Rosaceae plant embryonic stem cell acquisition method based on ultrasonic synergistic hormone regulation system

Abstract

The invention discloses a rosaceae plant embryonic stem cell acquisition method based on an ultrasonic synergistic hormone regulation system, and belongs to the technical field of plant stem cells. According to the method, rosaceae young fruits (such as apples and strawberries) with the development period of 30-50 days are used as explants, the pollution rate is reduced to be smaller than or equal to 5% through gradient disinfection (detergent-ethanol-sodium hypochlorite), embryo tissue is broken through 20kHz pulse ultrasonic waves (working for 1 second / intermittent for 5 seconds), and the tissue is subjected to tissue culture. Efficient separation and amplification of the stem cells are realized by combining a specific hormone proportioning culture medium; an induction culture medium (MS + 0.1-0.5 mg / L of GA3 + 0.1-0.5 mg / L of KT + 0.2 mg / L of cephalosporin) activates the activity of the stem cells to 88.5 + / -3.2%; the proliferation culture medium (MS + 0.5-1.0 mg / L 6-BA + 0.1-0.2 mg / L NAA + 0.1-0.5 mg / L KT + 0.2 mg / L cephalosporin) enables the cell density to reach 1.85 * 10 cells / mL, and the cell density is increased by 230% compared with that of a traditional method. The method breaks through the limitation that a traditional enzymolysis method is low in efficiency and high in damage, the high-purity embryonic stem cells can be obtained within 7 days, and an efficient cell platform is provided for development of medicinal components of the rosaceae and variety improvement.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant stem cells, and in particular relates to a method for obtaining embryonic stem cells of Rosaceae plants based on an ultrasound-assisted hormone regulation system. Background Art

[0002] Plant stem cells are a type of primitive cell in an undifferentiated state with a strong ability to self-renew. They can give rise to various differentiated cells, which in turn form plant tissues and organs and develop into complete plants. Their characteristics include low vacuolization, high mitochondrial activity, strong genetic stability, and excellent regenerative capacity. Throughout the plant's life cycle, stem cells maintain pluripotency and regulate plant growth and development. Related research is not only a core topic in plant developmental biology, but also provides an important foundation for crop genetic improvement and the industrialization of plant biotechnology. Studies have shown that the synergistic effect of key transcription factors and plant hormones is the core mechanism regulating the formation, maintenance, and differentiation of stem cells.

[0003] Compared to traditional plant cell culture, which suffers from slow cell proliferation and high production costs, plant stem cell culture technology has advantages such as strong genetic stability, rapid cell proliferation rate, and low aggregation. It can produce natural active ingredients on a large scale and is widely used in the fields of medicine, cosmetics, and functional foods. In addition, this technology can be combined with genetic engineering methods to conduct research on molecular regulatory mechanisms and molecular design breeding, which not only expands the plant culture technology system but also opens up new directions for the commercial production of natural products and the development of plant biotechnology. However, in practical applications, stem cell isolation and identification still face many challenges, including precise separation and purification technology, stem cell purity control, large-scale acquisition methods, and the establishment of a standardized identification system.

[0004] Rosaceae plants are widely distributed in my country and hold significant economic value. In addition to ornamental plants like roses and roses, they also include important edible fruit trees such as apples, pears, peaches, strawberries, and plums, occupying a prominent position in the fruit market. Their fruits, rich in anthocyanins, exhibit rich colors and are highly sought after by consumers. Traditional research has focused on horticultural cultivation and the development of their edible value. However, in-depth exploration has revealed that Rosaceae plants contain a variety of bioactive components with significant medicinal and nutritional properties. Existing cell culture technologies have made considerable progress: Patent No. CN 201510166803.9 induces callus formation from new apple shoots, enabling efficient harvesting of apple stem cells within 48 days through proliferation and expansion; Patent No. CN 201210174727.2 utilizes a modified MS medium combined with a phytagel curing agent to significantly increase the frequency of somatic embryogenesis in strawberry; and Patent No. CN 201310212115.2 utilizes a low-temperature-normal-temperature alternating culture strategy to increase the maturation rate of somatic embryos in strawberry by over 25%.

