Method for efficiently inducing peony underground bud polyploidy and application thereof

Abstract

The present application relates to the technical field of plant breeding, and discloses a method for efficiently inducing peony subterranean bud polyploidy and application thereof, comprising subterranean bud disinfection, cell synchronization, minimally invasive pretreatment, preparation of CS- Alg / PPy photothermal response slow-release microspheres, loading of composite inducer, microsphere implantation and photothermal regulation induction, polyploidy identification screening and transplanting, wherein the composite inducer comprises colchicine, 6-BA and graphene quantum dots, can block chromosome separation, realize cell chromosome doubling, regulate growth point cell division, simultaneously repair cell damage, improve bud survival rate, assist photothermal regulation to realize precise drug release, and promote expression of polyploid plant excellent traits. The method has high induction rate, high homozygosity and high bud survival rate, is suitable for cultivation of ornamental, cut flower, medicinal and stress-resistant peonies, and is advanced and practical in technology.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and more specifically, to a method for efficiently inducing polyploidy in underground buds of peony and its application. Background Technology

[0002] Peony (Paeonia lactiflora Pall.) is a traditional Chinese flower, valued for its ornamental, medicinal, and economic uses. Polyploid breeding is a key technology for improving peony quality; polyploid plants typically exhibit advantages such as larger flower diameter, thicker stems, enhanced stress resistance, and accumulation of medicinal components.

[0003] However, existing peony polyploid induction techniques have the following drawbacks: The induction rate using colchicine soaking (0.1%-0.5% concentration, soaking for 12-24 hours) or injection (direct injection into buds) is generally between 35% and 47%, with the drug having difficulty penetrating the thick scales of the underground buds, resulting in poor penetration efficiency; large fluctuations in drug concentration lead to asynchronous cell division, resulting in a chimerism rate exceeding 30%, making homozygous polyploid selection difficult; the injection method easily damages the vascular bundles, while the soaking method causes drug poisoning, with a bud survival rate of only 50%-60%; more than 60% of the inducing agent in traditional methods is not effectively absorbed, causing resource waste and soil pollution; and traditional soaking and smearing methods are difficult to apply precisely to the bud meristem, limiting efficiency.

[0004] Microspheres are typically composed of photothermal conversion materials (such as gold nanorods, polypyrrole, and indocyanine green ICG) combined with thermosensitive polymers (such as PLGA and PNIPAm). In existing technologies, photothermal responsive microspheres are used for the controlled release of plant growth regulators such as pesticides and auxins (e.g., IBA). This has verified the photothermal conversion, thermosensitive release, and biocompatibility of the carrier. Under near-infrared light excitation, the photothermal agent converts light energy into heat energy, and the localized heating causes a phase transition / structural loosening of the thermosensitive material, enabling the controlled release of the growth regulator. Release is triggered by light exposure and resumes slow release after light is turned off. The release rate can also be controlled by light intensity / duration. However, this technology has not yet been applied to the study of polyploid induction in peony underground buds.

[0005] Therefore, developing a method for inducing polyploidy in peony underground buds with low damage, high induction rate, and low chimerism rate is of great practical significance. Summary of the Invention

[0006] In view of this, the present invention proposes a method for efficiently inducing polyploidy in underground buds of peony and its application, aiming to solve the problems of low induction efficiency, asynchronous cell division, difficulty in screening homozygous polyploids, severe bud damage, and low drug utilization in the current technology.

[0007] This invention proposes a method for efficiently inducing polyploidy in peony underground buds, comprising the following steps: (1) Pretreatment: Select underground buds of healthy peony mother plants, disinfect, refrigerate and synchronize cells, and make a minimally invasive incision 0.5 cm away from the growth point on the side of the bud; (2) Preparation of photothermal response sustained-release microspheres: CS-Alg is used as a substrate, PPy is used to form porous microspheres, and after loading a composite inducer, they are vacuum dried to obtain microspheres loaded with the inducer; (3) Induction treatment: Microspheres loaded with inducing agent are embedded in a minimally invasive incision, moistened and fixed, and then induced to obtain induced plants; (4) Polyploid identification and screening: Identify and screen suitable homozygous polyploid plants from the induced plants; (5) Transplanting and planting: Transplant the selected polyploid seedlings to the field and perform routine field management.

[0008] Furthermore, the underground buds mentioned in step (1) are selected at the end of the dormancy period of the underground buds in January-February, with a diameter of 0.8-1.2cm and 3-4 layers of outer scales retained.

[0009] Furthermore, the disinfection process described in step (1) is as follows: after rinsing with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, then rinse with sterile water 5 times, and then absorb the surface moisture with filter paper; the depth of the minimally invasive incision is 0.3 cm and the width is 0.1 cm, and the incision direction is parallel to the bud growth direction.

