Breeding method for improving drought resistance of blueberries

Abstract

The invention discloses a breeding method for improving the drought resistance of blueberries, which comprises the following step of: overexpressing poplar mevalonate diphosphate decarboxylase genes in the blueberries by an agrobacterium-mediated method. The overexpression plant can increase the root length, the root diameter and the root volume of the blueberry under the drought stress condition; the conductivity of the blueberry roots is reduced; the content of malondialdehyde in the blueberries is reduced; the hydrogen peroxide content of blueberries is reduced; the carbon dioxide assimilation rate of the blueberries is increased; the proline content of the blueberries is increased; the superoxide dismutase of the blueberries is improved; the blueberry peroxidase is improved. The method has an application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering, and relates to a breeding method for improving drought resistance of blueberries. BACKGROUND

[0002] Blueberry is a deciduous shrub of the family Ericaceae, the genus Vaccinium, small branches green, with ribs or cylindrical, usually with rows of soft hair; leaf blades are leathery, ovate to elliptical, acute at the apex, cherry-shaped to nearly round at the base, with serrated edges to entire edges, dark green, hairless on the upper surface, hairy on the lower surface; racemose inflorescences are born on last year's branches, flowers are green and hairless; corolla is white to pink, nearly cylindrical; filaments are usually hairy. The berry is spherical, dark blue, hairless, covered with white powder; the flowering period is from June to July; the fruiting period is from August to September.

[0003] Blueberry is originally from the eastern part of Canada and the eastern and southern parts of the United States, and has been introduced in Japan, China, New Zealand and Europe. Blueberry prefers warm climate, is relatively resistant to high temperature, likes semi-sunny environment, likes humid environment, is generally resistant to drought and waterlogging, and is suitable for planting in fertile, loose and organic-rich acid soil. The main propagation method of blueberry is cutting.

[0004] Blueberry tastes sweet and sour, is rich in nutrients, and has the functions of preventing brain aging, protecting vision, strengthening heart, resisting cancer, softening blood vessels, and enhancing human immunity.

[0005] Drought is a major environmental stress factor affecting plant growth and development. Water deficiency can cause a series of changes at morphological, physiological, biochemical and molecular levels, and has adverse effects on plant growth, health and productivity. Drought can cause physiological damage to blueberry, affect economic performance, and even death. SUMMARY

[0006] In order to solve the problems in the prior art, the present application provides a breeding method for improving drought resistance of blueberry, which is that a poplar mevalonate diphosphate decarboxylase gene is overexpressed in blueberry by genetic engineering means.

[0007] In some embodiments, the amino acid sequence of the protein encoded by the poplar mevalonate diphosphate decarboxylase gene is shown in SEQ ID NO. 2.

[0008] In some embodiments, the coding sequence of the poplar mevalonate diphosphate decarboxylase gene is shown in SEQ ID NO. 1.

[0009] In some embodiments, the variety of the blueberry is Duke.

[0010] In some embodiments, the improvement of drought resistance of blueberry includes:

[0011] Increasing root length, root diameter and root volume of blueberry;

[0012] Increasing carbon dioxide assimilation rate of blueberry;

[0013] Decreasing root electrical conductivity of blueberry;

[0014] Decreasing malondialdehyde content of blueberry;

[0015] Decreasing hydrogen peroxide content of blueberry;

[0016] Increasing proline content of blueberry;

[0017] Increasing superoxide dismutase content of blueberry;

[0018] Increasing peroxidase content of blueberry.

[0019] In some embodiments, the method comprises the following steps:

[0020] S1: transferring the coding sequence of the poplar mevalonate diphosphate decarboxylase gene into an expression vector to obtain a recombinant vector containing the poplar mevalonate diphosphate decarboxylase gene;

[0021] S2: transforming Agrobacterium with the recombinant vector containing the poplar mevalonate diphosphate decarboxylase gene to obtain recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene;

[0022] S3: infecting the sterilized blueberry leaves with the recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene to obtain infected blueberry leaves;

[0023] S4: placing the infected blueberry leaves on a co-culture medium to obtain pre-cultured leaves;

[0024] The co-culture medium is a WPM-based medium, and the co-culture medium further contains 30-40 g / L sucrose, 4-7 g / L agar, 4-6 mg / L TDZ, and has a pH of 5.0-6.0;

