A method for efficiently regenerating potato protoplasts

Abstract

The invention discloses a potato protoplast efficient regeneration method, comprising the steps of: S1, test tube seedling culture; S2, protoplast preparation; S3, callus proliferation; S4, callus synchronization treatment; S5, callus induction regeneration, etc. The invention induces the protoplasts formed after potato leaf treatment to form callus, achieves high efficiency and synchronous sprouting through synchronization treatment and induced regeneration, greatly shortens the protoplast regeneration time, significantly improves the protoplast sprout regeneration rate, provides a large amount of experimental materials for potato genetic improvement and variety breeding, ensures the consistency and stability of regenerated plants, and provides technical support for subsequent genetic theory research, high-throughput virus-free seedling regeneration, potato gene editing breeding and other applications.

Description

Technical Field

[0001] The invention belongs to the technical field of potato cultivation, and in particular relates to a method for efficiently regenerating potato protoplasts. Background Art

[0002] Plant protoplast regeneration technology involves removing the cell walls of plant cells to form protoplasts, and regenerating them into complete plants through specific culture conditions. Potato is one of the earliest crops that was successfully cultured in vitro and obtained somatic hybrid plants. Protoplast culture and regeneration are key links in somatic hybridization. Somatic hybridization technology can avoid the difficulties in conventional breeding of potatoes due to ploidy level and endosperm balance number. The existing technology is mainly aimed at preparing active potato protoplasts. The technical achievements are concentrated in the protoplast preparation stage. The characteristics of protoplasts without cell walls are used to carry out transient genetic transformation, somatic hybridization and other research. The receptor materials are mostly leaves, and a few use stem segments, pollen, hypocotyls, etc. to obtain protoplasts for subsequent genetic transformation.

[0003] At present, there are two major defects in the potato leaf protoplast regeneration technology: one is the asynchronous regeneration, which leads to a large span of bud regeneration time in the same batch of protoplasts and serious asynchronous development of the regenerated buds; the other is the low regeneration rate. The highest reported regeneration rate of potato leaf protoplast induced buds is 22.9%, but most studies do not involve the analysis of the regeneration rate of a single batch. Therefore, it is urgent to develop a technology that can improve the synchronization level and regeneration rate of potato protoplasts. Summary of the invention

[0004] The purpose of the present invention is to provide a potato protoplast efficient regeneration method with short cycle and high regeneration rate in view of the shortcomings in the prior art.

[0005] The purpose of the present invention is implemented by the following technical solutions:

[0006] A method for efficiently regenerating potato protoplasts comprises the following steps:

[0007] S1, test tube seedling culture;

[0008] S2, protoplast preparation;

[0009] S3, callus proliferation;

[0010] S4, callus synchronization treatment: from the callus obtained in S3, the callus with a diameter of 100-1000 μm (relatively synchronized metabolic synthesis level and strong regeneration ability) is selected, and transferred into a callus proliferation medium, and then a synchronized proliferation culture solution is added, and cultured at 4-7°C for 1-3 days to increase the synchronization level, and then cultured at 20-23°C for 16 hours under light conditions and at 18-20°C for 8 hours under dark conditions. After 25 days of alternating light and dark culture, green callus is formed; the callus proliferation medium is a solid culture medium, and the synchronized proliferation medium is a liquid culture medium. The advantage of the solid-liquid double-layer culture method is that the nutrients in the callus proliferation medium can promote callus induction into seedlings, and at the same time, the synchronized proliferation liquid culture medium is added without changing the culture medium to avoid excessive division and proliferation of seedling tissue cells, achieve synchronization, and help shorten the culture time;

[0011] S5. Callus regeneration induction: The green callus obtained in S4 is transferred to a bud regeneration medium for culture. The culture conditions are culture at 20-23°C for 16 h under light conditions and culture at 18-20°C for 8 hours under dark conditions. After 40 days of alternating light and dark conditions, regenerated buds are differentiated.

[0012] Furthermore, the pH value of the synchronized proliferation culture medium is 5.6~6.0; the synchronized proliferation culture fluid comprises the following components: MS culture medium 4.33 g / L, glycine 2 mg / L, inositol 150 mg / L, vitamin B1 thiamine hydrochloride 0.5 mg / L, vitamin B6 vitamin B6 0.5 mg / L, niacin 5 mg / L, folic acid 0.5 mg / L, biotin 0.05 mg / L, sucrose 30 g / L, mannitol 3 g / L, adenine sulfate 40 mg / L, naphthylacetic acid 1 mg / L, 6-benzylaminoadenine 0.4~1.0 mg / L, indoleacetic acid 0.5 mg / L, abscisic acid 0~5 mg / L, and the solvent is sterile water.

