Cross-genus hybridization method for tulip

Abstract

The invention relates to the technical field of plant distant hybridization, and discloses a tulip trans-genus hybridization method, which comprises the following steps: firstly, coating a female parent tulip stigma with a first chemical composition containing a specific competitive inhibitor and a nano beacon, the composition being used for constructing a bionic affinity environment to overcome pre-fertilization obstacles, and implanting a space marker at a target site; then pollinating the pollen of the heterogeneity male parent to the pollen of the heterogeneity male parent; and finally, injecting a second chemical composition into the female parent daughter room at a specific time after pollination. The second chemical composition comprises a targeting nano-carrier of which the surface is modified with a docking ligand, and the carrier has a multi-layer core-shell structure, so that programmed release of active substances required in different development stages can be realized. By means of the established positioning targeting programmed release collaborative system, reproduction isolation recognition of female parents can be actively closed, meanwhile, precise targeting and dynamic nutrition support are conducted on successfully fertilized hybrid embryos, and the problems of incompatibility before fertilization and embryo abortion after fertilization are solved.

Description

Technical Field

[0001] The invention relates to the technical field of plant distant hybridization, in particular to a tulip inter-genus hybridization method. Background Art

[0002] Tulips, a world-renowned bulbous flower, have long focused their breeding goals on improving flower color, shape, and resistance. Inter-genus hybridization is an important way to obtain breakthrough new traits, introducing excellent genes from other genera into tulips to create entirely new horticultural varieties. However, due to the complex reproductive isolation mechanisms between species, the success rate of inter-genus hybridization of tulips is extremely low. The obstacles to distant hybridization are a continuous chain that runs through the entire process from pollination to seed maturation. Systematically solving a series of problems in the two major stages of pre-fertilization and post-fertilization is a technical bottleneck that urgently needs to be broken through in this field.

[0003] To rescue hybrid embryos, which are prone to abortion after distant hybridization, researchers have tried various methods. For example, a mixed solution containing multiple hormones and nutrients is injected into the ovary after pollination. However, these methods generally suffer from a lack of targeting. The ovary is a relatively large space, and the injected active substances are rapidly diluted and diffused throughout the ovarian cavity. This prevents the substances from reaching an effective concentration around the target ovule to support its development. Significantly increasing the overall concentration of the injection solution to compensate for this deficiency can easily cause physiological toxicity to the mother plant and the embryo, posing a high risk. This extensive delivery method is inefficient and a major factor contributing to the high embryo abortion rate.

[0004] Existing technologies often treat obstacles before and after fertilization separately. Some technologies are dedicated to solving the incompatibility on the stigma, such as using special culture media or chemical agents for pretreatment to promote the germination of pollen from different species. However, there is a lack of effective connection between these pollination technologies and subsequent pregnancy preservation measures. Even if a few pollen tubes are lucky enough to reach the ovule and complete fertilization, they will still face the problem of abortion due to insufficient nutrition supply. In addition, the traditional nutrition supply model is static. The mixture applied at one time cannot match the dynamic needs of embryonic development. The key substances required for early cell division and later organ construction are not the same. This supply itself is contrary to the inherent rhythm of life development and it is difficult to provide scientific and effective support. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a tulip cross-genus hybridization method, which solves the problems in the existing technology that the pre-fertilization and post-fertilization obstacle treatment measures are disconnected from each other, and the post-fertilization nutritional support lacks targeting and cannot be dynamically matched with the needs of embryonic development, thereby resulting in an extremely low hybridization success rate.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for inter-genus hybridization of tulips, comprising the following steps: Step S1, applying a first chemical composition containing nanobeacons to the stigma of a tulip serving as a female plant; Step S2, pollinating the stigma coated with the first chemical composition with pollen from a male parent of a different genus; Step S3: After pollination, applying a second chemical composition to the ovary of the female parent, wherein the second chemical composition comprises a carrier modified with a docking ligand, the docking ligand specifically binds to the nanobeacon, and the carrier contains at least one substance that promotes the development of hybrid embryos.

[0007] Through the above scheme, the present invention has established a two-stage chemical synergistic intervention system. In the first stage, a biomimetic affinity microenvironment is constructed in situ on the maternal stigma through the first chemical composition. This microenvironment can not only overcome the reproductive isolation barrier between species, but also implant a traceable nanobeacon during the process of pollen tube moving towards the ovule. In the second stage, the second chemical composition is applied to the ovary. The docking ligand on its surface can actively seek out and bind to the nanobeacons enriched around the successfully fertilized ovule through molecular recognition, thereby achieving precise targeting of the hybrid embryo. The carrier continuously releases its encapsulated substances at the target location, providing the fragile hybrid embryo with the support needed for development, thereby systematically solving the two major technical obstacles before and after fertilization.

[0008] As a preferred embodiment of the present invention, the first chemical composition is a biomimetic affinity environment formed in situ on the stigma surface to overcome pre-fertilization barriers. The composition comprises: a thermosensitive hydrogel matrix; a specific competitive inhibitor for blocking the maternal stigma incompatibility recognition receptor; a pollen tube active guidance complex for promoting pollen tube germination and directional growth; and quantum dots serving as the nanobeacon. The specific competitive inhibitor is a synthetic short peptide molecule that specifically binds to and temporarily blocks the pistil incompatibility recognition receptor through spatial conformational specificity. The short peptide molecule mimics the glycoprotein structure on the surface of pollen from a different genus. This structure can effectively reduce or eliminate the maternal rejection of pollen from a different genus.