[0025] S5: placing the pre-cultured leaves into a bacterial solution containing the recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene to obtain infected leaves;

[0026] S6: placing the infected leaves on the co-culture medium to obtain co-cultured leaves;

[0027] S7: placing the co-cultured leaves on a callus induction medium to obtain blueberry callus;

[0028] The callus induction medium is a WPM-based medium, and the callus induction medium further comprises 15-25 g / L sucrose, 4-6 g / L agar, 0.8-1.2 mg / L zeatin, 0.8-1.2 mg / L TDZ, 0.4-0.6 mg / L 2,4-D, 20-40 mg / L kanamycin, and has a pH of 5.0-6.0.

[0029] S8: placing the blueberry callus on a bud induction medium to obtain a callus with buds;

[0030] The bud induction medium is a WPM-based medium, and the bud induction medium further comprises 15-25 g / L sucrose, 4-6 g / L agar, 2-4 mg / L zeatin, 0.8-1.2 mg / L TDZ, 20-40 mg / L kanamycin, and has a pH of 5.0-6.0.

[0031] S9: inserting the callus with buds into a rooting medium to obtain a rooted blueberry plant;

[0032] The rooting medium is a 1 / 2WPM-based medium, and the rooting medium further comprises 15-25 g / L sucrose, 4-6 g / L agar, 0.5-0.7 mg / L IBA, 20-40 mg / L kanamycin, and has a pH of 5.0-6.0.

[0033] In some embodiments, the rooted blueberry plant is transplanted into soil to obtain a blueberry plant.

[0034] In some embodiments, the backbone of the expression vector is a pK2GW7 vector.

[0035] In some embodiments, the Agrobacterium is Agrobacterium GV3101.

[0036] In some embodiments, in step S4, the culture condition is dark culture at a temperature of 23-25℃ for 2.5-3.5 days.

[0037] In step S5, the time for the infiltration is 30-50 minutes.

[0038] In step S7, the culture condition is dark culture at a temperature of 23-25℃ for 13-17 days, and then light culture at a light intensity of 2000-2500 lx for 18-22 days.

[0039] In step S8, the light culture is performed at a light intensity of 2000-2500 lx for 18-22 days.

[0040] In step S9, the light culture is performed at a light intensity of 2000-2500 lx for 18-22 days. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 1 is a photo of blueberry callus.

[0042] Figure 2 Figure 2 is a photo of drought stress phenotype control of plants overexpressing mevalonate diphosphate decarboxylase and control plants. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0044] Materials and instruments not described in the present application are conventional materials and instruments in the art, and operation details not described in the present application are conventional operations in the art.

[0045] Example 1: Preparation of plants overexpressing Populus trichocarpa mevalonate diphosphate decarboxylase in blueberry

[0046] The mevalonate diphosphate decarboxylase gene of Populus trichocarpa in the plant genie platform (website: https: / / plantgenie.org / ) has the accession number Potri.019G048900 in the Populus trichocarpa reference genome (variety name Populus trichocarpa v3.1).

[0047] The coding sequence of mevalonate diphosphate decarboxylase is as follows (SEQ ID NO. 1):

[0048]

[0049] The protein sequence of mevalonate diphosphate decarboxylase is as follows (SEQ ID NO. 2):

[0050] MAEKTWVRMVTAQTPTNIAVIKYWGKRDETLILPVNDSISVTLDPAHLCTTTTVAVSPSFDQDRMWLNGKEISLSGGRYQNCLREIRARACAVEDKEKGIKIAKKDWEKLHLHVASYNNFPTAAGLASSAAGFACLVFALAKLMNAKEDNSELSAIARQGSGSACRSLFGGFVKWIMGKAEDGSDSLAVQLVDEKHWDELVIIIAVVSSRQKETSSTTGMRDSVETSLLLQHRAKEVVPKRIKQMEEAIKNRDFGSFAQLSCADSNQFHAVCLDTCPPIFYMNDTSHRIISCVEKWNCSEGTPQVAYTFDAGPNAVLIAHNRKAATQLMQKLLFCFPPSSDADLNSYVIGDKSILKDAGIEDIKDVEALPPPPEIKDAQRCKGDVSYFICTKPGRGPALLSDESQALLHPETGLPK

