Vitrification ultralow-temperature preservation method for calluses of anthurium andraeanum
Through the vitrification method of hyperosmotic pretreatment, loading treatment, dehydration treatment and liquid nitrogen preservation, the genetic trait variation and high cost problems in callus preservation were solved, and the long-term stable preservation of callus and new varieties were achieved. breeding.
Patent Information
- Application Number
- CN202411342087.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2024-12-31
AI Technical Summary
The prior art is difficult to effectively preserve callus, resulting in genetic trait variation and high cost, and the loss of cell function after ultra-low temperature preservation, and lack of vitrification ultra-low temperature preservation system suitable for callus.
The vitrification method of hypertonic pretreatment, loading treatment, dehydration treatment and liquid nitrogen preservation is adopted to quickly cool callus through high-concentration vitrification liquid to avoid ice crystal formation, and provide rapid thawing and recovery culture methods.
The long-term and stable preservation of callus tissue has been achieved, the biological activity and genetic stability of cells have been maintained, the preservation cost has been reduced, and an effective way to breed new varieties has been provided.
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Abstract
Description
Technical Field
[0001] The invention relates to a method in the technical field of ultra-low temperature preservation, in particular to an ultra-low temperature preservation method for anthurium callus tissue by vitrification. Background Art
[0002] Anthurium andraeanum Lind. is a perennial evergreen herbaceous plant belonging to the genus Anthurium in the family Araceae. Its aliases include red anthurium, anthurium, and flamingo flower. It has high economic and ornamental value. Because of its unique spathe, bright colors, evergreen all year round, and monthly flowering, it is the first choice for indoor ornamental plants in today's society. Anthurium is native to tropical rainforests such as Costa Rica and Colombia. Today, with the continuous development of plant introduction technology, Anthurium is widely cultivated in Europe, Asia, Africa and other regions. It was introduced to my country in the 1970s and mass-produced in the 1990s. It is cultivated in large quantities in southeastern my country. As of the beginning of the 21st century, there are about 900 varieties. There are more and more varieties of Anthurium, but because Anthurium is native to tropical rainforests and cannot tolerate low temperatures, the cost of using greenhouses to produce and cultivate it is high. The traditional nursery seed preservation method requires a lot of manpower, material and financial resources; the in vitro tissue culture method requires continuous subculture, and its genetic traits are prone to mutation. Therefore, developing a new method for long-term and stable preservation of anthurium germplasm resources is of great significance for anthurium production and the breeding of new varieties.
[0003] Cryopreservation technology is currently the only method for long-term, effective and stable preservation of plant germplasm resources. Cryopreservation refers to the preservation of plant cells, tissues or organs at a temperature below -80°C (generally using liquid nitrogen as a cold source, -196°C). Cryopreservation technology can keep the preserved plant germplasm resources at this temperature, so that certain metabolic activities in the cells are in a stopped state. When they are needed, through a proper thawing process, these germplasm resources can restore their regeneration ability and maintain normal biological activity and genetic stability. It can also avoid genotype and chromosome mutations in genetic materials during long-term succession. As early as the 1990s, cryopreservation technology has been widely used in the preservation of germplasm resources of different plants, such as Ginkgo biloba L., ginseng (Panax ginseng C.A.Mey.), citrus (citrus reticulata Blanco), etc. At present, more than 10,000 resources have been preserved in the established cryopreservations in countries around the world. Studies have found that cells that have been cryopreserved at ultra-low temperatures will experience serious death, and that cells that have been cryopreserved at ultra-low temperatures will experience long-term functional loss during the recovery culture process. To address the drawbacks of cryopreservation, vitrification was subsequently developed for cryopreservation. Vitrification refers to the direct conversion of water into a "glassy state" without forming ice crystals. Rapid cooling from the liquid phase to the glass phase is used to avoid the formation of ice crystals inside and outside the cells, thereby reducing damage to callus tissue or cells when ice crystals form. After pretreatment, the material is loaded and dehydrated at 0°C or room temperature using a high-concentration vitrification solution, and then the material is quickly placed in liquid nitrogen for cryopreservation. High-concentration vitrification solution can quickly form a vitrified state in liquid nitrogen. Vitrification cryopreservation has the characteristics of wide adaptability, high freezing rate, and simple operation, and is widely used in the preservation of plant germplasm resources. At present, there are only a few literatures on the cryopreservation system of Anthurium, among which there are studies on the cryopreservation of leaves, petioles, and stem segments (including axillary buds) of Anthurium using the droplet vitrification method. The materials are obtained from seedlings, and the preservation effect of leaves and petioles is not as good as that of stem segments (including axillary buds). After preservation, callus tissue needs to be further induced to obtain regenerated seedlings; there are also studies on the cryopreservation system of embryonic suspension cell fluid of Anthurium. The research materials are liquid suspension cells, and the materials after preservation need to go through more steps before regenerated seedlings can be induced or used as biological breeding materials. Compared with the existing literature, Anthurium callus with cell totipotency is an important genetic resource carrier. A large number of regenerated seedlings can be obtained through induction. In genetic transformation experiments, new varieties can be bred by biological breeding methods. Therefore, its long-term preservation value is higher.Anthurium callus tissue is the main material for Anthurium breeding, propagation and production, and its cultivation technology is simple and low-cost. However, there is currently no vitrification ultra-low temperature preservation system for it. Summary of the invention
[0004] The purpose of the present invention is to provide an ultra-low temperature preservation method for anthurium callus tissue vitrification method to address the shortcomings of the existing in vitro culture technology of anthurium, which is used for the preservation of anthurium germplasm resources, and will provide an effective way to preserve the resources of Anthurium and even Araceae plants and to cultivate new varieties by utilizing plant diversity.