[0010] Furthermore, the preparation of the photothermal responsive sustained-release microspheres in step (2) includes: ① Preparation of base solution: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution, and dissolve 2g of sodium alginate in 100mL of deionized water; ② Polypyrrole composite: Add 0.5g of pyrrole monomer to the chitosan solution, add 10mL of 0.1mol / L ferric chloride solution, and polymerize at room temperature for 2h; ③ Microsphere molding: PPy / CS composite solution and sodium alginate solution are mixed at a volume ratio of 1:1, and 5% calcium chloride solution is dripped into the microfluidic chip for cross-linking and curing for 30 min to prepare porous microspheres with a diameter of 50-80 μm.

[0011] Furthermore, the composite inducer in step (2) is a mixture of 0.2%-0.3% colchicine, 0.8 mg / L 6-benzylaminopurine, and 0.1% graphene quantum dots; the composite inducer is loaded by adsorption under a vacuum of -0.05 MPa for 2 hours.

[0012] Furthermore, in step (3), the amount of microspheres implanted with the loading inducer is 3-5; the moisturizing treatment is to wrap the microspheres with sterile degreased cotton and fix them with a plastic wrap with pre-reserved air holes, and the sterile substrate is humus: perlite = 3:1.

[0013] Furthermore, the induction treatment in step (3) involves irradiating the microspheres with a 600-800nm ​​light source for 4 hours daily, controlling the local temperature of the microspheres to 32-35℃, and culturing them for 30 days at 15-20℃ and 60-70% humidity. The light source is a 50W simulated sunlight source with an irradiation distance of 20cm. During the culturing period, sterile water is added to the absorbent cotton every 3 days, and the plastic wrap is removed after 30 days.

[0014] Furthermore, the identification described in step (4) includes: ① Initial screening: 30 days after treatment, 0.1g of leaf tissue was taken for DNA content detection, and plants with a peak value twice that of diploid plants were screened. ② Secondary screening: 60 days after germination, the root tips were pretreated with 0.1% colchicine, fixed with Carnoy's solution, dissociated with hydrochloric acid, stained with acetic carmine, and the chromosome number was observed under a microscope.

[0015] Furthermore, the transplanting time mentioned in step (5) is before spring budding, with a plant spacing of 50cm×60cm. After transplanting, the plants are thoroughly watered to settle the roots and covered with mulch.

[0016] This invention also provides the application of the method for efficiently inducing polyploidy in underground buds of peony described in the above technical solution. Specifically, the application is in the cultivation of new ornamental peony varieties, in the improvement of cut peony varieties, in the improvement of medicinal peony varieties, and in the cultivation of stress-resistant peonies.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs cell synchronization and precise drug delivery, increasing the homozygous polyploid rate to over 80% and reducing the chimeric rate to below 12%, thus solving the screening challenges inherent in traditional technologies.

[0018] This invention uses minimally invasive incisions to avoid damage to vascular bundles, and a slow-release carrier to reduce drug toxicity. It results in less damage to buds, higher bud survival rate, and improved transplant survival rate.

[0019] This invention employs photothermal responsive sustained-release microsphere carriers, enabling controllable drug release. The synergistic effect of composite inducers improves the induction rate compared to traditional methods. Furthermore, through targeted delivery of sustained-release microspheres and absorption-enhancing effects of graphene quantum dots, drug utilization is improved, reducing inducer waste and environmental pollution, which aligns with the concept of green breeding.

[0020] The molecular transport channel effect of graphene quantum dots in this invention can more efficiently break through the permeation barrier of peony underground bud scales than DMSO, thereby increasing the concentration of the inducer in the cells at the growth point; at the same time, photothermal regulation enables long-term sustained release of the drug, avoiding the problems of large fluctuations in drug concentration and insufficient local concentration in the culture medium in the prior art.

[0021] This invention does not require tissue culture and can be implemented through minimally invasive implantation and field light and heat regulation. It does not require special culture media or sterile facilities, simplifying the operation process and making it more suitable for the actual production scenario of peony division propagation. It can be directly applied to large-scale field breeding and also solves the pain point of existing technologies that are difficult to transform laboratory technologies into production applications. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0023] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] This invention proposes a method for efficiently inducing polyploidy in peony underground buds, comprising the following steps: (1) Pretreatment: Select underground buds of healthy peony mother plants, disinfect, refrigerate and synchronize cells, and make a minimally invasive incision 0.5 cm away from the growth point on the side of the bud; (2) Preparation of photothermal response sustained-release microspheres: CS-Alg is used as a substrate, PPy is used to form porous microspheres, and after loading a composite inducer, they are vacuum dried to obtain microspheres loaded with the inducer; (3) Induction treatment: Microspheres loaded with inducing agent are embedded in a minimally invasive incision, moistened and fixed, and then induced to obtain induced plants; (4) Polyploid identification and screening: Identify and screen suitable homozygous polyploid plants from the induced plants; (5) Transplanting and planting: Transplant the selected polyploid seedlings to the field and perform routine field management.

