Method capable of improving oat tissue culture efficiency

Abstract

The invention discloses a method capable of improving the oat tissue culture efficiency. The method comprises the steps of oat seed disinfection, embryo taking, callus induction culture, callus subculture, callus differentiation culture, callus rooting culture, seedling hardening treatment, oat regeneration plant obtaining and the like. The method has the remarkable effects that by optimizing the proportion of hormones in the callus induction culture medium, the subculture medium, the differentiation induction culture medium and the rooting culture medium, the capability of performing dedifferentiation and redifferentiation on the oat embryos to form new plants can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and in particular to a method capable of improving the efficiency of oat tissue culture. Background Art

[0002] Oats (Avena sativa) are a globally important dual-use crop for both food and feed. Their production ranks fourth among cereal crops worldwide, behind wheat (Triticum aestivum), rice (Oryza sativa), and corn (Zea mays). They are considered the most complete and nutritious cereal food. The amino acid profile of the protein in naked oat kernels is well-balanced, containing all eight essential amino acids. In recent years, the nutritional, health, feed, and environmental benefits of oats have been rapidly gaining recognition. In addition to being a high-quality grain, oats can also serve as livestock forage. Their soft stems and thick leaves are rich in nutrients, with higher levels of protein, fat, and digestible fiber in their stems and leaves than crops like wheat, barley, and corn, while containing less crude fiber. This makes oats highly palatable and a high-quality green fodder or silage for livestock such as dairy cows. Oats are cold-resistant, drought-resistant, and moderately saline-alkali-tolerant, making them a key cultivar for grasslands in alpine regions. They play a significant role in addressing forage shortages in my country's high-altitude and high-latitude regions. Furthermore, oats are windproof, erosion-resistant, and water- and soil-retaining, effectively increasing soil nutrient levels and fertilizer retention, thus contributing to ecological restoration. Therefore, oats possess exceptionally high ecological value.

[0003] Breeding new oat varieties is a key technical approach to achieving high, high-quality, stable, and efficient yields. With the advancement of biotechnology, the application of plant genetic engineering in crop improvement is gaining increasing attention. The primary challenge in implementing molecular breeding methods such as marker-assisted selection, transgenic technology, and gene editing is the establishment of tissue culture and a stable in vitro plant regeneration system. Tissue culture is the foundation for successful introduction of foreign genes, and plant regeneration is crucial for successful genetic transformation. Oat tissue culture has been reported both domestically and internationally. While overseas work began in the 1960s, reports of oat tissue culture research in China only began in the 1980s. However, compared to other grass crops such as corn, wheat, and rice, reports on oat tissue culture research remain relatively rare. Currently, the most common genetic transformation method for oats, both domestically and internationally, is biolistic bombardment. A major advantage of biolistic bombardment is its flexibility in accepting recipient plant species, and vector construction is also relatively simple. However, due to the random nature of gene bombardment, the integration of exogenous genes into the host genome is relatively complex, resulting in unstable integration sites and a high copy number. Transgenic offspring are prone to mutations and loss of the exogenous gene, resulting in unstable expression of the exogenous gene in the host plant. Agrobacterium-mediated transformation, on the other hand, offers high transformation frequency, low copy number, and simple instrumentation. In recent years, Agrobacterium-mediated transformation has become widely used in monocotyledonous plants such as rice and maize, making Agrobacterium-mediated transformation of oats possible.

[0004] The genetic transformation capacity of oats primarily depends on their regeneration capacity, which in turn is strongly dependent on genotype. This variability in oat regeneration capacity due to genotype differences has severely hampered research on the biological mechanisms of oats and hampered the development of oat biotechnology breeding. Although there have been previous reports of regenerating plants through oat tissue culture, the requirements for tissue culture conditions vary significantly between oat varieties, and with the continuous emergence of new oat varieties, the universality of the same culture conditions has decreased.

