Cryopreservation method for sterile stem tips of terpinen-4 alcohol type melaleuca alternifolia

Abstract

The invention discloses a cryopreservation method of a sterile stem tip of terpinen-4 alcohol type melaleuca alternifolia. Comprising the following steps: (1) obtaining sterile stem tips; (2) pretreatment of sterile stem tips; (3) loading the sterile stem tips; (4) dehydrating the sterile stem tips; (5) cryopreservation; (6) thawing in a water bath; (7) unloading; and (8) carrying out recovery culture and rooting culture. According to the method disclosed by the invention, the minimization of genetic variation is ensured, the time and labor cost of explant disinfection, primary culture and the like are greatly saved, the problem of'senescence 'phenomenon caused by long-term mass subculture in the current production is avoided, and the long-term preservation of the sterile stem tips is realized for the first time.

Description

Technical Field

[0001] The invention relates to an in vitro preservation method for forest germplasm resources, and in particular to a cryopreservation method for sterile stem tips of terpinene-4-ol type Melaleuca alternifolia. Technical Background

[0002] Melaleuca alternefolia, native to Australia, is also known as Australian tea tree. It is an important economic tree species for medicinal and spice use in the Myrtaceae family. The essential oil extracted from its branches and leaves, namely Australian tea tree oil (TTO), is world-renowned and known as "Australian gold". It has anti-inflammatory, bactericidal, hemostatic, analgesic, anti-prickly heat and itching, oil control and acne removal, mosquito repellent, immune activation, antioxidant, and anti-cancer effects. It is widely used in the pharmaceutical, biopesticide, daily chemical, and food industries and has extremely high economic value (Yadav et al., 2016; Chidi et al., 2020; Lee et al., 2020; Redondo-Blanco et al., 2020).

[0003] Melaleuca alternifolia was introduced to China in the 1990s. However, due to its extremely small seeds, many of which were aborted, low germination rate, obvious interspecific differentiation, and lack of high-quality family seedlings, the quality of its essential oil was unstable, which seriously restricted its development (Guo Yicheng, 2007; Zhou Lihua et al., 2001).

[0004] Ultra-low temperature preservation can preserve excellent genes for a long time at the lowest cost and for the longest time, and is currently the ideal method for long-term preservation of germplasm resources. Ultra-low temperature preservation is a biotechnology that preserves biological materials at temperatures below -80°C (usually liquid nitrogen) (Engelmann, 2004; Baust, 2002). At such a low temperature, the metabolic process of plant cells basically stops, and the genetic stability of the material can be maintained to the maximum extent. It has the advantages of long-term storage and stability, and can store a large amount of material in a very small space, saving space and labor, and is economical and effective. It is currently the safest and most economical choice for the preservation of germplasm resources (Wang et al., 2012; Ozudagru et al., 2010). An important concept in this technology is the glass state, which refers to a state in which a substance exists in an amorphous form, and its viscosity is extremely high (about 10 12 -10 14Pa·s), the fluidity is small, the molecular structure is difficult to rearrange in this state, the water is non-freezable, the formation of ice crystals is avoided, and the stability of the tissue is increased. The role of the cryoprotectant CPA is to increase the permeability of cells to water, effectively improve the movement of water between samples, so as to achieve the purpose of increasing the freezing rate and reducing the glass transition temperature (Walters, 2008&2010). The most important problems faced by this technology are: first, dehydrating the material to make it reach a vitrified state to avoid irreversible mechanical damage caused by the formation of ice crystals in the cells during freezing and thawing; second, oxidative damage caused by some high concentrations of cryoprotectants (CPA) used in the dehydration process; third, CPA produces different toxicity due to different types and action time. Fourth, woody plants, especially some tropical and subtropical forest germplasms, are still difficult to preserve due to their high water content and inability to tolerate dehydration. Most of them are preserved in seeds and callus tissues, and stem tips are used as preservation objects, so it is difficult to obtain regenerated plants.

[0005] Therefore, it is crucial to strictly maintain the balance between the protective and toxic effects of CPA, properly dehydrate and allow cells to enter a vitrified state, and prevent and alleviate the oxidative damage caused by CPA for successful cryopreservation. CPA includes permeable ethylene glycol, and non-permeable macromolecules such as polyacrylpyrrolidone (PVP), Ficoll and dextran, as well as some small molecule sugars. While osmotic dehydration, CPA enters the cell and changes the thermodynamic properties of the cell fluid. Rapid cooling can achieve intracellular and extracellular vitrification, maintain the integrity of the cell structure and regeneration ability, and achieve the preservation of some tropical plants.

[0006] Melaleuca alternifolia is mainly planted in a few provinces in the south. Production practice and scientific experiments have shown that it has high requirements for water, is resistant to flooding, but not resistant to dehydration and cold. Even a tissue culture seedling about 30 cm long transplanted for one month will turn yellow and dry up if it is subjected to 0℃ low temperature stress for 48 hours; it will be irreversibly damaged if it is subjected to -5℃ stress for 24 hours. It cannot grow again if it is returned to normal temperature. Therefore, the ultra-low temperature preservation of the sterile stem tips of the less cold-resistant terpinene-4-ol type is of great significance and quite challenging from a technical perspective, whether in terms of production, scientific research or germplasm resource security. Summary of the invention

