Ultralow-temperature preservation and recovery method for larch olgensis embryonic callus

Abstract

A method for cryopreservation and recovery of larch olgensis embryogenic callus relates to the biotechnology field of forest breeding, and comprises the following steps: selecting an embryogenic cell line induced by immature seeds of larch olgensis, and subculturing the embryogenic cell line for 3-5 days to obtain embryogenic callus as a standby material; carrying out gradient pre-culture at the molar concentrations of 0.2 mol / L and 0.4 mol / L in sequence, wherein the culture is carried out at each concentration for 20-48 hours; after pre-culture, putting the embryogenic callus into a freezing tube, and adding a sterile freezing protection solution; and putting the freezing pipe into a programmed cooling box for programmed cooling, and immediately putting the freezing pipe into a liquid nitrogen biological container for storage. The method disclosed by the invention is simple and convenient in operation steps and obvious in effect, the subculture age limit of the larix olgensis embryonic callus is slowed down, and relatively good cell viability and development potential in the initial stage of induction are maintained, so that the occurrence of variation is effectively reduced, manpower and material resources consumed by long-term subculture materials can be saved, and the production cost is reduced. The method is of great significance to theoretical and practical application research of larix olgensis.

Description

Technical Field

[0001] The invention belongs to the biotechnical field of forest tree breeding and relates to a method for ultra-low temperature preservation of embryonic callus of Larix olgensis. Background Art

[0002] Larix olgensis, also known as yellow pine, yellow larches, Korean larches, is a plant of the genus Larix (Larix spp.) of the Pinaceae family. It is an important conifer species in Northeast my country, especially in the Changbai Mountain area. It has important ecological value. It can maintain water and soil, purify air, reduce noise, provide biological habitats, etc., and plays an irreplaceable role in maintaining ecological balance. Somatic embryogenesis (abbreviated as somatic embryogenesis) refers to the process of directly producing zygotic embryo structures from somatic cells without gamete fusion. Compared with other breeding methods, somatic embryogenesis has the advantages of high reproduction coefficient, strong genetic stability, short breeding cycle, and low production cost. In addition, the process of somatic embryogenesis is highly controllable and easy to observe, and has important application value in theoretical and practical research. At present, somatic embryogenesis has been applied in the rapid propagation, germplasm resource preservation and genetic improvement of Larix olgensis. However, in in vitro culture, embryonic callus will cause a series of adverse effects on later development due to long-term subculture, including decreased cell viability, loss of callus regeneration ability, changes in gene expression patterns, morphological and physiological abnormalities of regenerated plants, etc., which seriously affect the stability of the system and tissue regeneration efficiency.

[0003] Cryopreservation refers to the long-term preservation of plant organs, tissues or cells under liquid nitrogen conditions (-196℃). At this temperature, the organelles that control the growth and development of various cells and the activities of various biological enzymes involved in them are greatly inhibited, the metabolic activities in the plant materials are basically stopped, the cells no longer divide, and the entire plant material is in a state of "life stagnation" or "suspended death". This technology is usually combined with in vitro culture technology, which can achieve long-term and safe preservation of pollen, callus, somatic embryos, zygotic embryos, seeds, in vitro meristems and dormant buds. It is currently recognized internationally as the most effective and ideal long-term preservation method for plant germplasm resources. Since the first proposal of cryopreservation technology, with the development of science and technology, cryopreservation methods have been gradually improved. The most common methods are slow freezing, rapid freezing, vitrification, solid freezing, two-step freezing and freeze-drying, among which slow freezing is often used to preserve embryo cultures of coniferous species. So far, the slow freezing method has been successfully applied to coniferous tree species such as red pine, Larix principis-rupprechtii, Picea koraiensis, and Pinus elliottii.