[0028] In this invention, the underground buds in step (1) are selected at the end of the dormancy period of the underground buds in January-February, with a diameter of 0.8-1.2cm and 3-4 layers of outer scales retained.

[0029] In this invention, the disinfection process in step (1) is as follows: after rinsing with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, then rinse with sterile water 5 times, and then absorb the surface moisture with filter paper; the depth of the minimally invasive incision is 0.3 cm and the width is 0.1 cm, and the incision direction is parallel to the bud growth direction.

[0030] In this invention, the preparation of the photothermal responsive sustained-release microspheres in step (2) includes: ① Preparation of base solution: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution, and dissolve 2g of sodium alginate in 100mL of deionized water; ② Polypyrrole composite: Add 0.5g of pyrrole monomer to the chitosan solution, add 10mL of 0.1mol / L ferric chloride solution, and polymerize at room temperature for 2h; ③ Microsphere molding: PPy / CS composite solution and sodium alginate solution are mixed at a volume ratio of 1:1, and 5% calcium chloride solution is dripped into the microfluidic chip for cross-linking and curing for 30 min to prepare porous microspheres with a diameter of 50-80 μm.

[0031] In the preparation process of the photothermal responsive sustained-release microspheres, chitosan (CS) is added. Chitosan can provide a microsphere framework and crosslink with sodium alginate to form a porous structure, providing space for the loading of inducers. It has excellent biocompatibility and can avoid toxicity to peony underground bud cells. The surface of chitosan is rich in amino groups, which can bind to colchicine and 6-BA through hydrogen bonds to enhance the drug adsorption stability.

[0032] In the preparation process of the photothermal responsive sustained-release microspheres, sodium alginate (Alg) is added. The sodium alginate (Alg) forms an ionic cross-linking network with chitosan, which improves the mechanical strength of the microspheres (compressive strength ≥ 0.3 MPa) and prevents breakage during implantation. Its strong hydrophilicity facilitates drug swelling and release while maintaining the moisture content of the microspheres, making it suitable for the microenvironment of the bud incision. The sodium alginate (Alg) has controllable degradation properties, slowly degrading in the soil microenvironment without residual pollution.

[0033] In the preparation process of the photothermal responsive sustained-release microspheres, the present invention adds pyrrole monomer (Py), which is oxidatively polymerized to form polypyrrole (PPy), giving the microspheres photothermal responsive properties; PPy can convert light energy into heat energy under 600-800nm ​​light irradiation, causing the microspheres to locally heat up to 32-35℃, triggering drug sustained release; it can also be combined with CS-Alg substrate without affecting the porous structure and biocompatibility of the microspheres.

[0034] In the preparation process of the photothermal responsive sustained-release microspheres, calcium chloride is added. On the one hand, the calcium chloride works synergistically with sodium alginate to enable the microspheres to cross-link and form rapidly. On the other hand, it can adjust the hardness of the microspheres, balance the drug release rate and mechanical stability, and ensure that they do not collapse after implantation.

[0035] In this invention, the composite inducer in step (2) is a mixture of 0.2%-0.3% colchicine, 0.8 mg / L 6-benzylaminopurine, and 0.1% graphene quantum dots; the composite inducer is loaded by adsorption under a vacuum of -0.05 MPa for 2 hours.

