuts were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto water chestnut stem tip initiation medium (LS + 6-BA 1.0 mg / L + sucrose 30 g / L + agar 6 g / L) with a pH of 5.8. The culture temperature was 26℃, the light intensity was 2500 lx, and the light intensity was 10 hours per day. Buds or clustered buds appeared after 30 days.

[0060] 2. Subculture: Explants were transferred to a subculture medium containing LS + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L for subculture. The pH of the subculture medium was 5.8. Under a culture temperature of 26℃ and a light intensity of 2500 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0061] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the explants are transferred to MS medium for in vitro preservation of test-tube seedlings, which is composed of MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L, with a pH of 5.8.

[0062] 4. Low-temperature storage: Place the culture container containing the explants in a refrigerator at 4°C for 24 weeks (168 days).

[0063] 5. Room temperature culture: The culture container that had been stored at low temperature for 24 weeks was taken out of the refrigerator and placed at a temperature of 25℃ and a light intensity of 2500 lx, with 10 hours of light per day. After 4 weeks at 25℃, about 42.22% of the explants germinated.

[0064] 6. In vitro preservation: After the explants germinate again, place the culture container in a light incubator at a temperature of 15℃ and a light intensity of 2500 lx for 10 hours a day. The explants will grow into test-tube seedlings and can be preserved under these culture conditions for 10 months without transfer.

[0065] Example 4: In vitro preservation of water chestnut germplasm seedlings

[0066] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants were in vitro preserved through shoot tip initiation, subculture, and induction to form test-tube plantlets. The steps included:

[0067] 1. Stem tip initiation: Using Tuanfeng water chestnut as the experimental material, clean, disease-free apical or lateral buds of Tuanfeng water chestnuts were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto water chestnut stem tip initiation medium (LS + 6-BA 1.0 mg / L + sucrose 30 g / L + agar 6 g / L) with a pH of 5.8. The culture temperature was 26℃, the light intensity was 2500 lx, and the light intensity was 10 hours per day. Buds or clustered buds appeared after 30 days.

[0068] 2. Subculture: Explants were transferred to a subculture medium containing LS + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L for subculture. The pH of the subculture medium was 5.8. Under a culture temperature of 26℃ and a light intensity of 2500 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0069] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the explants are transferred to MS medium for in vitro preservation of test-tube seedlings, which is composed of MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L, with a pH of 5.8.

[0070] 4. Low-temperature storage: Place the culture container containing the transplanted explants in a refrigerator at 4°C and store for 30 weeks (i.e., 210 days).

[0071] 5. Room temperature culture: The culture container that had been stored at low temperature for 30 weeks was taken out of the refrigerator and placed at a temperature of 25℃ and a light intensity of 2500 lx, with 10 hours of light per day. After 4 weeks at 25℃, about 4.44% of the explants germinated.

[0072] 6. In vitro preservation: After the explants germinate again, place the culture container in a light incubator at a temperature of 15℃ and a light intensity of 2500 lx for 10 hours a day. The explants will grow into test-tube seedlings and can be preserved under these culture conditions for 10 months without transfer.

[0073] Example 5: In vitro preservation of water chestnut germplasm seedlings

[0074] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants underwent shoot tip initiation, subculture, and induction to form in vitro corms for in vitro preservation. The process included the following steps:

[0075] 1. Stem tip initiation: Using *Tamarix kusnezoffii* as the experimental material, clean, disease-free terminal or lateral buds of *Tamarix kusnezoffii* were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto *Tamarix kusnezoffii* stem tip initiation medium (LS + 6-BA 0.8 mg / L + sucrose 30 g / L + agar 6 g / L). Buds or clusters of buds appeared after 30 days. The pH of the *Tamarix kusnezoffii* stem tip initiation medium was 6.0.

[0076] 2. Subculture: Explants were transferred to a subculture medium (LS + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L) for subculture. The pH of the subculture medium was 6.0. Under a culture temperature of 24℃ and a light intensity of 2000 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0077] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the explants are transferred to MS medium for in vitro preservation of test-tube seedlings, which consists of MS + 6-BA 0.5 mg / L + AC 0.5 mg / L + sucrose 80 g / L + agar 6 g / L, with a pH of 6.0.

[0078] 4. Low-temperature storage: Place the culture container containing the explants in a refrigerator at 4°C and store for 12 weeks (84 days).

[0079] 5. Room temperature culture: Take out the culture container that has been stored at low temperature for 12 weeks from the refrigerator and place it at a temperature of 24℃ and a light intensity of 2000 lx, with 10 hours of light per day, and place it at room temperature for 2 to 4 weeks. About 95% of the explants will germinate.