[0004] Patent Publication No. CN111466294B, Invention Name "A Method for Ultra-low Temperature Preservation of Embryonic Tissue of Larix principis-ruppreparata", discloses: Larix principis-ruppreparata embryonic tissue (embryonic callus of cell line of Larix principis-ruppreparata) is subjected to liquid gradient pre-culture treatment with gradually increasing osmotic agent concentration. The Larix principis-ruppreparata embryonic tissue after gradient pre-culture treatment is subjected to ultra-low temperature cryopreservation, and then thawed when used. This invention method is suitable for long-term ultra-low temperature preservation of excellent embryonic cell lines of Larix principis-ruppreparata. After long-term preservation of Larix principis-ruppreparata embryonic cells by this invention method, the cell survival rate is high, the embryonic tissue recovery culture is rapid, and the embryonic callus recovery growth rate is high, reaching more than 66%. However, the liquid gradient pre-culture operation is cumbersome, easy to be contaminated with bacteria, and some materials may be damaged during the liquid shaking culture process. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for ultra-low temperature preservation and recovery of embryonic callus of Larix olgensis.

[0006] The present invention provides a method for cryopreservation of embryonic callus of Larix olgensis, which is carried out according to the following steps:

[0007] 1. Material selection:

[0008] The embryonic cell lines induced from immature seeds of Larix olgensis were selected, and the embryonic callus tissues of the cells were subcultured for 3-5 days as backup materials.

[0009] 2. Pre-cultivation treatment:

[0010] The spare material in step 1 is pre-cultured in a solid proliferation medium, and the pre-culture method is: gradient pre-culture at a molar concentration of 0.2 mol / L and 0.4 mol / L in sequence, and each concentration is cultured for 20-48 hours;

[0011] Among them, the pre-culture process at a molar concentration of 0.2 mol / L is as follows:

[0012] Under sterile conditions, spread the spare materials in step 1 on the first preculture medium and culture them in the dark at 25°C ± 2°C for 20-48 hours;

[0013] The pre-culture process at a molar concentration of 0.4 mol / L is as follows:

[0014] Under sterile conditions, the embryonic callus on the first preculture medium was transferred to the second preculture medium and cultured in the dark at 25°C ± 2°C for 20-48h;

[0015] 3. Freezing treatment:

[0016] After pre-culture, the embryonic callus tissue is placed in a cryovial, and a sterile cryoprotectant solution is added; the cryovial is placed in a programmed cooling box for programmed cooling, and then immediately placed in a liquid nitrogen biological container for storage.

[0017] Further, the solid proliferation medium described in step 2 is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / LMgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / LNa2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L It is made of CoCl2·6H2O, 10-15mg / L FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / L CaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.0.

[0018] Further, the pre-culture medium described in step 2 is

[0019] From 100~200mg / L NH4NO3, 900~910mg / L KNO3, 130~140mg / L KH2PO4, 200~250mg / LMgSO4·7H2O, 250~260mg / L Mg(NO3)2·6H2O, 40~60mg / L MgCl2·6H2O, 15~20mg / LH3BO3, 10~15mg / LZnSO4·7H2O, 5~10mg / L MnSO4·H2O, 0.1~0.5mg / L Na2MoO4·2H2O, 4~5mg / L KI, 0.1~0.5mg / LCuSO4·5H2O, 0.1~0.5mg / L CoCl2·6H2O, 10~15mg / L It is made of FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / L CaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 0.2mol / L or 0.4mol / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT, distilled water and 0.2mol / L or 0.4mol / L sucrose, with a pH value of 6.0.

[0020] Furthermore, the cryoprotectant solution described in step three is prepared by adding 0.4 mol / L sucrose to 2%-5% by weight of dimethyl sulfoxide and 5%-10% by weight of polyethylene glycol as basic protective agents.

[0021] Furthermore, the volume mass ratio of the embryonic callus tissue to the sterile cryoprotectant solution in step three is 1 mL: (0.3-0.6) g.

[0022] Furthermore, the programmed cooling rate in step three is 1°C / min, and the programmed cooling time is more than 4 hours.