[0036] This invention prepares the composite inducer using a mixture of 0.2%-0.3% colchicine, 0.8 mg / L 6-benzylaminopurine, and 0.1% graphene quantum dots. The graphene quantum dots (GQDs) are small in size and possess both the high permeability and surface activity of nanomaterials. They can penetrate the cell walls and cell membranes of peony underground bud scale cells, forming molecular transport channels. Furthermore, their surface functional groups can bind with colchicine and 6-BA (6-benzylaminopurine) through hydrogen bonds and hydrophobic interactions, forming a stable inducer GQDs complex. This prevents drug degradation or loss outside the cell, increasing the concentration of the inducer within the growing point cells. On the other hand, colchicine is highly toxic to plant cells, and traditional methods often result in high concentrations... It can easily lead to bud necrosis; graphene quantum dots can adsorb colchicine molecules through surface charge, reducing its "instantaneous burst concentration" in cells. At the same time, its excellent biocompatibility can reduce the damage of colchicine to cell membranes; furthermore, graphene quantum dots can slightly regulate the stability of microtubules in the cell spindle, forming a synergy with the effect of colchicine in inhibiting spindle formation. Colchicine mainly blocks chromosome segregation, while GQDs assist in enhancing the spindle damage effect, thus increasing the success rate of chromosome doubling. At the same time, GQDs can remove reactive oxygen species (ROS) generated during the induction process, reducing the damage of oxidative stress to cells and ensuring the normal division and growth of polyploid cells; In addition, 6-BA was added to the compound inducer. 6-BA, as a synthetic cytokinin, can specifically activate division-related genes in the growth point cells of underground buds, causing the underground bud cells at the end of dormancy to rapidly enter an active division state. Colchicine damages the microtubule system of plant cells and inhibits cell metabolism. 6-BA can reduce the damage of colchicine to cell membranes and organelles by promoting ribosome synthesis and enhancing the activity of antioxidant enzymes (SOD, POD), thus accelerating tissue healing at minimally invasive incision sites. 6-BA regulates cell metabolic repair, and graphene quantum dots reduce the instantaneous concentration of colchicine, avoiding the "high induction" phenomenon in traditional induction methods. The contradiction of "high mortality rate accompanied by high efficiency" is addressed by 6-BA, which selectively promotes the growth advantages of polyploid cells. Polyploid cells are larger and have a relatively slower division rate. 6-BA compensates for the division disadvantage of polyploid cells by regulating cell cycle protein expression and inhibits the excessive proliferation of undoubled diploid cells. In the later stages of induction, 6-BA can promote stem cell elongation and thickening, as well as leaf chlorophyll synthesis, enabling polyploid plants to quickly exhibit the excellent phenotype of "thick stems, thick leaves, and large flowers." 6-BA can alleviate the slight stress on cells caused by local temperature (32-35℃) during photothermal regulation, maintain the normal metabolic rhythm of cells, and avoid abnormal growth caused by high temperature. The present invention also adds colchicine to the composite inducer. Colchicine can specifically inhibit the formation of the spindle in metaphase of mitosis, block chromosome separation, and achieve chromosome doubling, which is the basis for polyploid induction.

[0037] In this invention, the amount of microspheres implanted with the loading inducer in step (3) is 3-5; the moisturizing treatment is to wrap with sterile degreased cotton and fix with a plastic wrap with pre-reserved air holes, and the sterile substrate is humus: perlite = 3:1.

[0038] In this invention, the induction treatment in step (3) involves irradiating the microspheres with a 600-800nm ​​light source for 4 hours daily, controlling the local temperature of the microspheres to 32-35℃, and culturing them for 30 days at 15-20℃ and 60-70% humidity. The light source is a 50W simulated sunlight source with an irradiation distance of 20cm. During the culturing period, sterile water is added to the absorbent cotton every 3 days, and the plastic wrap is removed after 30 days.

[0039] In this invention, photothermal microspheres loaded with inducers are directly embedded into the underground buds at a distance of 0.5 cm from the growth point through a minimally invasive incision. This avoids the vascular bundles and allows the drug to act directly on the highly active division area, avoiding the problems of drug penetration through scales in traditional soaking methods and drug diffusion and loss in injection methods. This results in increased concentrations of colchicine and 6-BA in the target cells.

[0040] In this invention, daily 600-800nm ​​light irradiation (4h / day) triggers the photothermal effect of PPy microspheres, stabilizing the local temperature at 32-35℃. This temperature accelerates the slow release of the inducer in the microspheres while avoiding cytotoxicity caused by drug burst release, thus synchronizing the drug release rate with the cell division cycle. During the division phase, cells continuously obtain an effective concentration of colchicine, significantly improving the chromosome doubling success rate.

[0041] In this invention, the release of drugs and cell division are uniformly regulated by photothermal microspheres, avoiding the heterogeneity of some cells contacting the drug and others not in contact with it, which is common in traditional methods. At the same time, the local effect of photothermal microspheres reduces the diffusion of drugs to non-target tissues, reduces the source of diploid-polyploid chimerism, and, together with 6-BA, enhances the growth advantage of polyploid cells, thereby increasing the homozygous polyploid rate and reducing the chimerism rate.

[0042] In this invention, the cultivation conditions of 15-20℃ room temperature and 60-70% humidity simulate the microenvironment of natural germination of peony underground buds, avoiding growth stress caused by extreme temperature or humidity; the management method of supplementing sterile water every 3 days balances moisture retention and aeration, ensuring normal metabolism of buds during induction.

