Efficient in-vitro regeneration and rapid propagation method for Shannong No.1 rootstock

Abstract

The invention relates to the technical field of agricultural biology, and discloses an efficient in-vitro regeneration and rapid propagation method for Shannong No.1 rootstocks, and the method comprises the steps of explant selection and pretreatment, sterile disinfection, axillary bud germination and induction, adventitious bud proliferation, rooting culture and acclimatization and transplanting. The browning rate and the pollution rate of the explants are effectively reduced by combining full-dark culture at the initial induction stage; gibberellin A3 is creatively added into a proliferation culture medium to be matched with 6-benzylamino adenine and indole-3-butyric acid, so that a bud seedling vitrification phenomenon is prevented, and stem elongation is promoted; in the rooting stage, dark treatment is combined with high-concentration auxin induction, so that the rooting rate and the root system quality are improved. According to the method, an efficient and stable regeneration system is established, the method has the advantages of high propagation coefficient, robust seedling, high transplanting survival rate and the like, and reliable technical support is provided for large-scale propagation of the Shannong No.1 rootstock.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock. Background Technology

[0002] Peach is an important deciduous fruit tree in my country, and its high-quality cultivation highly depends on excellent rootstocks. Shannong No. 1, as a newly bred excellent rootstock variety, has outstanding resistance to root-knot nematodes, salt and alkali tolerance, and drought tolerance. Its grafting compatibility with the main cultivated varieties exceeds 95%, and it can achieve a dwarfing effect of 30%-40%. It is suitable for intensive dense planting and has broad application prospects. In order to realize its large-scale seedling production and germplasm innovation, it is crucial to establish an efficient in vitro regeneration and rapid propagation technology system. This technology can break through seasonal limitations and maintain the excellent traits of the parent plants with a high propagation coefficient. It is the key to promoting the industrialization of this rootstock and subsequent genetic improvement.

[0003] However, the in vitro rapid propagation technology for this rootstock still faces bottlenecks: First, browning and contamination of explants are prominent issues. Traditional disinfection processes struggle to balance sterilization effectiveness and tissue activity, easily leading to contamination rates as high as 30%-50% or browning rates as high as 25%-40%, severely impacting explant survival and callus induction. Second, bud proliferation efficiency is low and the cycle is long. Existing culture medium hormone ratios largely borrow from other varieties and have not been optimized for Shannong No. 1, resulting in an imbalance between cytokinin and auxin, insufficient proliferation multiples, and subculture easily leads to problems such as bud aging and vitrification, with a cycle as long as 30-40 days, making it difficult to meet the needs of rapid propagation. Third, rooting culture is unstable, resulting in low transplant survival rates. Unreasonable auxin concentrations in the rooting medium often lead to poor root development and an imbalanced root-to-shoot ratio, affecting the subsequent absorption and adaptation capabilities of test-tube seedlings. Finally, the reproducibility of the technology system is poor. Existing methods rely on specific experimental conditions and personnel experience. Key parameters such as culture medium formulation and temperature and light conditions lack standardization, and results vary greatly between different batches. A stable and scalable standardized process has not yet been formed.

[0004] In summary, overcoming the aforementioned technical bottlenecks and establishing an efficient, stable, and standardized in vitro rapid propagation system is an urgent need to accelerate the promotion and application of the superior rootstock Shannong No. 1 and improve the economic and ecological benefits of the peach industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, which solves the problems of high explant browning and contamination rates, low bud proliferation coefficient, unstable rooting, low transplant survival rate, and poor genotype compatibility in existing technologies.

[0006] To achieve the above objectives, the present invention provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, comprising the following steps: Branches with axillary buds were collected from Shannong No. 1 rootstock, washed, and prepared into stem segments; Soak the stem segments in a 75% ethanol solution for 25-30 seconds, rinse, then soak in a 0.1% mercuric chloride solution for 5-6 minutes, rinse and set aside. The sterilized stem segments were inoculated into the bud induction medium and cultured in complete darkness first, and then transferred to light culture to obtain germinating adventitious buds. Adventitious shoots are inoculated into an adventitious shoot proliferation medium and subcultured to obtain shoot clusters. The plant hormones added to the adventitious shoot proliferation medium include 6-benzylaminopurine, indole-3-butyric acid and gibberellin A3. The bud clusters were cut into individual buds and inoculated into a rooting medium. They were first cultured in complete darkness for 3 days, and then transferred to light culture until roots grew. The plant hormones added to the rooting medium included indole-3-butyric acid and α-naphthaleneacetic acid. After the test-tube seedlings have rooted, they are hardened off and then transplanted into the substrate.