[0023] The present invention provides a method for thawing embryonic callus of Larix olgensis after ultra-low temperature storage, and the thawing method is as follows:

[0024] The embryonic callus of Larix olgensis preserved in claim 1 is taken out from the freezing tube and thawed in a 37°C water bath; after the tissue is thawed, the cryoprotectant is filtered out, the embryonic callus is washed 3-4 times with a liquid proliferation culture medium, the liquid culture medium on the surface of the tissue is dried with filter paper, and then the embryonic callus is placed in a solid proliferation culture medium, and after culturing, it is transferred to a new solid proliferation culture medium, thereby completing the thawing method.

[0025] Further, the liquid proliferation culture medium is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / L MgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / L Na2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L CoCl2·6H2O, 10-15 mg / L It is made of FeSO4·7H2O, 15-20mg / LNa2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / L CaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.0.

[0026] Further, the solid proliferation culture medium is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / L MgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / L Na2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L CoCl2·6H2O, 10-15 mg / L It is made of FeSO4·7H2O, 15-20mg / LNa2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / L CaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.0.

[0027] Furthermore, the recovery rate of the thawing method reaches 100%.

[0028] The present invention has the following beneficial effects:

[0029] The invention uses embryonic cell lines induced by seeds of Larix olgensis as experimental materials to study the ultra-low temperature preservation and recovery of embryonic callus of Larix olgensis. The invention has simple operation steps, short recovery time and high recovery rate.

[0030] The embryonic callus of Larix olgensis, the research object of the present invention, suffers from problems such as decreased cell viability, loss of callus regeneration ability, and abnormal morphology and physiology of regenerated plants due to long-term subculture, which seriously affects the stability and utilization efficiency of the system.

[0031] The method of the present invention selects a suitable preculture medium and culture time, and screens the type and content of cryoprotectant to obtain a method for ultra-low temperature preservation and recovery of Larix olgensis embryonic callus. The material after ultra-low temperature preservation can be recovered within 2-4 weeks after thawing, and the recovery rate reaches 100%, the cell activity after recovery reaches 83.77%, and the amount of somatic embryogenesis of the embryonic callus after recovery reaches 129% of that of the tissue not treated with low temperature.

[0032] Compared with the patent CN111466294B, the present invention performs solid gradient pre-culture treatment on the embryonic callus of Larix olgensis, which is easy to operate, not easy to be infected with bacteria, and can avoid the problem of material damage during the culture process, and the recovery rate reaches 100%. A long-term and stable method for ultra-low temperature preservation of Larix olgensis embryonic callus is established to extend the storage time of the material and maintain its genetic stability, and solve the adverse effects of long-term subculture on the embryonic callus of Larix olgensis.

[0033] The method of the present invention has simple operation steps and obvious effects, reduces the subculture period of the embryonic callus of Larix olgensis, maintains good cell viability and development potential in the early stage of induction, thereby effectively reducing the occurrence of mutations, further avoiding a series of problems such as reduced cell viability and loss of callus regeneration ability caused by long-term subculture, greatly prolongs the preservation time and genetic stability of the embryonic callus of Larix olgensis, and can save manpower and material resources consumed by long-term subculture materials, which is of great significance to theoretical and practical application research of Larix olgensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The embryonic cell line induced from seeds of Larix olgensis 73-37 family in step 1 of Example 1;

[0035] Figure 2 This is a photo of the pre-culture process in step 2 of Example 1;

[0036] Figure 3 This is a photo of the freezing process in step 3 of Example 1;

[0037] Figure 4 The comparison photos are of thawing and recovery after 30 days of cryopreservation in step 4 of Example 1, wherein A is the embryonic callus of Larix olgensis that has just been thawed, and B is the embryonic callus of Larix olgensis that has recovered for 2-4 weeks after thawing;