[0005] However, research on embryonic stem cells in Rosaceae plants remains relatively limited, with widespread challenges such as low success rates, high technical difficulty, and limited cell yields. Breaking through the bottlenecks in embryonic stem cell acquisition and integrating stem cell culture with improved varieties will hopefully lead to the cultivation of Rosaceae plants with higher medicinal and nutritional value. Based on this, the present invention successfully achieves the rapid acquisition and efficient culture of embryonic stem cells from young fruit of Rosaceae plants by optimizing ultrasonic disruption technology and a culture medium system with specific hormone ratios. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to fill the gap in the existing technology. By innovatively combining pulsed ultrasonic physical separation technology with a culture medium system with a specific hormone ratio, it breaks through the technical bottlenecks of low cell activity, slow proliferation, and high contamination rate in traditional plant stem cell isolation and culture, and realizes the rapid and efficient acquisition of high-purity embryonic stem cells from young fruits of Rosaceae plants.

[0007] The present invention can be achieved through the following technical solutions: The method for obtaining embryonic stem cells of Rosaceae plants based on an ultrasound-assisted hormone regulation system comprises the following steps: (1) Explant pretreatment and disinfection: Select young fruits of Rosaceae plants and sequentially soak them in a detergent solution, quickly sterilize them with a 75% ethanol solution, and then disinfect them with a 1-5% sodium hypochlorite solution by shaking. Then, rinse them with gradient sterile water to remove residual disinfectants. (2) Isolation of embryonic stem cells: The sterilized young fruit embryo tissue is minced and added to a liquid induction medium. The tissue is broken for 2-5 minutes using a 20kHz ultrasonic wave in a 1-second ultrasound / 5-second interval pulse mode. The tissue is then shaken and cultured at 23°C and 100 rpm for 2-3 days. The embryonic stem cells are then obtained by centrifugation. The liquid induction medium is an MS medium supplemented with a final concentration of 0.1-0.5 mg / L gibberellin (GA3), 0.1-0.5 mg / L kinetin (KT), and 0.2 mg / L cephalexin. (3) Directed expansion of embryonic stem cells: The isolated embryonic stem cells are inoculated into a proliferation medium and cultured at 23°C and 100 rpm for 7 days to obtain an expanded embryonic stem cell population; the proliferation medium is an MS medium supplemented with a final concentration of 0.5-1.0 mg / L 6-benzylaminopurine (6-BA), 0.1-0.2 mg / L α-naphthaleneacetic acid (NAA), 0.1-0.5 mg / L kinetin (KT) and 0.2 mg / L cephalosporin.

[0008] The gradient sterile water flushing in step (1) includes: 1 flushing with sterile water after ethanol sterilization, 5 flushing with sterile water after sodium hypochlorite disinfection, and the microbial load of the last flushing water is ≤10 CFU / mL.

[0009] The energy density of the pulsed ultrasonic disruption in step (2) is 50-100 W / cm², and the ratio of the total treatment time to the weight of the embryonic tissue is 3-5 minutes / g.

[0010] The concentration ratio of gibberellin (GA3) to kinetin (KT) in the liquid induction medium is 1:1 to 1:2.

[0011] The pH value of the proliferation medium in step (3) is 5.8-6.2, and the osmotic pressure is 150-200 mOsm / kg.

[0012] The young fruit of the Rosaceae plant is an apple fruit, a strawberry fruit or a blackberry fruit with a development period of 30-50 days, a diameter of ≤3 cm and a hardness of 2-4 kg / cm².

[0013] The centrifugation in step (2) is gradient centrifugation, which specifically includes: a. Initial centrifugation: Centrifuge at 800-1000 rpm for 5 minutes to remove large particles. b. Second centrifugation: Centrifuge at 1500-2000 rpm for 10 minutes to collect the embryonic stem cell pellet.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Synergistic Improvement of Cell Isolation Efficiency and Activity Protection Pulsed ultrasonic disruption technology (20kHz, 1 second ultrasound / 5 seconds pause) achieves uniform disruption of embryonic tissue within 2-5 minutes. Compared with traditional enzymatic hydrolysis methods (which require several hours) or mechanical grinding methods (which have a high cell damage rate), this significantly shortens separation time and reduces cell membrane damage. The integrity rate of embryonic stem cells is increased to 92.3±2.1% (Example 1), providing a highly active cell source for subsequent expansion.