[0005] The invention firstly discloses a method for ultra-low temperature preservation of anthurium callus tissue by vitrification, which comprises the steps of performing hypertonic pretreatment on the anthurium callus tissue, sequentially placing the anthurium callus tissue in a loading liquid and a vitrification solution for dehydration treatment, and finally adding the dehydrated callus tissue back into a new vitrification solution and rapidly placing the callus tissue into liquid nitrogen for preservation.
[0006] Preferably, the method for ultra-low temperature preservation of Anthurium callus tissue by vitrification method has the following specific steps:
[0007] 1) Hyperosmotic pretreatment: placing the Anthurium callus in a pretreatment medium for culture;
[0008] 2) loading treatment: placing the anthurium callus tissue pretreated in step 1) in a loading liquid for dehydration treatment;
[0009] 3) Dehydration treatment: removing the loading solution in step 2), placing the Anthurium callus in a vitrification solution, and further dehydrating the solution at 0° C.;
[0010] 4) Liquid nitrogen storage: The anthurium callus tissue further dehydrated in step 3) is quickly transferred into liquid nitrogen for storage.
[0011] The anthurium callus tissue in step 1) refers to anthurium callus tissue that has been subcultured for 50 to 70 days and has a good growth state.
[0012] Preferably, the anthurium callus tissue in step 1) refers to anthurium callus tissue that has been subcultured for 60 days and is in good growth condition.
[0013] Step 1) The pretreatment culture medium is a 1 / 2MS solid culture medium with a sucrose content of 0 to 0.5 M; the pretreatment culture time is 1 to 3 days; and the pretreatment culture temperature is 5°C or 25°C.
[0014] Preferably, the pretreatment culture medium in step 1) is a 1 / 2MS solid culture medium containing 0.5M sucrose; the pretreatment culture time is 1 day, and the pretreatment temperature is 25°C.
[0015] Step 2) The loading solution is 1 / 2MS culture solution containing 0.4M sucrose and 2M glycerol. The dehydration time of the loading solution is 20 to 60 minutes.
[0016] Preferably, the time for dehydration treatment of the loading liquid in step 2) is 60 minutes.
[0017] Step 3) The vitrification solution is a 1 / 2MS culture medium containing 30 wt% glycerol, 15 wt% ethylene glycol, 15 wt% dimethyl sulfoxide, and 0.4 M sucrose. The dehydration time of the vitrification solution is 30 to 90 minutes.
[0018] Preferably, the time for dehydration treatment of the vitrification solution in step 3) is 60 minutes.
[0019] On the other hand, the present invention discloses a thawing and recovery culture method for the ultra-low temperature preservation of the anthurium callus tissue by vitrification method, which comprises taking out the ultra-low temperature preservation callus tissue by vitrification method from liquid nitrogen, placing it at 35-40°C for rapid thawing, then washing it with 1-1.2M sucrose washing solution, absorbing the washing solution on the surface, and finally transferring it to a recovery culture medium for recovery culture at 24-26°C.
[0020] Preferably, thaw quickly in a 40°C water bath for 90 seconds.
[0021] Preferably, the washing solution is 1 / 2MS culture solution containing 1.2M sucrose.
[0022] Preferably, wash with washing solution three times, 10 minutes each time.
[0023] The recovery culture medium used in the present invention is the same as the 1 / 2MS solid culture medium.
[0024] Preferably, the recovery culture condition is a dark environment, and the recovery culture temperature is 25°C.
[0025] The length of storage time in liquid nitrogen has no effect on the survival rate of callus tissue, therefore, the purpose of long-term storage of Anthurium callus tissue can be achieved.