[0038] Figure 5 Comparison of cytological morphology of Larix olgensis 73-37 family before and after cryopreservation, where A is the cell morphology before cryopreservation, and B is the cell morphology recovered after cryopreservation;

[0039] Figure 6 Comparative photos of thawing and recovery after 30 days of cryopreservation in step 4 of Example 3, wherein A is the embryonic callus of Larix olgensis that has just been thawed, and B is the embryonic callus of Larix olgensis that has recovered for 2-4 weeks after thawing;

[0040] Figure 7The photographs are comparative photos of somatic embryogenesis of embryonic callus preserved at low temperature and not preserved at low temperature in step 6 of Example 3, wherein the left side shows somatic embryogenesis of embryonic callus preserved at low temperature, and the right side shows somatic embryogenesis of embryonic callus not preserved at low temperature;

[0041] Figure 8 This is a diagram of cell viability determined by the TTC method in Example 2. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0043] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0044] Embodiment 1:

[0045] The method for screening the cryopreservation and recovery of embryonic callus of Larix olgensis in this embodiment comprises the following steps:

[0046] 1. Selection of test materials:

[0047] The experimental materials were embryonic cell lines induced from immature seeds of Larix olgensis 73-37 family, and the embryonic callus tissues ( Figure 1 ) to conduct research on cryopreservation and recovery.

[0048] 2. Pre-cultivation treatment:

[0049] The present invention is carried out by adding high concentration of sucrose for gradient culture. The pre-cultured culture fluid is pre-cultured with solid proliferation culture medium for gradient pre-culture ( Figure 2 ).

[0050] 3. Freezing treatment:

[0051] After pre-culture, the embryonic callus was placed in a 2 ml cryovial and 1.2 ml of sterile cryoprotectant solution ( Figure 3 ). Place the cryotube in a programmed cooling box for programmed cooling at a rate of -1°C / min and place it in a -80°C refrigerator. After programmed cooling for at least 4 hours, take out the cryotube and quickly store it in a liquid nitrogen biological container.

[0052] The type of preculture medium and culture time in step 2 and the type of cryoprotectant in step 3 are shown in Table 1.

[0053] Table 1 Optimal conditions for cryopreservation and recovery of embryonic callus of Larix olgensis screened by orthogonal experimental design

[0054]

[0055] 4. Thawing process:

[0056] Take out the cryovials stored in the liquid nitrogen biological container and thaw them in a 37°C water bath. After the tissues are thawed, filter out the cryoprotectant solution, wash the embryonic callus in each cryovial 3-4 times with liquid proliferation medium, absorb the liquid medium on the surface of the tissue with filter paper, and then place the embryonic callus in solid proliferation medium.

[0057] 5. Cell activity assay:

[0058] The TTC method was used to determine the activity of embryonic callus cells of Larix olgensis. TTC (2,3,5-triphenyltetrazolium chloride) is a commonly used biochemical reagent that can be reduced by hydrogen to insoluble red triphenylformazan (TTF). The TTC method is mainly used to detect the activity of dehydrogenases in organisms, and the vitality of organisms is judged by observing the color change of TTC. After TTC staining, remove the TTC solution, wash with distilled water, and add ethanol for decolorization. Take the supernatant to measure the absorbance value. The larger the absorbance value, the stronger the cell activity. The specific operation is as follows:

[0059] One day later, 0.1 g of embryonic callus tissue placed on the solid proliferation medium in step 4 was randomly selected and transferred to a 5 mL centrifuge tube, 1.5 mL of 0.5% TTC solution was added, and the mixture was cultured in the dark at 25°C for 24 h; after staining, the TTC solution was removed and the residual solution was washed with distilled water; 3 mL of 95% ethanol was added, and the mixture was incubated at 65°C for 30 min, and then the absorbance of the supernatant at 485 nm was measured. Each treatment was repeated three times. The results are shown in Table 2.