[0009] Furthermore, in the first chemical composition, the concentrations of the components are as follows: the thermosensitive hydrogel matrix is ​​chitosan gel at a mass / volume concentration of 1.5% to 2.5%; the concentration of the specific competitive inhibitor is 10µM to 20µM; the pollen tube active guidance complex contains boric acid at a final concentration of 1.0mM to 1.5mM, calcium chloride at 2.5mM to 3.5mM, and gamma-aminobutyric acid at 0.5mM to 1.0mM; and the concentration of the quantum dots is 0.1µM to 0.5µM. This concentration range ensures the effective function of each component while avoiding potential toxicity to plant tissues.

[0010] In a preferred embodiment of the present invention, the carrier in the second chemical composition is a nanoparticle made of biodegradable PLGA material. The nanobeacon is a quantum dot with stable optical properties and a chemically modified surface. The docking ligand is a substance that binds with high affinity to the surface of the quantum dot through molecular recognition, selected from single-chain antibody fragments, nucleic acid aptamers, and combinations thereof. The specific binding of the nanobeacon and the docking ligand forms the basis of the targeting system of the present invention.

[0011] Furthermore, to dynamically support embryonic development, the PLGA nanoparticles possess a multilayered core-shell structure. This structure is designed to respond to the physiological needs of hybrid embryos at different developmental stages, enabling the programmed, phased release of substances promoting hybrid embryo development. The nanoparticle's core contains first-stage substances, including auxins and cytokinins, designed to promote early embryonic cell division and differentiation; the middle layer of the nanoparticles contains second-stage substances, including gibberellins and a vitamin complex, designed to promote organ development in mid- and late embryonic stages. This programmed release mechanism mimics the natural endosperm delivery pattern, enhancing the effectiveness of nutrient delivery.

[0012] Furthermore, in the first-stage and second-stage materials, the concentrations of the components are as follows: the auxin concentration is 5µM to 10µM; the cytokinin concentration is 10µM to 15µM; the gibberellin concentration is 5µM to 10µM; and the second chemical composition further comprises an amino acid at a concentration of 1mM to 2mM, selected from glutamine, serine, and combinations thereof. These substances are all key factors required for normal embryonic development.

[0013] In a specific implementation, step S1 is performed during the receptive period of the female parent's stigma reaching physiological maturity to ensure optimal pollination conditions. Step S3 is performed during the early embryonic development window of 72 to 120 hours after pollination, a critical period when hybrid embryos are most in need of exogenous nutritional support. The second chemical composition is administered by targeted injection into the ovary cavity via a microinjector, ensuring that the nutrients are accurately delivered to the site of action.

[0014] The preparation method of the PLGA nanoparticles modified with docking ligands and having a multi-layer core-shell structure may include the following steps: PLGA nanoparticles were prepared using the W1 / O / W2 double emulsification method: the first-stage substance containing auxin and cytokinin was dissolved in the inner aqueous phase (W1); PLGA and the second-stage substance containing gibberellins and multivitamins were dissolved in an organic solvent as the oil phase (O); the above oil phase was added to the outer aqueous phase (W2) containing an emulsifier for primary emulsification, and then a W1 / O / W2 double emulsion was formed by high-pressure homogenization or ultrasonic treatment; finally, the nanoparticles were solidified by solvent evaporation.

[0015] Surface modification of the docking ligand: The carboxyl groups on the surface of the purified PLGA nanoparticles are activated using the EDC / NHS chemical coupling method, and then a single-chain antibody fragment or a nucleic acid aptamer is added to react its amino group with the activated carboxyl group to form a stable amide bond, thereby covalently linking the docking ligand to the surface of the nanoparticles; finally, the second chemical composition is obtained after washing and purification.

[0016] The artificially synthesized short peptide molecule that simulates the glycoprotein structure on the surface of pollen of a heterogeneous male parent can be obtained by the following methods: screening the S nuclease (SRNase) or receptor-like protein kinase that serves as an incompatible recognition receptor through a phage display peptide library or a yeast two-hybrid system to obtain a short peptide that can specifically bind to the receptor; or designing a short peptide sequence that can effectively occupy the active site of the receptor through computer-assisted molecular docking simulation, and then chemically synthesizing it.

[0017] The present invention provides a method for inter-genus hybridization of tulips. It has the following beneficial effects: 1. This invention systematically overcomes the two major obstacles in distant hybridization—pre-fertilization and post-fertilization—through a two-stage synergistic intervention mechanism, significantly improving hybridization success rates. Specifically, the first chemical composition creates a biomimetic affinity environment on the surface of the maternal stigma, effectively addressing the issues of non-germination and stunted pollen tube growth in heterogeneous pollen caused by reproductive isolation. Furthermore, the second chemical composition provides nutritional support to successfully fertilized hybrid embryos through a targeted delivery system, addressing the problem of embryonic abortion caused by developmental defects. These two stages are closely linked, comprehensively improving the overall efficiency from pollination to mature seed production.