[0013] Furthermore, the pH value of the bud regeneration medium is 5.6-6.0; the bud regeneration medium comprises the following components: 4.4 g / L MS medium, 10 g / L sucrose, 1 mg / L zeatin or t-zeatin, 0.01 mg / L naphthaleneacetic acid, 30-0.1 mg / L GA, 6 g / L agar, and the solvent is sterile water.

[0014] Furthermore, S1 comprises: culturing the potato tissue culture seedlings for 3-4 weeks under the following conditions: culturing at 20-23° C. for 16 hours under light conditions and culturing at 18-20° C. for 8 hours under dark conditions, and culturing in light and dark conditions alternately.

[0015] Further, the S2 includes:

[0016] S21, pre-culture: under sterile conditions, take 2-3 fully expanded leaves from the upper part of the potato tissue culture seedlings obtained by the culture in S1, put them into pre-treatment I medium, and culture them in the dark at 24°C for 1-5 days; then remove the pre-treatment I medium, put the leaves cultured in the pre-treatment I medium into pre-treatment II medium, and incubate them in a refrigerator at 4°C for 24 hours; the pre-treatment II medium includes ingredients such as inositol and MES, inositol is an osmotic pressure regulator, which helps to maintain the balance of osmotic pressure inside and outside the cells, and MES is a buffer that can adjust and stabilize the pH value of the solution; the pre-culture step can provide a basis for the efficient and stable preparation of protoplasts, which is conducive to the subsequent full enzymatic release of protoplast cells in the leaves; low-temperature culture is conducive to maintaining enzyme activity in the cells, slowing down the cell metabolism rate, and maintaining the integrity of the cell structure;

[0017] S22, enzymatic hydrolysis: cutting the leaves pre-cultured in S21 into thin strips, immersing them in enzymatic hydrolysis solution, and incubating them in the dark at 28° C. for 9 to 10 h; filtering after enzymatic hydrolysis, centrifuging the filtrate and removing the supernatant;

[0018] S23, protoplast purification: add MR culture medium to the precipitate obtained after centrifugation in S22, suspend the protoplasts, suck them out with a pipette, gently add them along the tube wall to another centrifuge tube containing MP culture medium, centrifuge, and collect the annular protoplasts; rinse the annular protoplasts twice with MPS washing solution; then suspend and wash once with E culture medium; resuspend with E culture medium and adjust the dilution to 5×10 4 / mL to obtain a protoplast suspension.

[0019] Further, the pH value of the E culture solution is 5.6-6.0; the E culture solution comprises the following components: 1900 mg / L potassium nitrate, 440 mg / L calcium chloride dihydrate, 370 mg / L magnesium sulfate heptahydrate, 170 mg / L potassium dihydrogen phosphate, 3.1 mg / L boric acid, 0.42 mg / L potassium iodide, 0.13 mg / L sodium molybdate dihydrate, 0.013 mg / L cobalt chloride hexahydrate, 11.16 mg / L manganese sulfate tetrahydrate, 4.3 mg / L zinc sulfate heptahydrate, 0.013 mg / L copper sulfate pentahydrate, 13.94 mg / L ferrous sulfate heptahydrate, 18.64 mg / L disodium ethylenediaminetetraacetic acid, 0.5 mg / L vitamin B1, 0.5 mg / L vitamin B6, 5.0 mg / L niacin, 0.05 mg / L biotin, 2.0 mg / L glycine, 0.5 mg / L, hydrolyzed casein 100 mg / L, pyruvic acid 10 mg / L, DL-malic acid 20 mg / L, citric acid 20 mg / L, BSA 500 mg / L, naphthylacetic acid 0-1.0 mg / L, 6-benzylaminoadenine 0.4 mg / L, 2,4-dichlorophenoxyacetic acid 0-2.0 mg / L, D-mannitol 0.15M mol / L, inositol 50 mol / L, xylitol 125 mol / L, sorbitol 125 mol / L, D-cellobiose 125 mol / L, glucose 125 mol / L, sucrose 0.15 mol / L, and the solvent was sterile water.

[0020] Furthermore, S3 comprises: statically culturing the protoplast suspension prepared in S2 at 21-24° C. in the dark, and proliferating into small cell clusters to form callus tissue after culturing for 25 days.

[0021] Beneficial effects: The present invention provides a method for efficient regeneration of potato protoplasts, using potato leaves as donor materials, designing a special culture medium and culture conditions for synchronization treatment and induced regeneration, and performing efficient regeneration of potato protoplasts through test tube seedling culture, protoplast preparation, callus proliferation, callus synchronization treatment, callus induction regeneration and other steps. By improving the synchronization level and bud regeneration rate of callus induced by protoplasts, protoplasts with target traits are more likely to form regenerated plants, and the plant regeneration rate is increased while shortening the time span of induced regeneration, which is beneficial to improving potato breeding efficiency, and is not limited by seasons and regions, and can also be used for high-throughput potato virus-free seedling production, reducing production costs such as multi-generation propagation and basic virus-free seedling preservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a photograph of the test tube seedling culture in Example 1.