[0060] Among them, the embryonic callus that has not been cryopreserved is used as the control, and the conversion formula between absorbance value and cell viability is:

[0061]

[0062] Table 2 Cell activity of embryonic callus of Larix olgensis after cryopreservation

[0063]

[0064]

[0065] Note: K i represents the sum of observations at the same factor level, X iIt represents the average value of observations at different levels of the same factor, and R represents the range. Among them, T1 = 12h, T2 = 24h, T3 = 36h, T4 = 48h; M1 = 0.2mol / L + 0.4mol / L sucrose, M2 = 0.2mol / L sorbitol + 0.4mol / L sorbitol, M3 = 0.4mol / L sucrose, M4 = 0.4mol / L sorbitol; D1 = 2.5%, D2 = 5%, D3 = 10%, D4 = 15%; P1 = 0%, P2 = 5%, P3 = 10%, P4 = 15%.

[0066] As shown in Table 2, the second level of culture time, the first level of pre-culture medium type and DMSO concentration, and the third level of PEG concentration were significantly higher than the cell viability (K value) of the other three levels, and the pre-culture medium type had the most significant effect on the post-freezing cell viability (R value).

[0067] Compared with embryonic callus that was pre-cultured and cryoprotected, the cell structure of embryonic callus that was directly frozen was destroyed, the cell viability was only 1.111% and it could not proliferate. In summary, 0.2mol / L sucrose and 0.4mol / L sucrose gradient pre-culture for 24h, and cryoprotection with 2.5% dimethyl sulfoxide and 10% polyethylene glycol as basic protective agents were the most suitable for ultra-low temperature preservation of embryonic callus of Larix olgensis, and the cell viability after thawing could reach 5.111%.

[0068] 6. Determination of the value-added rate:

[0069] One day later, 0.1 g of the embryonic callus placed on the solid proliferation medium in step 4 was randomly selected and transferred to a new solid proliferation medium. After 4 weeks of recovery, it was weighed and the proliferation rate of the embryonic callus was calculated. The results are shown in Table 3.

[0070] The calculation formula for the proliferation rate of embryonic callus is:

[0071]

[0072] Table 3 Proliferation rate of embryonic callus after ultra-low temperature cryopreservation

[0073]

[0074]

[0075] Note: T1=12h, T2=24h, T3=36h, T4=48h; M1=0.2mol / L+0.4mol / L sucrose, M2=0.2mol / L sorbitol+0.4mol / L sorbitol, M3=0.4mol / L sucrose, M4=0.4mol / L sorbitol; D1=2.5%, D2=5%, D3=10%, D4=15%; P1=0%, P2=5%, P3=10%, P4=15%.

[0076] Among the 16 treatments, the highest proliferation rate can reach 480%. The embryonic callus of Larix olgensis recovers quickly, and a large amount of embryonic callus can be proliferated in 2-4 weeks. The newly proliferated embryonic callus is white and transparent in appearance ( Figure 4 ), the cell morphology was normal, and the appearance and microstructure were not significantly different from those of the embryonic callus of normally proliferating Larix olgensis ( Figure 5 ).

[0077] Embodiment 2:

[0078] This example screens the best thawing method for cryopreservation of embryonic callus of Larix olgensis, including the following steps:

[0079] 1. Selection of test materials:

[0080] Same as Example 1.

[0081] 2. Pre-cultivation treatment:

[0082] The first stage of pre-culture treatment: Under sterile conditions, 0.6 g of embryonic callus tissue of 3-5 days old was spread on a solid medium containing 0.2 mol / L sucrose and cultured in the dark at 25℃±2℃ for 24 hours;

[0083] Second stage pre-culture treatment: Under sterile conditions, the embryonic callus treated in the first stage pre-culture was transferred to a solid culture medium containing 0.4 mol / L sucrose and cultured in the dark at 25°C ± 2°C for 24 h.