[0007] In view of the above problems, the present invention discloses a method for ultra-low temperature preservation of sterile stem tips of terpinene-4-ol type Melaleuca alternifolia, including the steps of obtaining, pre-culturing, dehydrating, freezing, thawing and recovering culturing of sterile stem tips. When the tissue culture seedlings used for production are subcultured for a certain number of times and the phenomenon of "aging" occurs, the method is used to take out the sterile stem tips stored in liquid nitrogen at any time for rapid subculture and proliferation, which greatly saves the time and labor costs of explant disinfection, primary culture, etc. while minimizing genetic variation. Proliferation buds of 4-15 generations of subculture are collected, and sterile stem tips with uniform size and good stability are quickly obtained through certain pretreatment, and then the stress resistance of the material is improved through pre-culturing, and then antifreeze and antioxidant substances are added for dehydration, and vitrification is performed for freezing, and finally stored in liquid nitrogen. Through the technology of the present invention, the stem apex meristem after cryopreservation turns from black to green within 2-3 weeks after thawing, and resumes normal growth within 4-5 weeks, thereby realizing the ultra-low temperature preservation of the stem apex of the cold-sensitive woody plant Melaleuca alternifolia, which is of great significance for the preservation and utilization of its germplasm resources and further research on the mechanism of cryopreservation.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] A method for freezing and preserving sterile stem tips of terpinene-4-ol type Melaleuca alternifolia, comprising the following steps:

[0010] (1) Obtaining sterile stem tips: Using the tissue culture test tube seedlings established above as materials, a cluster of 4-5 cm high terpinene-4-ol-type Melaleuca alternifolia tissue culture seedlings of 30-45 days old were taken, cut into individual plants, and placed in proliferation medium 1 for 10-20 days. The plants were shaded for the first 3-5 days, and then the light was gradually increased. The temperature was 25-30°C. When axillary buds grew out and were 1-3 mm in length, the excess tissue at the bottom of the stem was cut off using a sterile surgical blade and stainless steel high-precision ultra-tip tweezers, and the stem apex meristem and 2-5 leaf primordia were retained, and the plants were cut into 1-3 mm pieces.

[0011] (2) Pretreatment of sterile stem tips: Place the sterile stem tips in medium 2 and culture in an incubator for 3-4 days;

[0012] (3) Loading of sterile stem tips: using glycerol + sucrose for 20-30 min;

[0013] (4) Dehydration of sterile shoot tips: Place the shoot tips in a cryovial containing 1.5-2 mL of optimized PVS2 solution at 2-6°C and then dehydrate at 150-250 rpm for 40-60 min.

[0014] (5) Cryopreservation: Place the stem tip in a cryopreservation tube and place in liquid nitrogen for more than 48 hours;

[0015] (6) Rapid thawing in a 38-42°C water bath for 1-2 minutes;

[0016] (7) Unloading with sucrose for 20-26 min;

[0017] (8) Recovery Culture: Transfer the shoot tip to the recovery culture medium and carry out recovery culture for 15-30 days. The shoot tip will turn from black to green. Continue to culture and regenerate plants.

[0018] Preferably, the tissue culture seedling cluster subcultured for 30-45 days in step (1) is a tissue culture seedling subcultured for 4-15 generations.

[0019] Preferably, the proliferation medium 1 described in step (1) comprises: modified MS + 2.0-4.0 mg / L 6-benzylaminoadenine + 0.5-1.0 mg / L naphthaleneacetic acid + 30000-80000 mg / L sucrose + 3500-4500 mg / L agar + 0-100 g / L polyethylene glycol PEG-8000 + 100 ug / ml proanthocyanidin B2 + 0-0.1 g / L glutathione.

[0020] Preferably, the formula of the culture medium 2 described in step (2) is: modified MS + 0.25 g / L activated carbon + 0.03 g / L riboflavin + 50 g / L polyethylene glycol PEG-8000 + 0.05 g / L glutathione + 50 g / L dimethyl sulfoxide + 102.6 g / L sucrose.

[0021] Preferably, the culture conditions described in step (2) are: temperature of 4-10°C and illumination of 7000-9000 Lux.

[0022] Preferably, the glycerol concentration in step (3) is 1.8-2.2M.

[0023] Preferably, the concentration of sucrose in step (3) is 0.4-0.5M.

[0024] Preferably, the optimized PVS2 formula described in step (4) is 10% coconut water + 300g / L glycerol + 130g / L sucrose + 100g / L ethylene glycol + 75g / L dimethyl sulfoxide.

[0025] Preferably, the concentration of sucrose in step (7) is 1.1-1.3M.

[0026] Preferably, the recovery medium formula described in step (8) is: modified MS + 6-benzylaminopurine 2.0 mg / L + naphthaleneacetic acid 0.5 mg / L + sucrose 30000 mg / L + agar 3500 mg / L + 100 ug / ml proanthocyanidin B2 + glutathione 0.05 g / L + 10% coconut water.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are not only profound but also significant, which are specifically reflected in the following aspects:

[0028] 1. Revolutionary sterile stem tip ultra-low temperature preservation technology: The present invention successfully constructs an ultra-low temperature preservation method for sterile stem tips of Melaleuca alternifolia. This breakthrough means that after cryopreservation, the sterile stem tips of Melaleuca alternifolia can achieve a transition from black to green in just 2-3 weeks after thawing, and fully recover to a normal growth state in the next 4-5 weeks. More importantly, these stem tips have almost no significant differences in morphological characteristics compared with those before freezing, and maintain a high degree of genetic stability and physiological activity. Using this method, the present invention can take out the preserved sterile stem tips from liquid nitrogen at any time for rapid and efficient subculture proliferation. This technology not only greatly reduces the time and labor costs required for explant disinfection and primary culture, but also fundamentally avoids the "aging" phenomenon that may occur in the long-term and large-scale subculture process, and realizes the long-term, stable and efficient preservation of sterile stem tips for the first time. This achievement not only provides strong technical support for the protection of genetic resources of Melaleuca alternifolia, but also opens up new ways for the preservation of sterile materials of other plants.