[0024] Figure 2 This is a photo of the leaf pre-cultivation in Example 1.

[0025] Figure 3 This is a photo of the annular protoplasts in Example 1.

[0026] Figure 4 This is a microscope image of the protoplast suspension in Example 1.

[0027] Figure 5 This is a microscopic image of the proliferating cell cluster in Example 1.

[0028] Figure 6 This is a photo of the green callus in Example 1.

[0029] Figure 7 This is a photograph of the regenerated buds differentiated in Example 1.

[0030] Figure 8 This is a bright field image of the protoplasts in Example 3 under a fluorescent microscope.

[0031] Fig. 9 This is a fluorescence microscopic image of the protoplast suspension in Example 3 after FDA staining.

[0032] Fig.10 This is a photo taken after 100 days of differentiation and regeneration in Example 4.

[0033] Fig.11 This is a photo of the regenerated buds in Example 4 after 15 days of propagation.

[0034] Fig.12 The figure shows the effect of the synchronized proliferation medium with different abscisic acid concentrations on the callus induction budding rate in Comparative Example 1.

[0035] Fig.13 The results are as follows: callus induced bud regeneration in the synchronized proliferation medium with different abscisic acid concentrations in Comparative Example 1; wherein A, 0 mg / L, B, 1 mg / L, C, 3 mg / L, D, 5 mg / L, and E, 7 mg / L. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The experiments in the embodiments of the present invention were repeated three times, and the results were averaged.

[0038] Example 1: A method for efficient regeneration of potato protoplasts.

[0039] A method for efficiently regenerating potato protoplasts comprises the following steps S1-S5.

[0040] S1. Test tube seedling culture: Figure 1 As shown, potato tissue culture seedlings were placed in MS medium, cultured at 23°C for 16 h under light conditions and at 20°C for 8 hours under dark conditions, and cultured alternately in light and dark for 3 weeks; the potato tissue culture seedlings used were Ganyin No. 9.

[0041] S2, protoplast preparation; including steps S21-S23:

[0042] S21, pre-culture: Under sterile conditions, take 2-3 fully expanded leaves from the upper part of the potato tissue culture seedlings obtained by culture in S1 and place them in the pre-treatment I medium, such as Figure 2 As shown, the leaves were incubated in the dark at 24°C for 72 h. Then, the pretreatment medium I was removed, and the leaves incubated in the pretreatment medium II were placed in the pretreatment medium II and incubated in a 4°C refrigerator for 24 h.

[0043] S22, enzymatic hydrolysis: cut the leaves pre-cultured in S21 into 0.5-1.0 mm strips, immerse in 15 mL enzymatic hydrolysis solution, and incubate in the dark at 28°C for 9-10 h; after enzymatic hydrolysis, filter with 240-mesh nylon gauze, collect the filtrate, centrifuge at 800 r / min for 8 min, and remove the supernatant;

[0044] S23. Protoplast purification: Add 1 mL of MR culture medium to the precipitate obtained after centrifugation in S22 to suspend the protoplasts. Aspirate with a pipette and gently add along the tube wall to another 5 mL centrifuge tube containing 2.5 mL of MP culture medium. Centrifuge at 500 rpm for 5 min. Figure 3 As shown, the ring-shaped protoplasts were collected; the ring-shaped protoplasts were rinsed twice with MPS washing solution; then the ring-shaped protoplasts were suspended and washed once with E culture medium; and then the ring-shaped protoplasts were resuspended with E culture medium and diluted to 5×10 4 / mL, and obtain a protoplast suspension, such as Figure 4 shown.

[0045] S3. Callus proliferation: The protoplasts prepared in S2 were cultured at 24°C in the dark. After 25 days of culture, they proliferated into small cell clusters and formed callus. Figure 5 shown.

[0046] S4. Callus synchronization treatment: From the callus obtained in S3, callus with a diameter of 100-1000 μm was selected and transferred to callus proliferation medium. Synchronized proliferation medium was then added. After culturing at 4°C for 1 day, it was cultured at 23°C for 16 h under light conditions and at 20°C for 8 hours under dark conditions. This light and dark alternation culture was repeated for 25 days to form green callus of about 1.5 mm. Figure 6 shown.

[0047] S5. Callus induction regeneration: The green callus obtained in S4 was transferred to the bud regeneration medium for culture. The culture conditions were cultured at 23°C for 16 h under light conditions and at 20°C for 8 h under dark conditions. After 40 days of alternating light and dark conditions, regenerated buds were differentiated. Figure 7 shown.