[0007] Preferably, the branches are semi-lignified branches of the current year collected from early May to early June; The stem segment retains 0.5 cm of the base of the petiole and has a length of 2.0-3.0 cm.

[0008] By adopting the above technical solutions, the semi-lignified branches in May and June have the strongest branching ability and the lowest content of endogenous inhibitors; retaining the base of the petiole can protect the axillary buds from mechanical damage, and at the same time, it can serve as a nutrient buffer zone to further improve the induced survival rate.

[0009] Preferably, the bud induction medium contains the following components in appropriate amounts: MS medium dry powder: 4.74 g / L; Sucrose: 30.0 g / L; Agar powder: 7.0 g / L; pH value: 5.8-6.0.

[0010] By adopting the above technical solution, using MS medium of standard concentration with 30g / L sucrose, the macro-element and carbon source requirements for axillary bud germination can be met, promoting the rapid transformation of dormant buds into the growth state.

[0011] Preferably, the adventitious bud proliferation medium contains the following components in appropriate amounts: MS medium dry powder: 4.74 g / L; 6-Benzylaminopurine: 0.5 mg / L; Indole-3-butyric acid: 0.1-0.3 mg / L; Gibberellin A3: 0.1-0.2 mg / L; Sucrose: 30.0 g / L; Agar powder: 7.0 g / L; pH value: 5.8-6.0.

[0012] By adopting the above technical solution, the specific formula ratio achieves a dynamic balance between cytokinins and auxins. The concentration of 6-benzylaminopurine is fixed at 0.5 mg / L to ensure a high proliferation coefficient, while the lower concentration can also prevent vitrification. The 0.2-0.3 mg / L GA3 works synergistically with IBA to ensure the proliferation multiple while making the obtained seedlings have dark green leaves and thick stems, thus solving the problem of weak seedlings in subculture.

[0013] Preferably, the method for preparing the adventitious bud proliferation culture medium includes: Dissolve MS medium powder, sucrose, and the corresponding amount of 6-benzylaminopurine stock solution in distilled water and stir well. Adjust the pH to 5.8-6.0 using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution, add agar powder and stir thoroughly until dissolved, then add distilled water to bring the volume to 1L. Autoclave at 121℃ and 0.1 MPa for 20 minutes. After the temperature drops to 50-60℃, add the appropriate amount of indole-3-butyric acid and gibberellin A3 stock solution, stir well, dispense into culture flasks, and cool for later use.

[0014] By adopting the above technical solutions, the well-defined preparation process ensures the stability of the physicochemical properties of the culture medium. In particular, the standardization of the volume adjustment and pH adjustment steps ensures the consistency of ion concentration and hormone activity in each batch of culture medium, providing a standardized environment for the synchronous growth of tissue culture seedlings.

[0015] Preferably, the rooting medium contains the following components in appropriate amounts: MS medium dry powder: 2.37 g / L; Indole-3-butyric acid: 4.0-5.0 mg / L; α-Naphthaleneacetic acid: 0.5 mg / L; Sucrose: 30.0 g / L; Agar powder: 7.0 g / L; pH value: 5.8-6.0.

[0016] By adopting the above technical solution, the amount of MS dry powder in the culture medium is halved (i.e., 1 / 2 MS), which reduces the concentration of inorganic salts and reduces the inhibitory effect of high osmotic pressure on new root systems. At the same time, the combination of high concentration of IBA (4.0-5.0 mg / L) and NAA (0.5 mg / L) can induce cortical cells to quickly restore their division ability, form callus tissue and differentiate adventitious roots, thus solving the key bottleneck of rooting difficulty in the in vitro regeneration of woody plants.

[0017] Preferably, the matrix is ​​a mixture of peat moss, perlite and vermiculite in a volume ratio of 2:1:1.

[0018] By adopting the above technical solution, peat moss provides organic matter and water retention, perlite provides aeration, and vermiculite provides fertilizer retention. The 2:1:1 ratio creates a loose, breathable, water- and fertilizer-retaining rhizosphere environment, which is conducive to the transition of tissue culture seedling roots from heterotrophic to autotrophic and improves the transplant survival rate.