[0029] 2. An efficient and convenient method for obtaining stem apex: Traditional stem apex ultra-low temperature preservation technology often requires the use of a microscope for delicate peeling operations. This process is not only time-consuming and labor-intensive, but also easily causes damage to the stem apex. The present invention, however, optimizes the bud proliferation and growth characteristics of Melaleuca alternifolia through a carefully designed culture medium formula and culture conditions. This innovation enables a large number of vigorous and uniform stem apex to be obtained in a short period of time, greatly improving the efficiency of material acquisition. More importantly, under the naked eye, the present invention can easily obtain stem apex of appropriate size with just a little cutting, without the need for tedious operations using a microscope, thereby significantly saving time and energy and improving work efficiency.

[0030] 3. Optimize and screen the culture medium formula to improve stress resistance: After a large number of repeated experiments and explorations, the present invention finally optimized and screened the culture medium formula, which can not only effectively promote the growth and development of Melaleuca alternifolia, but also significantly improve the stress resistance of the material. This discovery provides a solid foundation and favorable conditions for subsequent successful preservation. By enhancing the stress resistance of the material, the present invention enables Melaleuca alternifolia to better resist the influence of various adverse factors during ultra-low temperature storage, thereby ensuring the stability and reliability of the preservation effect.

[0031] 4. Synergistic effect to reduce oxidative damage: During the cryopreservation process, oxidative damage is a problem that cannot be ignored. The present invention creatively adds two substances, anthocyanin B2 and glutathione, which can synergistically reduce the oxidative damage generated during the preservation process. Anthocyanin B2, as a powerful antioxidant, can scavenge free radicals and protect cells from oxidative stress; while glutathione can enhance the antioxidant capacity of cells and promote cell repair and regeneration. The combined effect of these two substances effectively controls the oxidative damage of Melaleuca alternifolia during the cryopreservation process, thereby further improving the success rate of preservation and the survival rate of the material. This discovery not only provides new ideas and methods for cryopreservation technology, but also provides useful reference and reference for the preservation of other plant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure shows the effect of different pre-culture media on the survival rate and regeneration rate. Note: 1. represents the basic medium of modified MS + dimethyl sulfoxide + sucrose; 2. basic medium + riboflavin; 3. basic medium + glutathione; 4. basic medium + polyethylene glycol PEG-8000; 5. basic medium + riboflavin + glutathione + PEG.

[0033] Figure 2 The figure shows the effect of pre-culture time on the survival rate and regeneration rate.

[0034] Figure 3 This is a graph showing the effect of pre-culture temperature on freezing performance.

[0035] Figure 4 This figure shows the effect of loading time on the cryopreservation effect.

[0036] Figure 5 This is the effect of cryoprotectant composition on freezing effect. Note: 1. No anthocyanidin and glutathione added; 2. Add proanthocyanidin 100ug / ml; 3. Add proanthocyanidin 200ug / ml; 4. Add glutathione 0.025g / L; 5. Add glutathione 0.05g / L; 6. Add glutathione 0.1g / L; 7. Add proanthocyanidin 200ug / ml + glutathione 0.05g / L.

[0037] Figure 6 This is a graph showing the effect of dehydration time on freezing performance.

[0038] Figure 7 This is a picture of sterile test tube seedlings of Melaleuca alternifolia.

[0039] Figure 8 This is a picture of a plant with axillary buds.

[0040] Fig. 9 This is a picture of a sterile stem apex with 3 cotyledons.

[0041] Fig.10 This is a picture of the stem apex at the early stage of recovery culture after freezing.

[0042] Fig.11 The picture shows the shoot tip that has been incubated for 15 days after thawing, turning from black to green.

[0043] Fig.12 The picture shows the regenerated plant seedlings (right) that were cultured for one month after the stem tip turned from black to green during recovery culture, and the original control seedlings with normal growth (left). DETAILED DESCRIPTION

[0044] The specific implementation modes of the present invention are described below in conjunction with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0045] 1. Materials

[0046] The test material is the sterile stem tip of Melaleuca alternifolia, which was obtained from the excellent asexual line of Sanjiaolou Nursery of Guangxi Academy of Forestry Sciences in October 2021, and the seedling age is more than 2 years. On a sunny morning, collect the newly grown shoots, wrap them with a wet towel, and bring them back to the laboratory; use pointed tweezers to pull out the leaves on the shoots, disinfect them, and place them in the primary culture medium for culture. After the culture medium and solution are adjusted to pH 5.6, they are sterilized in an autoclave at 121°C for 20 minutes to establish sterile test tube seedlings, and the following operations are performed using sterile test tube seedlings as materials.

[0047] 2. Reagents and Methods

[0048] In an embodiment of the present invention, a method for freezing and preserving sterile stem tips of terpinen-4-ol type Melaleuca alternifolia comprises the following steps:

[0049] (1) Obtaining sterile shoot tips: Using the tissue culture test tube seedlings established above as materials, take the 4-5 cm tall pinene-4-ol-type Melaleuca alternifolia tissue culture seedling clusters that were subcultured for 30-45 days, cut them into individual plants, and place them in proliferation medium 1: modified MS + 2.0-4.0 mg / L 6-benzylaminoadenine + 0.5-1.0 mg / L naphthaleneacetic acid + 30000-80000 mg / L sucrose + 3500-4500 mg / L agar + 0-100 g / L polyethylene glycol PEG-8000 + 100 ug / ml proanthocyanidin B2 + 0-0.1 g / L glutathione, culture for 10-15 days, shade the first 3-5 days, then gradually increase the light intensity, and the temperature is 25-30°C. When axillary buds grow out and are 1-3 mm in length, use a sterile surgical blade and stainless steel high-precision ultra-tip tweezers (the tip of the chuck is ≤ 0.1 mm) to cut off excess tissue at the bottom of the stem, retain the apical meristem and 2-5 leaf primordia, and cut into 1-3 mm sizes.