[0043] In this invention, the minimally invasive incision exposes only local tissue without damaging the vascular bundles and growth points. Combined with sterile absorbent cotton for moisturizing and plastic wrap for fixation (with pre-reserved ventilation holes), it avoids the buds from losing water and wilting or the wound from becoming infected. This solves the problems of vascular bundle breakage caused by puncture wounds in traditional injection methods and scale rot caused by water accumulation in soaking methods.

[0044] In this invention, the identification in step (4) includes: ① Initial screening: 30 days after treatment, 0.1g of leaf tissue was taken and the DNA content was detected by flow cytometry. Plants with a peak value of twice that of diploid plants were screened. ② Secondary screening: 60 days after germination, the root tips were pretreated with 0.1% colchicine, fixed with Carnoy's solution, dissociated with hydrochloric acid, stained with acetic carmine, and the chromosome number was observed under a microscope.

[0045] In this invention, the transplanting time in step (5) is before spring budding, with a plant spacing of 50cm×60cm. After transplanting, the plants are thoroughly watered to settle the roots and covered with mulch.

[0046] This invention also provides the application of the method for efficiently inducing polyploidy in underground buds of peony described in the above technical solution. Specifically, the application is in the cultivation of new ornamental peony varieties, in the improvement of cut peony varieties, in the improvement of medicinal peony varieties, and in the cultivation of stress-resistant peonies.

[0047] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] The specific implementation method is as follows: Example 1 1. Preprocessing procedure (1) Material selection: In January, select healthy 3-year-old peony mother plants, dig out 100 underground buds with a diameter of 0.8cm and free from pests and diseases, and retain 3 layers of outer scales; (2) Disinfection: Rinse with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, rinse with sterile water 5 times, and dry the surface with filter paper; (3) Cell synchronization: Refrigerate at 4℃ for 72 hours to allow underground bud cells to enter interphase synchronously; (4) Minimally invasive preparation: Under sterile conditions, use a sharp blade to make an incision 0.3 cm deep and 0.1 cm wide on the side of the bud, 0.5 cm from the growth point. The incision direction is parallel to the growth direction of the bud.

[0050] 2. Preparation of photothermal responsive sustained-release microspheres (1) Preparation of base solution: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution and stir until completely dissolved; separately dissolve 2g of sodium alginate in 100mL of deionized water to prepare sodium alginate solution; (2) Polypyrrole composite: Add 0.5g of pyrrole monomer to the chitosan solution, stir evenly, then add 10mL of 0.1mol / L ferric chloride solution dropwise, and polymerize at room temperature for 2h to form PPy / CS composite solution; (3) Microsphere molding: PPy / CS composite solution and sodium alginate solution are mixed at a volume ratio of 1:1, and 5% calcium chloride solution is dripped into the microfluidic chip. After cross-linking and curing for 30 min, porous microspheres with a diameter of 50 μm are prepared. (4) Inducer loading: The microspheres were immersed in a composite inducer solution made of 0.25% colchicine, 0.8 mg / L 6-BA and 0.1% graphene quantum dots, and adsorbed under vacuum negative pressure at -0.05 MPa for 2 h. After centrifugation, the microspheres were collected and vacuum dried to constant weight.

[0051] 3. Induction treatment (1) Microsphere implantation: Use sterile forceps to take 3 microspheres loaded with inducing agent and embed them into the minimally invasive incision, ensuring that the microspheres are in close contact with the inner wall of the incision; (2) Moisturizing treatment: Wrap the incision with sterile absorbent cotton, add a small amount of sterile water to keep it moist, and wrap it with plastic wrap to fix it, leaving ventilation holes; (3) Photothermal regulation: The treated underground buds were transplanted into a sterile substrate composed of humus and perlite in a ratio of 3:1. The substrate was irradiated with a light source with a wavelength of 600nm and a power of 50W for 4 hours a day at a distance of 20cm. The microspheres were locally heated to 32-35℃ through the photothermal effect of PPy, which promoted the sustained release and penetration of drugs. (4) Cultivation management: Keep the room temperature at 15℃ and the air humidity at 60%. Add a small amount of sterile water to the cotton every 3 days to avoid water accumulation in the substrate. Remove the plastic wrap after 30 days of cultivation.

[0052] 4. Polyploid identification and screening (1) Initial screening (30 days after treatment): After the buds sprout new leaves, take 0.1g of leaf tissue and use flow cytometry to detect DNA content, and screen plants with DNA peak value twice that of diploid plants. (2) Secondary screening (60 days after germination): Take the root tips of the positive plants in the initial screening, pretreat them with 0.1% colchicine solution for 2 hours, fix them with Carnoy's solution for 24 hours, dissociate them with 1 mol / L hydrochloric acid for 10 minutes, stain them with acetic carmine, cut them by hand, and observe the chromosome number under a microscope to confirm polyploidy. (3) Trait screening: After transplanting to the field, observe the growth of the plants, retain the homozygous polyploid plants with thick stems, thick leaves and no deformities, and eliminate chimeras (some tissues are diploid) and individuals with abnormal growth.

