Ultralow-temperature preservation method of lycoris bulblets

Abstract

The invention provides an ultralow-temperature preservation method of lycoris bulblets, and belongs to the technical field of plant tissue culture. The method comprises the following steps: 1, treating an explant; 2, establishing a tissue culture system; 3, subculturing and inducing the bulblets; 4, pre-culturing the bulblets; 5, cutting bulblets and fixing loading strips; 6, loading and vitrification treatment; 7, freezing, unfreezing and washing; and 8, recovering culture. The rapid tissue propagation and ultralow-temperature preservation method of the lycoris radiate is convenient and efficient, and has reference significance on ultralow-temperature preservation of the lycoris radiate and other bulb and perennial root plant genetic resources.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method for cryopreservation of Lycoris radiata bulbs. Background Technology

[0002] Lycoris genus ( Lycoris Lycoris radiata is a type of perennial herbaceous plant with important ornamental and medicinal value, belonging to the Amaryllidaceae family. This genus includes several species such as *Lycoris chinensis*, *Lycoris longituba*, *Lycoris incarnata*, and *Lycoris radiata*, which mostly grow in shady and damp places on hillsides. Due to their unique flower shapes and rich colors, Lycoris plants have extremely high ornamental value and occupy an important position in landscaping and the floriculture industry. At the same time, their bulbs contain abundant alkaloids, such as lycorine, lycoramine, galantamine, and lycoramine, which have important medicinal value in the treatment of Alzheimer's disease, anti-inflammatory and analgesic applications.

[0003] For plant genetic resources that are difficult to propagate through vegetative means, such as Lycoris radiata, garlic, strawberries, and tubers, preservation is generally achieved through field cultivation in seed nurseries or conservation nurseries. This method not only requires arable land, is time-consuming and labor-intensive, but also carries the risk of germplasm variation or destruction due to natural disasters such as drought, floods, low temperatures, heat damage, and pests, as well as unsuitable artificial domestication and cultivation. Therefore, it is not suitable for the long-term preservation of germplasm resources.

[0004] Cryopreservation refers to the preservation of plant genetic resources at liquid nitrogen (-196℃). Under these conditions, the biochemical activity of plants almost ceases, and physiological and genetic changes during storage can be controlled to a minimum, making it the optimal choice for long-term preservation of plant genetic resources. It avoids the vulnerability of field storage to natural disasters leading to germplasm variation or destruction, as well as the risks of genetic trait variation or contamination caused by multiple generations in tissue culture preservation. Research on plant cryopreservation has made continuous progress in diversification and simplification of preservation techniques since the 1990s, and the number of plant species that can be preserved has been steadily increasing. However, most research focuses on the cryopreservation of plant genetic resources, primarily woody plants such as apples, cherries, citrus, bananas, and cassava, as well as tuber plants such as potatoes, sweet potatoes, and strawberries. Currently, there are few international reports on cryopreservation techniques for Lycoris radiata. Lin Tian et al. first adopted a series of measures to improve shoot tip resistance, reduce damage in each step, and used direct liquid nitrogen injection instead of a programmed cooling process, successfully preserving Lycoris radiata (red Lycoris) for the first time. Lycoris radiate HerbUsing it as a model plant, a shoot tip cryopreservation system was established, and a patent was obtained in 2008 (a cryopreservation method for vegetatively propagated Lycoris radiata, patent number: ZL200610113832.X).

[0005] However, the original patent still has the following drawbacks: 1. During initial aseptic inoculation, the explants sampled are mostly 3-4 year old bulbs. The act of digging up the bulbs from the soil itself damages the germplasm resources and the living environment. Furthermore, due to the soil sampling, mold and endophytic bacteria contamination is severe; 2. The stem tip peeling step requires high technical skills, needing to peel off the tightly surrounding scales layer by layer and cut the stem tip of appropriate size (see attached). Figure 1 1) The cutting process can easily cause secondary damage to the delicate stem tip tissue; 2) It is difficult to obtain usable stem tips of the same length (it takes 3 months for small bulbs suitable for peeling stem tips to grow), and it is also difficult to obtain a large number of uniform materials. Therefore, it is necessary to further optimize the cryopreservation materials, simplify the operation steps, and reduce the difficulty of operation. Summary of the Invention