[0005] Therefore, the inventors explored the callus induction and plant regeneration efficiency of mature oat embryos under different culture conditions, found out the culture conditions that are more suitable for oat regeneration, and established an efficient and stable tissue culture regeneration system. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for improving the efficiency of oat tissue culture, which can effectively improve the ability of oat embryos to dedifferentiate and redifferentiate to form new plants, and improve the differentiation rate of callus tissue.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] One of the objects of the present invention is to provide a method for improving the efficiency of oat tissue culture, comprising the following steps:

[0009] Step 1: Disinfection of oat seeds;

[0010] Step 2: embryo extraction;

[0011] Step 3: Callus induction culture: The removed mature embryos are inoculated into callus induction medium and cultured in the dark at room temperature;

[0012] The callus induction medium contains the following substances and concentrations: 2,4-D 3 mg / L, NAA 1 mg / L, IAA 1 mg / L, sucrose 25 g / L, MS powder 4.43 mg / L, phytogels 3.5 mg / L, and hydrolyzed casein 500 mg / L;

[0013] Step 4: Subculture of callus tissue: transfer the callus tissue grown in step 3 to a subculture medium and subculture it at room temperature in a dark environment;

[0014] Step 5, callus differentiation culture: inoculate the subcultured callus onto differentiation induction medium and culture under light at room temperature;

[0015] Step 6: Callus rooting culture: The callus that has differentiated into leaves is transferred to a rooting medium and cultured under light at room temperature;

[0016] Step 7: Hardening: Hardening oat regenerated seedlings that grow well in the rooting medium to obtain oat regenerated plants.

[0017] Furthermore, the specific steps of disinfecting the oat seeds in step 1 are as follows:

[0018] Select mature oat seeds of similar size and full grains, and rinse them with tap water for 3-5 minutes;

[0019] Sterilize with 75% anhydrous ethanol in a clean bench for 3 minutes;

[0020] Rinse with sterile water 3 times;

[0021] Disinfection with 10% sodium hypochlorite for 5 minutes;

[0022] The oat seeds were rinsed 5 times with sterile water to obtain sterilized oat seeds.

[0023] Furthermore, the specific steps for embryo extraction in step 2 are as follows:

[0024] Soak the sterilized oat seeds in sterile water and place them in a 4°C refrigerator for 24 hours;

[0025] After the seeds mature, use sterile tweezers to take out the oat seeds and spread them flat on sterile filter paper. Let them air dry for 15 to 20 minutes to allow the water to be absorbed by the filter paper.

[0026] Use tweezers to peel off the mature embryo of the oat seed.

[0027] Furthermore, the subculture medium contains the following substances and concentrations: 2,4-D 3 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, and plant gel 3.5 g / L.

[0028] Furthermore, the differentiation induction medium contains the following substances and concentrations: MS powder 4.43 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, IAA 0.5 mg / L, and plant gel 3.5 g / L.

[0029] Furthermore, the rooting medium contains the following substances and concentrations: MS powder 0.221 g / L, NAA 0.5 mg / L, 0.1% rooting powder solution, and plant gel 3.5 g / L.

[0030] Furthermore, the duration of callus induction culture in step 3 is 15 days.

[0031] Furthermore, the callus subculture in step 4 includes at least two subcultures, with a subculture interval of 15 days.

[0032] Furthermore, the duration of light culture in step 5 and step 6 is 16 hours.

[0033] The second object of the present invention is to provide a culture medium for oat tissue culture, wherein the culture medium consists of a callus induction medium, a subculture medium, a differentiation induction medium and a rooting medium;

[0034] The callus induction medium contains the following substances and concentrations: 2,4-D 3 mg / L, NAA 1 mg / L, IAA 1 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, phytogels 3.5 g / L, and hydrolyzed casein 500 mg / L;

[0035] The subculture medium contained the following substances and concentrations: 2,4-D 3 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, and phytagel 3.5 g / L;

[0036] The differentiation induction medium contains the following substances and concentrations: MS powder 4.43 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, IAA 0.5 mg / L, and phytagel 3.5 g / L;

[0037] The rooting culture medium contains the following substances and concentrations: MS powder 0.221 g / L, NAA 0.5 mg / L, 0.1% rooting powder solution, and plant gel 3.5 g / L.