[0050] The modified MS formula is as follows: KNO3 1900mg / L, NH4NO3 1650mg / L, KH2PO4 170mg / L, MgSO4·7H2O 370mg / L, CaCl2·2H2O 440mg / L, KI 0.83mg / L, H3BO3 6.2mg / L, MnSO4·H2O 15.6mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, EDTA-2Na 37.3mg / L, FeSO4·7H2O 27.8mg / L, inositol 100mg / L, niacin VB50.5mg / L, pyridoxine hydrochloride VB60.5mg / L, thiamine hydrochloride VB10.4mg / L, glycine 2.0mg / L.

[0051] This step is very important, and the physiological state of the material is the key to the success of ultra-low temperature preservation. Melaleuca alternifolia is mainly used to extract essential oils from its branches and leaves, and the yield of branches and leaves is one of the determining factors of its economic benefits. Nitrate provides N elements for plants, which plays an important role in their growth. In the present invention, it can provide the necessary nitrogen elements for the synthesis of various stress-resistant proteins and amino acids during cryopreservation while promoting development. Boron is one of the important trace elements necessary for plant growth. While providing boron, boric acid also has certain anti-inflammatory, bactericidal and antiseptic effects to help repair some mechanical damages produced during the acquisition of the stem tip of Melaleuca alternifolia. The role of inositol in plant tissue culture mainly includes participating in carbohydrate metabolism, phospholipid metabolism and ion balance, promoting rapid tissue growth, and having a good effect on the formation of buds. At the same time, physiological and transcriptome studies have shown that after cryopreservation, the cell membrane is damaged to a certain extent, and the genes in the phospholipid metabolic pathway related to cell membrane repair are significantly enriched, so an appropriate amount of inositol can promote the repair of cell membranes in addition to promoting the formation of buds. The increase of organic substances such as inositol, pyridoxine hydrochloride, and thiamine hydrochloride plays an important role in promoting the growth of Melaleuca alternifolia and improving related metabolism. The polyethylene glycol PEG-8000 in the proliferation medium has the effect of osmotic stress, which helps the plant cope with osmotic dehydration in the later stage and improves the overall stress resistance.

[0052] The tissue culture seedling clusters subcultured for 30-45 days in the above step (1) are tissue culture seedlings with 4-15 subculture times.

[0053] Subculture of tissue culture seedlings will produce "domestication" and "decline". As the subculture time increases, the plant's reproductive efficiency and rooting ability increase, which is manifested as a "domestication" phenomenon. This may be related to the accumulation of plant growth regulators in the body. As these growth regulators accumulate in the body, the ability of cell proliferation and differentiation gradually increases until it reaches a stable level, which is crucial for the success of cryopreservation. If subcultured for a long time, the subculture will decline, manifested as poor growth, decreased regeneration ability, and even loss of morphogenesis ability, which may also significantly affect its cryopreservation. The survival rate of 4-15 generations after freezing is significantly higher than that of other generations.

[0054] (2) Pretreatment of sterile stem tips: Place the sterile stem tips in culture medium 2 and culture in an incubator for 3-4 days.

[0055] The formula of the culture medium 2 described in the above step (2) is: modified MS + 0.25g / L activated carbon + 0.03g / L riboflavin + 50g / L polyethylene glycol PEG-8000 + 0.05g / L glutathione + 50g / L dimethyl sulfoxide + 102.6g / L sucrose.

[0056] The culture conditions described in the above step (2) are low temperature (4-10° C.) and high light (7000-9000 Lux).

[0057] (3) Loading of sterile shoot tips: 1.8-2.2 M glycerol + 0.4-0.5 M sucrose for 20-30 min.

[0058] (4) Dehydration of sterile shoot tips: Place the shoot tips in a cryovial containing 1.5-2 mL of optimized PVS2 solution at 2-6°C for precooling, and then dehydrate at 150-250 rpm for 40-60 min.

[0059] The optimized PVS2 formula described in step (4) above is 10% coconut water + 300g / L glycerol + 130g / L sucrose + 100g / L ethylene glycol + 75g / L dimethyl sulfoxide. Cells are relatively sensitive to dimethyl sulfoxide, and 7.5% dimethyl sulfoxide reduces the toxicity to cells to a certain extent compared with the conventional 15% dimethyl sulfoxide.

[0060] (5) Cryopreservation: Place the stem tip in a cryopreservation tube and store in liquid nitrogen for more than 48 hours.

[0061] (6) Rapidly thaw in a 38-42℃ water bath for 1-2 minutes.

[0062] (7) 1.1-1.3 M sucrose unloading for 20-26 min.

[0063] (8) Recovery Culture: The shoot tips were transferred to the recovery culture medium for recovery culture for 15-30 days. The shoot tips were observed to turn from black to green. The survival rate was measured by TTC. The plants were cultured in the rooting medium to obtain regenerated plants.

[0064] The recovery medium formula described in the above step (8) is: modified MS + 6-benzylaminoadenine 2.0 mg / L + naphthaleneacetic acid 0.5 mg / L + sucrose 30000 mg / L + agar 3500 mg / L + 100 ug / ml proanthocyanidin B2 + glutathione 0.05 g / L + 10% coconut water.