[0026] Compared with the prior art, the present invention achieves ultra-low temperature preservation of Anthurium callus tissue by specially designing a combination of processing methods, conditions, and reagents during ultra-low temperature preservation by vitrification, which provides a technical guarantee for the preservation of Anthurium germplasm resources, and will provide an effective way to preserve Anthurium and even Araceae plant resources and to utilize plant diversity to cultivate new varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:
[0028] Figure 1 This is the effect curve diagram of the orthogonal design experiment. DETAILED DESCRIPTION
[0029] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.
[0030] The present invention relates to a method for ultra-low temperature preservation of Anthurium callus tissue by vitrification; the specific steps are as follows:
[0031] 1) Hyperosmotic pretreatment: placing the Anthurium callus in a pretreatment medium for culture;
[0032] 2) loading treatment: placing the anthurium callus tissue pretreated in step 1) in a loading liquid for dehydration treatment;
[0033] 3) Dehydration treatment: removing the loading solution in step 2), placing the Anthurium callus in a vitrification solution, and further dehydrating the solution at 0° C.;
[0034] 4) Liquid nitrogen storage: The anthurium callus tissue further dehydrated in step 3) is quickly transferred into liquid nitrogen for storage.
[0035] Example 1
[0036] 1. Experimental Materials
[0037] 1.1 Experimental object: Using young leaves of Anthurium candidum as materials, 1 / 2MS solid medium was used for 60 days of induction culture in a dark environment at 25℃ to obtain Anthurium candidum callus. Anthurium candidum callus blocks with good growth conditions were selected as experimental objects for ultra-low temperature preservation by vitrification method.
[0038] 1.2 Experimental Reagents
[0039] 1.2.1 The loading solution is 1 / 2MS culture solution containing 0.4M sucrose and 2M glycerol.
[0040] 1.2.2 The vitrification solution is a 1 / 2MS culture medium containing 30wt% glycerol, 15wt% ethylene glycol, 15wt% dimethyl sulfoxide, and 0.4M sucrose.
[0041] 1.2.3 The washing solution is 1 / 2MS culture solution containing 1.2M sucrose.
[0042] 1.2.4 The pretreatment culture medium is a 1 / 2MS solid culture medium with a sucrose content of 0.3M or 0.5M; that is, 1 / 2MS + sucrose (0.3M or 0.5M) + agar + 1mg / L TDZ (thidiazuron) + 1mg / L 2,4-D (2,4-dichlorophenoxyacetic acid).
[0043] 1.2.5 The recovery culture medium is 1 / 2MS solid culture medium.
[0044] 1.2.6 1 / 2MS solid culture medium: 1 / 2MS + 3% sucrose + agar + 1mg / L TDZ (thidiazuron) + 1mg / L 2,4-D (2,4-dichlorophenoxyacetic acid). The present invention uses the ratio with the highest induction rate among the following five hormone concentration ratios for this experiment:
[0045] ①0.4mg / L TDZ+0.5mg / L 2,4-D,
[0046] ②0.4mg / L TDZ+1mg / L 2,4-D,
[0047] ③1mg / L TDZ+0.5mg / L 2,4-D,
[0048] ④1mg / L TDZ+1mg / L 2,4-D,
[0049] ⑤0.4mg / L TDZ.
[0050] 2. Experimental Methods
[0051] Based on the analysis of the cryopreservation systems of multiple species, a method for cryopreservation of Anthurium candidum callus by vitrification was established through orthogonal design experiments (Table 1), and the experimental operation was carried out according to the following steps.
[0052] Table 1 Four-factor three-level orthogonal design experiment
[0053]
[0054] Note: The pre-culture sucrose concentration is 0, which means there is no pre-culture step.
[0055] (1) Pre-culture: Experiments 4-9 selected callus pieces of Anthurium with good growth conditions, inoculated them on 1 / 2MS solid medium containing sucrose at a concentration of (0.3, 0.5 mol / L), and cultured them in the dark at 25°C (1, 2, 3 days). Example 1-3 did not undergo this pre-culture step.
[0056] (2) Loading: The pre-cultured Anthurium callus tissue was cut into small pieces of about 0.1 g with a knife and placed in a 2 mL cryovial. 1.8 mL of loading solution (1 / 2MS + 0.4 M sucrose + 2 M glycerol) was added and treated at room temperature (20, 40, 60 min).
[0057] (3) Dehydration: Aspirate the loading solution, add 1.8 mL of vitrification solution (1 / 2MS + 0.4 M sucrose + 30% glycerol w / v + 15% ethylene glycol w / v + 15% dimethyl sulfoxide w / v) into the cryovial, and dehydrate at 0°C (30, 60, 90 min).