[0048] Example 2: Reagents and culture media used in potato protoplast regeneration.

[0049] 1) Pretreatment I medium: pH 5.6~6.0, including the following components:

[0050] NH4NO380 mg / L, calcium chloride dihydrate 147 mg / L, naphthylacetic acid 2.0 mg / L, 6-benzylaminoadenine 1.0 mg / L, MES 3mmol / L, the solvent is sterile water.

[0051] 2) Pretreatment II medium: pH 5.6~6.0, including the following components:

[0052] Potassium nitrate 190 mg / L, calcium chloride dihydrate 44 mg / L, magnesium sulfate heptahydrate 37 mg / L, potassium dihydrogen phosphate 17 mg / L, boric acid 0.62 mg / L, potassium iodide 0.083 mg / L, sodium molybdate dihydrate 0.025 mg / L, cobalt chloride hexahydrate 0.0025 mg / L, manganese sulfate tetrahydrate 2.23 mg / L, zinc sulfate heptahydrate 0.86 mg / L, copper sulfate pentahydrate 0.0025 mg / L, ferrous sulfate heptahydrate 2.79 mg / L, disodium ethylenediaminetetraacetic acid 3.73 mg / L, vitamin B1 0.05 mg / L, vitamin B6 0.05mg / L, niacin 0.5 mg / L, biotin 0.005 mg / L, glycine 0.2 mg / L, folic acid 0.05 mg / L, hydrolyzed casein 100 mg / L, naphthylacetic acid 2.0 mg / L, 6-benzylaminopurine 0.5 mg / L, inositol 10 mol / L, MES 3mmol / L, and the solvent was sterile water.

[0053] 3) Enzyme solution: pH 5.6~6.0, including the following components: 1 / 10 RA solution, 0.7% cellulose enzyme R-10, 0.5% pectinase, 2% PVP, 0.2% BSA, 3 mmol / L MES, 0.4 M mannitol, and the rest is sterile water; among which:

[0054] RA solution includes the following components:

[0055] Potassium nitrate 1900 mg / L, calcium chloride dihydrate 440 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 3.1 mg / L, potassium iodide 0.42 mg / L, sodium molybdate dihydrate 0.13 mg / L, cobalt chloride hexahydrate 0.013 mg / L, manganese sulfate tetrahydrate 11.16 mg / L, zinc sulfate heptahydrate 4.3 mg / L, copper sulfate pentahydrate 0.013 mg / L, ferrous sulfate heptahydrate 13.94 mg / L, disodium ethylenediaminetetraacetic acid 18.64 mg / L, vitamin B1 0.5 mg / L, vitamin B6 0.5mg / L, niacin 5.0 mg / L, biotin 0.05 mg / L, glycine 2.0 mg / L, folic acid 0.5 mg / L, hydrolyzed casein 100 mg / L, the solvent is sterile water.

[0056] 4) MR culture medium: pH 5.6~6.0, including the following components:

[0057] Potassium nitrate 1900 mg / L, calcium chloride dihydrate 440 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 3.1 mg / L, potassium iodide 0.42 mg / L, sodium molybdate dihydrate 0.13 mg / L, cobalt chloride hexahydrate 0.013 mg / L, manganese sulfate tetrahydrate 11.16 mg / L, zinc sulfate heptahydrate 4.3 mg / L, copper sulfate pentahydrate 0.013 mg / L, ferrous sulfate heptahydrate 13.94 mg / L, disodium ethylenediaminetetraacetic acid 18.64 mg / L, vitamin B1 0.5 mg / L, vitamin B6 0.5mg / L, niacin 5.0 mg / L, biotin 0.05 mg / L, glycine 2.0 mg / L, folic acid 0.5 mg / L, hydrolyzed casein 100 mg / L, naphthylacetic acid 1.0 mg / L, 6-benzylaminoadenine 0.4 mg / L, D-mannitol 0.35 mol / L, and the solvent was sterile water.

[0058] 5) MP culture medium: pH 5.6~6.0, including the following components:

[0059] Potassium nitrate 1900 mg / L, calcium chloride dihydrate 440 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 3.1 mg / L, potassium iodide 0.42 mg / L, sodium molybdate dihydrate 0.13 mg / L, cobalt chloride hexahydrate 0.013 mg / L, manganese sulfate tetrahydrate 11.16 mg / L, zinc sulfate heptahydrate 4.3 mg / L, copper sulfate pentahydrate 0.013 mg / L, ferrous sulfate heptahydrate 13.94 mg / L, disodium ethylenediaminetetraacetic acid 18.64 mg / L, vitamin B1 0.5 mg / L, vitamin B6 0.5mg / L, niacin 5.0 mg / L, biotin 0.05 mg / L, glycine 2.0 mg / L, folic acid 0.5 mg / L, hydrolyzed casein 100 mg / L, naphthylacetic acid 1.0 mg / L, 6-benzylaminoadenine 0.4 mg / L, sucrose 23% mol / L, and the solvent was sterile water.