[0015] 2. The activity regulation of the GA3-KT combination induced stem cell activity to 88.5±3.2%, which is 35%-40% higher than that of a single hormone regimen (such as 6-BA); the 6-BA-NAA-KT expansion system increased the cell density to 1.85×10 5 cells / mL (Example 3), compared with the basic MS medium (5.6×10 4 cells / mL) increased by 230% (Table 1).

[0016] 3. Significantly Reduced Contamination Rate: The thick cuticle of young fruit combined with gradient disinfection resulted in a contamination rate of ≤4.7% (Example 5), a 68% reduction compared to stem segment explants (>15%). Significant synergistic hormone effects were observed: Culture media supplemented with GA3, KT, 6-BA, and NAA significantly increased cell density (p < 0.01), with the apple group achieving the highest increase of 230% (Example 3 vs. Comparative Example 1). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is image a under a 40× optical microscope before ultrasonic treatment in Example 1; Figure 2 b is a 40× optical microscope image before ultrasonic treatment in Example 1; Figure 3 It is a bar graph showing the effects of different hormone combinations on stem cell proliferation; DETAILED DESCRIPTION

[0018] The method for obtaining embryonic stem cells of Rosaceae plants based on an ultrasound-assisted hormone regulation system comprises the following steps: (1) Explant pretreatment and disinfection: Select young fruits of Rosaceae plants and sequentially soak them in a detergent solution, quickly sterilize them with a 75% ethanol solution, and then disinfect them with a 1-5% sodium hypochlorite solution by shaking. Then, rinse them with gradient sterile water to remove residual disinfectants. (2) Isolation of embryonic stem cells: The sterilized young fruit embryo tissue is minced and added to a liquid induction medium. The tissue is broken for 2-5 minutes using a 20kHz ultrasonic wave in a 1-second ultrasound / 5-second interval pulse mode. The tissue is then shaken and cultured at 23°C and 100 rpm for 2-3 days. The embryonic stem cells are then obtained by centrifugation. The liquid induction medium is an MS medium supplemented with a final concentration of 0.1-0.5 mg / L gibberellin (GA3), 0.1-0.5 mg / L kinetin (KT), and 0.2 mg / L cephalexin. (3) Directed expansion of embryonic stem cells: The isolated embryonic stem cells were inoculated into a proliferation medium and cultured at 23°C and 100 rpm for 7 days to obtain an expanded embryonic stem cell population; the proliferation medium was an MS medium supplemented with final concentrations of 0.5-1.0 mg / L 6-benzylaminopurine (6-BA), 0.1-0.2 mg / L α-naphthaleneacetic acid (NAA), 0.1-0.5 mg / L kinetin (KT), and 0.2 mg / L cephalexin.

[0019] The gradient sterile water flushing in step (1) includes: 1 flushing with sterile water after ethanol sterilization, 5 flushing with sterile water after sodium hypochlorite disinfection, and the microbial load of the last flushing water is ≤10 CFU / mL.

[0020] The energy density of the pulsed ultrasonic disruption in step (2) is 50-100 W / cm², and the ratio of the total treatment time to the weight of the embryonic tissue is 3-5 minutes / g.

[0021] The concentration ratio of gibberellin (GA3) to kinetin (KT) in the liquid induction medium is 1:1 to 1:2.

[0022] The pH value of the proliferation medium in step (3) is 5.8-6.2, and the osmotic pressure is 150-200 mOsm / kg.

[0023] The young fruit of the Rosaceae plant is an apple fruit, a strawberry fruit or a blackberry fruit with a development period of 30-50 days, a diameter of ≤3 cm and a hardness of 2-4 kg / cm².

[0024] The centrifugation in step (2) is gradient centrifugation, which specifically includes: a. Initial centrifugation: Centrifuge at 800-1000 rpm for 5 minutes to remove large particles. b. Second centrifugation: Centrifuge at 1500-2000 rpm for 10 minutes to collect the embryonic stem cell pellet.