[0065] Technical principle of the present invention:

[0066] The high-efficiency cryopreservation method of the terpinene-4-ol type Melaleuca alternifolia aseptic stem tips of the present invention, with its sophisticated process design and precise proportion of each raw material, together constitutes the key technology to ensure the high survival rate and regeneration ability of the stem tips. This method is not only scientific and rigorous, but also each step and its components play an indispensable role. The synergy between them and the dosage control are the key to achieving technological breakthroughs. The following is an in-depth analysis of the innovative method of the present invention:

[0067] (I) Raw material effects and synergistic mechanisms

[0068] 1. Proliferation Medium 1:

[0069] Modified MS: As a basal culture medium, it provides the basic nutrients required for plant cell growth.

[0070] 6-Benzylaminoadenine: promotes cell division and accelerates the formation of axillary buds.

[0071] Naphthaleneacetic acid: induces rooting and promotes cell elongation.

[0072] Sucrose: As a carbon source, it supports cell metabolism and growth.

[0073] Agar: acts as a solidifying agent to stabilize the structure of the culture medium.

[0074] Polyethylene glycol PEG-8000: Polyethylene glycol PEG-8000 plays the role of osmotic stress, regulates osmotic pressure, and reduces cell damage. It can be similar to creating adverse stress treatment to improve plant resistance.

[0075] Procyanidin B2: The procyanidin used in the present invention exists in the leaves and seeds of fruits such as grapes and blueberries. It has very strong anti-free radical and antioxidant capabilities, which are 18-20 times that of vitamin C. The procyanidin B2 (Procyanidin B2 PCB2) therein is a B-type procyanidin dimer, which has been proven to have stronger antioxidant and anti-inflammatory effects than other anthocyanins, and can protect cells from oxidative stress damage.

[0076] Glutathione: The glutathione used in the present invention is an antioxidant that can help remove free radicals and peroxides in the body and improve the antioxidant function of organisms; it can also promote growth, enhance the antioxidant capacity of cells, and improve the ability of organisms to resist adverse environments. It is also a good detoxifier. Glutathione interacts with proanthocyanidins, and compared with the control or single use, it can significantly reduce oxidative damage and improve the survival rate after freezing.

[0077] These ingredients work together to promote the healthy growth of axillary buds and lay a good foundation for subsequent cryopreservation.

[0078] 2. Pretreatment medium 2:

[0079] Activated carbon: Activated carbon can adsorb high concentrations of hormones in the early stage of axillary bud acquisition, which can prevent too many hormones, excessive plant growth, larger cells, higher water content, and poorer resistance.

[0080] Riboflavin: Vitamin B2, promotes cell metabolism.

[0081] Polyethylene glycol PEG-8000 and glutathione: further regulate osmotic pressure, enhance cell adaptability and antioxidant capacity.

[0082] Dimethyl sulfoxide: a cell cryoprotectant that promotes vitrification, lowers the cell freezing point, and reduces freezing damage.

[0083] Sucrose: provides energy and osmotic pressure to promote cell dehydration and support cell survival at low temperatures.

[0084] This step gradually acclimates the shoot tip to the upcoming freezing conditions through gentle environmental adjustments.

[0085] 3. Loading fluid:

[0086] Glycerol and sucrose: form a hypertonic environment, promote the discharge of intracellular water, reduce ice crystal formation, and protect cell structure. The present invention uses a loading solution of 1.8-2.2M glycerol + 0.4-0.5M sucrose for treatment, and then uses a PVS2 solution. The present invention found that the stem tip of Melaleuca alternifolia is more sensitive to 15% dimethyl sulfoxide, and the effect is better when soaked in low concentration dimethyl sulfoxide for a relatively longer time.

[0087] 4. Dehydrate PVS2:

[0088] Physiological tests show that the intracellular soluble sugar and amino acid content will increase during the cryopreservation of Melaleuca alternifolia. It is speculated that the cells synthesize such substances to cope with the osmotic stress caused by the freezing process. Coconut water is rich in free sugars, which enter the cells and play an important role in increasing the osmotic pressure of cells and avoiding osmotic damage caused by dehydration. At the same time, it is rich in vitamins, amino acids and trace elements, which have certain effects on cell growth recovery and stress resistance. The most important thing is that coconut water also has certain antioxidant properties to help cells cope with oxidative damage caused by cryopreservation. The results of the previous physiological index tests related to redox showed that oxidative damage is one of the important damages in the cryopreservation process of Melaleuca alternifolia. Antioxidant enzymes such as SOD, CAT, POD and some antioxidant substances such as VC, VE, proanthocyanidins, AsA, GSH were added. Proanthocyanidin B2 is the best exogenous additive screened from a large number of antioxidant enzymes and antioxidant substances.

[0089] Coconut water is combined with glycerin, sucrose, ethylene glycol and dimethyl sulfoxide to form a more efficient dehydration system and improve freezing tolerance.

[0090] 5. Cryopreservation:

[0091] Liquid nitrogen: provides an extremely low temperature environment, inhibits cell activity, and achieves long-term preservation.

[0092] 6. Defrosting and unloading:

[0093] Water bath: Rapid and even heating to reduce cell damage during thawing.

[0094] Sucrose: restores osmotic pressure balance and promotes cell water recovery.

[0095] 7. Restore the culture medium:

[0096] Coconut Water: Promotes rapid cell recovery and growth.

[0097] 6-Benzylaminoadenine, naphthylacetic acid, sucrose, agar, proanthocyanidin B2, glutathione, etc.: synergistically support cell division and growth, realize the transition of the stem tip from black to green, and improve the survival rate.