[0006] In view of this, the present invention provides a method for rapid tissue culture propagation and cryopreservation of Lycoris radiata. The present invention selects small bulbs of 2-3 mm for cryopreservation to solve the above-mentioned defects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for cryopreservation of Lycoris radiata bulbs, comprising the following steps: S1. Explant treatment: Take unopened flowers of Lycoris radiata, disinfect them with alcohol solution, then disinfect them with sodium hypochlorite solution, and then rinse them with water 3 to 5 times. S2. Establishment of tissue culture system: The receptacle and ovary were separated and inoculated onto a culture medium to induce small bulbils. The culture medium was MS + 0.4~0.6 mg / L NAA + 2.8~3.2 mg / L 6-BA, the culture temperature was 23~27℃, the photoperiod was 12L:12D, and the light intensity was 34~38 μmol / (m²). 2 ·s), small bulbs are obtained in 90-120 days, and subculture is carried out every 30-60 days; S3. Subculture and induction of small bulbs: Small bulbs subcultured for 90-100 days were longitudinally cut into 3-4 pieces and inoculated with stem discs onto MS medium containing 0.5-2 mol / L 6-BA and 0.1-0.5 mol / L NAA. Small bulbs of 2-3 mm were induced in 10-25 days. S4. Pre-culture of small bulbs: Stem discs with small bulbs were inoculated into MS medium containing 0.4~0.6 mol / L sucrose and cultured in the dark at 24~26℃ for 5 days. Then, they were transferred to MS medium containing 0.6~0.8 mol / L sucrose and 0.5~5 mg / LABA and pre-cultured for 1~3 days. S5. Bulb cutting and fixation on loading strips: Cut 2-3 mm bulbs, dip them in 2-4% sodium alginate solution, place them on aluminum foil loading strips, and immerse the loading strips in MS medium containing 0.1-0.3% calcium chloride, 0.3-0.5 mol / L sucrose, and 1.8-2.2 mol / L glycerol for fixation; S6. Loading and vitrification: After loading the fixed loading strips with small bulbs in the pretreatment solution for 55-65 minutes, transfer them to the vitrification protectant for water bath treatment. S7. Freezing and Thawing Washing: Place the loading strips with small bulbs into liquid nitrogen for storage. If long-term storage is required, transfer the loading strips to cryovials. When thawing, place the loading strips into liquid culture medium containing MS + 1.2 mol / L sucrose to thaw and wash. S8. Recovery culture: Dry the liquid on the surface of the loading strip, peel the small bulbs from the loading strip, inoculate them into the recovery culture medium, and culture in the dark at 24~26℃ for 7~8 days. Then, transfer them to the conditions of light intensity of 34~38μmol / (m²·s) and light-dark cycle of 12L:12D for culture.

[0008] Preferably, the volume fraction of the alcohol solution is 68-72%.

[0009] Preferably, the concentration of the sodium hypochlorite solution is 1.8-2.2%, and the disinfection time of the sodium hypochlorite solution is 13-17 minutes.

[0010] Preferably, the small bulbs in step S5 are cut under a microscope.

[0011] Preferably, the aluminum foil loading strip has a specification of 5mm × 20m, with 5 small bulbs on each loading strip.

[0012] Preferably, the fixed time in S5 is 10-30 min; the water bath treatment time in S6 is 1-2 h; and the washing time in S7 is 18-22 min.

[0013] Preferably, the pretreatment solution in S6 is MS liquid culture medium containing 2 mol / L glycerol and 0.4 mol / L sucrose.

[0014] Preferably, the vitrifying agent for S6 is PVS2.

[0015] Preferably, the recovery culture medium contains 0.5~2 mg / L BA + 0.1~0.5 mg / L MS semi-solid medium containing NAA + 0.5 mg / L GA3.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: (1) The present invention induces clustered buds by using the receptacle and ovary tissue of the above-ground part, thus avoiding damage to the bulbs of Lycoris radiata by the digging step and reducing fungal and bacterial contamination of the soil; (2) The small bulbs or stem tips used as ultra-low temperature preservation materials are propagated under the existing tissue culture system. In this invention, the small bulbs are used as explants for induction and a large number of uniform sterile materials can be obtained in just 15 to 25 days, while the stem tips require 3 months, thus shortening the cultivation cycle. (3) The present invention uses the induced small bulbs as cryopreservation material, which eliminates the cumbersome technical means of peeling the stem tip and further avoids damage to the stem tip growth point during the cutting operation; thereby further improving the survival rate.

[0017] (4) The method provided by this invention provides technical support for establishing a convenient, stable and efficient cryopreservation system for Lycoris radiata, which is conducive to the large-scale preservation of Lycoris radiata resources and has great reference value for the construction of a cryopreservation bank for wild resources. Attached Figure Description

[0018] Figure 1 This is a diagram illustrating the peeling of the stem tip of Lycoris radiata.

[0019] Figure 2 This is a cross-section of a Lycoris bulb after 3 months of growth.