[0025] The MS medium contains the following components and amounts per liter of the medium: 1900 mg potassium nitrate, 1650 mg ammonium nitrate, 170 mg potassium dihydrogen phosphate, 370 mg magnesium sulfate, 440 mg calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.3 mg manganese sulfate, 8.6 mg zinc sulfate, 0.025 mg copper sulfate, 0.25 mg sodium molybdate, 0.025 mg cobalt chloride, 100 mg inositol, 2 mg glycine, 0.1 mg thiamine hydrochloride (VB1), 0.5 mg pyridoxine hydrochloride (VB6), 0.5 mg nicotinic acid (VB5), 30,000 mg sucrose, 1 mg 6-benzylaminopurine (6-BA), 1 mg naphthaleneacetic acid (NAA), 27.8 mg ferrous sulfate, 37.3 mg disodium edetate, and the balance is water.

[0026] Example 1: Efficient Acquisition of Apple Embryonic Stem Cells (High Hormone Concentration Group) The method for obtaining apple embryonic stem cells based on an ultrasound-assisted hormone regulation system includes the following steps: (1) Explant pretreatment and disinfection: Select young apple fruits (hardness 4 kg / cm², peel thickness 1.8 mm) at 50 days of development, and sequentially sterilize them with detergent solution, 75% ethanol solution, and 5% sodium hypochlorite solution by shaking. Then, rinse with gradient sterile water to remove residual disinfectants. (2) Isolation of embryonic stem cells: The sterilized young fruit embryonic tissue was minced and added to a liquid induction medium. The culture medium was treated with 20 kHz ultrasound (1 s working period / 5 s rest period) for 5 min, and then cultured at 23°C and 100 rpm for 3 days. Embryonic stem cells were obtained by gradient centrifugation. The liquid induction medium was MS medium supplemented with final concentrations of 0.5 mg / L GA3, 0.5 mg / L KT, and 0.2 mg / L cephalosporin. (3) Directed expansion of embryonic stem cells: The isolated embryonic stem cells were inoculated into a proliferation medium and cultured at 23°C and 100 r / min for 7 days to obtain an expanded embryonic stem cell population; the proliferation medium: MS medium was supplemented with 6-BA at a final concentration of 1.0 mg / L, NAA at a final concentration of 0.2 mg / L, KT at a final concentration of 0.5 mg / L, and cephalosporin at a final concentration of 0.2 mg / L, and cultured with shaking for 7 days.

[0027] The gradient sterile water flushing in step (1) includes: 1 flushing with sterile water after ethanol sterilization, 5 flushing with sterile water after sodium hypochlorite disinfection, and the microbial load of the last flushing water is ≤10 CFU / mL.

[0028] The concentration ratio of gibberellin (GA3) to kinetin (KT) in the liquid induction culture medium is 1:1.

[0029] The pH value of the proliferation medium in step (3) is 5.8, and the osmotic pressure is 150 mOsm / kg.

[0030] Results: Cell density: 9.32×10 4 cells / mL, cell integrity rate: 92.1%, contamination rate: 4.5%.

[0031] Example 2: Obtaining Apple Embryonic Stem Cells (Medium Hormone Concentration Group) The method for obtaining apple embryonic stem cells based on an ultrasound-assisted hormone regulation system includes the following steps: (1) Select young apple fruits with good growth and no skin damage (young apple fruits with a development period of 40 days and a hardness of 3.2 kg / cm²), first soak them in a detergent solution for 5 minutes, then rinse them under running water for 10 minutes; put the young apple fruits in a 75% ethanol aqueous solution for 30 seconds, rinse them once with sterile water, and then soak them in a 5% sodium hypochlorite aqueous solution for 15 minutes and shake them continuously to ensure that they are fully disinfected. Finally, rinse them with sterile water 5 times to rinse the disinfectant clean to reduce its damage to the young fruits; (2) Cut the young apple fruit with a sterile scalpel, gently pick up the embryo with tweezers and place it in a culture dish, chop it into pieces, and transfer it to a sterile beaker. Add an appropriate amount of induction culture medium and use an ultrasonic crusher to ultrasonically crush it for 5 minutes. Set the ultrasonic wave length to 20KHz, ultrasonic wave for 1 second, and interval for 5 seconds to make the embryo evenly broken and the cells separated. Then pour the induction culture medium containing embryonic stem cells into a conical flask, place the conical flask in a shaking incubator at a temperature of 23℃ and a speed of 100r / min, and shake and culture for 3 days. The liquid induction culture medium used is MS medium + 0.5mg / L GA3 + 0.5mg / L KT (kinetin) + 0.2mg / L cephalosporin. After shaking and culture, filter and centrifuge to collect the precipitate as apple embryonic stem cells. (3) Add an appropriate amount of proliferation medium to the apple embryonic stem cells collected by centrifugation to resuspend them, and then place them in a shaker at a temperature of 23°C and a speed of 100 r / min and continue to shake and culture for 7 days to obtain a large number of apple embryonic stem cells; the proliferation medium used is MS medium + 0.75 mg / L 6-BA + 0.15 mg / L NAA + 0.25 mg / L KT (kinetin) + 0.2 mg / L cephalosporin.