[0038] The remarkable effect of the present invention is that by optimizing the ratio of each hormone in the callus induction medium, subculture medium, differentiation induction medium and rooting medium, the present invention can effectively improve the ability of oat embryos to dedifferentiate and redifferentiate to form new plants, and increase the callus differentiation rate by 5% to 15%. It has broad application prospects in oat gene editing, molecular design breeding and gene function verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a comparison chart of the effects of different hormone ratio culture media on the induction rate and diameter of oat mature embryo callus;

[0040] Figure 2 This is a comparison chart of the effects of different hormone ratio culture media on the bud formation rate and differentiation rate of oat callus;

[0041] Figure 3 This is a schematic diagram of the results of oat callus induction;

[0042] Figure 4 Schematic diagram of the results of oat callus induction differentiation and seedling hardening and transplanting. DETAILED DESCRIPTION

[0043] The specific implementation manner and working principle of the present invention will be further described in detail below with reference to the accompanying drawings.

[0044] Example 1:

[0045] This embodiment provides a culture medium for oat tissue culture, which consists of a callus induction medium, a subculture medium, a differentiation induction medium, and a rooting medium;

[0046] The callus induction medium contains the following substances and concentrations: 3 mg / L 2,4-D, 1 mg / L NAA, 1 mg / L IAAA, 25 g / L sucrose, 4.43 g / L MS powder, 3.5 g / L phytagel, and 500 mg / L hydrolyzed casein;

[0047] The subculture medium contained the following substances and concentrations: 3 mg / L 2,4-D, 25 g / L sucrose, 4.43 g / L MS powder, and 3.5 g / L phytagel;

[0048] The differentiation induction medium contains the following substances and concentrations: 4.43 g / L MS powder, 1.5 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L IAA, and 3.5 g / L phytagel;

[0049] The rooting medium contains the following substances and concentrations: 0.221 g / L MS powder, 0.5 mg / L NAA, 0.1% rooting powder, and 3.5 g / L plant gel.

[0050] Example 2:

[0051] This example proposes a method for improving the efficiency of oat tissue culture by using the culture medium described in Example 1. The specific steps are as follows:

[0052] Step 1: Disinfection of oat seeds:

[0053] The seeds of Baiyan No. 7 oat were collected, and mature oat seeds of similar size and full grains were selected and rinsed with tap water for 3-5 minutes; then disinfected with 75% anhydrous ethanol for 3 minutes in an ultra-clean workbench; rinsed with sterile water 3 times; disinfected with 10% sodium hypochlorite for 5 minutes; and rinsed with sterile water 5 times to obtain the disinfected oat seeds.

[0054] Step 2: Embryo retrieval:

[0055] Soak the sterilized oat seeds in sterile water and place them in a 4°C refrigerator for 24 hours. After the seeds mature, use sterile tweezers to take out the oat seeds and spread them flat on sterile filter paper. Let them air dry for 15 to 20 minutes to allow the moisture to be absorbed by the filter paper. Use tweezers to peel off the mature embryos of the oat seeds.

[0056] Step 3: Callus induction culture:

[0057] The removed mature embryos were inoculated into callus induction medium. In this embodiment, four callus induction mediums ASC1, ASC2, ASC3, and ASC4 with different hormones added were designed, as shown in Table 1. ASC3 is the callus induction medium described in Example 1, and the specific ratio is: 3 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 1 mg / L NAA (naphthaleneacetic acid), 1 mg / L IAA (auxin), 25 g / L sucrose, 4.43 g / L MS powder, 3.5 g / L phytogel, and 500 mg / L hydrolyzed casein.

[0058] Nine mature embryos were placed in each culture dish, and 15 culture dishes were placed in each material. After inoculation, the culture dishes were placed in the dark at 25±1°C for 15 days. The number of callus blocks formed and the size of the callus diameter were counted every 3 days.

[0059] Effects of different hormone ratios on the induction rate and diameter of mature embryo callus from oats Figure 1 As shown, Figure 1 A shows the callus induction rate of mature embryos of oat; Figure 1 B shows the diameter of mature embryo callus of oat.

[0060] from Figure 1 The experimental results shown in the figure show that all four callus induction media can induce mature oat embryos to grow callus tissue. Among them, under the hormone ratio of ASC3 medium, the callus formation rate of mature oat embryos was the highest, which was 78.5%, followed by ASC1 70.4%, ASC2 68.2% and ASC4 65.1%.