[0098] (II) Dosage control and process parameter optimization

[0099] Precisely control the dosage of ingredients: Each ingredient has its optimal concentration range. Too high or too low a concentration may affect cell survival and regeneration. For example, too high a sucrose concentration may cause excessive osmotic pressure and damage cells; too low a concentration may not provide sufficient energy support.

[0100] The order of steps cannot be changed: from obtaining sterile stem tips, proliferation culture to pretreatment, loading, dehydration, freezing, thawing, unloading and recovery culture, each step is a scientific arrangement based on the physiological characteristics of cells. Changing the order may cause cells to be unable to adapt to changes and reduce survival rate.

[0101] Optimize process parameters: such as light intensity, temperature, shaking speed, time, etc., are carefully adjusted to achieve the best results. For example, suitable light and temperature can promote healthy cell growth, while appropriate shaking dehydration conditions can effectively reduce intracellular water and improve freezing efficiency.

[0102] In summary, the method for freezing and preserving aseptic stem tips of terpinene-4-ol-type Melaleuca alternifolia of the present invention achieves effective synergy among the raw materials through carefully designed steps and precisely controlled ingredient dosages, significantly improves the survival rate and regeneration capacity of the stem tips, and provides strong technical support for the long-term preservation and utilization of plant genetic resources.

[0103] In order to make the disclosure of the present invention more complete, the following are some steps of exploration:

[0104] 1) Pretreatment of sterile stem tips

[0105] The sterile shoot tips were placed in culture medium 2 (modified MS + 0.25 g / L activated carbon + 0.03 g / L riboflavin + 50 g / L polyethylene glycol PEG-8000 + 0.05 g / L glutathione + 50 g / L dimethyl sulfoxide + 102.6 g / L sucrose) and cultured in an incubator at low temperature (10°C) and high light (8000 Lux) for 2 days, 3 days, 4 days, 5 days, and 6 days.

[0106] 2) Dehydration of sterile shoot tips

[0107] The shoot tip was placed in a 4°C precooled cryotube containing 300 g / L glycerol + 130 g / L sucrose + 100 g / L ethylene glycol + 75 g / L dimethyl sulfoxide (DMSO) with 10% coconut water instead of water, and was shaken at 200 rpm for 20 min, 40 min, 60 min, and 80 min. Since the shoot tip is sensitive to DMSO, 15% DMSO will cause a certain degree of damage.

[0108] 3) Rapid thawing in a 40℃ water bath

[0109] Remove the cap of the cryopreservation tube containing the shoot tip from liquid nitrogen and place it in a container filled with 40°C warm water. Thaw quickly for 1-2 minutes until the cryoprotectant melts.

[0110] 4) 1.2M sucrose unloading

[0111] The shoot tips thawed in a water bath were unloaded with 1.2 M sucrose solution for 10 min, and then replaced with fresh 1.2 M sucrose unloading solution and continued unloading for 10 min to remove the toxic effect of residual PVS on the cells.

[0112] 5) Resume training

[0113] Transfer the shoot tip to recovery medium (modified MS + 6-benzylaminoadenine 2.0 mg / L + naphthaleneacetic acid 0.5 mg / L + sucrose 30000 mg / L + agar 3500 mg / L + 100 ug / ml proanthocyanidin B2 + glutathione 0.05 g / L + 10% coconut water) and restore culture for 15-30 days. It can be seen that the shoot tip turns from black to green. Continue to culture to obtain regenerated plants.

[0114] 6) Vitality detection

[0115] Survival rate = stem tips that turned from black to green after freezing / total number of stem tips × 100%. Regeneration rate = sprouted stem tips / total number of stem tips × 100%. The above experiments were repeated 3 times and SPSS was used for statistical analysis.

[0116] 3. Test results

[0117] (1) Effect of adding exogenous antioxidants to pre-culture medium on cryopreservation effect

[0118] Obtaining shoot tips with consistent physiological status in a short period of time provides the necessary material conditions for cryopreservation. The quality and physiological status of the shoot tips are crucial to the effect of cryopreservation (i.e., germination rate after freezing), and directly determine whether the experiment can be successful. The composition of the culture medium determines the physiological state of the shoot tips and is also one of the key factors affecting the cryopreservation effect. High concentrations of cytokinins and auxins help accelerate cell division and growth. The addition of exogenous antioxidants helps to resist oxidative damage during freezing and thawing. Figure 1 As shown, the survival rate and regeneration rate were significantly increased by exogenous addition of glutathione, riboflavin and polyethylene glycol PEG-8000.

[0119] (2) Effect of pre-culture time on cryopreservation effect

[0120] Pre-culture plays an important role in enhancing cell resistance. Adding cryoprotectants such as dimethyl sulfoxide can reduce the free water content in cells and prevent cell damage caused by free water crystallization during freezing. Figure 2 As shown, adding appropriate concentrations of polyethylene glycol PEG-8000, glutathione, and riboflavin significantly improves the overall freezing effect. It is speculated that polyethylene glycol PEG-8000 has the effect of osmotic stress, which helps the plant cope with osmotic dehydration in the later stage and improves the overall stress resistance. Low temperature training can also induce the expression of stress resistance genes in Melaleuca alternifolia, thereby improving its ability to tolerate low temperatures.

[0121] The study showed that the survival rate and regeneration rate of 3d pre-culture were the highest, at 53% and 41%, respectively, followed by 4d, with a survival rate and regeneration rate of 37.67% and 31.33%, respectively.