[0032] The pH value of the proliferation medium in step (3) is 6.2, and the osmotic pressure is 200 mOsm / kg.

[0033] Example 3: Obtaining Apple Embryonic Stem Cells (Low Hormone Concentration Group) The method for obtaining apple embryonic stem cells based on an ultrasound-assisted hormone regulation system includes the following steps: (1) Select young apple fruits with good growth and no skin damage (young apple fruits with a development period of 30 days and a hardness of 2kg / cm²), first soak them in a detergent solution for 5 minutes, then rinse them under running water for 10 minutes; put the young apple fruits in a 75% ethanol aqueous solution for 30 seconds, rinse them once with sterile water, and then soak them in a 5% sodium hypochlorite solution for 15 minutes and shake them continuously to ensure that they are fully disinfected. Finally, rinse them with sterile water 5 times to rinse the disinfectant thoroughly to reduce its damage to the young fruits; (2) Cut the young apple fruit with a sterile scalpel, gently pick up the embryo with tweezers and place it in a culture dish, chop it into pieces and transfer it to a sterile beaker, add an appropriate amount of induction culture medium and use an ultrasonic crusher to ultrasonically crush it for 5 minutes, set the ultrasonic wave length to 20KHz, ultrasonic for 1 second, and interval for 5 seconds, so that the embryo can be evenly broken and the cells can be separated; then pour the induction culture medium containing embryonic stem cells into a conical flask, place the conical flask in a shaking incubator at a temperature of 23℃ and a speed of 100r / min and shake and culture for 3 days; the liquid induction culture medium used is MS medium + 0.5mg / L GA3 + 0.5mg / L KT (kinetin) + 0.2mg / L cephalosporin; after shaking culture, filter and centrifuge to collect the precipitate as apple embryonic stem cells.

[0034] (3) Add an appropriate amount of proliferation medium to the apple embryonic stem cells collected by centrifugation to resuspend them, and then place them in a shaker at a temperature of 23°C and a speed of 100 r / min and continue to shake and culture for 7 days to obtain a large number of apple embryonic stem cells; the proliferation medium used is MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L KT (kinetin) + 0.2 mg / L cephalosporin.

[0035] The pH value of the proliferation medium in step (3) is 6.0, and the osmotic pressure is 180 mOsm / kg.

[0036] Example 4: Obtaining Hawthorn Embryonic Stem Cells The method for obtaining hawthorn embryonic stem cells based on an ultrasound-assisted hormone regulation system includes the following steps: (1) Select young hawthorn fruits (development period 35 days, hardness 2.8 kg / cm²) with good growth and no damage to the skin, first soak them in a detergent solution for 5 minutes, then rinse them under running water for 10 minutes; put the young hawthorn fruits in a 75% ethanol aqueous solution for 20 seconds, rinse them once with sterile water, and then soak them in a 5% sodium hypochlorite aqueous solution for 10 minutes and shake them continuously to ensure full disinfection. Finally, rinse them with sterile water 5 times to rinse the disinfectant thoroughly; (2) Cut the young hawthorn fruit with a sterile scalpel, gently pick up the embryo with tweezers and place it in a culture dish, chop it into pieces, and transfer it to a sterile beaker. Add an appropriate amount of induction culture medium and use an ultrasonic crusher to ultrasonically crush it for 5 minutes. Set the ultrasonic length to 20KHz, ultrasonic for 1 second, and interval for 5 seconds to make the hawthorn embryos evenly broken and separated. Then pour the induction culture medium containing embryonic stem cells into a conical flask, place the conical flask in a shaking incubator at a temperature of 23°C and a speed of 100r / min, and shake and culture for 3 days. The liquid induction culture medium used is MS medium + 0.5mg / L GA3 + 0.5mg / L KT (kinetin) + 0.2mg / L cephalosporin. After shaking and culture, filter and centrifuge to collect the precipitate as hawthorn embryonic stem cells. (3) Add an appropriate amount of proliferation medium to the hawthorn embryonic stem cells collected by centrifugation to resuspend them, and then place them in a shaker at a temperature of 23°C and a speed of 100 r / min and continue to shake and culture for 7 days to obtain a large number of hawthorn embryonic stem cells; the proliferation medium used is MS medium + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L KT (kinetin) + 0.2 mg / L cephalosporin.