[0051] II. Cloning of mevalonate diphosphate decarboxylase from Populus trichocarpa

[0052] According to the sequence of the mevalonate diphosphate decarboxylase gene of Populus trichocarpa, cloning primers F1 and R1 were designed. F1 (SEQ ID NO: 3): ATGGCAGAGAAAACATGGGTGA

[0053] R1 (SEQ ID NO: 4): TTTAGGCAGCCCAGTTTCGGGA

[0054] Total RNA was extracted from the stems of Populus trichocarpa cutting seedlings (about 60 cm in height) using the TRIZOL method, and the total RNA was reverse transcribed into cDNA. Using primers F1 and R1, the cDNA was amplified using high-fidelity enzyme Phanta Max Master Mix. The amplification thermal cycle program was set in the PCR instrument as 95°C pre-denaturation for 3 min, 95°C denaturation for 20 s, 56°C annealing for 30 s, 72°C extension for 2 min, denaturation-annealing-extension cycle for 30 times, and finally 72°C complete extension for 15 min. The PCR product was gel purified and recovered to connect a cloning vector and sequenced by Sanger method, and it was verified that the coding sequence was the same as that disclosed by the plantgenie platform.

[0055] III. Construction of the diphosphomevalate decarboxylase gene expression vector

[0056] The pK2GW7 vector was double digested with restriction enzymes PmeI and SpeI (purchased from NEB), and the target band was detected by agarose gel electrophoresis. The linearized pK2GW7 vector fragment was recovered from the gel. The primers F2 and R2 were designed for recombination of the diphosphomevalate decarboxylase gene, and the PCR amplification product was obtained using the recombinant cloning vector containing the aforementioned diphosphomevalate decarboxylase gene as a template. The diphosphomevalate decarboxylase gene PCR amplification product was ligated with the linearized pK2GW7 vector fragment using the homologous recombination enzyme Exnase II (purchased from Nanjing Vazyme), incubated at 37°C for 45 min, and placed on ice for 8 min. The competent E. coli DH5a cells were transformed, and the LB solid medium containing 60 mg / L spectinomycin was used for screening and culture. The primers F3 and R2 were used for PCR detection, and the target band was obtained. The amplification product was amplified with primers F3 and R3, and Sanger sequencing was performed to verify the amplification product. The recombinant pK2GW7 vector with the diphosphomevalate decarboxylase gene sequence was transformed into the Agrobacterium GV3101 competent cells. The primers F3 and R2 were used for single colony detection PCR, and the successful transformation of the single colony was determined by electrophoresis. The positive single colony was cultured to obtain the recombinant Agrobacterium containing the diphosphomevalate decarboxylase gene (bacterial solution 1), which was stored at -80°C for later use.

[0057] F2 (SEQ ID NO: 5): TCGACCTGCAGGCGGCCGCAATGGCAGAGAAAACATGGGT

[0058] R2 (SEQ ID NO: 6): TCCTTGTAATCGTTTGTTTGTTTAGGCAGCCCAGTTTCGG

[0059] F3 (SEQ ID NO: 7): GGACTCCGGTATTTTTACAACAA

[0060] R3 (SEQ ID NO: 8): GTTTACCCGCCAATATATCCTGTCA

[0061] IV. Control Agrobacterium

[0062] The pK2GW7 vector was transformed into the Agrobacterium GV3101 competent cells. The total DNA of the Agrobacterium was extracted, and the positive band was obtained after amplification with primers F3 and R3, indicating successful transformation. The positive single colony was cultured to obtain the recombinant Agrobacterium containing the empty pK2GW7 vector (bacterial solution 2), which was stored at -80°C for later use.

[0063] V. Preparation of blueberry with overexpression of mevalonate diphosphate decarboxylase gene

[0064] (I) Preparation of culture medium

[0065] (1) Co-culture medium

[0066] According to the final concentration of added materials, 35 g / L sucrose, 5.5 g / L agar, 5 mg / L TDZ were added to the WPM basic medium, adjusted to pH 5.4, autoclaved, and cooled for dispensing.