[0122] (3) Effect of pre-culture temperature on cryopreservation effect

[0123] Effect of pre-culture temperature on cryopreservation effect Figure 3 As shown, the pre-culture cryopreservation effect at 10℃ was the best, with the survival rate and regeneration rate reaching 50% and 39%, respectively, followed by 4℃ and 20℃.

[0124] (4) Effect of loading time on freezing effect

[0125] On the one hand, loading can dehydrate tissue cells, and on the other hand, it can increase the tolerance of cryoprotectants such as PVS2 during the next cryoprotectant treatment. Figure 4 As shown in the figure, the survival rate and regeneration rate were the highest when the LS loading solution (basic medium + 2M glycerol + 0.4M sucrose) was loaded for 20 minutes. Prolonging or shortening the loading time will affect the freezing effect.

[0126] (5) Effect of cryoprotectant composition on cryopreservation effect

[0127] like Figure 5 As shown, different cryoprotectant components were added to explore the effect on the freezing effect. When 100ug / ml proanthocyanidins and 0.05g / L glutathione were added, the survival rate and regeneration rate could reach 71.33% and 63%, which were significantly better than no addition or single addition.

[0128] (6) Effect of dehydration time on cryopreservation effect

[0129] The vitrification solution treatment further dehydrates the cells and lowers the vitrification temperature, which is an extremely important step and the last step before the stem tip is placed in liquid nitrogen. The present invention conducted a single factor experiment of dehydration time (20, 40, 60, 80 min), and the experimental results are as follows Figure 6 As shown in the figure, after 40 minutes of dehydration, the survival rate and regeneration rate reached the maximum value, which were 63.33% and 52% respectively. Increasing or shortening the dehydration time significantly decreased the survival rate and regeneration rate.

[0130] The present invention obtains the following preferred embodiments through the above-mentioned process parameters optimized by exploration:

[0131] A method for freezing and preserving sterile stem tips of terpinen-4-ol type Melaleuca alternifolia, comprising the following steps:

[0132] (1) Obtaining sterile stem tips: The established tissue culture test tube seedlings were used as materials (see Figure 7), take the 4.1cm-high terpinene-4-ol-type Melaleuca alternifolia tissue culture seedling clusters (tissue culture seedling clusters are tissue culture seedlings with 10 subcultures) after 32 days of subculture, cut them into individual plants, and place them in proliferation medium 1: modified MS + 4.0mg / L 6-benzylaminoadenine + 1.0mg / L naphthaleneacetic acid + 60000mg / L sucrose + 4500mg / L agar + 50g / L polyethylene glycol PEG-8000 + proanthocyanidin B2 + 0.05g / L glutathione, culture for 15 days, shade treatment for the first 4 days, and then gradually increase the light, the temperature is 25-30℃. Wait for axillary buds to grow (see Figure 8 ), when the length is 2 mm, use a sterile surgical blade and stainless steel high-precision ultra-tip tweezers (the tip of the chuck is 0.1 mm) to cut off the excess tissue at the bottom of the stem, retaining the stem apical meristem and three leaf primordia (see Fig. 9 ), cut into 1mm.

[0133] (2) Pretreatment of sterile shoot tips: Place the sterile shoot tips in culture medium 2 and culture in an incubator for 3 days.

[0134] The formula of the culture medium 2 described in the above step (2) is: modified MS + 0.25g / L activated carbon + 0.03g / L riboflavin + 50g / L polyethylene glycol PEG-8000 + 0.05g / L glutathione + 50g / L dimethyl sulfoxide + 102.6g / L sucrose.

[0135] The culture conditions described in the above step (2) are: temperature of 10°C and illumination of 8000 Lux.

[0136] (3) Loading of sterile shoot tips: 2 M glycerol + 0.4 M sucrose for 20 min.

[0137] (4) Dehydration of sterile shoot tips: Place the shoot tips in a cryotube containing 1.5 mL of optimized PVS2 solution and precool at 4°C, then dehydrate by shaking at 200 rpm for 40 min.

[0138] The optimized PVS2 formula described in step (4) above is 10% coconut water + 300g / L glycerol + 130g / L sucrose + 100g / L ethylene glycol + 75g / L dimethyl sulfoxide.

[0139] (5) Cryopreservation: Place the shoot tip in a cryopreservation tube and store in liquid nitrogen for 48 hours.

[0140] (6) Rapidly thaw in a 40°C water bath for 2 min.

[0141] (7) 1.2 M sucrose unloading for 20 min.

[0142] (8) Revert the stem tip (see Fig.10) were transferred to recovery medium and cultured for 15 days. The stem tip was observed to turn from black to green (see Fig.11 ), and continue to culture for 1 month to obtain regenerated plants (see Fig.12 ).

[0143] The recovery medium formula described in the above step (8) is: modified MS + 6-benzylaminoadenine 2.0 mg / L + naphthaleneacetic acid 0.5 mg / L + sucrose 30000 mg / L + agar 3500 mg / L + 100 ug / ml proanthocyanidin B2 + glutathione 0.05 g / L + 10% coconut water.

[0144] Although in theory, biological materials can be preserved for a long time at ultra-low temperatures, many factors such as temperature and osmotic pressure during the freezing process can easily cause damage or even death to biological materials. The main damage caused by ultra-low temperature preservation includes but is not limited to the toxic effects of cryoprotectants, osmotic stress caused by excessive dehydration, ice crystal damage, and oxidative damage caused by ROS accumulation. Among the many damage theories, oxidative damage has been proven to be one of the important reasons affecting the effect of ultra-low temperature preservation. Oxidative damage can not only cause cell membrane rupture, protein denaturation, carbohydrates and nucleic acids (extravasation), but also cause DNA damage, which seriously affects the survival rate and regeneration ability of plant tissues.