[0060] 6) MPS washing solution: pH 5.6~6.0, including the following components:

[0061] Potassium nitrate 1900 mg / L, calcium chloride dihydrate 440 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 3.1 mg / L, potassium iodide 0.42 mg / L, sodium molybdate dihydrate 0.13 mg / L, cobalt chloride hexahydrate 0.013 mg / L, manganese sulfate tetrahydrate 11.16 mg / L, zinc sulfate heptahydrate 4.3 mg / L, copper sulfate pentahydrate 0.013 mg / L, ferrous sulfate heptahydrate 13.94 mg / L, disodium ethylenediaminetetraacetic acid 18.64 mg / L, vitamin B1 0.5 mg / L, vitamin B6 0.5mg / L, niacin 5.0 mg / L, biotin 0.05 mg / L, glycine 2.0 mg / L, folic acid 0.5 mg / L, hydrolyzed casein 100 mg / L, naphthylacetic acid 1.0 mg / L, 6-benzylaminoadenine 0.4 mg / L, D-mannitol 0.25 mol / L, inositol 0.025 mol / L, sorbitol 0.025 mol / L, glucose 0.025 mol / L, sucrose 0.025 mol / L, and the solvent was sterile water.

[0062] 7) E culture medium: pH 5.6~6.0, including the following components:

[0063] Potassium nitrate 1900 mg / L, calcium chloride dihydrate 440 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 3.1 mg / L, potassium iodide 0.42 mg / L, sodium molybdate dihydrate 0.13 mg / L, cobalt chloride hexahydrate 0.013 mg / L, manganese sulfate tetrahydrate 11.16 mg / L, zinc sulfate heptahydrate 4.3 mg / L, copper sulfate pentahydrate 0.013 mg / L, ferrous sulfate heptahydrate 13.94 mg / L, disodium ethylenediaminetetraacetic acid 18.64 mg / L, vitamin B1 0.5 mg / L, vitamin B6 0.5mg / L, niacin 5.0 mg / L, biotin 0.05 mg / L, glycine 2.0 mg / L, folic acid 0.5 mg / L, hydrolyzed casein 100 mg / L, pyruvic acid 10 mg / L, DL-malic acid 20 mg / L, citric acid 20 mg / L, BSA 500 mg / L, naphthaleneacetic acid 1.0 mg / L, 6-benzylaminoadenine 0.4 mg / L, 2,4-dichlorophenoxyacetic acid 2.0 mg / L, D-mannitol 0.15 mol / L, inositol 50 mol / L, xylitol 125 mol / L, sorbitol 125 mol / L, D-cellobiose 125 mol / L, glucose 125 mol / L, sucrose 0.15 mol / L, and the solvent was sterile water.

[0064] 8) Callus proliferation medium: pH 5.6~6.0, including the following components:

[0065] MS medium 4.33 g / L, vitamin NN solution 1 mL / L, sucrose 30 g / L, mannitol 3 g / L, adenine sulfate 40 mg / L, inositol 50 mg / L, agar 6 g / L, naphthaleneacetic acid 1 mg / L, 6-benzylaminoadenine 0.5 mg / L, and the solvent is sterile water.

[0066] 9) Synchronized proliferation medium: pH 5.6~6.0, including the following components:

[0067] MS medium 4.33 g / L, glycine 2 mg / L, inositol 150 mg / L, vitamin B1 thiamine hydrochloride 0.5 mg / L, vitamin B6 vitamin B6 0.5 mg / L, niacin 5 mg / L, folic acid 0.5 mg / L, biotin 0.05 mg / L, sucrose 30 g / L, mannitol 3 g / L, adenine sulfate 40 mg / L, naphthylacetic acid 1 mg / L, 6-benzylaminoadenine 0.4~1.0 mg / L, indoleacetic acid 0.5 mg / L, abscisic acid 5 mg / L, and the solvent is sterile water.

[0068] 10) Shoot regeneration medium: pH 5.6~6.0, including the following components:

[0069] MS medium 4.4 g / L, sucrose 10 g / L, zeatin or t-zeatin 1 mg / L, naphthaleneacetic acid 0.01 mg / L, GA30~0.1 mg / L, agar 6 g / L, and the solvent is sterile water.

[0070] Other solutions, reagents, and culture media are commercially available.

[0071] Example 3: Protoplast viability assay.