[0084] 3. Freezing treatment:

[0085] Same as Example 1;

[0086] The cryoprotectant solution is made of 2.5% dimethyl sulfoxide and 10% polyethylene glycol as basic protective agents, and 0.4 mol / L sucrose is added.

[0087] 4. Thawing process:

[0088] Take out the cryovials stored in the liquid nitrogen biological container and thaw them in a 4℃ refrigerator, room temperature (25℃), and a 37℃, 42℃, or 50℃ water bath. After the tissues are thawed, filter out the cryoprotectant solution, wash the embryonic callus in each cryovial with liquid proliferation medium 3-4 times, dry the liquid medium on the surface of the tissue with filter paper, and then place the embryonic callus in solid proliferation medium. After 1 day, measure the cell viability by TTC method. The results are as follows: Figure 8 .

[0089] Depend on Figure 8 It can be seen that with the increase of thawing temperature, cell viability shows a trend of first increasing and then decreasing, among which 37°C is the best, which is significantly higher than the other temperatures.

[0090] Embodiment 3:

[0091] The verification of the cryopreservation and recovery scheme of embryonic callus of Larix olgensis in this embodiment and somatic embryogenesis include the following steps:

[0092] 1. Selection of test materials:

[0093] The experimental material was the embryonic cell line induced from immature seeds of Larix olgensis 73-22 family, and the cryopreservation and recovery of the embryonic callus after 3-5 days of subculture were studied.

[0094] 2. Pre-cultivation treatment:

[0095] Same as Example 2.

[0096] 3. Freezing treatment:

[0097] Same as Example 2.

[0098] 4. Thawing process:

[0099] Take out the cryovials stored in the liquid nitrogen biological container and thaw them in a 37°C water bath. After the tissues are thawed, filter out the cryoprotectant solution, wash the embryonic callus in each cryovial 3-4 times with liquid proliferation medium, dry the liquid medium on the surface of the tissue with filter paper, and then place the embryonic callus in solid proliferation medium. Transfer the callus to a new solid proliferation medium after 1 day.

[0100] 5. Embryonic callus recovery culture:

[0101] After 2-4 weeks, the surface of the cryopreserved callus proliferates with white transparent filamentous embryonic callus ( Figure 6 ), and transfer the newly proliferated embryonic callus to a new solid proliferation medium for subculture.

[0102] 6. Somatic embryogenesis of callus after recovery:

[0103] The recovered embryonic callus was cultured in transition medium for 10 days and then transferred to somatic embryogenesis medium until somatic embryos ( Figure 7 ).

[0104] The transition medium 1 / 4BM in step six is ​​composed of 37.5mg / L NH4NO3, 227.475mg / L KNO3, 34.025mg / L KH2PO4, 61.625mg / L MgSO4·7H2O, 64.125mg / L Mg(NO3)2·6H2O, 12.5mg / L MgCl2·6H2O, 3.875mg / L H3BO3, 3.6mg / L ZnSO4·7H2O, 2.3725mg / L MnSO4·H2O, 0.03125mg / L Na2MoO4·2H2O, 1.0375mg / L KI, 0.03125mg / L CuSO4·5H2O, 0.03125mg / L It is made of CoCl2·6H2O, 3.475mg / LFeSO4·7H2O, 4.6625mg / L Na2-EDTA, 59.05mg / L Ca(NO3)2·4H2O, 10.05mg / L CaCl2·2H2O, 2.0mg / L glycine, 0.5mg / L niacin, 0.5mg / L vitamin B6, 1mg / L vitamin B1, 60g / L sucrose, 5.2g / L agar, 1g / L glutamine, 10g / L inositol, 0.5g / L acid-hydrolyzed casein and distilled water, with a pH value of 6.0.