[0019] This invention provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock. It has the following beneficial effects: 1. This invention solves the problems of severe browning of explants and contamination by limiting the disinfection time parameters of ethanol soaking and mercuric chloride soaking, and combining it with complete darkness culture in the early stage of induction. This disinfection combination can thoroughly kill surface and latent pathogens while avoiding vascular bundle damage caused by overtreatment. The initial complete darkness environment can inhibit polyphenol oxidase activity and block the oxidation of phenolic substances, thereby significantly improving the survival rate of explants and the uniformity of axillary bud germination.

[0020] 2. This invention introduces gibberellin A3 into the adventitious bud proliferation medium, forming a specific hormone ratio with 6-benzylaminopurine and indole-3-butyric acid. This effectively overcomes the vitrification phenomenon that easily occurs in the subculture of Shannong No. 1 rootstock. At this concentration, gibberellin A3 can regulate the water metabolism of cells and promote internode elongation, resulting in proliferating seedlings with normal morphology, strong stems, and dark green leaves, thus ensuring the quality of seedlings required for subsequent rooting culture.

[0021] 3. This invention employs a rooting strategy that combines dark treatment induction with light culture. By utilizing a completely dark environment for the first 3 days after inoculation, combined with high concentrations of indole-3-butyric acid and α-naphthaleneacetic acid, the rooting rate is significantly improved. Short-term dark treatment can prevent the photodegradation of exogenous auxin, which is conducive to the transport and accumulation of auxin to the lower morphological end, promoting the rapid dedifferentiation of basal cells to form root primordia. This solves the problems of difficult in vitro regeneration and low root quantity of this variety. Detailed Implementation

[0022] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0023] The explants for the Shannong No. 1 peach rootstock were taken from the current year's branches of healthy, disease-free, and mature plants.

[0024] MS medium powder (containing macro-elements, micro-elements, and organic components, but excluding agar and sucrose), plant tissue culture grade agar powder (gel strength ≥1200g / cm³). 2Sucrose, mercuric chloride, and anhydrous ethanol are all commercially available products.

[0025] Preparation Examples 1-7: Preparation Example 1: This preparation example provides a bud induction medium, the formulation of which includes: 4.74 g / L MS medium powder, 30.0 g / L sucrose, 7.0 g / L agar powder, with the balance being distilled water, and a pH value of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder and sucrose in approximately 900 mL of distilled water and stir well. Adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Add agar powder and stir thoroughly until the agar dissolves. Make up to 1 L and autoclave at 121 °C and 0.1 MPa for 20 minutes. Dispense into culture flasks and cool for later use.

[0026] Preparation Example 2: This preparation example provides an adventitious bud proliferation medium, the formulation of which includes: 4.74 g / L MS medium powder, 0.5 mg / L 6-benzylaminopurine, 0.1 mg / L indole-3-butyric acid, 0.1 mg / L gibberellin A, 30.0 g / L sucrose, 7.0 g / L agar powder, with the balance being distilled water, and a pH value of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder, sucrose, and an appropriate amount of 6-benzylaminopurine stock solution in approximately 900 mL of distilled water, stir well, adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, add agar powder, stir until the agar is completely dissolved, and bring the volume to 1 L. Autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amounts of indole-3-butyric acid and gibberellin A3 stock solution, stir well, dispense into culture flasks, and cool for later use.

[0027] Preparation Example 3: This preparation example provides an adventitious bud proliferation medium, the formulation of which includes: 4.74 g / L MS medium powder, 0.5 mg / L 6-benzylaminopurine, 0.2 mg / L indole-3-butyric acid, 0.15 mg / L gibberellin A, 30.0 g / L sucrose, 7.0 g / L agar powder, with the balance being distilled water, and a pH value of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder, sucrose, and an appropriate amount of 6-benzylaminopurine stock solution in approximately 900 mL of distilled water, stir well, adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, add agar powder, stir until the agar is completely dissolved, and bring the volume to 1 L. Autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amounts of indole-3-butyric acid and gibberellin A3 stock solution, stir well, dispense into culture flasks, and cool for later use.