[0018] 2. This invention utilizes nanobeacons that arrive at the ovule along with the pollen tube as spatial positioning markers, and a docking ligand on the surface of the second chemical composition carrier as a targeting probe. The specific combination of these two ensures that nutrients and hormones are precisely concentrated around the target ovule. This targeted release method avoids the dispersal and waste of nutrients throughout the ovary, achieving a more optimal effect of promoting pregnancy and development at a lower application rate.

[0019] 3. This invention utilizes a multi-layered core-shell nanocarrier structure to achieve a programmed, phased release of key substances required for embryonic development, dynamically mimicking the nutrient supply pattern of the natural endosperm. The first-phase substances (auxin and cytokinin) released from the carrier's core precisely address the cell division and differentiation requirements of early embryonic development, while the second-phase substances (gibberellins and multivitamins) released from the middle layer meet the requirements for organ development in the middle and late stages of embryonic development. This sequential delivery method closely matches the dynamic physiological needs of hybrid embryos, ensuring their healthy development in a more scientific and effective manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the present specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Please see the attached Figure 1 : Example 1

[0023] This embodiment provides a specific preparation and implementation process of a tulip inter-genus hybridization method, wherein each parameter is selected as an intermediate value within the required range.

[0024] Preparation of chemical compositions Preparation of the first chemical composition (bionic affinity environment): Take 100 mL of buffer solution and add and dissolve the following components in sequence: Chitosan: 2.0 g, forming a gel matrix with a mass / volume concentration of 2.0%.

[0025] Artificially synthesized short peptide molecules (as specific competitive inhibitors): added at a concentration of 15µM.

[0026] Boric acid: added at a concentration of 1.25 mM.

[0027] Calcium chloride: Add at a concentration of 3.0 mM.

[0028] γ-Aminobutyric acid: added at a concentration of 0.75 mM.

[0029] Carboxyl-modified quantum dots (as nanobeacons): Add at a concentration of 0.3 µM. Mix the mixture thoroughly at 4°C and set aside.

[0030] The second chemical composition (targeted nutrient carrier) was prepared using a W1 / O / W2 double emulsion solvent evaporation method to prepare drug-loaded PLGA nanoparticles. First, auxin (final concentration 7.5µM) and cytokinin (final concentration 12.5µM) were dissolved in 2mL of deionized water to form the inner aqueous phase (W1). Simultaneously, 200mg of PLGA, gibberellin (final concentration 7.5µM), and a vitamin complex were dissolved in 10mL of dichloromethane to form the oil phase (O). The inner aqueous phase was added to the oil phase and subjected to high-speed shearing to form colostrum (W1 / O). This colostrum was then added to 100mL of the outer aqueous phase (W2) containing 2% polyvinyl alcohol (PVA) and subjected to high-pressure homogenization to form a W1 / O / W2 double emulsion. The dichloromethane was evaporated by magnetic stirring at room temperature to solidify the nanoparticles. Finally, PLGA nanoparticles with a multilayer core-shell structure were obtained by centrifugation and washing.

[0031] The surface of the nanoparticles was then modified with a docking ligand. Using the EDC / NHS chemical conjugation method, the purified PLGA nanoparticles were resuspended in MES buffer and EDC and NHS were added to activate the surface carboxyl groups. Following the reaction, the activated nanoparticles were incubated with a single-chain antibody fragment (scFv, serving as a docking ligand) in PBS buffer to achieve covalent attachment.

[0032] Finally, the final composition was prepared. The washed and purified targeted nanoparticles were resuspended in 100 mL of buffer solution, and glutamine and serine were added to bring the total amino acid concentration to 1.5 mM. After thorough mixing, the second chemical composition was obtained.

[0033] Hybridization method implementation steps S1: When the stigma of a tulip (the female parent) enters the physiological receptive period, 5 μL of the first chemical composition is evenly applied to the stigma surface. Step S2: After the composition forms a gel on the stigma surface, mature pollen from a male parent of a different genus is evenly applied to the gel surface. Step S3: 96 hours after pollination, 20 μL of the second chemical composition is drawn up using a microsyringe and precisely injected into the ovary cavity of the female parent. Example 2

[0034] In this embodiment, each parameter is selected as the lower limit value within the required range.

[0035] Preparation of chemical compositions Preparation of the first chemical composition (bionic affinity environment): Take 100 mL of buffer solution and add and dissolve the following components in sequence: Chitosan: 1.5 g, forming a gel matrix with a mass / volume concentration of 1.5%.

[0036] Artificially synthesized short peptide molecules (as specific competitive inhibitors): added at a concentration of 10µM.

[0037] Boric acid: added at a concentration of 1.0 mM.

[0038] Calcium chloride: Add at a concentration of 2.5 mM.

[0039] γ-Aminobutyric acid: added at a concentration of 0.5 mM.

[0040] Carboxyl-modified quantum dots (as nanobeacons): Add at a concentration of 0.1 µM. Mix the mixture thoroughly at 4°C and set aside.