[0020] Figure 3 Flowchart for rapid propagation and cryopreservation of Lycoris radiata tissue culture.

[0021] Figure 4 The induction and cryopreservation of Lycoris bulbils are shown in the following images: a) clustered buds induced by the receptacle; b) bulbils induced for 20 days; c) bulbils fixed in a loading strip; and d) regenerated buds after cryopreservation and recovery culture. Detailed Implementation

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1 1. Disinfection of Lycoris radiata and Lycoris radiata Take unopened flowers of Lycoris radiata, disinfect the surface with 68% alcohol, disinfect with 1.8% sodium hypochlorite for 17 minutes, and rinse 5 times with sterile water.

[0024] 2. Establishment of tissue culture system The receptacle and ovary were separated and inoculated onto MS medium supplemented with 0.4 mg / L NAA and 3.2 mg / L 6-BA to induce small bulblets; the culture temperature was 23–27 °C, the photoperiod was 12 L:12 D, and the light intensity was 34 μmol / (m²). 2·s), small bulbs are obtained in 112 days, and the generation is repeated every 30 days.

[0025] 3. Induction of small bulbs of Lycoris radiata Take the small bulbs from step 2, subcultured for 90 days, and cut them longitudinally into four pieces. Place them, along with their stem discs, into MS medium containing 0.5 mol / L 6-BA and 0.5 mol / L NAA, and culture at 24–26°C in the dark. After 20 days of culture, small white granular bulbs of 2–3 mm in size will be visible.

[0026] 4. Pre-culture of small bulbs Stem discs with small bulbs were inoculated into MS medium containing 0.5 mol / L sucrose and cultured in the dark at 24-26°C for 5 days. Then, they were transferred to MS medium containing 0.6 mol / L sucrose and 5 mg / LABA and pre-cultured for 3 days.

[0027] 5. Cutting and securing small bulbs to loading strips Tissue blocks with small bulbils obtained in Example 2 were used. 2-3 mm bulbils were cut under a dissecting microscope, lightly dipped in a 2-4% calcium-free sodium alginate solution, and neatly arranged on 5 mm × 20 mm aluminum foil loading strips, with 5 bulbils on each strip. The loading strips were then fixed in MS medium containing 0.1% calcium chloride, 0.3 mol / L sucrose, and 1.8 mol / L glycerol. After 30 min of fixation, excess liquid was absorbed onto sterile filter paper.

[0028] 6. Loading and vitrification The fixed loading strips with small bulbs were placed in the pretreatment solution and loaded for 55 min, then transferred to the vitrification protectant PVS2 and treated in a water bath for 1 h. The pretreatment solution was MS liquid medium containing 2 mol / L glycerol and 0.4 mol / L sucrose.

[0029] 7. Freezing, thawing, and washing The loading strips with small bulbs were placed in liquid nitrogen for storage. For long-term storage, the loading strips were transferred to 2 mL cryovials. When thawing, the loading strips were placed in liquid culture medium containing MS + 1.2 mol / L sucrose and washed for 20 min.

[0030] 8. Resume culture After blotting the liquid off the loading strip with filter paper, the bulblets were peeled off the loading strip and inoculated into the recovery medium, which contained 2 mg / L BA + 0.1 mg / L BA. The medium was prepared in MS semi-solid medium containing NAA and 0.5 mg / L GA3 and cultured in the dark at 25°C for 7 days. Then, it was transferred to a light source with an intensity of 34 μmol / (m²) 2 Cultured under conditions of 12 / 12 h light / dark cycle (·s), the survival rate was 65% after 14 days.

[0031] Example 2 1. Disinfection of Lycoris radiata and Lycoris radiata Take unopened flowers of Lycoris radiata, disinfect the surface with 70% alcohol, disinfect with 2% sodium hypochlorite for 15 minutes, and rinse 5 times with sterile water.

[0032] 2. Establishment of tissue culture system The receptacle and ovary were separated and inoculated onto MS medium supplemented with 0.5 mg / L NAA and 3 mg / L 6-BA to induce small bulblets; the culture temperature was 23–27 °C, the photoperiod was 12 L:12 D, and the light intensity was 36 μmol / (m²). 2 ·s), small bulbs are obtained in 100 days, and the generation is repeated every 45 days.

[0033] 3. Induction of small bulbs of Lycoris radiata Take the small bulbs from step 2, subcultured for 90 days, and cut them longitudinally into four pieces. Place them with the stem disc in MS medium containing 1 mol / L 6-BA and 0.3 mol / L NAA, and culture at 24-26℃ in the dark. After 15 days of culture, small white granular bulbs of 2-3 mm in size will be visible.