[0067] (2) Callus induction medium

[0068] According to the final concentration of added materials, 20 g / L sucrose, 5 g / L agar, 1 mg / L zeatin, 1 mg / L TDZ, 0.5 mg / L 2,4-D were added to the WPM basic medium, adjusted to pH 5.4, autoclaved, and 30 mg / L kanamycin was added after cooling, and then dispensed.

[0069] (3) Bud induction medium

[0070] According to the final concentration of added materials, 20 g / L sucrose, 5 g / L agar, 3 mg / L zeatin, 1 mg / L TDZ were added to the WPM basic medium, adjusted to pH 5.4, autoclaved, and 30 mg / L kanamycin was added after cooling, and then dispensed.

[0071] (4) Rooting induction medium

[0072] According to the final concentration of added materials, 20 g / L sucrose, 5 g / L agar, 0.6 mg / L IBA were added to the 1 / 2 WPM basic medium, adjusted to pH 5.8, autoclaved, and 30 mg / L kanamycin was added after cooling, and then dispensed.

[0073] (II) Treatment of explants

[0074] Blueberry (variety: Duke) tender leaves were used as explants. First, the blueberry leaves were rinsed with water, then treated with 15% (w / v) sodium hypochlorite aqueous solution for 25 min, rinsed with sterile distilled water for 5 times, and the excess distilled water was absorbed with sterile filter paper.

[0075] (III) Activation of recombinant Agrobacterium

[0076] The foregoing bacterial solution 1 and bacterial solution 2 were streaked on LB solid medium containing 100 mg / L kanamycin and 50 mg / L rifampicin, respectively, and cultured at 30°C for 3 days. One well-grown single colony from each plate was inoculated into 50 mL of LB liquid medium containing 100 mg / L kanamycin and 50 mg / L rifampicin, and cultured at 30°C in a shaking incubator at 200 rpm. When the OD600 The value is 0.4. At room temperature, centrifuge at 5000 rpm for 10 minutes to remove the supernatant and collect the bacterial precipitate. Resuspend the bacterial precipitate with 100 mL of WPM liquid medium, and shake culture to reach OD 600 The value is between 0.2 and 0.3 to obtain bacterial liquid 3 (from bacterial liquid 1) and bacterial liquid 4 (from bacterial liquid 2).

[0077] (Four) pre-culture

[0078] Place the sterilized blueberry leaves on the co-culture medium, and pre-culture the leaves at 24°C in the dark for 3 days to soften the leaves.

[0079] (Five) infection

[0080] Place the pre-cultured leaves in bacterial liquid 3 and bacterial liquid 4, respectively, and shake gently at 160 rpm in a shaker for 40 minutes at 24°C. After infection, discard the bacterial liquid and absorb the surface of the infected leaves on sterile filter paper.

[0081] (Six) co-culture

[0082] Inoculate the infected leaves in the co-culture medium and culture in the dark at 24°C for 3 days. After co-culture, wash the leaves with sterile water containing 150 mg / L kanamycin and 300 mg / L cephalosporin three times.

[0083] (Seven) callus induction

[0084] Inoculate the co-cultured blueberry leaves on the callus induction medium, and make the wound of the leaves fully contact with the medium. Culture in the dark at 25°C for 15 days, then light culture under the condition of light intensity 2000-2500 lx, with 16 hours of light per day. After 20 days of light culture, callus tissue grows on the edge of the blueberry leaves. See Figure 1 .

[0085] (Eight) bud induction culture

[0086] Inoculate the callus tissue on the bud induction medium, and light culture under the condition of light intensity 2000-2500 lx, with 16 hours of light per day. After 20 days of culture, the resistant plants grow to about 3-5 cm.

[0087] (Nine) root induction culture

[0088] Transfer the resistant plants to the rooting induction medium for rooting culture, and light culture under the condition of light intensity 2000-2500 lx, with 16 hours of light per day. After 20 days of culture, complete resistant seedlings are obtained,

[0089] (Ten) transplanting

[0090] After washing the culture medium with clean water, the blueberry seedlings with roots and buds were transplanted into flowerpots with mixed soil of grass carbon and vermiculite (4:6 by weight) and placed in a plastic greenhouse. The temperature inside the cover was kept at 20-25°C and the humidity was kept at 80±5% within 10 days after transplantation. After 10 days, the plants could be placed in a natural environment.