[0028] Preparation Example 4: This preparation example provides an adventitious bud proliferation medium, the formulation of which includes: 4.74 g / L MS medium powder, 0.5 mg / L 6-benzylaminopurine, 0.3 mg / L indole-3-butyric acid, 0.2 mg / L gibberellin A3, 30.0 g / L sucrose, 7.0 g / L agar powder, with the balance being distilled water, and a pH value of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder, sucrose, and an appropriate amount of 6-benzylaminopurine stock solution in approximately 900 mL of distilled water, stir well, adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution, add agar powder, stir until the agar is completely dissolved, and bring the volume to 1 L. Autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amounts of indole-3-butyric acid and gibberellin A3 stock solution, stir well, dispense into culture flasks, and cool for later use.

[0029] Preparation Example 5: This preparation example provides a rooting medium, the formulation of which includes: 2.37 g / L MS medium powder, 4.0 mg / L indole-3-butyric acid, 0.5 mg / L α-naphthaleneacetic acid, 30.0 g / L sucrose, 7.0 g / L agar powder, and the balance being distilled water, with a pH of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder and sucrose in approximately 900 mL of distilled water and stir well. Adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Add agar powder and stir until the agar is completely dissolved. Make up to 1 L and autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amount of indole-3-butyric acid and α-naphthaleneacetic acid stock solution and stir well. Dispense into culture flasks and cool for later use.

[0030] Preparation Example 6: This preparation example provides a rooting medium, the formulation of which includes: 2.37 g / L MS medium powder, 4.5 mg / L indole-3-butyric acid, 0.5 mg / L α-naphthaleneacetic acid, 30.0 g / L sucrose, 7.0 g / L agar powder, and the balance being distilled water, with a pH of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder and sucrose in approximately 900 mL of distilled water and stir well. Adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Add agar powder and stir until the agar is completely dissolved. Make up to 1 L and autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amount of indole-3-butyric acid and α-naphthaleneacetic acid stock solution and stir well. Dispense into culture flasks and cool for later use.

[0031] Preparation Example 7: This preparation example provides a rooting medium, the formulation of which includes: 2.37 g / L MS medium powder, 5.0 mg / L indole-3-butyric acid, 0.5 mg / L α-naphthaleneacetic acid, 30.0 g / L sucrose, 7.0 g / L agar powder, with the balance being distilled water, and a pH value of 5.8-6.0. The preparation method includes the following steps: Dissolve MS medium powder and sucrose in approximately 900 mL of distilled water and stir well. Adjust the pH to 5.8-6.0 using 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid solution. Add agar powder and stir until the agar is completely dissolved. Make up to 1 L and autoclave at 121 °C and 0.1 MPa for 20 minutes. After the temperature drops to 50-60 °C, add the appropriate amount of indole-3-butyric acid and α-naphthaleneacetic acid stock solution and stir well. Dispense into culture flasks and cool for later use.

[0032] Examples 1-3: Example 1: This embodiment provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, including the following steps: Selection and pretreatment of explants: On a sunny morning in early May at 9:00, select healthy, disease-free mature plants of Shannong No. 1 rootstock, collect one-year-old semi-lignified branches with axillary buds, remove the leaves, retain 0.5 cm at the base of the petiole, and cut the stem into 2.0 cm long sections with one plump axillary bud; rinse with tap water for 10 minutes to remove surface dust, and place in sterile water for later use.

[0033] Aseptic disinfection: In a clean bench, immerse the pretreated stem segments in a 75% ethanol solution for 25 seconds, gently shaking them during the process. Rinse them three times with sterile water for one minute each time. Then, immerse them in a 0.1% mercuric chloride solution for 5 minutes, stirring continuously during the process. Discard the mercuric chloride solution and rinse them six times with sterile water for two minutes each time.

[0034] Axillary bud germination and induction culture: The morphological lower end of the sterilized stem segment was inserted into the bud induction culture medium provided in Preparation Example 1, with the axillary bud facing upward. Three stem segments were inoculated in each bottle. After inoculation, the culture was first carried out in complete darkness for 3 days, and then transferred to light culture. The culture temperature was 24℃, the light intensity was 2000 lux, the light time was 24h, and the culture period was 25 days to obtain germinating adventitious buds.