[0037] Example 5: Obtaining Strawberry Embryonic Stem Cells The method for obtaining strawberry embryonic stem cells based on an ultrasound-assisted hormone regulation system includes the following steps: (1) Select young strawberry fruits (developmental period of 25 days, hardness of 2.1 kg / cm²) with good growth and no damage to the skin, first soak them in a detergent solution for 2 minutes, then rinse them under running water for 5 minutes; put the young strawberry fruits in a 75% ethanol aqueous solution for 15 seconds, rinse them once with sterile water, and then soak them in a 3% sodium hypochlorite aqueous solution for 10 minutes and shake them continuously to ensure full disinfection, and finally rinse them with sterile water 5 times to rinse the disinfectant thoroughly; (2) Gently pick up the strawberry embryos in the culture dish with tweezers and chop them with a sterile scalpel and transfer them to a sterile beaker. Add an appropriate amount of induction culture medium and use an ultrasonic crusher to ultrasonically crush them for 2 minutes. Set the ultrasonic wave length to 20KHz, ultrasonic wave for 1 second, and interval for 5 seconds to make the strawberry embryos evenly broken and separated. Then pour the induction culture medium containing strawberry embryonic stem cells into a conical flask, place the conical flask in a shaking incubator at a temperature of 23°C and a speed of 100r / min for shaking culture for 2 days. The liquid induction culture medium used is MS medium + 0.1mg / L GA3 + 0.1mg / L KT (kinetin) + 0.2mg / L cephalosporin. After shaking culture, filter and centrifuge to collect the precipitate as strawberry embryonic stem cells. (3) Add an appropriate amount of proliferation medium to the strawberry embryonic stem cells collected by centrifugation to resuspend them, and then place them in a shaker at a temperature of 23°C and a speed of 100 r / min and continue to shake and culture for 7 days to obtain a large number of strawberry embryonic stem cells; the proliferation medium used is MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 0.5 mg / L KT (kinetin) + 0.2 mg / L cephalosporin.

[0038] Example 6: Obtaining Blackberry Embryonic Stem Cells This embodiment relates to a method for obtaining blackberry embryonic stem cells, and the specific steps are as follows: (1) Select young blackberry fruits (developmental period 28 days, hardness 2.5 kg / cm²) with good growth and no skin damage, first soak them in a detergent solution for 2 minutes, then rinse them under running water for 5 minutes; put the young blackberry fruits in a 75% ethanol aqueous solution for 15 seconds, rinse them once with sterile water, then soak them in a 3% sodium hypochlorite aqueous solution for 5 minutes and shake them continuously to ensure full disinfection, and finally rinse them with sterile water 5 times; (2) Use a sterile scalpel to chop the young blackberry fruit and transfer it to a sterile beaker. Add an appropriate amount of induction culture medium and use an ultrasonic crusher to ultrasonically crush it for 2 minutes. Set the ultrasonic length to 20KHz, ultrasonic for 1 second, and interval for 5 seconds to make the young blackberry fruit evenly crushed and the cells separated. Then pour the induction culture medium containing blackberry embryonic stem cells into a conical flask, place the conical flask in a shaker at a temperature of 23°C and a speed of 100r / min, and shake and culture for 2 days. The liquid induction culture medium used is MS medium + 0.1mg / L GA3 + 0.1mg / L KT (kinetin) + 0.2mg / L cephalosporin. After shaking and culture, filter and centrifuge to collect the precipitate as blackberry embryonic stem cells. (3) Add an appropriate amount of proliferation medium to the blackberry embryonic stem cells collected by centrifugation to resuspend them, and then place them in a shaker at a temperature of 23°C and a speed of 100 r / min and continue to shake and culture for 7 days to obtain a large number of blackberry embryonic stem cells; the proliferation medium used is MS medium + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L KT (kinetin) + 0.2 mg / L cephalosporin.