[0041] The second chemical composition (targeted nutrient carrier) was prepared using a W1 / O / W2 double emulsion solvent evaporation method to prepare drug-loaded PLGA nanoparticles. First, auxin (final concentration 5µM) and cytokinin (final concentration 10µM) were dissolved in 2mL of deionized water to form the inner aqueous phase (W1). Simultaneously, 200mg of PLGA, gibberellins (final concentration 5µM), and multivitamins were dissolved in 10mL of dichloromethane to form the oil phase (O). The inner aqueous phase was added to the oil phase and subjected to high-speed shearing to form colostrum (W1 / O). This colostrum was then added to 100mL of the outer aqueous phase (W2) containing 2% polyvinyl alcohol (PVA) and subjected to high-pressure homogenization to form a W1 / O / W2 double emulsion. The dichloromethane was evaporated under magnetic stirring at room temperature to solidify the nanoparticles. Finally, PLGA nanoparticles with a multilayer core-shell structure were obtained by centrifugation and washing.

[0042] The surface of the ligand was then modified using the EDC / NHS chemical coupling method. Purified PLGA nanoparticles were resuspended in MES buffer, and EDC and NHS were added to activate the surface carboxyl groups. After the reaction, the activated nanoparticles were incubated with the aptamer (serving as the docking ligand) in PBS buffer to achieve covalent attachment.

[0043] Finally, the final composition was prepared. The washed and purified targeted nanoparticles were resuspended in 100 mL of buffer solution, and glutamine was added to a concentration of 1 mM. After thorough mixing, the second chemical composition was obtained.

[0044] Hybridization method implementation steps S1: When the stigma of a tulip plant (the female parent) enters the physiological receptive period, 5 μL of the first chemical composition is evenly applied to the stigma surface. Step S2: After the composition forms a gel on the stigma surface, mature pollen from a male parent of a different genus is evenly applied to the gel surface. Step S3: 72 hours after pollination, 20 μL of the second chemical composition is drawn up using a microsyringe and precisely injected into the ovary cavity of the female plant. Example 3

[0045] In this embodiment, each parameter is selected as the upper limit value within the required range.

[0046] Preparation of chemical compositions Preparation of the first chemical composition (bionic affinity environment): Take 100 mL of buffer solution and add and dissolve the following components in sequence: Chitosan: 2.5 g, forming a gel matrix with a mass / volume concentration of 2.5%.

[0047] Artificially synthesized short peptide molecules (as specific competitive inhibitors): added at a concentration of 20µM.

[0048] Boric acid: added at a concentration of 1.5 mM.

[0049] Calcium chloride: Add at a concentration of 3.5 mM.

[0050] γ-Aminobutyric acid: added at a concentration of 1.0 mM.

[0051] Carboxyl-modified quantum dots (as nanobeacons): Add at a concentration of 0.5 µM. Mix the mixture thoroughly at 4°C and set aside.

[0052] The second chemical composition (targeted nutrient carrier) was prepared using a W1 / O / W2 double emulsion solvent evaporation method to prepare drug-loaded PLGA nanoparticles. First, auxin (final concentration 10µM) and cytokinin (final concentration 15µM) were dissolved in 2mL of deionized water to form the inner aqueous phase (W1). Simultaneously, 200mg of PLGA, gibberellin (final concentration 10µM), and a vitamin complex were dissolved in 10mL of dichloromethane to form the oil phase (O). The inner aqueous phase was added to the oil phase and subjected to high-speed shearing to form colostrum (W1 / O). This colostrum was then added to 100mL of the outer aqueous phase (W2) containing 2% polyvinyl alcohol (PVA) and homogenized under high pressure to form a W1 / O / W2 double emulsion. The dichloromethane was evaporated under magnetic stirring at room temperature to solidify the nanoparticles. Finally, PLGA nanoparticles with a multilayer core-shell structure were obtained by centrifugation and washing.

[0053] The surface of the nanoparticles was then modified with a docking ligand. Using the EDC / NHS chemical conjugation method, the purified PLGA nanoparticles were resuspended in MES buffer and EDC and NHS were added to activate the surface carboxyl groups. Following the reaction, the activated nanoparticles were incubated with a combination of a single-chain antibody fragment and a nucleic acid aptamer (as a docking ligand) in PBS buffer to achieve covalent attachment.

[0054] Finally, the final composition was prepared. The washed and purified targeted nanoparticles were resuspended in 100 mL of buffer solution, and a combination of glutamine and serine was added to bring the total amino acid concentration to 2 mM. After thorough mixing, the second chemical composition was obtained.

[0055] Hybridization method implementation steps S1: When the stigma of a tulip (the female parent) enters the physiological receptive period, apply 5 μL of the first chemical composition evenly to the stigma surface. Step S2: After the composition forms a gel on the stigma surface, apply mature pollen from a male parent of a different genus evenly to the gel surface. Step S3: 120 hours after pollination, use a microsyringe to draw up 20 μL of the second chemical composition and precisely inject it into the ovary cavity of the female parent.

[0056] Comparative Example 1 Compared to Example 1, the second chemical composition differs in that the docking ligand modification step is omitted in the preparation of the second chemical composition. Specifically, after the multilayer core-shell PLGA nanoparticles are prepared, the subsequent EDC / NHS chemical coupling reaction is not performed. Therefore, the surface of the resulting PLGA nanoparticles is not modified with a single-chain antibody fragment (scFv). All other procedures remain the same.