[0035] Proliferation culture of adventitious buds: Select robust adventitious buds without vitrification, remove old stems, retain new buds, and inoculate them into the adventitious bud proliferation medium provided in Preparation Example 2. The culture temperature is 24 degrees Celsius, the light intensity is 2500 lux, the light duration is 24 h, the culture cycle is 25 days, and the subculture is repeated 3 times to obtain proliferating bud clusters.

[0036] Rooting culture: Select robust seedlings with a height of 3 cm, remove the callus tissue at the base, and inoculate them into the rooting medium provided in Preparation Example 5. After inoculation, culture them in complete darkness for 3 days, and then transfer them to light culture. The culture temperature is 22℃, the light intensity is 1500 lux, and the light time is 12 hours / day. After 20 days of culture, white roots grow from the base.

[0037] Hardening off and transplanting: When the roots of the test-tube seedlings reach 1 cm in length, open the bottle and place them under diffused light in a greenhouse for 3 days to harden off, spraying them with sterile water to keep them moist every day; take out the test-tube seedlings, wash the culture medium off the roots, and transplant them into sterilized substrate (peat soil: perlite: vermiculite volume ratio of 2:1:1), water them thoroughly, cover them with plastic wrap, and place them in a greenhouse with a temperature of 20℃ and a shading rate of 50%. After 7 days, gradually remove the plastic wrap, and after 15 days, transfer them to normal management.

[0038] Example 2: This embodiment provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, including the following steps: Selection and pretreatment of explants: On a sunny morning in mid-May at 10:00, select healthy, disease-free mature plants of Shannong No. 1 rootstock, collect one-year-old semi-lignified branches with axillary buds, remove the leaves, retain 0.5 cm at the base of the petiole, and cut the stem into 2.5 cm long sections with 2 plump axillary buds; rinse with tap water for 12 minutes to remove surface dust, and place in sterile water for later use.

[0039] Aseptic disinfection: In a clean bench, immerse the pretreated stem segments in a 75% ethanol solution for 28 seconds, gently shaking them during the process. Rinse them three times with sterile water, then transfer them to a 0.1% mercuric chloride solution and immerse them for 5.5 minutes, stirring continuously. Discard the mercuric chloride solution and rinse them six times with sterile water.

[0040] Axillary bud germination and induction culture: The morphological lower end of the sterilized stem segment was inserted into the bud induction culture medium provided in Preparation Example 1, with the axillary bud facing upward. Four stem segments were inoculated in each bottle. After inoculation, the culture was first carried out in complete darkness for 3 days, and then transferred to light culture. The culture temperature was 25 degrees Celsius, the light intensity was 2250 lux, the light duration was 24 hours, and the culture period was 28 days to obtain germinating adventitious buds.

[0041] Adventitious bud proliferation culture: Select robust, non-vitrified adventitious buds, remove old stems, and inoculate them into the adventitious bud proliferation medium provided in Preparation Example 3; the culture temperature is 25℃, the light intensity is 2800 lux, the light duration is 24h, and the culture period is 22 days; repeat the subculture 3 times to obtain proliferating bud clusters.

[0042] Rooting culture: Select robust seedlings with a height of 3.5 cm, remove the callus tissue at the base, and inoculate them into the rooting medium provided in Preparation Example 6. After inoculation, culture them in complete darkness for 3 days, and then transfer them to light culture. The culture temperature is 23℃, the light intensity is 1800 lux, and the light time is 12 hours / day. After 22 days of culture, white roots grow from the base.

[0043] Hardening off and transplanting: When the roots of the test-tube seedlings reach 1.5 cm in length, open the bottle and place them under diffused light in a greenhouse for 4 days to harden off, spraying them with sterile water to keep them moist every day; take out the test-tube seedlings, wash the culture medium off the roots, and transplant them into sterilized substrate (peat soil: perlite: vermiculite volume ratio of 2:1:1), water them thoroughly, cover them with plastic wrap, and place them in a greenhouse with a temperature of 22 degrees Celsius and a shading rate of 50%. After 7 days, gradually remove the plastic wrap, and after 15 days, transfer them to normal management.