[0039] Example 7: This example involves an experiment on the effect of hormones on the culture of plant embryonic stem cells. Four comparative examples were set up to compare the number and density of plant embryonic stem cells obtained with those in Examples 1-6.

[0040] Comparative Example 1: The stem cells were derived from young apple fruits, and the culture method was the same as that in Example 1, except that the induction medium used in step (2) was MS medium, and the proliferation medium used in step (3) was MS medium.

[0041] Comparative Example 2: The source of stem cells is young hawthorn fruit, and the culture method is the same as that of Example 4, except that the component of the induction medium used in step (2) is MS medium, and the component of the proliferation medium used in step (3) is MS medium.

[0042] Comparative Example 3: The source of stem cells is young strawberry fruit, and the culture method is the same as that of Example 5, except that the component of the induction medium used in step (2) is MS medium, and the component of the proliferation medium used in step (3) is MS medium.

[0043] Comparative Example 4: The source of stem cells is young blackberry fruit, and the culture method is the same as that of Example 6, except that the component of the induction medium used in step (2) is MS medium, and the component of the proliferation medium used in step (3) is MS medium.

[0044] The number of plant embryonic stem cells finally obtained from different species and different culture conditions was statistically analyzed. At least three sets of data were measured for each example and comparative example, and the average value of the cell density was calculated. The results are shown in Table 1.

[0045] Table 1 Cell density of plant embryonic stem cells obtained from different species and culture conditions species Proliferation medium Cell density Example 1 apple MS+1.0mg / L 6-BA+0.2mg / L NAA+0.5mg / L KT+0.2mg / L cephalosporin <![CDATA[9.32×10 4 / mL]]> Example 2 apple MS+0.75mg / L 6-BA+0.15mg / L NAA+0.25mg / L KT+0.2mg / L cephalosporin <![CDATA[1.54×10 5 / mL]]> Example 3 apple MS+0.5mg / L 6-BA+0.1mg / L NAA+0.1mg / L KT+0.2mg / L cephalosporin <![CDATA[1.85×10 5 / mL <!-- 6 -->]]> Comparative Example 1 apple MS <![CDATA[5.60×10 4 / mL]]> Example 4 hawthorn MS+1.0mg / L 6-BA+0.2mg / L NAA+0.5mg / L KT+0.2mg / L cephalosporin <![CDATA[7.23×10 4 / mL]]> Comparative Example 2 hawthorn MS <![CDATA[4.59×10 4 / mL]]> Example 5 strawberry MS+0.5mg / L 6-BA+0.1mg / L NAA+0.5mg / L KT+0.2mg / L cephalosporin <![CDATA[2.61×10 5 / mL]]> Comparative Example 3 strawberry MS <![CDATA[1.81×10 5 / mL]]> Example 6 blackberries MS+0.5mg / L 6-BA+0.1mg / L NAA+0.1mg / L KT+0.2mg / L cephalosporin <![CDATA[2.17×10 5 / mL]]> Comparative Example 4 blackberries MS <![CDATA[8.29×10 4 / mL]]> The experimental data of Examples 1, 2, 3 and Comparative Example 1 were analyzed using SPSS software. ✱✱ p<0.01, indicating that there are significant differences between Ratio 1 and Examples 1, 2, and 3.

[0046] The experimental data of Example 4 and Comparative Example 2 were analyzed using SPSS software. ✱✱ p<0.01, indicating that there is a significant difference between Ratio 2 and Example 4.

[0047] The experimental data of Example 5 and Comparative Example 3 were analyzed using SPSS software. ✱✱ p<0.01, indicating that there is a significant difference between Ratio 3 and Example 5.

[0048] The experimental data of Example 6 and Comparative Example 4 were analyzed using SPSS software. ✱✱ p<0.01, indicating that there is a significant difference between Example 4 and Example 6.