[0105] The somatic embryogenesis medium in step six is ​​composed of 150mg / L NH4NO3, 909.9mg / L KNO3, 136.1mg / L KH2PO4, 246.5mg / L MgSO4·7H2O, 256.5mg / L Mg(NO3)2·6H2O, 50mg / L MgCl2·6H2O, 15.5mg / L H3BO3, 14.4mg / L ZnSO4·7H2O, 9.49mg / L MnSO4·H2O, 0.125mg / L Na2MoO4·2H2O, 4.15mg / L KI, 0.125mg / L CuSO4·5H2O, 0.125mg / L CoCl2·6H2O, 13.9mg / L It is made of FeSO4·7H2O, 18.65mg / LNa2-EDTA, 236.2mg / LCa(NO3)2·4H2O, 40.2mg / L CaCl2·2H2O, 2.0mg / L glycine, 0.5mg / L niacin, 0.5mg / L vitamin B6, 1mg / L vitamin B1, 60g / L sucrose, 2g / L gellan gum, 0.5g / L glutamine, 0.5g / L inositol, 0.25g / L acid-hydrolyzed casein, 20mg / LABA, 80g / L PEG and distilled water, with a pH value of 6.0.

[0106] Plant embryos are good carriers for the continuation of plant genetic resources. Ultra-low temperature preservation is an important technical guarantee for the long-term preservation of germplasm resources. The present invention uses embryonic callus tissue of embryonic cell lines induced by Changbai larch seeds for 3-5d as material. After 20-48h pre-culture and protection of cryoprotectant, the embryonic callus tissue can be recovered within 2-4 weeks, and the recovery rate reaches 100%. The cell activity after recovery reaches 83.77%, and the amount of somatic embryogenesis of embryonic callus tissue after recovery reaches 129% of the tissue not treated with low temperature. The method of the present invention has simple operation steps and obvious effects, slows down the subculture period of Changbai larch embryonic callus tissue, maintains good cell viability and development potential in the early stage of induction, thereby effectively reducing the occurrence of variation, further avoiding a series of problems such as reduced cell viability and loss of callus regeneration ability caused by long-term subculture, greatly prolonging the storage time and genetic stability of Changbai larch embryonic callus tissue, and at the same time, it can save manpower and material resources consumed by long-term subculture materials, which is of great significance to the theoretical and practical application research of Changbai larch.

Claims

1. A method for cryopreservation of embryonic callus of Larix olgensis, characterized in that It is carried out according to the following steps:

1. Material selection: The embryonic cell lines induced from immature seeds of Larix olgensis were selected, and the embryonic callus tissues of the cells were subcultured for 3-5 days as backup materials.

2. Pre-cultivation treatment: The spare material in step 1 is pre-cultured in a solid proliferation medium, and the pre-culture method is: gradient pre-culture at a molar concentration of 0.2 mol / L and 0.4 mol / L in sequence, and each concentration is cultured for 20-48 hours; Among them, the pre-culture process at a molar concentration of 0.2 mol / L is as follows: Under sterile conditions, spread the spare materials in step 1 on the first preculture medium and culture them in the dark at 25°C ± 2°C for 20-48 hours; The pre-culture process at a molar concentration of 0.4 mol / L is as follows: Under sterile conditions, the embryonic callus on the first preculture medium was transferred to the second preculture medium and cultured in the dark at 25°C ± 2°C for 20-48h; 3. Freezing treatment: After pre-culture, the embryonic callus tissue is placed in a cryovial, and a sterile cryoprotectant solution is added; the cryovial is placed in a programmed cooling box for programmed cooling, and then immediately placed in a liquid nitrogen biological container for storage.

2. The method for cryopreservation of embryonic callus of Larix olgensis according to claim 1, characterized in that The solid proliferation medium described in step 2 is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / LMgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / L Na2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L CoCl2·6H2O, 10-15 mg / L It is made of FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / LCaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.

0.