[0057] Comparative Example 2 Compared with Example 1, the difference is that the first chemical composition lacks the key specific competitive inhibitor. Specifically, no artificially synthesized short peptide molecule is added when preparing the first chemical composition. All other steps are the same.

[0058] Comparative Example 3 Compared to Example 1, the nanocarriers in the second chemical composition lack a programmed release structure. Specifically, the drug-loaded PLGA nanoparticles of the second chemical composition were prepared using an O / W single emulsion solvent evaporation method, dissolving the auxin, cytokinin, gibberellin, and multivitamin all at once in the oil phase for encapsulation. Therefore, the prepared nanoparticles lack a multilayer core-shell structure, and all active substances are co-encapsulated. All other aspects remain the same.

[0059] Comparative Example 4 Compared to Example 1, the difference lies in the concentration of the active substance in the second chemical composition exceeding the optimized range defined by the present invention. Specifically, when preparing the second chemical composition, the final concentration of auxin in the kernel was increased to 50 µM, and the final concentration of cytokinin was increased to 50 µM, both of which are significantly higher than the upper limit defined by the present invention. All other conditions remained the same.

[0060] Test Example 1: Verification of the effectiveness of the targeting system Experimental Description This experimental example aims to verify the effectiveness of the nanobeacon docking ligand targeting system in this technical solution. Tulips (Apeldoorn variety) with robust growth and uniform flowering age were selected as the female parent, and pollen from Fritillaria thunbergii (Fritillariathunbergii) was selected as the male parent.

[0061] Experimental Procedures The selected female plants were randomly divided into four groups. Experimental groups A, B, and C were treated using the full set of methods described in Example 1, Example 2, and Example 3, respectively; and control group D was treated using the method described in Comparative Example 1.

[0062] Pre-pollination treatment: When the stigma of each group of female plants reached the physiologically mature receptive stage, the first chemical composition prepared in Example 1, Example 2, and Example 3 was evenly applied to the stigma of the female plants in experimental groups A, B, and C, respectively. Control group D was also treated with the first chemical composition prepared in Example 1 to ensure that all groups were implanted with nanobeacons.

[0063] Pollination: After the composition on the stigma surface forms a gel, the collected Fritillaria pollen is evenly applied to the stigma gel of each group of female parents.

[0064] Post-pollination treatment: Experimental group A: 96 hours after pollination, the second chemical composition prepared in Example 1 (the surface of the carrier was modified with a single-chain antibody fragment) was injected into the ovary cavity.

[0065] Experimental group B: 72 hours after pollination, the second chemical composition prepared in Example 2 (the surface of the carrier is modified with nucleic acid aptamers) was injected into the ovary cavity.

[0066] Experimental group C: 120 hours after pollination, the second chemical composition prepared in Example 3 (a combination of a carrier surface modified with a single-chain antibody fragment and a nucleic acid aptamer) was injected into the ovary cavity.

[0067] Control group D: 96 hours after pollination, the second chemical composition prepared in Comparative Example 1 (the surface of the carrier was not modified with any docking ligand) was injected into the ovary cavity.

[0068] Data Collection: 25 days after pollination, 20 treated flowers were randomly selected from each group. Ovaries were dissected and the total number of ovules and the number of normally developed embryos (enlarged in size and regular in morphology) were counted under a stereomicroscope. Another portion of plants were cultured until seed maturity, and the final seed set rate was calculated.

[0069] Experimental data Table 1 Comparative data of hybridization effects between experimental groups and control groups .

[0070] Experimental Summary As can be seen from the experimental data in Table 1, the number of normally developed embryos and the final fruit set rate in experimental groups A, B, and C treated using the methods of Examples 1, 2, and 3 were significantly higher than those in control group D treated using the method of Comparative Example 1. This fully demonstrates that modification of the docking ligand on the surface of the second chemical composition carrier is crucial for improving the hybridization success rate and is the core element for achieving the significant beneficial effects of this technical solution.

[0071] The dual-stage synergistic intervention system established by this technical solution works by achieving precise spatial localization and targeted delivery of active substances. In the first stage, nanobeacons, arriving in the ovule along with the affinity pollen tube, establish a unique, identifiable spatial address around the successfully fertilized ovule. In the second stage, the second chemical composition used in the experimental group, whose PLGA nanocarriers are modified with docking ligands (such as single-chain antibody fragments or nucleic acid aptamers) on the surface, acts as precise navigation probes. Through high-affinity molecular recognition, these nanobeacons actively seek out and bind to these nanobeacons, thereby achieving aggregation in the complex ovary environment and efficiently enriching the encapsulated substances such as auxins and cytokinins around the hybrid embryos where nutritional support is most needed.