[0044] Example 3: This embodiment provides a method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, including the following steps: Selection and pretreatment of explants: On a sunny morning in early June at 11:00, select healthy, disease-free mature plants of Shannong No. 1 rootstock and collect one-year-old semi-lignified branches with axillary buds; cut off the leaves, leaving 0.5 cm at the base of the petiole, and cut the stem into 3.0 cm long sections with 2 plump axillary buds; rinse with tap water for 15 minutes to remove surface dust, and place in sterile water for later use.

[0045] Aseptic disinfection: In a clean bench, immerse the pre-treated stem segments in a 75% ethanol solution for 30 seconds, gently shaking them during the process, and rinse them three times with sterile water; then transfer them to a 0.1% mercuric chloride solution and immerse them for 6 minutes, stirring continuously during the process, discarding the mercuric chloride solution, and rinsing them six times with sterile water.

[0046] Axillary bud germination and induction culture: The morphological lower end of the sterilized stem segment was inserted into the bud induction culture medium provided in Preparation Example 1, with the axillary bud facing upward. Four stem segments were inoculated in each bottle. After inoculation, the culture was first carried out in complete darkness for 3 days, and then transferred to light culture. The culture temperature was 26 degrees Celsius, the light intensity was 2500 lux, the light duration was 24 hours, and the culture period was 30 days to obtain germinating adventitious buds.

[0047] Proliferation culture of adventitious buds: Select robust adventitious buds without vitrification, remove old stems, and inoculate them into the adventitious bud proliferation medium provided in Preparation Example 4. The culture temperature is 26℃, the light intensity is 3000 lux, the light duration is 24h, the culture period is 25 days, and the culture is repeated 3 times to obtain proliferating bud clusters.

[0048] Rooting culture: Select robust seedlings with a height of 4 cm, remove the callus tissue at the base, and inoculate them into the rooting medium provided in Preparation Example 7. After inoculation, culture them in complete darkness for 3 days, and then transfer them to light culture. The culture temperature is 24 degrees Celsius, the light intensity is 2000 lux, and the light time is 12 hours / day. After 25 days of culture, white roots grow from the base.

[0049] Hardening off and transplanting: When the roots of the test-tube seedlings reach 2 cm in length, open the bottle and place them under diffused light in a greenhouse for 5 days to harden off, spraying them with sterile water to keep them moist every day; take out the test-tube seedlings, wash the culture medium off the roots, and transplant them into a sterilized substrate (peat soil: perlite: vermiculite in a volume ratio of 2:1:1), water them thoroughly, cover them with plastic wrap, and place them in a greenhouse with a temperature of 25 degrees Celsius and a shading rate of 50%. After 7 days, gradually remove the plastic wrap, and after 15 days, transfer them to normal management.

[0050] Comparative Examples 1-7: Comparative Example 1: Compared with Example 2, the difference is that in the aseptic disinfection process, the soaking time in 0.1% mercuric chloride solution for 5.5 minutes is changed to soaking time in 0.1% mercuric chloride solution for 3 minutes, while the rest are the same.

[0051] Comparative Example 2: Compared with Example 2, the difference is that in the aseptic disinfection process, the soaking time in 0.1% mercuric chloride solution for 5.5 minutes is changed to soaking time in 10% sodium hypochlorite solution for 10 minutes, while the rest are the same.

[0052] Comparative Example 3: Compared with Example 2, the difference is that gibberellin A3 (GA3) was not added to the adventitious bud proliferation medium, while the other components and amounts were the same.

[0053] Comparative Example 4: Compared with Example 2, the difference is that the concentration of 6-benzylaminopurine (6-BA) in the adventitious shoot proliferation medium was adjusted to 1.5 mg / L, and gibberellin A3 was not added; all other aspects were the same.

[0054] Comparative Example 5: Compared with Example 2, the difference is that in the rooting culture step, after inoculation, the culture is not carried out in complete darkness, but is directly placed under light conditions for culture; all other steps are the same.

[0055] Comparative Example 6: Compared to Example 2, the difference is that the concentration of indole-3-butyric acid (IBA) in the rooting medium was adjusted to 0.5 mg / L, while all other aspects remained the same.

[0056] Comparative Example 7: Compared to Example 2, the difference is that the rooting medium did not contain α-naphthaleneacetic acid (NAA) but only indole-3-butyric acid (IBA), while the rest were the same.

[0057] Test Example 1-2: Test Example 1: Experimental objective: This test aims to verify the stability of the in vitro regeneration and rapid propagation methods of Shannong No. 1 rootstock provided in Examples 1 to 3 in actual operation and the physiological indicators at each stage.