[0053] 5. Transplanting and planting The selected polyploid seedlings were transplanted to the field with a spacing of 50cm×60cm. The transplanting time was before spring budding. After transplanting, the seedlings were thoroughly watered to help them settle down, covered with mulch to keep them warm and moist, and weeded and fertilized regularly, and were managed as usual in the field.

[0054] Example 2 1. Preprocessing procedure (1) Material selection: In January, select 5-year-old healthy peony mother plants, dig out 100 underground buds with a diameter of 1.2cm and free from pests and diseases, and retain 4 layers of outer scales; (2) Disinfection: Rinse with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, rinse with sterile water 5 times, and dry the surface with filter paper; (3) Cell synchronization: Refrigerate at 4℃ for 72 hours to allow underground bud cells to enter interphase synchronously; (4) Minimally invasive preparation: Under sterile conditions, use a sharp blade to make an incision 0.3 cm deep and 0.1 cm wide on the side of the bud, 0.5 cm from the growth point. The incision direction is parallel to the growth direction of the bud.

[0055] 2. Preparation of photothermal responsive sustained-release microspheres (1) Preparation of base solution: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution and stir until completely dissolved; dissolve 2g of sodium alginate in 100mL of deionized water to prepare sodium alginate solution; (2) Polypyrrole composite: Add 0.5g of pyrrole monomer to the chitosan solution, stir evenly, then add 10mL of 0.1mol / L ferric chloride solution dropwise, and polymerize at room temperature for 2h to form PPy / CS composite solution; (3) Microsphere molding: PPy / CS composite solution and sodium alginate solution are mixed at a volume ratio of 1:1, and 5% calcium chloride solution is dripped into the microfluidic chip. After cross-linking and curing for 30 min, porous microspheres with a diameter of 80 μm are prepared. (4) Inducer loading: The microspheres were immersed in a composite inducer solution made of 0.25% colchicine, 0.8 mg / L 6-BA and 0.1% graphene quantum dots, and adsorbed under vacuum negative pressure at -0.05 MPa for 2 h. After centrifugation, the microspheres were collected and vacuum dried to constant weight.

[0056] 3. Induction treatment (1) Microsphere implantation: Use sterile forceps to take 5 microspheres loaded with inducing agent and embed them into the minimally invasive incision, ensuring that the microspheres are in close contact with the inner wall of the incision; (2) Moisturizing treatment: Wrap the incision with sterile absorbent cotton, add a small amount of sterile water to keep it moist, and wrap it with plastic wrap to fix it, leaving ventilation holes; (3) Photothermal regulation: The treated underground buds were transplanted into a sterile substrate made of humus and perlite in a ratio of 3:1. The substrate was irradiated with a light source with a wavelength of 800nm ​​and a power of 50W for 4 hours a day at a distance of 20cm. The microspheres were locally heated to 32-35℃ through the photothermal effect of PPy, which promoted the sustained release and penetration of drugs. (4) Cultivation management: Maintain room temperature at 20℃ and air humidity at 70%. Add a small amount of sterile water to the absorbent cotton every 3 days to avoid water accumulation in the substrate. Remove the plastic wrap after 30 days of cultivation.

[0057] 4. Polyploid identification and screening (1) Initial screening (30 days after treatment): After the buds sprout new leaves, take 0.1g of leaf tissue and use flow cytometry to detect DNA content, and screen plants with DNA peak value twice that of diploid plants. (2) Secondary screening (60 days after germination): Take the root tips of the positive plants in the initial screening, pretreat them with 0.1% colchicine solution for 2 hours, fix them with Carnoy's solution for 24 hours, dissociate them with 1 mol / L hydrochloric acid for 10 minutes, stain them with acetic carmine, cut them by hand, and observe the chromosome number under a microscope to confirm polyploidy. (3) Trait screening: After transplanting to the field, observe the growth of the plants, retain the homozygous polyploid plants with thick stems, thick leaves and no deformities, and eliminate chimeras (some tissues are diploid) and individuals with abnormal growth.

[0058] 5. Transplanting and planting The selected polyploid seedlings were transplanted to the field with a spacing of 50cm×60cm. The transplanting time was before spring budding. After transplanting, the seedlings were thoroughly watered to help them settle down, covered with mulch to keep them warm and moist, and weeded and fertilized regularly, and were managed as usual in the field.