[0072] 0.1 mL of the protoplast suspension obtained in step S2 of Example 1 was taken, and 2 μL of FDA dye solution was added. After standing at room temperature for 5 minutes, the protoplast activity was observed. Figure 8 , Fig. 9 As shown, it indicates that most of the protoplast cells in the protoplast suspension obtained in S2 are active and can further proliferate into callus tissue to induce budding.

[0073] Example 4: Detection of bud induction and regeneration effect.

[0074] In step S5 of Example 1, the green callus obtained in S4 was transferred to a bud regeneration medium and cultured for 40 days. After the regenerated buds appeared, the callus induction sprouting was counted. The test results showed that the callus could be induced to sprout after 40 days of culture, which was more than 25 days earlier than the sprouting induction time in the prior art.

[0075] After 40 days of callus induction and regeneration culture, the culture was continued for 30 days and 60 days, and the regeneration rate was calculated (regeneration rate = number of calli that produced regeneration buds / 500 calli*100%, with 500 calli as the base number). The results showed that after the first regeneration bud appeared, the regeneration rate reached 32.7% after 30 days of culture, and 67.9% after 60 days of culture (the differentiation and regeneration after 40 days of callus induction and regeneration culture + 60 days of culture for a total of 100 days is shown in Figure 2). Fig.10 as shown).

[0076] The regenerated shoots differentiated in step S5 of Example 1 were transferred to MS culture medium for rooting or propagation. After 15 days of culture, Fig.11 As shown, the plants can take root and grow normally.

[0077] Comparative Example 1: Synchronized proliferation medium component selection test.

[0078] A synchronized proliferation culture medium: pH 5.6-6.0, comprising the following components: 4.33 g / L MS culture medium, 2 mg / L glycine, 150 mg / L inositol, 0.5 mg / L vitamin B1 thiamine hydrochloride, 0.5 mg / L vitamin B6, 5 mg / L nicotinic acid, 0.5 mg / L folic acid, 0.05 mg / L biotin, 30 g / L sucrose, 3 g / L mannitol, 40 mg / L adenine sulfate, 1 mg / L naphthaleneacetic acid, 0.4-1.0 mg / L 6-benzylaminoadenine, 0.5 mg / L indoleacetic acid, the solvent is sterile water, and also comprises abscisic acid.

[0079] The present invention conducts experiments on the concentration selection of abscisic acid in the components of the synchronized proliferation culture medium.

[0080] Five ABA concentration gradients were set, namely 0 mg / L, 1 mg / L, 3 mg / L, 5 mg / L, and 7 mg / L, and the other components and concentrations of the synchronized proliferation medium remained unchanged. The efficient regeneration method of potato protoplasts was the same as that in Example 1, in order to screen the ABA concentration that was beneficial to improving the synchronization level.

[0081] The results show that if Fig.12As shown in the data, callus tissue can be induced to sprout under the condition of ABA concentration of 0-7 mg / L, but the budding rate of callus tissue treated with ABA concentration of 5 mg / L is significantly higher than that of other concentrations, and its synchronization level is also better than other concentration gradients in the same time, which not only promotes the rapid proliferation of callus tissue, but also increases the overall yield.

[0082] In addition, if Fig.13 As shown in the figure, by comparing the bud regeneration of callus under different concentrations, it was found that under the condition of ABA concentration of 5 mg / L, the bud regeneration ability of callus tissue was relatively strong.

[0083] Comparative Example 2: Comparison between the present invention and the prior art.

[0084] The test group (method of Example 1) was compared with the control group ① (prior art, reference: Li Xiao. "Study on potato protoplast culture regeneration and transient transformation using CRISPR / Cas9", wherein the culture medium formula is: MS medium + 1 mg / L ZT + 0.01 mg / L naphthaleneacetic acid + 0.01 mg / L GA3 + 1 mg / L KT + 2% sucrose) and the control group ② (prior art, reference: Cai Xingkui. "Protoplast fusion to create new potato germplasm resistant to bacterial wilt and its genetic analysis", wherein the culture medium formula is 0.5 mg / L indoleacetic acid + 2.5 mg / L zeatin), and the results are shown in Table 1.

[0085] Table 1 Comparison of callus induction regeneration time and regeneration rate of leaf protoplasts

[0086]