[0080] 6. In vitro preservation: Place the culture container in a light incubator at 15℃ with a light intensity of 2000 lx for 10 hours per day. The explants will grow into test-tube plantlets, which can be preserved under these culture conditions for 12 months without subculturing.

[0081] Example 6: In vitro preservation of water chestnut germplasm seedlings

[0082] Using the terminal or lateral buds of *Centipeda minima* as explants, the plants underwent shoot tip initiation, subculture, and induction to form in vitro corms for in vitro preservation. The process included the following steps:

[0083] 1. Stem tip initiation: Using *Tamarix kusnezoffii* as the experimental material, clean, disease-free terminal or lateral buds of *Tamarix kusnezoffii* were soaked in 75% alcohol for 30 seconds, then soaked in 0.1% HgCl2 solution for 0.5–1 minute, and rinsed 3–5 times with sterile water for 3–5 minutes each time. The buds were then inoculated onto a stem tip initiation medium consisting of LS + 6-BA 0.8 mg / L + sucrose 30 g / L + agar 6 g / L. Buds or clusters of buds appeared after 30 days. The pH of the stem tip initiation medium was 5.8.

[0084] 2. Subculture: Explants were transferred to a subculture medium containing LS + 0.5 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar for subculture. The pH of the subculture medium was 5.8. Under a culture temperature of 25℃ and a light intensity of 2200 lx, with 10 hours of light per day, new shoots or clusters of shoots appeared in 25–30 days.

[0085] 3. Transfer to in vitro preservation medium for test-tube seedlings: After the water chestnut germplasm resources have been subcultured to a certain number, the buds are transferred to MS medium for in vitro preservation of test-tube seedlings, which consists of MS + 6-BA 0.3 mg / L + AC 0.8 mg / L + sucrose 80 g / L + agar 6 g / L, with a pH of 5.8.

[0086] 4. Low-temperature storage: Place the culture container containing the grafted buds in a refrigerator at 4°C for 21 weeks (i.e., 147 days).

[0087] 5. Room temperature cultivation: The culture container that has been stored at low temperature for 21 weeks was taken out of the refrigerator and placed at a temperature of 25℃ and a light intensity of 2200lx, with 10 hours of light per day, and placed at room temperature for 4 weeks. About 74% of the plants germinated.

[0088] 6. In vitro preservation: Place the culture container in a light incubator at 15℃ with a light intensity of 2200 lx for 10 hours per day. The explants will grow into test-tube plantlets, which can be preserved under these culture conditions for 6 months without subculturing.

[0089] Experiment Example 1: Low-Temperature Storage Experiment of Water Chestnut Germplasm Resources

[0090] 1. Materials and Methods

[0091] 1.1 Experimental materials: Water chestnut tissue culture seedlings

[0092] 1.2 Test Methods:

[0093] The water chestnut tissue culture seedlings, after subculture in Example 1, were transferred to in vitro preservation medium (MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L, pH 5.8–6.0) for in vitro seedling preservation. 210 bottles were transferred, with 3 seedlings per bottle, for a total of 630 seedlings. The transferred seedlings were stored at 4–5°C under dark conditions. Periodically, 42–45 seedlings (14–15 bottles) were randomly removed from the refrigerator and placed in a culture environment of 24–26°C, 2000–2500 lx light intensity, and 10 hours of light per day to allow for regeneration. One month after regeneration, the number of seedlings that regenerated and sprouted new shoots was investigated, and the regeneration and sprouting rate was calculated for each batch.

[0094] 2. Test Results

[0095] Table 1 shows the recovery and germination of water chestnut germplasm resources after different periods of low-temperature storage at 4℃.

[0096] Table 1. Recovery and sprouting of water chestnut germplasm resources after different periods of low-temperature storage at 4℃.

[0097]

[0098]

[0099] As shown in Table 1, short-term low-temperature storage followed by revival at 24–26℃ has little impact on the germination rate of water chestnut germplasm resources. When water chestnut tissue culture seedlings are stored at 4℃ for 12 weeks (approximately 3 months) and then placed in culture conditions of 24–26℃, light intensity of 2000–2500 lx, and 10 hours of light per day, the germination rate remains as high as 97.78%. Subsequently, with prolonged low-temperature storage, the germination rate of water chestnut tissue culture seedlings gradually decreases. After being stored at 4℃ for 21 weeks (approximately 5 months), the germination rate of water chestnut tissue culture seedlings decreased to 76.19%. After being stored at 4℃ for 24 weeks, the germination rate decreased to 42.22%. After being stored at 4℃ for 28 weeks, the germination rate decreased to 28.99%. After being stored at 4℃ for 30 weeks, the germination rate decreased to 4.44%, indicating that the vast majority of water chestnut tissue culture seedlings had died and could not recover.