[0072] In sharp contrast, in control group D, although the second chemical composition also contained substances that promoted embryonic development, its carrier was unable to recognize the nanobeacons around the ovules due to the lack of surface docking ligands, and could only diffuse aimlessly and randomly in the ovary cavity. This resulted in the active substance being unable to form an effective concentration at the key target site, and the vast majority of hybrid embryos were aborted in the early stages of development due to the lack of necessary and timely nutritional support. This result profoundly reveals the innovativeness of the present invention, that is, by constructing a strategy of nanobeacon positioning and docking ligand targeting, the traditional, inefficient diffuse nutrient supply is transformed into efficient and precise targeted fertilization, thereby fundamentally solving the key technical bottleneck of embryo abortion after fertilization in distant hybridization.

[0073] Test Example 2: Verification of effectiveness in overcoming pre-fertilization obstacles Experimental Description This experimental example aims to verify the effectiveness of the first chemical composition in this technical solution in overcoming pre-fertilization reproductive isolation. Tulips (Apeldoorn variety) with robust growth and uniform flowering age were selected as the female parent, and pollen from Fritillaria thunbergii was selected as the male parent.

[0074] Experimental Procedures The selected female plants were randomly divided into four groups. Experimental groups A, B, and C were treated with the first chemical composition described in Example 1, Example 2, and Example 3, respectively; and control group D was treated with the first chemical composition described in Comparative Example 2.

[0075] Pre-pollination treatment: when the stigma of each group of female parents reaches the physiologically mature receptive period, the corresponding first chemical composition is evenly applied to the surface of the stigma.

[0076] Experimental group A: The first chemical composition prepared in Example 1 was applied.

[0077] Experimental Group B: The first chemical composition prepared in Example 2 was applied.

[0078] Experimental Group C: The first chemical composition prepared in Example 3 was applied.

[0079] Control group D: the first chemical composition prepared in Comparative Example 2 (lacking a specific competitive inhibitor) was applied.

[0080] Pollination: After the composition on the stigma surface forms a gel, the collected Fritillaria pollen is evenly applied to the stigma gel of each group of female parents.

[0081] Data collection: 72 hours after pollination, 20 treated flowers were randomly selected from each group, and the pistils were removed. The growth of pollen tubes within the style was observed under a fluorescence microscope using aniline blue staining. The ovaries were dissected, and the number of pollen tubes that entered the ovules and completed fertilization was counted to calculate the fertilization rate.

[0082] Experimental data Table 2 Comparison of fertilization effects between experimental groups and control group .

[0083] Experimental Summary As shown in the experimental data in Table 2, the fertilization rates of experimental groups A, B, and C, treated using the methods of Examples 1, 2, and 3, all reached over 65%, while the fertilization rate of control group D, treated using the method of Comparative Example 2, was only 2.1%. This significant difference clearly demonstrates that the first chemical composition of this technical solution can effectively overcome the pre-fertilization barriers of distant hybridization and is the prerequisite and foundation for achieving inter-generic hybridization.

[0084] The core mechanism of this technical solution's success lies in the in situ creation of a multifunctional biomimetic affinity environment by the first chemical composition on the surface of the maternal pollen plant's stigma. The key innovative component—a synthetic short peptide molecule (i.e., a specific competitive inhibitor)—mimics the spatial conformation of specific glycoproteins on the surface of the paternal pollen plant. This short peptide molecule preemptively binds with high affinity to non-affinity recognition receptors (such as S nuclease) on the maternal pollen plant's stigma, temporarily blocking these receptors and preventing the maternal plant's defense system from recognizing and rejecting heterologous pollen. This proactive, source-directed intervention removes the primary obstacle to heterologous pollen germination and the smooth growth of pollen tubes.

[0085] In contrast, the control group D lacked this key specific competitive inhibitor, and the incompatibility recognition system on the surface of its stigma remained active. When pollen of a different genus fell, it was immediately recognized by the mother plant as a foreign object and a rejection reaction was initiated, resulting in the pollen being unable to germinate normally, or the growth of the germinated pollen tube was rapidly blocked, deformed, or degraded, and ultimately unable to pass through the style to reach the ovary for fertilization. The results of this experimental example fundamentally verified the innovativeness of this solution: by constructing a bionic affinity environment containing specific competitive inhibitors, the core problem of incompatibility in distant hybridization was solved at the molecular recognition level, creating the necessary conditions for subsequent fertilization and embryonic development. Test Example 3: Verification of the effectiveness of programmed release structure Experimental Description This experimental example aims to verify the technical advantages of the multilayer core-shell structure carrier with programmed release function used in the second chemical composition of this technical solution. Tulips (Apeldoorn variety) with robust growth and uniform flowering age were selected as the female parent, and pollen from Fritillaria thunbergii was selected as the male parent.

[0086] Experimental Procedure: The selected female plants were randomly divided into four groups. To ensure identical pre-fertilization conditions across groups, all groups were pretreated and pollinated with the first chemical composition prepared in Example 1. The key variable in the experiment was the different nanocarrier structures of the second chemical composition applied after pollination.

[0087] Pre-pollination treatment and pollination: When the stigma of each group of female parents reaches the physiologically mature receptive period, the first chemical composition prepared in Example 1 is uniformly coated, and then Fritillaria pollen is evenly applied to the stigma gel of each group of female parents.

[0088] Post-pollination treatment: Experimental group A: 96 hours after pollination, the second chemical composition prepared in Example 1 (a carrier with a multi-layer core-shell structure) was injected into the ovary cavity.