[0145] Especially for some woody plants that are not naturally cold-resistant, since they have relatively weak adaptability to extreme environmental conditions, the success rate of preservation under the above-mentioned various stresses is often low, and even if successful, it is difficult to obtain complete regenerated plants. In response to these existing problems, the present invention has carefully screened the components and dosage of the culture medium through in-depth innovation and optimization. In this process, the present invention has carefully proportioned and tested a variety of nutrients in order to find the optimal combination that can maximize the success rate of plant tissue preservation. At the same time, the present invention also optimizes a series of key process parameters, including storage temperature, light conditions, etc., to ensure that the plant tissue can maintain the best physiological state during storage.

[0146] By using the cryopreservation method provided by the present invention, plant tissues treated with dehydration, cold-resistance and antioxidant treatment can still show strong vitality in the recovery culture stage. They can not only take root and sprout smoothly, but also grow into healthy plants with a survival rate of more than 70%. This achievement not only provides an extremely effective method for the long-term preservation of plant germplasm resources, but also enables these valuable genetic resources to be safely preserved in the next few years.

[0147] More importantly, when certain species are facing the threat of extinction or endangerment, or when there is an urgent need for specific plant resources in the fields of scientific research and breeding, the innovative method of the present invention can be used to achieve the recovery growth and seedling formation of plant tissues. This has extremely important value and significance for the long-term preservation and efficient utilization of plant genetic resources. In addition, the successful practice of the present invention also provides useful reference and inspiration for the long-term preservation and utilization of other organisms (such as animals, microorganisms, etc.), and has made important contributions to promoting the development of biodiversity conservation and research.

Claims

1. A method for freezing and preserving sterile stem tips of 4-terpinene-1-ol type Melaleuca alternifolia, characterized in that: The following steps are involved: (1) Obtaining sterile stem tips: Using the tissue culture test tube seedlings established above as materials, a cluster of 4-5 cm high terpinene-4-ol-type Melaleuca alternifolia tissue culture seedlings of 30-45 days old were taken, cut into individual plants, and placed in proliferation medium 1 for 10-20 days. The plants were shaded for the first 3-5 days, and then the light was gradually increased. The temperature was 25-30°C. When axillary buds grew out and were 1-3 mm in length, the excess tissue at the bottom of the stem was cut off using a sterile surgical blade and stainless steel high-precision ultra-tip tweezers, and the stem apex meristem and 2-5 leaf primordia were retained, and the plants were cut into 1-3 mm pieces. (2) Pretreatment of sterile stem tips: Place the sterile stem tips in medium 2 and culture in an incubator for 3-4 days; (3) Loading of sterile stem tips: using glycerol + sucrose for 20-30 min; (4) Dehydration of sterile shoot tips: Place the shoot tips in a cryovial containing 1.5-2 mL of optimized PVS2 solution at 2-6°C and then dehydrate at 150-250 rpm for 40-60 min. (5) Cryopreservation: Place the stem tip in a cryopreservation tube and store in liquid nitrogen for more than 48 hours; (6) Rapid thawing in a 38-42°C water bath for 1-2 minutes; (7) Unloading with sucrose for 20-26 min; (8) Recovery Culture: Transfer the shoot tip to the recovery culture medium and carry out recovery culture for 15-30 days. The shoot tip will turn from black to green. Continue to culture and regenerate plants.

2. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The tissue culture seedling clusters subcultured for 30-45 days in step (1) are tissue culture seedlings subcultured 4-15 times.

3. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: Proliferation medium 1 described in step (1): modified MS + 6-benzylaminoadenine 2.0-4.0 mg / L + naphthaleneacetic acid 0.5-1.0 mg / L + sucrose 30000-80000 mg / L + agar 3500-4500 mg / L + 0-100 g / L polyethylene glycol PEG-8000 + 100ug / ml proanthocyanidin B2+0-0.1g / L glutathione.

4. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The formula of the culture medium 2 described in step (2) is: modified MS + 0.25 g / L activated carbon + 0.03 g / L riboflavin + 50 g / L polyethylene glycol PEG-8000 + 0.05 g / L glutathione + 50 g / L dimethyl sulfoxide + 102.6 g / L sucrose.

5. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The culture conditions described in step (2) are: temperature of 4-10°C and illumination of 7000-9000 Lux.

6. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The glycerol concentration described in step (3) is 1.8-2.2M.

7. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The concentration of sucrose in step (3) is 0.4-0.5M.

8. The method for freezing and preserving the sterile stem tip of 4-terpinene-1-ol Melaleuca alternifolia according to claim 1, characterized in that: The optimized PVS2 formula described in step (4) is 10% coconut water + 300g / L glycerol + 130g / L sucrose + 100g / L ethylene glycol + 75g / L dimethyl sulfoxide.

9. The method for freezing and preserving the sterile stem tip of terpinene-4-ol type Melaleuca alternifolia according to claim 1, characterized in that: The concentration of sucrose in step (7) is 1.1-1.3M.

10. The method for freezing and preserving the sterile stem tips of 4-terpinene-1-ol Melaleuca alternifolia according to claim 1, characterized in that: The recovery medium formula described in step (8) is: modified MS + 6-benzylaminoadenine 2.0 mg / L + naphthaleneacetic acid 0.5 mg / L + sucrose 30000 mg / L + agar 3500 mg / L + 100 ug / ml proanthocyanidin B2 + glutathione 0.05 g / L + 10% coconut water.

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