[0087] The results show that the prior art does not involve the technology of synchronization treatment; and the potato protoplast regeneration method of the present invention can greatly shorten the cycle from cell to plant regeneration. The prior art generally takes about 4 months from callus proliferation to induction of regeneration and budding, while the present invention only takes 3 months. At the same time, the bud regeneration rate of the prior art is about 20%, which requires 6 months or even longer, while the present invention only takes 5 months to achieve more than 60% regeneration buds. The present invention is expected to obtain the highest regeneration rate in a relatively short period of time, which has significant advantages in scientific research and production practice applications.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficient regeneration of potato protoplasts, characterized in that: It includes the following steps: S1, test tube seedling culture; S2, protoplast preparation; S2 includes: S21, pre-culture: under sterile conditions, take 2-3 fully expanded leaves from the upper part of the potato tissue culture seedlings obtained by culture in S1, put them into pre-treatment I culture medium, and culture them in the dark at 24°C for 1-5 days; then remove the pre-treatment I culture medium, put the leaves cultured in the pre-treatment I culture medium into pre-treatment II culture medium, and incubate them in a refrigerator at 4°C for 24 hours; S22, enzymatic hydrolysis: cut the leaves pre-cultured in S21 into thin strips, immerse them in enzymatic hydrolysis solution, and incubate them in the dark at 28°C for 9-10 hours; filter after enzymatic hydrolysis, centrifuge the filtrate and remove the supernatant; S23, protoplast purification: add MR culture solution to the precipitate obtained after centrifugation in S22, suspend the protoplasts, then add it to MP culture solution, centrifuge, and collect the annular protoplasts; rinse the annular protoplasts twice with MPS washing solution; then suspend and wash once with E culture solution; then resuspend with E culture solution and adjust the dilution to 5×10 4 / mL, and a protoplast suspension was obtained; S3, callus proliferation: the protoplast suspension prepared in S2 is statically cultured at 21-24°C in the dark, and after culturing for 25 days, it proliferates into small cell clusters to form callus; S4, callus synchronization treatment: from the callus obtained in S3, the callus with a diameter of 100-1000 μm is selected, and transferred into a callus proliferation medium, and then a synchronized proliferation culture solution is added, and after culturing at 4-7° C. for 1-3 days, it is cultured at 20-23° C. for 16 hours under light conditions and at 18-20° C. for 8 hours under dark conditions, and after culturing alternately under light and dark conditions for 25 days, green callus is formed; S5, callus induction regeneration: the green callus obtained in S4 is transferred to a bud regeneration medium for culture, and the culture conditions are cultured at 20-23°C for 16 hours under light conditions and cultured at 18-20°C for 8 hours under dark conditions. After 40 days of alternating light and dark conditions, regenerated buds are differentiated; The pH value of the pretreatment I culture medium is 5.6-6.0; the pretreatment I culture medium is composed of 80 mg / L NH4NO3 + 147 mg / L calcium chloride dihydrate + 2.0 mg / L naphthaleneacetic acid + 1.0 mg / L 6-benzylaminoadenine + 3mmol / L MES + sterile water; The pH value of the pretreatment II culture medium is 5.6-6.0; the pretreatment II culture medium is composed of 190 mg / L potassium nitrate + 44 mg / L calcium chloride dihydrate + 37 mg / L magnesium sulfate heptahydrate + 17 mg / L potassium dihydrogen phosphate + 0.62 mg / L boric acid + 0.083 mg / L potassium iodide + 0.025 mg / L sodium molybdate dihydrate + 0.0025 mg / L cobalt chloride hexahydrate + 2.23 mg / L manganese sulfate tetrahydrate + 0.86 mg / L zinc sulfate heptahydrate + 0.0025 mg / L copper sulfate pentahydrate + 2.79 mg / L ferrous sulfate heptahydrate + 3.73 mg / L disodium ethylenediaminetetraacetic acid + 0.05 mg / L vitamin B1 + 0.05 mg / L vitamin B6 + 0.5 mg / L niacin + 0.005 mg / L biotin + 0.2 mg / L glycine + It is composed of folic acid 0.05 mg / L + hydrolyzed casein 100 mg / L + naphthylacetic acid 2.0 mg / L + 6-benzylaminoadenine 0.5 mg / L + inositol 10 mol / L + MES 3mmol / L + sterile water; The pH value of the MR culture medium