[0061] By measuring the diameter of callus tissue growth under four culture media, the results showed that the callus tissue growth diameter in ASC3 was the largest, which was 0.71 cm; followed by the callus tissue in ASC1, which was 0.62 cm; and the callus tissue growth diameters in ASC2 and ASC4 were smaller, which were 0.47 cm and 0.43 cm respectively.

[0062] In conclusion, the medium with ASC3 hormone ratio is most conducive to inducing the dedifferentiation of mature embryos of oats to form callus tissue.

[0063] Step 4: Subculture of callus tissue:

[0064] After about 15 days, the callus grown in step 3 was transferred to subculture medium ASS and cultured in the dark at 25±1°C. Subculture was performed every 15 days for a total of two subcultures.

[0065] The subculture medium described in this step is the subculture medium described in Example 1, and the specific composition is shown in Table 1, including: 3 mg / L 2,4-D, 25 g / L sucrose, 4.43 g / L MS powder, and 3.5 g / L plant gel.

[0066] Step 5: Callus differentiation culture:

[0067] The callus tissue after two subcultures was inoculated onto three different differentiation media ASD1, ASD2, and ASD3. The ratios of each culture medium are shown in Table 1. The culture was placed under light at 25±1°C for 16 hours per day. The differentiation rate of the oat callus was finally calculated. ASD2 is the differentiation induction medium described in Example 1 of the present invention, and the specific ratio is: 4.43 g / L MS powder, 1.5 mg / L 6-BA (6-benzylaminopurine), 0.5 mg / L NAA, 0.5 mg / L IAA, and 3.5 g / L phytogel.

[0068] Effects of different hormone ratio culture media on the bud formation rate and differentiation rate of oat callus Figure 2 As shown, Figure 2 A shows the bud formation rate of oat callus; Figure 2 B shows the differentiation rate of oat callus.

[0069] The experimental results showed that all three differentiation media with different hormone ratios could induce oat callus to form buds and differentiate. The bud formation rates of calli using ASD1 and ASD2 media were higher, at 51.81% and 53.48%, respectively, while the bud formation rate of calli using ASD3 medium was lower, at 48.24%. Further differentiation induction using the same differentiation induction medium revealed that the bud differentiation rates of ASD1 and ASD2 media were higher, at 47.52% and 48.27%, respectively, while the bud differentiation rate of ASD3 medium was lower, at 41.78%. This indicates that ASD2 can be used as an effective medium for inducing callus differentiation in oats.

[0070] Step 6: Callus rooting culture:

[0071] The callus tissue that has differentiated into leaves is transferred to a rooting medium and cultured under light at room temperature to allow the root buds to grow proportionally; the composition of the rooting medium is shown in Table 1, specifically comprising: 0.221 g / L MS powder, 0.5 mg / L NAA, 0.1% rooting powder, and 3.5 g / L plant gel.

[0072] Thus, the rooting medium is based on 1 / 2 MS powder, to which 1% rooting powder and 0.5 mg / L NAA are added to promote root growth. The culture is placed at 25±1°C and incubated with 16 hours of light, and the seedling rate is finally calculated.

[0073] Step 7: Hardening:

[0074] The oat regenerated seedlings that grow well in the rooting medium are subjected to seedling hardening treatment to obtain oat regenerated plants.

[0075] Take out the regenerated seedlings after hardening for 2-3 days, rinse the culture medium at their roots with tap water, and transplant them into the soil. Use sterile water containing 1% rooting powder for watering in the early stage. Place the seedlings at 16℃ under light / dark 16 / 8h to grow in the early stage of transplanting to promote their growth and tillering.

[0076] Table 1 Culture medium used for oat tissue regeneration

[0077]

[0078] The results of inducing oat callus using the method described in this example are as follows: Figure 3 As shown, Figure 3 A shows the mature embryo of oat. Figure 3 B and 3C show oat callus tissue; the results of differentiating, hardening and transplanting the obtained oat regenerated plants are shown in Figure 4 As shown, Figure 4 A shows the buds obtained by inducing differentiation of oat callus. Figure 4B and 4C show oat regenerated plants obtained by oat bud differentiation. Figure 4 D is the oat plant after hardening and transplanting.

