Method for establishing a high-efficiency regeneration system of oat germ tip callus induction based on small peptides

Abstract

The invention discloses a method for establishing an oat germ tip callus induction and efficient regeneration system based on small peptides, firstly, pretreated oat seeds are placed in a 1 / 2MS culture medium for culture; when the seeds germinate to a certain length, the germ tip is taken and placed in an induction culture medium with the addition of small peptides for induction, then transferred to a differentiation and proliferation culture medium with the addition of small peptides for continued culture, and then transferred to a rooting culture medium for culture to differentiated seedlings with a certain root length; finally, the seedlings are removed, the seedlings are hardened, and the vermiculite is cultured to obtain a complete regenerated plant. The invention selects oat germs as explants for plant tissue regeneration culture, the materials are cheap and easy to obtain, the operation is simple, and by introducing small peptides that can regulate the local wound response and regeneration ability of plants, the ability of the oat germ tip to induce callus in in vitro culture is enhanced, and the regeneration conditions of callus are optimized, thereby constructing an efficient and stable oat regeneration system, providing an important basis for gene editing and oat germplasm innovation.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing breeding, and in particular relates to a method for establishing an oat germ tip callus induction and efficient regeneration system based on small peptides. Background Art

[0002] Oats are annual herbaceous plants of the genus Avena in the Poaceae family, mainly distributed in Inner Mongolia, Hebei, Gansu and other regions of my country. As a dual-purpose crop for food and feed, oats have excellent characteristics such as high grass yield, drought resistance, cold resistance, and resistance to barrenness. They also have the advantages of high nutritional content, good palatability, high digestibility, and are suitable for green feed or hay preparation. At the same time, oats play an important role in ensuring national food security, protecting the ecological environment, and promoting agricultural efficiency. Therefore, oats are a grass species with great development value.

[0003] Tissue culture technology is an asexual reproduction technology that uses the theory of plant omnipotence. It separates the embryo tip from the plant tissue as an explant and then places it in different culture media for sterile culture. Tissue culture technology has significant advantages such as fast reproduction speed, high production efficiency, maintaining the inherent shape and characteristics of the original variety, saving land and saving reproduction materials.

[0004] The selection of suitable explants is the first step in establishing tissue culture technology, which is directly related to the success of subsequent culture. However, in the process of callus induction and regeneration system establishment of oat embryo tips, there are still problems of low efficiency and poor regeneration ability. Therefore, exploring new methods and strategies, such as introducing small peptides with biological activity, to optimize culture conditions, improve induction efficiency and regeneration ability, is of great significance for promoting genetic improvement of oat crops and the development of modern agriculture. Summary of the invention

[0005] In view of the problems existing in the above-mentioned background technology, the purpose of the present invention is to provide a method for establishing an oat germ tip callus induction and efficient regeneration system based on small peptides.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for establishing an oat germ tip callus induction and efficient regeneration system based on small peptides, the method comprising the following steps:

[0008] (1) sterilizing the peeled oat seeds and then soaking them in sterile water;

[0009] (2) The soaked oat seeds were rinsed with sterile water for several times, and then placed in 1 / 2MS medium and cultured in the dark at a constant temperature of 26±1°C for 2 days; the 1 / 2MS medium was: MS base salts 2.17 g / L + sucrose 30 g / L + agar 7 g / L, pH = 5.8;

[0010] (3) When the length of oat seeds germinated was 0.1-0.3 cm, the embryo tip was placed in an induction medium supplemented with a small peptide, and cultured in the dark at a constant temperature of 26±1°C for 29-31 days; the induction medium was: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + small peptide 1 nM, pH = 5.8;

[0011] (4) Transferring the cells to a differentiation and proliferation medium supplemented with a small peptide and continuing culturing until the seedlings are 2 to 3 cm long; the differentiation and proliferation medium is: basal medium + 6-BA 2 mg / L + NAA 0.5 mg / L + small peptide 1 nM, pH = 5.7;

[0012] (5) transferring the seedlings to be differentiated into a rooting medium and culturing the seedlings until the root length is 5 to 7 cm; the rooting medium is a basal medium;

[0013] (6) Remove the seedlings, open the bottle and harden them, then wash the roots and use sterilized vermiculite to culture them to obtain complete regenerated plants.

[0014] Preferably, in step (1), the disinfection treatment is: first soaking the oat seeds in 75% C2H5OH for 1 min, then washing the oat seeds with ultrapure water for 4 to 6 times; then soaking in 5% NaClO for 7 min, then washing the oat seeds with sterile ultrapure water for 5 to 7 times, and adding sterile ultrapure water after washing to cover the oat seeds.

[0015] Preferably, in step (2), the culture conditions of the 1 / 2MS culture medium are: cultured in the dark at a constant temperature of 26±1°C for 2 days.

[0016] Preferably, in step (3), the culture conditions of the induction culture medium are: cultured in the dark at a constant temperature of 26±1°C for 30 days.

[0017] Preferably, in step (4), the culture conditions of the differentiation and proliferation medium are: light culture, the light intensity is 4300-4800 Lux, and the culture time is 14 h·d -1 , the culture temperature is 26±1℃.