[0034] 4. Pre-culture of small bulbs Stem discs with small bulbs were inoculated into MS medium containing 0.5 mol / L sucrose and cultured in the dark at 24-26℃ for 5 days. Then, they were transferred to MS medium containing 0.7 mol / L sucrose and 3 mg / LABA and pre-cultured for 3 days.

[0035] 5. Cutting and securing small bulbs to loading strips Tissue blocks with small bulbils obtained in Example 2 were used. 2-3 mm bulbils were cut under a dissecting microscope, lightly dipped in 3% calcium-free sodium alginate solution, and neatly placed on 5 mm × 20 mm aluminum foil loading strips, with 5 bulbils on each strip. The loading strips were then fixed in MS medium containing 0.2% calcium chloride, 0.4 mol / L sucrose, and 2 mol / L glycerol. After 30 min of fixation, excess liquid was blotted off on sterile filter paper.

[0036] 6. Loading and vitrification The fixed loading strips with small bulbs were placed in the pretreatment solution and loaded for 60 min, then transferred to the vitrification protectant PVS2 and treated in a water bath for 1.5 h. The pretreatment solution was MS liquid medium containing 2 mol / L glycerol and 0.4 mol / L sucrose.

[0037] 7. Freezing, thawing, and washing The loading strips with small bulbs were placed in liquid nitrogen for storage. For long-term storage, the loading strips were transferred to 2 mL cryovials. When thawing, the loading strips were placed in liquid culture medium containing MS + 1.2 mol / L sucrose and washed for 20 min.

[0038] 8. Resume culture After blotting the liquid off the loading strip with filter paper, the bulblets were peeled off the loading strip and inoculated into the recovery medium, which contained 1 mg / L BA + 0.3 mg / L BA. The medium was prepared in MS semi-solid medium containing NAA and 0.5 mg / L GA3 and cultured in the dark at 25°C for 7 days. Then, it was transferred to a light source with an intensity of 36 μmol / (m²) 2 Cultured under conditions of 12 / 12 h light / dark cycle (·s), the survival rate was 80% after 14 days.

[0039] Example 3 1. Disinfection of Lycoris radiata and Lycoris radiata Take unopened flowers of Lycoris radiata, disinfect the surface with 72% alcohol, disinfect with 2.2% sodium hypochlorite for 15 minutes, and rinse 5 times with sterile water.

[0040] 2. Establishment of tissue culture system The receptacle and ovary were separated and inoculated onto MS medium supplemented with 0.6 mg / L NAA and 3.2 mg / L 6-BA to induce small bulblets; the culture temperature was 23–27 °C, the photoperiod was 12 L:12 D, and the light intensity was 38 μmol / (m²). 2 ·s), small bulbs are obtained in 106 days, and the generation is repeated every 60 days.

[0041] 3. Induction of small bulbs of Lycoris radiata Take the small bulbs from step 2, subcultured for 100 days, and cut them longitudinally into four pieces. Place them with the stem disc in MS medium containing 2 mol / L 6-BA and 0.1 mol / L NAA, and culture at 24-26℃ in the dark. After 17 days of culture, small white granular bulbs of 2-3 mm in size will be visible.

[0042] 4. Pre-culture of small bulbs Stem discs with small bulbs were inoculated into MS medium containing 0.5 mol / L sucrose and cultured in the dark at 24-26°C for 5 days. Then, they were transferred to MS medium containing 0.8 mol / L sucrose and 0.5 mg / LABA and pre-cultured for 3 days.

[0043] 5. Cutting and securing small bulbs to loading strips Tissue blocks with small bulbils obtained in Example 2 were used. 2-3 mm bulbils were cut under a dissecting microscope, lightly dipped in 4% calcium-free sodium alginate solution, and neatly arranged on 5 mm × 20 mm aluminum foil loading strips, with 5 bulbils on each strip. The loading strips were then fixed in MS medium containing 0.3% calcium chloride, 0.5 mol / L sucrose, and 2.2 mol / L glycerol. After 30 min of fixation, excess liquid was absorbed onto sterile filter paper.

[0044] 6. Loading and vitrification The fixed loading strips with small bulbs were placed in the pretreatment solution and loaded for 65 min, then transferred to the vitrification protectant PVS2 and treated in a water bath for 2 h. The pretreatment solution was MS liquid medium containing 2 mol / L glycerol and 0.4 mol / L sucrose.

[0045] 7. Freezing, thawing, and washing The loading strips with small bulbs were placed in liquid nitrogen for storage. For long-term storage, the loading strips were transferred to 2 mL cryovials. When thawing, the loading strips were placed in liquid culture medium containing MS + 1.2 mol / L sucrose and washed for 20 min.