[0058] (4) Freezing and thawing: The cryovials were quickly immersed in liquid nitrogen and frozen for 1 h before being rapidly thawed in a 40°C water bath for 90 s.
[0059] (5) Washing: Aspirate the vitrification solution PVS2 and wash with washing solution (1 / 2MS + 1.2M sucrose) at room temperature for 30 min, replacing the washing solution with fresh one every 10 min.
[0060] (6) Recovery culture: The treated callus tissue blocks were placed in basic solid culture medium (1 / 2MS + 3% sucrose + agar + 1 mg / L TDZ + 1 mg / L 2,4-D) and recovered incubated at 25°C in the dark for 24 h.
[0061] Table 2 Orthogonal design experimental results
[0062]
[0063] The relative survival rate of Anthurium callus was determined by TTC (triphenyltetrazolium chloride) method, and the experimental results are shown in Table 2. The results showed that under the treatment of Experiment 7, that is, when the pre-culture concentration was 0.5M, the time was 1 day, the loading time was 60 minutes, and the dehydration time was 60 minutes, the relative survival rate of the cells reached the highest of 48.65%.
[0064] The Orthogonal Design Assistant was used to analyze the nine completed orthogonal design experiments, and a set of theoretically optimal solutions were obtained based on the obtained effect curves ( Figure 1 ), recorded as experiment 10, that is, 0.5M pre-cultured for 2 days at 25°C, loading time of 60 minutes, and elution time of 60 minutes, the relative cell survival rate was 34.28%. It can be concluded that when the pre-culture concentration is 0.5M, the time is 1 day, the loading time is 60 minutes, and the dehydration time is 60 minutes, the relative cell survival rate reaches the highest.
[0065] Example 2
[0066] On the basis of Experiment 7, other treatment conditions were kept unchanged, and the pre-culture condition was changed to 5°C. After treatment, the relative survival rate of cells was 10.59%, which showed that the optimal pre-culture temperature was 25°C.
[0067] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. A method for ultra-low temperature preservation of anthurium callus tissue by vitrification, comprising: subjecting the anthurium callus tissue to hypertonic pretreatment, sequentially placing the anthurium callus tissue in a loading solution and a vitrification solution for dehydration treatment, and finally rapidly placing the dehydrated callus tissue into liquid nitrogen for preservation.
2. The cryopreservation method according to claim 1, characterized in that The method comprises the following steps: S1. Hyperosmotic pretreatment: placing the Anthurium callus in a pretreatment medium for culture; S2, loading treatment: placing the Anthurium callus tissue pre-treated in step S1 in a loading liquid for dehydration treatment; S3, dehydration treatment: removing the loading solution in step S2, placing the Anthurium callus in a vitrification solution, and further dehydrating it at 0°C; S4. Liquid nitrogen storage: The anthurium callus tissue further dehydrated in step S3 is quickly transferred into liquid nitrogen for storage.
3. The cryopreservation method according to claim 1 or 2, characterized in that: In step S1, the anthurium callus tissue refers to anthurium callus tissue that has been subcultured for 50 to 70 days and has a good growth state.
4. The cryopreservation method according to claim 3, characterized in that: The culture medium used in the subculture is 1 / 2MS solid culture medium.
5. The cryopreservation method according to claim 2, characterized in that: In step S1, the pretreatment culture medium is a 1 / 2MS solid culture medium with a sucrose content of 0 to 0.5 M; the pretreatment culture time is 1 to 3 days; and the pretreatment culture temperature is 5°C or 25°C.
6. The cryopreservation method according to claim 1 or 2, characterized in that: The loading solution is 1 / 2MS culture solution containing 0.4M sucrose and 2M glycerol; the vitrification solution is 1 / 2MS culture solution containing 30wt% glycerol, 15wt% ethylene glycol, 15wt% dimethyl sulfoxide and 0.4M sucrose.
7. The cryopreservation method according to claim 1 or 2, characterized in that: The dehydration time of the loading solution is 20 to 60 minutes, and the dehydration time of the vitrification solution is 30 to 90 minutes.
8. A method for thawing and recovering the Anthurium callus preserved by the method according to any one of claims 1 to 7, characterized in that: The anthurium callus tissue ultra-low temperature preserved by vitrification was taken out from liquid nitrogen, placed in a 35-40°C water bath for rapid thawing, then washed with a washing solution, the washing solution on the surface was then dried, and finally transferred to a recovery medium for recovery culture.
9. The thawing and recovery culture method according to claim 8, characterized in that: The washing solution is 1 / 2MS culture solution containing 1-1.2M sucrose.
10. The thawing and recovery culture method according to claim 8, characterized in that: The recovery culture medium is 1 / 2MS solid culture medium; the recovery culture condition is a dark environment, and the recovery culture temperature is 24-26°C.