[0059] Comparative Example 1 1. Preprocessing procedure (1) Material selection: In January, select healthy 3-year-old peony mother plants, dig out 100 underground buds with a diameter of 0.8cm and free from pests and diseases, and retain 3 layers of outer scales; (2) Disinfection: Rinse with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, rinse with sterile water 5 times, and dry the surface with filter paper; (3) Cell synchronization: Refrigerate at 4℃ for 72 hours to allow underground bud cells to enter interphase synchronously; (4) Minimally invasive preparation: Under sterile conditions, use a sharp blade to make an incision 0.3 cm deep and 0.1 cm wide on the side of the bud, 0.5 cm from the growth point. The incision direction is parallel to the growth direction of the bud.

[0060] 2. Induction treatment (1) Preparation of inducing agent: Prepare a single polyploid inducing agent solution by dissolving 0.2g of colchicine in 100mL of sterile water and stirring until completely dissolved to prepare a colchicine soaking solution with a concentration of 0.2%; (2) Soaking treatment: 100 underground buds that have been pretreated (disinfected, synchronized, minimally invasive) are completely immersed in the above-mentioned 0.2% colchicine soaking solution. The soaking environment is controlled as follows: room temperature 15℃, light-proof conditions, soaking time 12h, during which the soaking solution is gently stirred once every 3h to ensure that the buds are in full contact with the inducing agent. (3) Post-soaking treatment: After soaking, remove the underground buds and rinse them quickly with sterile water 3 times, 30 seconds each time, to remove the colchicine residue on the surface. Use filter paper to absorb the surface moisture. (4) Moisturizing and fixing: Wrap sterile absorbent cotton around the micro-invasive incision of the bud, add a small amount of sterile water to keep it moist, wrap it with plastic wrap and leave ventilation holes; (5) Cultivation and management: Transplant the treated underground buds into a sterile substrate made of humus and perlite in a ratio of 3:1, maintain a room temperature of 15℃ and an air humidity of 60%, add a small amount of sterile water to the cotton every 3 days to avoid water accumulation in the substrate, and remove the plastic wrap after 30 days of cultivation.

[0061] 3. Polyploid identification and screening (1) Initial screening (30 days after treatment): After the buds sprout new leaves, take 0.1g of leaf tissue and use flow cytometry to detect DNA content, and screen plants with DNA peak value twice that of diploid plants. (2) Secondary screening (60 days after germination): Take the root tips of the positive plants in the initial screening, pretreat them with 0.1% colchicine solution for 2 hours, fix them with Carnoy's solution for 24 hours, dissociate them with 1 mol / L hydrochloric acid for 10 minutes, stain them with acetic carmine, cut them by hand, and observe the chromosome number under a microscope to confirm polyploidy. (3) Trait screening: After transplanting to the field, observe the growth of the plants, retain the homozygous polyploid plants with thick stems, thick leaves and no deformities, and eliminate chimeras and individuals with abnormal growth.

[0062] 4. Transplanting and planting The selected polyploid seedlings were transplanted to the field with a spacing of 50cm×60cm. The transplanting time was before spring budding. After transplanting, the seedlings were thoroughly watered to help them settle down, covered with mulch to keep them warm and moist, and weeded and fertilized regularly, and were managed as usual in the field.

[0063] Comparative Example 2 The polyploid inducer is a mixture of 0.2% colchicine and 2% dimethyl sulfoxide, and the remaining steps are the same as in Comparative Example 1.

[0064] The methods for inducing polyploid buds in peony as described in Examples 1-2 and Comparative Examples 1-2, and the resulting polyploid seedlings, were uniformly identified. The same flow cytometer, microscope, and high-performance liquid chromatograph were used for all identification to ensure data comparability. The phenotypic tests in Examples 1-2 and Comparative Examples 1-2 involved comparing polyploid plants with their corresponding diploid plants. Flower diameter and flowering period were measured at peak flowering, while stem diameter was measured 15 days after flowering. Low-temperature tolerance was tested by simulating -20℃ in an artificial climate chamber for 72 hours, followed by a 15-day recovery period at normal temperature, after which the number of surviving plants was counted. The identification results are shown in Table 1 below. Table 1. Results of phenotypic testing in Examples 1-2 and Comparative Examples 1-2

[0065] In summary, this invention employs 0.25% colchicine, 0.8 mg / L 6-BA, and 0.1% graphene quantum dots, using photothermal sustained-release microspheres for precise controlled release. Triggered by 600-800 nm light, a local temperature field of 32-35℃ is achieved, resulting in an improved cumulative drug release rate over 48 hours. Compared to the single / compound inducers in Comparative Examples 1-2, the number of target dividing cells, drug utilization, and induction rate are all improved. Furthermore, the increases in flower diameter, number of double petal layers, and stem diameter in Examples 1-2 are significantly enhanced compared to Comparative Examples 1-2. The polyploids cultivated using the method described in this invention exhibit a survival rate of 85-89% at -20℃, with a flowering period extended by 5-7 days, meeting the requirements for cut flowers / ornamental displays.