[0058] Experimental steps: The parameters set in Examples 1 to 3 were selected for verification. The experimental materials were all current-year branches of mature plants of Shannong No. 1 collected in spring. Each example set up 3 parallel groups, with 100 explants initially inoculated in each group, for a total of 300 explants. The explants were cleaned, disinfected with ethanol and mercuric chloride, and subjected to primary induction culture, subculture proliferation culture, rooting culture, and hardening and transplanting in sequence according to the methods described in each example. The growth indicators of the whole cycle were counted. Among them, the contamination rate and browning rate were counted 15 days after the primary culture, the induction rate was counted at the end of the primary culture, the proliferation coefficient was counted at the end of the third subculture, and the calculation method was the total number of effective buds divided by the number of inoculated bud clusters. The rooting rate was counted 25 days after the rooting culture, and the standard was the growth of adventitious roots with a length of greater than or equal to 0.5 cm at the base. The transplant survival rate was counted 15 days after transplanting, and the standard was that the plant grew normally and new leaves emerged.

[0059] Experimental data: The experimental results are shown in Table 1. The data are presented as the mean ± standard deviation of the three parallel groups. Table 1. Statistics of growth indicators at various stages of the in vitro rapid propagation system of Shannong No. 1 rootstock. (Note: Data in the table are rounded to two decimal places, and the proliferation coefficient is the statistical mean after the third generation of culture.)

[0060] in conclusion: The contamination rate in Examples 1 to 3 was controlled below 3%, and the browning rate was below 9%, indicating that short-term ethanol soaking combined with mercuric chloride treatment for a specific duration can effectively kill endophytic fungi in woody plants and control heavy metal toxicity. The proliferation coefficient remained stable between 4.8 and 5.2, and no vitrification phenomenon occurred, indicating that the ratio of gibberellin A3, 6-BA, and IBA in the culture medium can promote stem elongation and prevent bud clustering. The rooting rate exceeded 89%, and the transplant survival rate exceeded 86%, confirming that the treatment method of dark-induced pulse combined with high-concentration IBA can initiate root primordia differentiation and inhibit excessive callus growth, establishing a stable regeneration system.

[0061] Test Example 2: Experimental objective: To clarify the impact of key process parameters in the technical solution of this invention on the regeneration system of Shannong No. 1 peach rootstock, a comparative experiment was set up.

[0062] Experimental steps: Parallel tests were conducted using Example 2 and Comparative Examples 1 to 7. The experiment consisted of three stages: disinfection, proliferation, and rooting. In the first stage, 100 stem segments were treated in each group, with three replicates. After 15 days of culture, the contamination rate and browning rate were calculated. In the second stage, sterile single buds were selected and inoculated into the culture medium. 50 bottles were inoculated in each group, with 3 buds per bottle, with three replicates. After 25 days of culture, the proliferation coefficient and vitrification rate were calculated. The criteria for vitrification were that the buds were water-soaked, semi-transparent, and the leaves were deformed. In the third stage, effective buds with a height of 3 to 4 cm were selected for rooting culture. 50 plants were inoculated in each group, with three replicates. After 25 days of culture, the rooting rate, average number of roots, and diameter of the basal callus were calculated.

[0063] Experimental data: The statistical results of key growth indicators for each treatment group are shown in Table 2. Data are expressed as mean ± standard deviation. Table 2. Effects of different process parameters on key indicators of in vitro culture of Shannong No. 1 peach rootstock Note: "—" indicates that the indicator is not the focus of the current comparison group, or that material loss due to previous processing was not included in subsequent statistics.

[0064] in conclusion: During the disinfection stage, the contamination rates of Comparative Examples 1 and 2 were 34.67% and 68.33%, respectively, higher than that of Example 2. This indicates that using sodium hypochlorite alone or shortening the treatment time of mercuric chloride is insufficient to eliminate endophytic bacteria in field woody materials. The treatment time in Example 2 is an effective parameter for balancing sterilization and tissue activity. During the proliferation stage, Comparative Example 3, without the addition of gibberellin A3, resulted in a proliferation coefficient of only 2.12 and bud clustering. Comparative Example 4, using a high concentration of 6-BA, achieved a vitrification rate of 42.67%. Example 2, by adding a trace amount of gibberellin A3 in combination with a low concentration of 6-BA, achieved bud elongation and proliferation without vitrification. During the rooting stage, Comparative Example 6, using a conventional concentration of IBA, resulted in a rooting rate of only 12.33%. Comparative Example 5, by omitting the dark culture step, resulted in callus tissue with a diameter of 8.45 mm at the base and a low rooting rate. Example 2, using a high concentration of IBA combined with 3 days of dark treatment, induced root primordia initiation and inhibited callus formation, achieving a rooting rate of 94.33%.