[0091] (xi) Gene identification

[0092] The genome of the leaves of the plants obtained by infection with bacterial solution 3 was extracted and subjected to PCR amplification using primers F3 and R3. The plants with amplified bands were identified by gel electrophoresis as plants with overexpression of the gene for mevalonate diphosphate decarboxylase, referred to as overexpression plants, and a total of 25 plants were obtained.

[0093] The genome of the leaves of the plants obtained by infection with bacterial solution 4 was extracted and subjected to PCR amplification using primers F3 and R3. The plants with amplified bands were identified by gel electrophoresis as transgenic control plants, referred to as control plants, and a total of 22 plants were obtained.

[0094] Example 2: Phenotypic identification of transgenic blueberries

[0095] I. Drought simulation in blueberry plants

[0096] After 15 days of transplantation of the tissue culture seedlings, 20 control plants and 20 overexpression plants were taken and the water potential of the soil in the flowerpots was measured using a PSYPRO Water Potential System (WESCOR, Utah, USA). The soil water potential was maintained at -1.80 MPa to simulate water deficit conditions, and the plants were kept under these conditions for 45 days.

[0097] II. Morphological observation of blueberry plants under drought stress

[0098] Figure 2 Figure 2 shows photographs of the morphology of blueberry plants under drought stress for 30 days. On the left, two plants are overexpression plants and on the right, two plants are control plants. After 30 days of drought, the leaves of the control plants began to turn yellow and wilt, while the leaves of the overexpression plants turned slightly yellow. This indicates that overexpression of the gene for mevalonate diphosphate decarboxylase improves drought resistance in blueberries.

[0099] III. Carbon dioxide assimilation test in blueberries

[0100] The carbon dioxide assimilation rate was measured in three overexpression plants and three control plants using a photosynthesis meter LI-COR 6400 (Lincoln, NE, USA) and the average value was taken (in μmol / m 2 / s). See Table 1 for the results.

[0101] Table 1. Change in carbon dioxide assimilation rate in overexpression plants and control plants under drought stress

[0102] Days 0 5 10 15 20 25 30 Overexpression plants 15.3 15.0 14.5 13.8 11.6 11.2 10.7 Control plants 10.2 8.6 6.2 6.5 5.1 2.3 1.5

[0103] As shown in Table 1, with the proceeding of drought, the decrease of carbon dioxide assimilation rate of the high expression plants is obviously slower than that of the control plants, which indicates that the high expression plants can make blueberry plants drought-resistant well.

[0104] Four, the test of blueberry roots

[0105] After 30 days of drought, the average root length, root diameter and root volume of the three overexpression plants are 2.1 times, 1.8 times and 2.8 times of those of the three control plants respectively, which indicates that the overexpression plants can significantly slow down the root shrinkage of blueberry caused by drought. The conductivity of the roots of the plants is determined by using DDS-307 (Lei Ji-DDS-307A, Shanghai) conductivity meter. After 30 days of drought, the average conductivity of the three overexpression plants is 0.4 times of that of the three control plants, which indicates that the damage of the overexpression spider to the drought stress is lower than that of the control plants.

[0106] Five, the biochemical detection of blueberry

[0107] The leaves of the three overexpression plants after 25 days of drought are mixed and used, and the leaves of the three control plants are mixed and used. The malondialdehyde content, proline content, hydrogen peroxide content, superoxide dismutase and peroxidase kits (all purchased from Nanjing Jiancheng Biological Engineering Institute) are used for the determination of the malondialdehyde content, proline content, hydrogen peroxide content, superoxide dismutase and peroxidase of the two kinds of mixed leaves according to the steps of the kit instruction.

[0108] Table 2. The determination results of biochemical indexes of overexpression plants and control plants under drought stress

[0109]

[0110]

[0111] As can be seen, compared with the control plants, the malondialdehyde content in the leaves of the overexpression plants is obviously reduced, which indicates that the damage of drought stress is small; the proline content is increased, which indicates that the osmotic pressure regulation ability is enhanced; the hydrogen peroxide content is increased, and the superoxide dismutase and peroxidase contents are increased, which indicates that the oxidative damage caused by drought of the high expression plants is weakened.

[0112] It can be known from the technical common sense that the present application can be realized by other embodiments without departing from the spirit or essential characteristics thereof. Therefore, the above disclosed embodiments are only illustrative in all aspects, and are not the only ones. All changes within the scope of the present application or within the scope equivalent to the present application are included in the present application.