is 5.6-6.0; the MR culture medium is composed of 1900 mg / L potassium nitrate + 440 mg / L calcium chloride dihydrate + 370 mg / L magnesium sulfate heptahydrate + 170 mg / L potassium dihydrogen phosphate + 3.1 mg / L boric acid + 0.42 mg / L potassium iodide + 0.13 mg / L sodium molybdate dihydrate + 0.013 mg / L cobalt chloride hexahydrate + 11.16 mg / L manganese sulfate tetrahydrate + 4.3 mg / L zinc sulfate heptahydrate + 0.013 mg / L copper sulfate pentahydrate + 13.94 mg / L ferrous sulfate heptahydrate + 18.64 mg / L disodium ethylenediaminetetraacetic acid + 0.5 mg / L vitamin B1 + 0.5 mg / L vitamin B6 + 5.0 mg / L niacin + 0.05 mg / L biotin + 2.0 mg / L glycine + 0.5 mg / L folic acid mg / L + hydrolyzed casein 100mg / L + naphthaleneacetic acid 1.0 mg / L + 6-benzylaminoadenine 0.4 mg / L + D-mannitol 0.35 mol / L + sterile water; The pH value of the MP culture medium is 5.6-6.0; the MP culture medium is composed of 1900 mg / L potassium nitrate + 440 mg / L calcium chloride dihydrate + 370 mg / L magnesium sulfate heptahydrate + 170 mg / L potassium dihydrogen phosphate + 3.1 mg / L boric acid + 0.42 mg / L potassium iodide + 0.13 mg / L sodium molybdate dihydrate + 0.013 mg / L cobalt chloride hexahydrate + 11.16 mg / L manganese sulfate tetrahydrate + 4.3 mg / L zinc sulfate heptahydrate + 0.013 mg / L copper sulfate pentahydrate + 13.94 mg / L ferrous sulfate heptahydrate + 18.64 mg / L disodium ethylenediaminetetraacetic acid + 0.5 mg / L vitamin B1 + 0.5 mg / L vitamin B6 + 5.0 mg / L niacin + 0.05 mg / L biotin + 2.0 mg / L glycine + 0.5 mg / L folic acid mg / L + hydrolyzed casein 100mg / L + naphthaleneacetic acid 1.0 mg / L + 6-benzylaminoadenine 0.4 mg / L + sucrose 23% mol / L + sterile water; The pH value of the E culture medium is 5.6-6.0; the E culture medium is composed of 1900 mg / L potassium nitrate + 440 mg / L calcium chloride dihydrate + 370 mg / L magnesium sulfate heptahydrate + 170 mg / L potassium dihydrogen phosphate + 3.1 mg / L boric acid + 0.42 mg / L potassium iodide + 0.13 mg / L sodium molybdate dihydrate + 0.013 mg / L cobalt chloride hexahydrate + 11.16 mg / L manganese sulfate tetrahydrate + 4.3 mg / L zinc sulfate heptahydrate + 0.013 mg / L copper sulfate pentahydrate + 13.94 mg / L ferrous sulfate heptahydrate + 18.64 mg / L disodium ethylenediaminetetraacetic acid + 0.5 mg / L vitamin B1 + 0.5 mg / L vitamin B6 + 5.0 mg / L niacin + 0.05 mg / L biotin + 2.0 mg / L glycine + 0.5 mg / L folic acid mg / L + hydrolyzed casein 100mg / L + pyruvic acid 10 mg / L + DL-malic acid 20 mg / L + citric acid 20 mg / L + BSA 500 mg / L + naphthylacetic acid 0~1.0 mg / L + 6-benzylaminoadenine 0.4 mg / L + 2,4-dichlorophenoxyacetic acid 0~2.0 mg / L + D-mannitol 0.15Mmol / L + inositol 50 mol / L + xylitol 125 mol / L + sorbitol 125 mol / L + D-cellobiose 125 mol / L + glucose 125 mol / L + sucrose 0.15mol / L + sterile water; The pH value of the callus proliferation medium is 5.6-6.0; the callus proliferation medium consists of 4.33 g / L MS medium + 1 mL / L vitamin NN solution + 30 g / L sucrose + 3 g / L mannitol + 40 mg / L adenine sulfate + 50 mg / L inositol + 6 g / L agar + 1 mg / L naphthaleneacetic acid + 0.5 mg / L 6-benzylaminoadenine + sterile water; The pH value of the synchronized proliferation culture medium is 5.6-6.0; the synchronized proliferation culture medium is composed of 4.33 g / L MS culture medium + 2 mg / L glycine + 150 mg / L inositol + 0.5 mg / L vitamin B1 thiamine hydrochloride + 0.5 mg / L vitamin B6 vitamin B6 + 5 mg / L nicotinic acid + 0.5 mg / L folic acid + 0.05 mg / L biotin + 30 g / L sucrose + 3 g / L mannitol + 40 mg / L adenine sulfate + 1 mg / L naphthaleneacetic acid + 0.4-1.0 mg / L 6-benzylaminoadenine + 0.5 mg / L indoleacetic acid + 0-5 mg / L abscisic acid + sterile water; The pH value of the bud regeneration medium is 5.6-6.0; the bud regeneration medium consists of 4.4 g / L MS medium + 10 g / L sucrose + 1 mg / L zeatin or t-zeatin + 0.01 mg / L naphthaleneacetic acid + 30-0.1 mg / L GA + 6 g / L agar + sterile water.

2. A method for efficient regeneration of potato protoplasts according to claim 1, characterized in that: The S1 comprises: culturing the potato tissue culture seedlings for 3-4 weeks under the following conditions: culturing at 20-23° C. for 16 hours under light conditions and culturing at 18-20° C. for 8 hours under dark conditions, and culturing in this way alternately under light and dark conditions.

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