[0089] Experimental Group B: 72 hours after pollination, the second chemical composition prepared in Example 2 (a carrier with a multi-layer core-shell structure) was injected into the ovary cavity.

[0090] Experimental Group C: 120 hours after pollination, the second chemical composition prepared in Example 3 (a carrier with a multi-layer core-shell structure) was injected into the ovary cavity.

[0091] Control group D: 96 hours after pollination, the second chemical composition prepared in Comparative Example 3 (the carrier had a non-layered structure, and all active substances were mixed and encapsulated) was injected into the ovary cavity.

[0092] Data collection: 25 days after pollination, 20 treated flowers were randomly selected from each group, and the ovaries were dissected. The total number of ovules and the number of normally developed embryos (enlarged in size and regular in morphology) were counted, and the normal embryo development rate was calculated.

[0093] Experimental data Table 3 Comparative data on the effects of different vector structures on hybrid embryo development .

[0094] Experimental Summary The experimental data in Table 3 clearly show that the normal embryo development rates of experimental groups A, B, and C treated using the methods described in Examples 1, 2, and 3 were much higher than those of control group D treated using the method of Comparative Example 3. This result strongly demonstrates that the multilayer core-shell nanocarrier with programmed release designed in this technical solution can more effectively promote the healthy development of hybrid embryos compared to traditional mixed encapsulation methods.

[0095] The core mechanism behind the superior results achieved by this technical solution lies in the precise timing of the supply of active substances and the dynamic developmental needs of hybrid embryos. The development of hybrid embryos is a dynamic process, with varying hormone and nutritional requirements at different stages. The nanocarriers in this solution preferentially release auxins and cytokinins from their inner core, precisely meeting the early embryonic developmental needs for rapid cell division and differentiation. As the carrier further degrades, the gibberellins and complex vitamins released from the middle layer provide critical support for organ development and structural improvement in the middle and late stages of the embryo. This phased, on-demand supply model scientifically mimics the nutrient supply rhythm of the natural endosperm.

[0096] In contrast, the simple mixed encapsulation method used in the control group D released all active substances at once, which led to a serious mismatch of resources. In the early stages of embryonic development, high concentrations of gibberellins and other substances required in the later stages may inhibit or interfere with the embryos that are still in the cell division stage; while in the later stages of embryonic development, the auxins and cytokinins required in the early stages have long been consumed or degraded and cannot provide support for the continued development of the embryo. The results of this experimental example deeply reveal the ingenuity and innovation of this scheme: that is, through the engineering design of the nanocarrier structure, the static material supply is transformed into dynamic, programmed support synchronized with the rhythm of life, thereby significantly improving the utilization efficiency of nutrients and providing an optimized developmental microenvironment for fragile hybrid embryos.

[0097] Test Example 4: Optimization and Verification of Active Substance Concentration Range Experimental Description This test example aims to verify the rationality and superiority of the active substance concentration range defined by this technical solution. Tulips (cultivar 'Apeldoorn') with robust growth and uniform flowering age were selected as the female parent, and pollen from Fritillaria thunbergii was selected as the male parent.

[0098] Experimental Procedure: The selected female plants were randomly divided into four groups. To ensure identical pre-fertilization conditions across the groups, all groups were pretreated and pollinated with the first chemical composition prepared in Example 1. The key variable in the experiment was the concentration of the active ingredient in the second chemical composition applied after pollination.

[0099] Pre-pollination treatment and pollination: When the stigma of each group of female parents reaches the physiologically mature receptive period, the first chemical composition prepared in Example 1 is uniformly coated, and then Fritillaria pollen is evenly applied to the stigma gel of each group of female parents.

[0100] Post-pollination treatment: Experimental group A: 96 hours after pollination, the second chemical composition (intermediate concentration) prepared in Example 1 was injected into the ovary cavity.

[0101] Experimental group B: 72 hours after pollination, the second chemical composition prepared in Example 2 (lower limit concentration) was injected into the ovary cavity.

[0102] Experimental group C: 120 hours after pollination, the second chemical composition prepared in Example 3 (upper limit concentration) was injected into the ovary cavity.

[0103] Control group D: 96 hours after pollination, the second chemical composition prepared in Comparative Example 4 (exceeding the upper limit concentration) was injected into the ovary cavity.

[0104] Data collection: 25 days after pollination, 20 treated flowers were randomly selected from each group, and the ovaries were dissected. The total number of ovules, the number of normally developed embryos, and the number of aborted or deformed embryos were counted, and the normal development rate of embryos was calculated.

[0105] Experimental data Table 4 Comparative data of the effects of different concentration treatments on hybrid embryo development .

[0106] Experimental Summary As shown in the experimental data of Table 4, the experimental groups A, B, and C treated by the methods described in Examples 1, 2, and 3 all achieved a higher normal embryo development rate. In sharp contrast, the control group D treated by the method of Comparative Example 4 had an extremely low normal embryo development rate, and a large number of embryonic abortions or deformities were observed. This result clearly shows that the active substance concentration range provided by this technical solution is reasonable and effective, and exceeding this range will have a serious negative impact on embryo development.