[0018] Preferably, in step (5), the culture conditions of the rooting medium are: light culture, the light intensity is 4300-4800 Lux, and the light duration is 14 h·d -1 , the culture temperature is 26±1℃.

[0019] Preferably, in steps (4) to (5), the basic culture medium is: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L, pH = 5.7.

[0020] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0021] (1) In the process of establishing the callus induction and efficient regeneration system of oat germ tips, the induction medium and the differentiation and proliferation medium used in the present invention are all added with small peptides with biological activity, which can regulate the local wound response and regeneration ability of plants, enhance the ability of oat germs to induce callus in in vitro culture, optimize the regeneration conditions of callus, and thus construct an efficient and stable oat regeneration system.

[0022] (2) The method for inducing oat germ tip callus tissue and establishing an efficient regeneration system based on small peptides provided by the present invention has the advantages of fast reproduction speed, high production efficiency, and maintaining the inherent shape and characteristics of the original variety, providing an important research basis for the smooth implementation of gene editing and the smooth advancement of oat germplasm innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 oat seeds in the 1 / 2MS medium provided in an embodiment of the present invention;

[0024] Figure 2 oat seeds in the early germination stage in the 1 / 2MS medium provided in an embodiment of the present invention;

[0025] Figure 3 The oat germ tip early stage induced callus tissue provided by the embodiment of the present invention;

[0026] Figure 4 The induced callus tissue of oat germ tip 15 days old provided by the embodiment of the present invention, wherein: Figure 4 a represents the culture carried out using the induction medium without adding small peptides. Figure 4 b is the culture carried out using the induction medium supplemented with small peptides;

[0027] Figure 5 The oat callus differentiated green seedlings provided by the embodiment of the present invention, wherein: Figure 5 a represents the differentiation and proliferation medium without the addition of small peptides. Figure 5 b is a differentiation and proliferation medium with added small peptides;

[0028] Figure 6 The rooting and differentiation seedlings are cultured in the rooting medium provided in the embodiment of the present invention;

[0029] Figure 7 The invention relates to a regenerated plant of oats after transplantation. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] The method for establishing a callus induction and efficient regeneration system of oat germ tip based on small peptides is as follows:

[0032] 1. Pretreatment of oat seeds

[0033] Peeling process: Select oat seeds that are free of mold and disease, have full grains, and are uniform in size, peel off the oat seed coat, and wait for the next step.

[0034] Disinfection: Take the peeled oat seeds and place them in a 50mL centrifuge tube. Soak them in 75% C2H5OH for 1 minute, then wash the oat seeds with ultrapure water for 4 to 6 times; soak them in 5% NaClO for 7 minutes, then place the centrifuge tube containing the oat seeds in a clean bench, wash the oat seeds with sterile ultrapure water for 5 to 7 times in the clean bench, and add sterile ultrapure water after washing to cover the oat seeds. During the two soaking processes, the 50mL centrifuge tube needs to be placed on a rotary culture mixer to keep rotating.

[0035] Soaking treatment: Soak the oat seeds in sterile ultrapure water for 24 hours, then wash the seeds 8 to 10 times with sterile ultrapure water in a clean bench. After washing, place the seeds on sterile filter paper to absorb moisture and proceed to the next step.

[0036] 2. Preparation of culture medium

[0037] 1 / 2MS medium: MS base salts 2.17 g / L + sucrose 30 g / L + agar 7 g / L; pH = 5.8; high pressure wet heat sterilization at 115°C for 30 min;

[0038] Induction medium: L3 base salts 4.49g / L + thiamine hydrochloride 10mg / L + L-glutamine 420mg / L + 2,4-D 2mg / L + hydrolyzed casein 0.25g / L + MES 1.95g / L + maltose 30g / L + plant gel 3.5g / L + small peptide 1nM, pH = 5.8; high pressure wet heat sterilization at 115℃ for 30min;

[0039] Basic culture medium: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L; high pressure wet heat sterilization at 115°C for 30 min.

[0040] Differentiation and proliferation medium: basal medium + 6-BA 2mg / L + NAA 0.5mg / L + small peptide 1nM, pH = 5.7;

[0041] Rooting medium: the above-mentioned basic medium, pH=5.7.

[0042] The small peptide used in the above-mentioned induction medium and differentiation and proliferation medium was synthesized by GenScript Biotech, named PtrCLE13A / B / C, with a sequence of RLVPTGPNPLHH. The detailed information is shown in Table 1:

[0043] Table 1 Information on small peptides used in induction medium and differentiation and proliferation medium

[0044]

[0045] 3. Treatment and differentiation induction of oat seeds

[0046] (1) The pretreated oat seeds were placed in 1 / 2MS culture medium and cultured in the dark in a tissue culture room at a constant temperature of 26±1°C for 2 days. Figure 1 During the dark culture period, observe in time until the seeds germinate and the bud length is 0.1 to 0.3 cm (refer to Figure 2 ) to proceed to the next step.