[0046] 8. Resume culture After blotting the liquid off the loading strip with filter paper, the bulblets were peeled off the loading strip and inoculated into the recovery medium, which contained 2 mg / L BA + 0.1 mg / L BA. The medium was prepared in MS semi-solid medium containing NAA and 0.5 mg / L GA3 and cultured in the dark at 25°C for 7 days. Then, it was transferred to a light source with an intensity of 38 μmol / (m²) 2 Cultured under conditions of 12 / 12 h light / dark cycle (·s), the survival rate was 73% after 14 days.

[0047] As can be seen from the above embodiments, the present invention provides a method for cryopreservation of Lycoris radiata bulbs, providing technical guidance for establishing a convenient, stable and efficient cryopreservation system for Lycoris radiata.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of Lycoris radiata bulbs, characterized in that, Includes the following steps: S1. Explant treatment: Take unopened flowers of Lycoris radiata, disinfect them with alcohol solution, then disinfect them with sodium hypochlorite solution, and then rinse them with water 3 to 5 times. S2. Establishment of tissue culture system: The receptacle and ovary were separated and inoculated onto a culture medium to induce small bulbils. The culture medium was MS + 0.4~0.6 mg / L NAA + 2.8~3.2 mg / L 6-BA, the culture temperature was 23~27℃, the photoperiod was 12L:12D, and the light intensity was 34~38 μmol / (m²). 2 ·s), small bulbs are obtained in 90-120 days, and subculture is carried out every 30-60 days; S3. Subculture and induction of small bulbs: Small bulbs subcultured for 90-100 days were longitudinally cut into 3-4 pieces and inoculated with stem discs onto MS medium containing 0.5-2 mol / L 6-BA and 0.1-0.5 mol / L NAA. Small bulbs of 2-3 mm were induced in 10-25 days. S4. Pre-culture of small bulbs: Stem discs with small bulbs were inoculated into MS medium containing 0.4~0.6 mol / L sucrose and cultured in the dark at 24~26℃ for 5 days. Then, they were transferred to MS medium containing 0.6~0.8 mol / L sucrose and 0.5~5 mg / LABA and pre-cultured for 1~3 days. S5. Bulb cutting and fixation on loading strips: Cut 2-3 mm bulbs, dip them in 2-4% sodium alginate solution, place them on aluminum foil loading strips, and immerse the loading strips in MS medium containing 0.1-0.3% calcium chloride, 0.3-0.5 mol / L sucrose, and 1.8-2.2 mol / L glycerol for fixation; S6. Loading and vitrification: After loading the fixed loading strips with small bulbs in the pretreatment solution for 55-65 minutes, transfer them to the vitrification protectant for water bath treatment; S7. Freezing and Thawing Washing: Place the loading strips with small bulbs into liquid nitrogen for storage. If long-term storage is required, transfer the loading strips to cryovials. When thawing, place the loading strips into liquid culture medium containing MS + 1.2 mol / L sucrose to thaw and wash. S8. Recovery culture: Dry the liquid on the surface of the loading strip, peel the small bulbs from the loading strip, inoculate them into the recovery culture medium, and culture in the dark at 24~26℃ for 7~8 days. Then, transfer them to the conditions of light intensity of 34~38μmol / (m²·s) and light-dark cycle of 12L:12D for culture.

2. The cryogenic preservation method according to claim 1, characterized in that, The volume fraction of the alcohol solution is 68-72%.

3. The cryogenic preservation method according to claim 1, characterized in that, The concentration of the sodium hypochlorite solution is 1.8-2.2%, and the disinfection time of the sodium hypochlorite solution is 13-17 minutes.

4. The cryogenic preservation method according to claim 1, characterized in that, In step S5, the small bulbs are cut off under a microscope.

5. The cryogenic preservation method according to claim 1, characterized in that, The aluminum foil loading strip has a specification of 5mm×20m, and each loading strip has 5 small bulbs.

6. The cryogenic preservation method according to claim 1, characterized in that, The fixed time in S5 is 10~30 min; the water bath treatment time in S6 is 1~2 h; the washing time in S7 is 18~22 min.

7. The cryogenic preservation method according to claim 1, characterized in that, The pretreatment solution described in S6 is MS liquid medium containing 2 mol / L glycerol and 0.4 mol / L sucrose.

8. The cryogenic preservation method according to claim 1, characterized in that, The glass transition protectant for S6 is PVS2.

9. The cryogenic preservation method according to claim 1, characterized in that, The recovery medium is an MS semi-solid medium containing 0.5~2 mg / L BA + 0.1~0.5 mg / L NAA + 0.5 mg / L GA3.

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