[0079] In summary, the method provided by the present invention can improve the regeneration ability of mature oat embryos. By optimizing the ratio of each hormone in the callus induction medium, subculture medium, differentiation induction medium and rooting medium, it can effectively improve the ability of oat embryos to dedifferentiate and redifferentiate to form new plants, and increase the callus differentiation rate by 5% to 15%. It has broad application prospects in oat gene editing, molecular design breeding and gene function verification.

[0080] The technical solution provided by the present invention is introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for improving oat tissue culture efficiency, characterized in that: The steps include: Step 1: Disinfection of oat seeds; Step 2, embryo extraction; Step 3, callus induction culture: inoculate the removed mature embryos into callus induction medium and culture them in the dark at room temperature; The callus induction medium contains the following substances and concentrations: 2,4-D 3 mg / L, NAA 1 mg / L, IAA 1 mg / L, sucrose 25 g / L, MS powder 4.43 mg / L, plant gel 3.5 mg / L, and hydrolyzed casein 500 mg / L; Step 4, callus subculture: transfer the callus grown in step 3 to a subculture medium and subculture at room temperature and in a dark environment; Step 5, callus differentiation culture: inoculating the subcultured callus onto a differentiation induction medium and culturing under light at room temperature; Step 6, callus rooting culture: the callus that differentiated into leaves was transferred to the rooting medium and cultured under light at room temperature; Step 7, seedling hardening treatment: hardening the oat regenerated seedlings that grow well in the rooting medium to obtain oat regenerated plants.

2. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The specific steps of oat seed disinfection described in step 1 are as follows: Select mature oat seeds of similar size and full grains, and rinse them with tap water for 3-5 minutes; Use 75% anhydrous ethanol to disinfect in a clean bench for 3 minutes; Rinse with sterile water 3 times; 10% sodium hypochlorite disinfection for 5 minutes; The oat seeds were rinsed 5 times with sterile water to obtain sterilized oat seeds.

3. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The specific steps for embryo extraction in step 2 are as follows: Soak the sterilized oat seeds in sterile water and place them in a 4°C refrigerator for 24 hours; After the seeds mature, use sterile tweezers to take out the oat seeds and spread them flat on sterile filter paper, and let them air dry for 15 to 20 minutes to allow the water to be absorbed by the filter paper; Use tweezers to peel off the mature embryo of the oat seed.

4. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The substances and concentrations contained in the subculture medium are as follows: 2,4-D 3 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, and plant gel 3.5 g / L.

5. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The substances and concentrations contained in the differentiation induction medium are as follows: MS powder 4.43 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, IAA 0.5 mg / L, and plant gel 3.5 g / L.

6. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The rooting medium contains the following substances and concentrations: 0.221 g / L MS powder, 0.5 mg / L NAA, 0.1% rooting powder solution, and 3.5 g / L plant gel.

7. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The duration of callus induction culture in step 3 is 15 days.

8. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The callus subculture in step 4 includes at least two subcultures, one subculture every 15 days.

9. The method for improving oat tissue culture efficiency according to claim 1, characterized in that: The duration of light culture in step 5 and step 6 is 16 hours.

10. A culture medium for oat tissue culture, characterized in that: The culture medium consists of a callus induction medium, a subculture medium, a differentiation induction medium and a rooting medium; The callus induction medium contains the following substances and concentrations: 2,4-D 3 mg / L, NAA 1 mg / L, IAA 1 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, plant gel 3.5 g / L, and hydrolyzed casein 500 mg / L; The substances and concentrations contained in the subculture medium are as follows: 2,4-D 3 mg / L, sucrose 25 g / L, MS powder 4.43 g / L, and plant gel 3.5 g / L; The differentiation induction medium contains the following substances and concentrations: MS powder 4.43 g / L, 6-BA 1.5 mg / L, NAA 0.5 mg / L, IAA 0.5 mg / L, and plant gel 3.5 g / L; The rooting medium contains the following substances and concentrations: 0.221 g / L MS powder, 0.5 mg / L NAA, 0.1% rooting powder solution, and 3.5 g / L plant gel.

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