3. The method for cryopreservation of embryonic callus of Larix olgensis according to claim 1, characterized in that The pre-culture medium described in step 2 is From 100~200mg / L NH4NO3, 900~910mg / L KNO3, 130~140mg / L KH2PO4, 200~250mg / LMgSO4·7H2O, 250~260mg / L Mg(NO3)2·6H2O, 40~60mg / L MgCl2·6H2O, 15~20mg / LH3BO3, 10~15mg / L ZnSO4·7H2O, 5~10mg / L MnSO4·H2O, 0.1~0.5mg / L Na2MoO4·2H2O, 4~5mg / LKI, 0.1~0.5mg / L CuSO4·5H2O, 0.1~0.5mg / L CoCl2·6H2O, 10~15mg / L It is made of FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / L CaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 0.2mol / L or 0.4mol / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT, distilled water and 0.2mol / L or 0.4mol / L sucrose, with a pH value of 6.

0.

4. The method for cryopreservation of embryonic callus of Larix olgensis according to claim 1, characterized in that The cryoprotectant solution described in step 3 is prepared by adding 0.4 mol / L sucrose to 2%-5% by weight of dimethyl sulfoxide and 5%-10% by weight of polyethylene glycol as basic protective agents.

5. A method for cryopreservation of embryonic callus of Larix olgensis according to claim 1 or 4, characterized in that The volume mass ratio of the embryonic callus tissue to the sterile cryoprotectant solution described in step 3 is 1 mL: (0.3-0.6) g.

6. The method for cryopreservation of embryonic callus of Larix olgensis according to claim 1, characterized in that The programmed cooling rate described in step 3 is 1°C / min, and the programmed cooling time is more than 4 hours.

7. A method for recovering embryonic callus of Larix olgensis after cryopreservation, characterized in that The recovery method is as follows: The embryonic callus of Larix olgensis preserved in claim 1 is taken out from the freezing tube and thawed in a 37°C water bath; after the tissue is thawed, the cryoprotectant is filtered out, the embryonic callus is washed 3-4 times with a liquid proliferation medium, the liquid medium on the surface of the tissue is dried with filter paper, and then the embryonic callus is placed in a solid proliferation medium, and after culturing, it is transferred to a new solid proliferation medium, thereby completing the recovery method.

8. The method for recovering embryonic callus of Larix olgensis after cryopreservation according to claim 7, characterized in that The liquid proliferation culture medium is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / LMgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / L Na2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L CoCl2·6H2O, 10-15 mg / L It is made of FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / LCaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.

0.

9. The method for recovering embryonic callus of Larix olgensis after cryopreservation according to claim 7, characterized in that The solid proliferation culture medium is composed of 100-200 mg / L NH4NO3, 900-910 mg / L KNO3, 130-140 mg / L KH2PO4, 200-250 mg / L MgSO4·7H2O, 250-260 mg / L Mg(NO3)2·6H2O, 40-60 mg / LMgCl2·6H2O, 15-20 mg / L H3BO3, 10-15 mg / L ZnSO4·7H2O, 5-10 mg / L MnSO4·H2O, 0.1-0.5 mg / L Na2MoO4·2H2O, 4-5 mg / L KI, 0.1-0.5 mg / L CuSO4·5H2O, 0.1-0.5 mg / L CoCl2·6H2O, 10-15 mg / L It is made of FeSO4·7H2O, 15-20mg / L Na2-EDTA, 200-250mg / L Ca(NO3)2·4H2O, 40-45mg / LCaCl2·2H2O, 1-5mg / L glycine, 0.1-1mg / L niacin, 0.1-1mg / L vitamin B6, 0.5-2mg / L vitamin B1, 20-30g / L sucrose, 5-10g / L agar, 0.5-2g / L glutamine, 0.5-2g / L inositol, 0.1-1g / L acid-hydrolyzed casein, 0.1-0.5mg / L 2,4-D, 0.01-0.1mg / L 6-BA, 0.01-0.1mg / L KT and distilled water, with a pH value of 6.

0.

10. The method for recovering embryonic callus of Larix olgensis after cryopreservation according to claim 7, characterized in that The recovery rate of the thawing method reaches 100%.

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