[0047] (2) Take the embryo tip of oat seeds with a germination length of 0.1 to 0.3 cm and place it in the induction medium. Figure 3 The cells were cultured in the dark in a tissue culture room at a constant temperature of 26±1℃ for 30 days, and the induced callus tissues were cultured for 15 days. Figure 4 As shown, Figure 4 a represents the culture carried out using the induction medium without adding small peptides. Figure 4 b is the culture using the induction medium with small peptides added. The callus formation rate, callus formation time and callus contamination rate were observed in time during the dark culture period.

[0048] (3) Transfer to differentiation and proliferation medium and continue culturing until the seedlings are 2 to 3 cm long. Figure 5 As shown, Figure 5 a represents the differentiation and proliferation medium without the addition of small peptides. Figure 5 b is the differentiation and proliferation medium with small peptides added; the culture process adopts light culture, the light intensity is 4300-4800 Lux, and the culture time is 14 h·d -1 , the culture temperature is 26±1℃.

[0049] (4) Transfer the seedlings to the rooting medium and culture until the root length of the seedlings to be differentiated is about 5 cm (refer to Figure 6 ); the culture process adopts light culture, the light intensity is 4300~4800Lux, and the culture time is 14h·d -1 , the culture temperature is 26±1℃.

[0050] (5) The seedlings were removed from the tissue culture room, filled with tap water, and opened under natural light to harden the seedlings. The rooting rate was then counted. The culture bottles were opened, the roots were cleaned, and the seedlings were planted in vermiculite sterilized in a high-pressure steam sterilizer to obtain complete regenerated plants (refer to Figure 7 ).

[0051] 4. Statistics and analysis of implementation results

[0052] Using the above disinfection method and induction method, the callus healing rate of oat seeds is 95% to 100%, the healing time is 4 to 5 days, the contamination rate is 0, the callus differentiation rate is 77% to 82%, and the rooting rate is 95% to 96%.

[0053] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a callus induction and efficient regeneration system of oat germ tips based on small peptides, characterized in that: The following steps are involved: (1) Sterilize the peeled oat seeds and then soak them in sterile water; (2) The soaked oat seeds were rinsed with sterile water for several times, and then placed in 1 / 2 MS medium and cultured in the dark at a constant temperature of 26±1°C for 2 days; the 1 / 2 MS medium was: MS base salts 2.17 g / L + sucrose 30 g / L + agar 7 g / L, pH = 5.8; (3) When the length of oat seeds germinated was 0.1-0.3 cm, the embryo tip was placed in an induction medium supplemented with a small peptide and cultured in the dark at a constant temperature of 26±1°C for 29-31 days; the induction medium was: L3 base salts 4.49 g / L + thiamine hydrochloride 10 mg / L + L-glutamine 420 mg / L + 2,4-D 2 mg / L + hydrolyzed casein 0.25 g / L + MES 1.95 g / L + maltose 30 g / L + plant gel 3.5 g / L + small peptide 1 nM, pH = 5.8; (4) Transferring the cells to a differentiation and proliferation medium supplemented with a small peptide and continuing culturing until the seedlings are 2-3 cm long; the differentiation and proliferation medium is: basal medium + 6-BA 2 mg / L + NAA 0.5 mg / L + small peptide 1 nM, pH = 5.7; (5) transferring the seedlings to a rooting medium and culturing until the root length of the seedlings to be differentiated is 5 to 7 cm; the rooting medium is a basal medium; (6) Remove the seedlings, open the bottle for seedling hardening, then wash the roots, and use sterilized vermiculite to culture to obtain complete regenerated plants; In steps (4) to (5), the basic culture medium is: MS base salts 4.33 g / L + maltose 30 g / L + plant gel 3.5 g / L, pH = 5.7; the sequence of the small peptide is RLVPTGPNPLHH.

2. The method for establishing a small peptide-based oat germ tip callus induction and efficient regeneration system as claimed in claim 1, characterized in that: In step (1), the disinfection treatment is as follows: first, the oat seeds are soaked in 75% C2H5OH for 1 min, and then the oat seeds are washed with ultrapure water for 4 to 6 times; then, they are soaked in 5% NaClO for 7 min, and then the oat seeds are washed with sterile ultrapure water for 5 to 7 times. After washing, sterile ultrapure water is added to cover the oat seeds.

3. The method for establishing a small peptide-based oat germ tip callus induction and efficient regeneration system as claimed in claim 1, characterized in that: In step (3), the culture conditions of the induction culture medium are: cultured in the dark at a constant temperature of 26±1°C for 30 days.

4. The method for establishing a small peptide-based oat germ tip callus induction and efficient regeneration system as claimed in claim 1, characterized in that: In step (4), the culture conditions of the differentiation and proliferation medium are: light culture, the light intensity is 4300~4800 Lux, and the culture time is 14 h·d -1 , the culture temperature is 26±1℃.

5. The method for establishing a small peptide-based oat germ tip callus induction and efficient regeneration system as claimed in claim 1, characterized in that: In step (5), the culture conditions of the rooting medium are: light culture, the light intensity is 4300~4800 Lux, and the light duration is 14 h·d -1 , the culture temperature is 26±1℃.

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