Claims

1. A breeding method to improve the drought resistance of blueberries, wherein the breeding method is: to overexpress the poplar mevalonate diphosphate decarboxylase gene in blueberries through genetic engineering.

2. The method as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the poplar mevalonate diphosphate decarboxylase gene is shown in SEQ ID NO.

2.

3. The method as described in claim 2, characterized in that, The coding sequence of the poplar mevalonate diphosphate decarboxylase gene is shown in SEQ ID NO.

1.

4. The method as described in claim 1, characterized in that, The blueberry variety mentioned is Duke.

5. The method as described in claim 1, characterized in that, The improvement in blueberry drought resistance includes: Increase blueberry root length, root diameter, and root volume; Increase the carbon dioxide assimilation rate of blueberries; Reduce the electrical conductivity of blueberry roots; Reduce malondialdehyde content in blueberries; Reduce the hydrogen peroxide content in blueberries; Increase the proline content of blueberries; Increase the superoxide dismutase content in blueberries; Increase the peroxidase content of blueberries.

6. The method according to any one of claims 1-5, characterized in that, The method includes the following steps: S1: The coding sequence of the poplar mevalonate diphosphate decarboxylase gene is transferred into an expression vector to obtain a recombinant vector containing the poplar mevalonate diphosphate decarboxylase gene; S2: Agrobacterium is transformed with a recombinant vector containing the poplar mevalonate diphosphate decarboxylase gene to obtain recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene; S3: Infect sterilized blueberry leaves with recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene to obtain infected blueberry leaves; S4: Place the infected blueberry leaves on a co-culture medium to obtain pre-cultured leaves; The co-culture medium is based on WPM and also contains 30-40 g / L sucrose, 4-7 g / L agar, 4-6 mg / L TDZ, and pH 5.0-6.

0. S5: The pre-cultured leaves are placed in a bacterial solution of recombinant Agrobacterium containing the poplar mevalonate diphosphate decarboxylase gene to obtain infected leaves; S6: Place the stained leaves on the co-culture medium to obtain co-cultured leaves; S7: Place the co-cultured leaves on a callus induction medium to obtain blueberry callus tissue; The callus induction medium is based on WPM and also contains 15-25 g / L sucrose, 4-6 g / L agar, 0.8-1.2 mg / L zeatin, 0.8-1.2 mg / L TDZ, 0.4-0.6 mg / L 2,4-D, 20-40 mg / L kanamycin, and pH 5.0-6.

0. S8: Place the blueberry callus on a bud induction medium to obtain callus that has sprouted; The bud induction medium is based on WPM and also contains 15-25 g / L sucrose, 4-6 g / L agar, 2-4 mg / L zeatin, 0.8-1.2 mg / L TDZ, 20-40 mg / L kanamycin, and pH 5.0-6.

0. S9: Insert the sprouted callus tissue into a rooting culture medium to obtain rooted blueberry plants; The rooting medium is based on 1 / 2 WPM and also contains 15-25 g / L sucrose, 4-6 g / L agar, 0.5-0.7 mg / L IBA, 20-40 mg / L kanamycin, and pH 5.0-6.

0.

7. The method as described in claim 6, characterized in that, Transplant the rooted blueberry plant into the soil to obtain a blueberry plant.

8. The method as described in claim 6, characterized in that, The backbone of the expression vector is the pK2GW7 vector.

9. The method as described in claim 6, characterized in that, The Agrobacterium is Agrobacterium GV3101.

10. The method as described in claim 6, characterized in that, In step S4, the culture conditions are dark culture, temperature 23-25℃, for 2.5-3.5 days; In step S5, the immersion time is 30-50 minutes; In step S6, the culture conditions are dark culture, temperature 23-25℃, for 2.5-3.5 days; In step S7, the culture conditions are: temperature 23-25℃, first dark culture for 13-17 days, then light culture at 2000-2500 lx for 18-22 days; In step S8, the cells are cultured under conditioned light with a light intensity of 2000-2500 lx for 18-22 days; In step S9, the cells are cultured under conditioned light with a light intensity of 2000-2500 lx for 18-22 days.

Images