Claims

1. A method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock, characterized in that, Includes the following steps: Branches with axillary buds were collected from Shannong No. 1 rootstock, washed, and prepared into stem segments; Soak the stem segments in a 75% ethanol solution for 25-30 seconds, rinse, then soak in a 0.1% mercuric chloride solution for 5-6 minutes, rinse and set aside. The sterilized stem segments were inoculated into the bud induction medium and cultured in complete darkness first, and then transferred to light culture to obtain germinating adventitious buds. The adventitious buds were inoculated into an adventitious bud proliferation medium and subcultured to obtain bud clusters. The plant hormones added to the adventitious bud proliferation medium included 6-benzylaminopurine, indole-3-butyric acid and gibberellin A3. The bud clusters were cut into individual buds and inoculated into a rooting medium. They were first cultured in complete darkness for 3 days, and then transferred to light culture until roots grew. The plant hormones added to the rooting medium included indole-3-butyric acid and α-naphthaleneacetic acid. After the test-tube seedlings have rooted, they are hardened off and then transplanted into the substrate.

2. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The branches were semi-lignified branches of the current year collected from early May to early June; the stem segments retained 0.5 cm of the base of the petiole and were 2.0-3.0 cm in length.

3. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The cleaning process after ethanol immersion involves rinsing with sterile water three times; the cleaning process after mercuric chloride immersion involves rinsing with sterile water six times.

4. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The bud induction medium contains the following components in varying amounts: MS medium dry powder: 4.74 g / L; Sucrose: 30.0 g / L; Agar powder: 7.0g / L pH value: 5.8-6.0; The culture time under completely dark conditions is 3 days; The conditions for light culture are: temperature 24-26℃, light intensity 2000-2500 lux, and light duration 24h.

5. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The adventitious bud proliferation medium contains the following components in varying amounts: MS medium dry powder: 4.74 g / L; 6-Benzylaminopurine: 0.5 mg / L; Indole-3-butyric acid: 0.1-0.3 mg / L; Gibberellin A3: 0.1-0.2 mg / L; Sucrose: 30.0 g / L; Agar powder: 7.0 g / L; pH value: 5.8-6.

0.

6. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 5, characterized in that, The method for preparing the adventitious bud proliferation culture medium includes: Dissolve MS medium powder, sucrose, and the corresponding amount of 6-benzylaminopurine stock solution in distilled water and stir well. Adjust the pH to 5.8-6.0 using a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution, add distilled water to a final volume of 1 L, and add agar powder and stir thoroughly until dissolved. Autoclaving at 121°C and 0.1 MPa for 20 minutes, followed by cooling to 50-60°C, is then performed. The appropriate amounts of indole-3-butyric acid and gibberellin A3 stock solution are added, and the solution is dispensed into culture flasks while still hot and then cooled for later use.

7. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The conditions for the proliferation culture are as follows: Temperature 24-26℃, light intensity 2500-3000 lux, light duration 24h, culture period 30-35 days.

8. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The rooting medium contains the following components in varying amounts: MS medium dry powder: 2.37 g / L; Indole-3-butyric acid: 4.0-5.0 mg / L; α-Naphthaleneacetic acid: 0.5 mg / L; Sucrose: 30.0 g / L; Agar powder: 7.0 g / L; pH value: 5.8-6.

0.

9. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The conditions for the light culture are as follows: Temperature 22-24℃, light intensity 1500-2000 lux, light duration 12 hours / day.

10. The method for efficient in vitro regeneration and rapid propagation of Shannong No. 1 rootstock according to claim 1, characterized in that, The seedling hardening period is 3-5 days; The matrix is ​​composed of peat moss, perlite and vermiculite in a volume ratio of 2:1:1.

Images