[0107] The underlying mechanism is that plant hormones (such as auxin and cytokinin) have a strict concentration-dependent effect on embryonic development, exhibiting a typical dual nature. Within the concentration range defined by this technical solution, these active substances can work synergistically to precisely regulate the division, differentiation, and morphogenesis of embryonic cells, providing appropriate physiological signals for the normal development of hybrid embryos. This constitutes a beneficial and supportive physiological window. However, once the hormone concentration is too high, it will disrupt the balance of endogenous hormones in the embryo, resulting in super-concentration inhibition and even cytotoxicity, interfering with normal cell cycle and metabolic activities, and ultimately leading to embryonic developmental arrest, structural deformities, and even direct death.

[0108] The results of Control Group D directly demonstrate the toxic effects of this super-optimal concentration, demonstrating that simply providing unlimited nutrition and hormones cannot successfully rescue hybrid embryos and instead accelerates their abortion. Therefore, a key innovation of this technical solution lies not only in the proposed use of certain substances but also in the extensive experimentation to identify the optimal concentration window within which these substances can maximize their positive effects while avoiding negative ones. Determining this optimal range is key to ensuring the stability, efficiency, and repeatability of the entire technical solution and is essential for translating the envisioned targeted nutritional support from theory into successful practice.

Claims

1. A method for inter-genus hybridization of tulips, characterized in that: The following steps are involved: Step S1, applying a first chemical composition containing nanobeacons to the stigma of a tulip serving as a female plant; Step S2, pollinating the stigma coated with the first chemical composition with pollen from a male parent of a different genus; Step S3: After pollination, applying a second chemical composition to the ovary of the female parent, wherein the second chemical composition comprises a carrier modified with a docking ligand, the docking ligand specifically binds to the nanobeacon, and the carrier contains at least one substance that promotes the development of hybrid embryos.

2. The tulip inter-genus hybridization method according to claim 1, characterized in that: The first chemical composition is a biomimetic affinity environment formed in situ on the stigma surface for overcoming pre-fertilization barriers, comprising: thermosensitive hydrogel matrix; Specific competitive inhibitor for blocking the inaffinity recognition receptor of the parent stigma; The pollen tube active guidance complex is used to promote pollen tube germination and directional growth; and quantum dots as the nanobeacons.

3. A tulip inter-genus hybridization method according to claim 1 or 2, characterized in that: In the first chemical composition, the concentration of each component is: The thermosensitive hydrogel matrix is ​​a chitosan gel with a mass / volume concentration of 1.5% to 2.5%; The concentration of the specific competitive inhibitor is 10M to 20M; The pollen tube active guidance complex comprises boric acid at a final concentration of 1.0 mM to 1.5 mM, calcium chloride at a final concentration of 2.5 mM to 3.5 mM, and gamma-aminobutyric acid at a final concentration of 0.5 mM to 1.0 mM; The concentration of the quantum dots is 0.1M to 0.5M.

4. The tulip inter-genus hybridization method according to claim 1, characterized in that: The carrier in the second chemical composition is nanoparticles made of biodegradable PLGA material.

5. The tulip inter-genus hybridization method according to claim 4, characterized in that: The PLGA nanoparticles have a multi-layer core-shell structure, which is used to respond to the physiological needs of hybrid embryos at different developmental stages and achieve programmed staged release of the substance that promotes hybrid embryo development, wherein: The inner core of the nanoparticle contains a first-stage substance containing auxin and cytokinin for promoting early embryonic cell division and differentiation; The middle layer of the nanoparticles is loaded with second-stage substances containing gibberellins and complex vitamins for promoting the construction of organs in the middle and late stages of the embryo.

6. The tulip inter-genus hybridization method according to claim 5, characterized in that: The concentrations of the components in the first-stage material and the second-stage material are: The concentration of the auxin is 5M to 10M; The concentration of the cytokinin is 10M to 15M; The concentration of the gibberellin is 5M to 10M; Furthermore, the second chemical composition further comprises an amino acid at a concentration of 1 mM to 2 mM, wherein the amino acid is selected from glutamine, serine, and a combination thereof.

7. The tulip inter-genus hybridization method according to claim 6, characterized in that: The step S1 is performed when the female parent stigma reaches the physiologically mature receptive stage.

8. The tulip inter-genus hybridization method according to claim 7, characterized in that: The step S3 is performed in the early embryonic development window of 72 to 120 hours after pollination, and the administration method is to inject the second chemical composition into the ovary cavity through a micro syringe.

9. The tulip inter-genus hybridization method according to claim 2, characterized in that: The specific competitive inhibitor is an artificially synthesized short peptide molecule that specifically binds to and temporarily blocks the pistil inaffinity recognition receptor through spatial conformation, and the short peptide molecule simulates the glycoprotein structure on the surface of pollen of the male parent of the opposite genus.

10. The tulip inter-genus hybridization method according to claim 1, characterized in that: The nanobeacon is a quantum dot with stable optical properties and a chemically modified surface; the docking ligand is a substance that binds to the surface of the quantum dot with high affinity through molecular recognition, and the substance is selected from single-chain antibody fragments, nucleic acid aptamers, and combinations thereof.

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