[0100] Therefore, water chestnut tissue culture seedlings can be stored at 4℃ for 28 weeks (196 days) and then allowed to resume growth under culture conditions of 24–26℃, 2000–2500 lx light intensity, and 10 hours of light per day. Preferably, water chestnut tissue culture seedlings can be stored at 4℃ for 21 weeks (147 days) and then allowed to resume growth under culture conditions of 24–26℃, 2000–2500 lx light intensity, and 10 hours of light per day. More preferably, water chestnut tissue culture seedlings are stored at 4°C for 18 weeks (i.e., 133 days) and then allowed to resume growth under culture conditions of 24–26°C, light intensity of 2000–2500 lx, and 10 hours of light per day; most preferably, water chestnut tissue culture seedlings are stored at 4°C for 12 weeks (i.e., 84 days) and then allowed to resume growth under culture conditions of 24–26°C, light intensity of 2000–2500 lx, and 10 hours of light per day.

Claims

1. A method for in vitro preservation of water chestnut germplasm seedlings, characterized in that, include: (1) After disinfecting the explants of water chestnuts, they were inoculated onto water chestnut shoot tip initiation culture medium for shoot tip initiation culture until they grew into single shoots or clusters of shoots; (2) Transfer the single bud or cluster of buds obtained in step (1) to the subculture medium for subculture and proliferation culture; (3) The shoot clusters obtained from subculture were divided into single shoots and transferred to the in vitro preservation medium of test-tube seedlings and stored at a low temperature of 4-6℃; wherein the in vitro preservation medium of test-tube seedlings is MS + 6-BA 0.1-0.5 mg / L + AC 0.0-1.0 g / L + sucrose 30-80 g / L + agar 5-6 g / L, and the pH value of the medium is 5.8-6.0; (4) After the low-temperature storage, the explants were cultured under normal temperature and light conditions to induce germination. (5) The germinated explants were cultured under light at 13-18℃ to form test tube seedlings, and the seedlings were cultured and preserved under the same conditions.

2. The in vitro preservation method according to claim 1, characterized in that, The shoot tip initiation culture medium mentioned in step (1) is: LS + 6-BA 0.5~1.0mg / L + NAA 0.0~0.5mg / L + sucrose 30g / L + agar 5~6g / L; the pH value of the culture medium is 5.8~6.0; the conditions for shoot tip initiation culture include: culture temperature of 24~26℃, light intensity of 2000~2500lx, and light exposure of 10~12h per day; preferably, the shoot tip initiation culture medium is: LS + 6-BA 0.8mg / L + sucrose 30g / L + agar 6g / L.

3. The in vitro preservation method according to claim 1, characterized in that, The subculture medium mentioned in step (2) is: LS + 6-BA 0.5-1.0 mg / L + sucrose 30 g / L + agar 5-6 g / L; the pH value of the medium is 5.8-6.0; the conditions for the subculture proliferation culture include: culture temperature of 24-26℃, light intensity of 2000-2500 lx, and light exposure of 10-12 h per day.

4. The in vitro preservation method according to claim 1, characterized in that, The low-temperature storage time in step (3) is 4 to 24 weeks, preferably 12 to 19 weeks, and most preferably 12 weeks.

5. The in vitro preservation method according to claim 1, characterized in that, No light is required during the low-temperature storage described in step (3).

6. The in vitro preservation method according to claim 1, characterized in that, The in vitro preservation medium for the test-tube seedlings mentioned in step (3) is: MS + 6-BA 0.5 mg / L + sucrose 50 g / L + agar 6 g / L.

7. The in vitro preservation method according to claim 1, characterized in that, The room temperature range mentioned in step (4) is 24 to 26°C.

8. The in vitro preservation method according to claim 1, characterized in that, The light cultivation conditions mentioned in step (4) include: light intensity of 2000-2500 lx, and light exposure of 10-12 h per day.

9. The in vitro preservation method according to claim 1, characterized in that, In step (5), the germinating explants are induced to form test-tube seedlings by light culture at 15°C.

10. The method for in vitro preservation according to claim 1, characterized in that, The light cultivation conditions mentioned in step (5) include: light intensity of 2000-2500 lx, and light exposure of 10-12 h per day.

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