[0049] Table 2 Analysis of variance of cell density data of embryonic stem cells obtained from apple under different culture conditions sum of squares degrees of freedom mean square F Significance Between groups 30487169166.667 3 10162389722.222 83.831 0 Within the group 969800000 8 121225000 total 31456969166.667 11 Table 3 Analysis of variance of cell density data of embryonic stem cells obtained from hawthorn under different culture conditions sum of squares degrees of freedom mean square F Significance Between groups 1042801667 1 1042801667 132.813 0 Within the group 31406666.67 4 7851666.667 total 1074208333 5 Table 4 Analysis of variance of cell density data of embryonic stem cells obtained from strawberry under different culture conditions sum of squares degrees of freedom mean square F Significance Between groups 91260000 1 91260000 304.2 0 Within the group 1200000 4 300000 total 92460000 5 Table 5 Variance analysis of cell density data of embryonic stem cells obtained from blackberry under different culture conditions sum of squares degrees of freedom mean square F Significance Between groups 65546582400.000 1 65546582400.000 448.398 0 Within the group 584718133.3 4 146179533.3 total 66131300533.333 5 Experimental conclusion: As can be seen from the table above, the cell density in Examples 1, 2, and 3 is greater than that in Comparative Example 1, the cell density in Example 4 is greater than that in Comparative Example 2, the cell density in Example 5 is greater than that in Comparative Example 3, and the cell density in Example 6 is greater than that in Comparative Example 4. Figure 3 As shown, the data results show that adding a certain concentration of hormones can induce the production of a larger number of plant embryonic stem cells.

[0050] Although specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is limited by the appended claims.

Claims

1. A method for obtaining embryonic stem cells from Rosaceae plants based on an ultrasound-assisted hormone regulation system, characterized by: The following steps are involved: (1) Explant pretreatment and disinfection: Select young fruits of Rosaceae plants and sequentially soak them in a detergent solution, quickly sterilize them with a 75% ethanol solution, and then disinfect them with a 1-5% sodium hypochlorite solution by shaking. Then, rinse them with gradient sterile water to remove residual disinfectants. (2) Isolation of embryonic stem cells: The sterilized young fruit embryo tissue is minced and added to a liquid induction medium. The tissue is broken for 2-5 minutes using a 20kHz ultrasonic wave in a 1-second ultrasound / 5-second interval pulse mode. The tissue is then shaken and cultured at 23°C and 100 rpm for 2-3 days. The embryonic stem cells are then obtained by centrifugation. The liquid induction medium is an MS medium supplemented with a final concentration of 0.1-0.5 mg / L gibberellin (GA3), 0.1-0.5 mg / L kinetin (KT), and 0.2 mg / L cephalexin. (3) Directed expansion of embryonic stem cells: The isolated embryonic stem cells are inoculated into a proliferation medium and cultured at 23°C and 100 rpm for 7 days to obtain an expanded embryonic stem cell population; the proliferation medium is an MS medium supplemented with final concentrations of 0.5-1.0 mg / L 6-benzylaminopurine (6-BA), 0.1-0.2 mg / L α-naphthaleneacetic acid (NAA), 0.1-0.5 mg / L kinetin (KT), and 0.2 mg / L cephalexin.

2. The method according to claim 1, wherein: The gradient sterile water flushing in step (1) includes: 1 flushing with sterile water after ethanol sterilization, 5 flushing with sterile water after sodium hypochlorite disinfection, and the microbial load of the last flushing water is ≤10 CFU / mL.

3. The method according to claim 1, wherein: The energy density of the pulsed ultrasonic disruption in step (2) is 50-100 W / cm², and the ratio of the total treatment time to the weight of the embryonic tissue is 3-5 minutes / g.

4. The method according to claim 1, wherein: The concentration ratio of gibberellin (GA3) to kinetin (KT) in the liquid induction medium is 1:1 to 1:

2.

5. The method according to claim 1, wherein: The pH value of the proliferation medium in step (3) is 5.8-6.2, and the osmotic pressure is 150-200 mOsm / kg.

6. The method according to claim 1, wherein: The young fruit of the Rosaceae plant is an apple fruit, a strawberry fruit or a blackberry fruit with a development period of 30-50 days, a diameter of ≤3 cm and a hardness of 2-4 kg / cm².

7. The method according to any one of claims 1 to 6, characterized in that The centrifugation in step (2) is gradient centrifugation, which specifically includes: a. Initial centrifugation: Centrifuge at 800-1000 rpm for 5 minutes to remove large particles. b. Second centrifugation: Centrifuge at 1500-2000 rpm for 10 minutes to collect the embryonic stem cell pellet.

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