[0066] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for efficiently inducing polyploidy in peony underground buds, characterized in that, Includes the following steps: (1) Pretreatment: Select underground buds of healthy peony mother plants, disinfect, refrigerate and synchronize cells, and make a minimally invasive incision 0.5 cm away from the growth point on the side of the bud; (2) Preparation of photothermal response sustained-release microspheres: CS-Alg is used as a substrate, PPy is used to form porous microspheres, and after loading a composite inducer, they are vacuum dried to obtain microspheres loaded with the inducer; (3) Induction treatment: Microspheres loaded with inducing agent are embedded in a minimally invasive incision, moistened and fixed, and then induced to obtain induced plants; (4) Polyploid identification and screening: Identify and screen suitable homozygous polyploid plants from the induced plants; (5) Transplanting and planting: Transplant the selected polyploid seedlings to the field and perform routine field management.

2. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The selection time for the underground buds mentioned in step (1) is the end of the dormancy period of the underground buds in January-February. The diameter of the underground buds is 0.8-1.2cm, and 3-4 layers of outer scales are retained.

3. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The disinfection process described in step (1) is as follows: after rinsing with clean water for 30 minutes, soak in 75% medical alcohol for 30 seconds, then rinse with sterile water 5 times, and then absorb the surface moisture with filter paper; the depth of the minimally invasive incision is 0.3 cm and the width is 0.1 cm, and the incision direction is parallel to the growth direction of the bud.

4. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The preparation of photothermal responsive sustained-release microspheres in step (2) includes: ① Preparation of base solution: Dissolve 2g of chitosan in 100mL of 1% acetic acid solution, and dissolve 2g of sodium alginate in 100mL of deionized water; ② Polypyrrole composite: Add 0.5g of pyrrole monomer to the chitosan solution, add 10mL of 0.1mol / L ferric chloride solution, and polymerize at room temperature for 2h; ③ Microsphere molding: PPy / CS composite solution and sodium alginate solution are mixed at a volume ratio of 1:1, and 5% calcium chloride solution is dripped into the microfluidic chip for cross-linking and curing for 30 min to prepare porous microspheres with a diameter of 50-80 μm.

5. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The composite inducer in step (2) is a mixture of 0.2%-0.3% colchicine, 0.8 mg / L 6-benzylaminopurine, and 0.1% graphene quantum dots; the composite inducer is loaded by adsorption under a vacuum of -0.05 MPa for 2 hours.

6. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, In step (3), the amount of microspheres implanted with the loading inducer is 3-5; the moisturizing treatment is to wrap the microspheres with sterile degreased cotton and fix them with a plastic wrap with pre-reserved air holes, and the sterile substrate is humus: perlite = 3:

1.

7. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The induction treatment described in step (3) involves irradiating the microspheres with a 600-800nm ​​light source for 4 hours daily, controlling the local temperature of the microspheres to 32-35℃, and culturing them for 30 days at 15-20℃ and 60-70% humidity. The light source is a 50W simulated sunlight source with an irradiation distance of 20cm. During the culture period, sterile water is added to the absorbent cotton every 3 days, and the plastic wrap is removed after 30 days.

8. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The identification described in step (4) includes: ① Initial screening: 30 days after treatment, 0.1g of leaf tissue was taken for DNA content detection, and plants with a peak value twice that of diploid plants were screened. ② Secondary screening: 60 days after germination, the root tips were pretreated with 0.1% colchicine, fixed with Carnoy's solution, dissociated with hydrochloric acid, stained with acetic carmine, and the chromosome number was observed under a microscope.

9. The method for efficiently inducing polyploidy in peony underground buds according to claim 1, characterized in that, The transplanting time mentioned in step (5) is before spring budding, with a plant spacing of 50cm×60cm. After transplanting, water thoroughly to settle the roots and cover with mulch.

10. The application of the method for efficiently inducing polyploidy in peony underground buds according to any one of claims 1-9, characterized in that, The specific application is as follows: (1) Application in the breeding of new ornamental peony varieties; (2) Application in the improvement of cut peony varieties; (3) Application in the improvement of medicinal peony varieties; (4) Application in the cultivation of stress-resistant peony.

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