Long-acting sterile ornamental and regeneration integrated culture method for in-vitro buds of Chinese roses

Abstract

According to the long-acting sterile ornamental and regeneration integrated culture method for the in-vitro Chinese rose buds, the pollution risk is low, and a fundamental guarantee is provided for long-term sterile culture of the Chinese rose bud explants through the broad-spectrum and lasting antibacterial effect of the in-vitro Chinese rose buds. Under the system, the complete period from flowering to decline of the Chinese rose bud explant is completed by means of the slow-release nutrition, and the viewing period of the cut Chinese rose flower is remarkably prolonged. In addition, after the ornamental period is finished, the cut Chinese rose flowers also have complete transplanting survival ability, and the transformation from disposable consumption to sustainable cultivation is realized. The scheme has the industrialization advantages of being short in production period, high in stability and low in maintenance cost, is suitable for all-season regional application, remarkably improves the ornamental value and practicability of the product, and provides an innovative product for the fields of home decoration, high-end gift presentation, medical health care, education and science popularization and the like.

Description

Technical Field

[0001] This invention relates to the field of plant culture technology, and in particular to a method for the long-term sterile ornamental and regeneration integrated culture of detached rose buds. Background Technology

[0002] Cut flowers, as consumer goods obtained from living plants and intended for decoration and gifting, rely heavily on their limited shelf life for their industrial value. However, constrained by internal physiological decay, external microbial contamination, and interrupted nutrient supply after detachment, cut flowers generally face a short shelf life (typically only 3-15 days), a core bottleneck in the industry. This limitation severely restricts their transportation radius and market expansion potential, and drives up consumer costs due to high spoilage rates. Taking the mainstream cut rose as an example, its natural vase life is only about 7 days, and even with current chemical preservation methods, its shelf life is difficult to consistently exceed 15 days. Current physical or chemical preservation technologies aim to slow down the decay process but cannot reverse the systemic physiological decline caused by detachment, ultimately rendering the product a disposable waste.

[0003] Plant tissue culture flowering induction technology offers another pathway to overcome the natural flowering period limitations of cut flowers. This technology, by controlling culture conditions in an in vitro sterile environment, can induce flowering in plants, potentially extending the ornamental period. However, traditional plant tissue culture techniques involve complex stages such as explant sterilization, multiple subcultures, and flower bud induction, with a cycle exceeding 140 days, resulting in cumbersome procedures and low efficiency. More importantly, this technology generally faces industrialization bottlenecks such as low flowering rates and unstable flower phenotypes, making it difficult to translate its technological advantages into stable commercial products.

[0004] Furthermore, in plant tissue culture, leaves and root tips are commonly used explants, while branches containing flower buds or blossoms are used very infrequently. This is mainly due to the following two technical challenges: First, the complex structure of flower buds, with their multi-layered wrapping, easily harbors microorganisms, leading to incomplete disinfection and a high risk of contamination. Second, the thin cell walls of petals and other tissues are extremely sensitive to disinfectants, easily causing irreversible damage such as discoloration and petal damage. Therefore, for ornamental plants such as roses, the disinfection process must achieve both thorough disinfection and zero damage to the ornamental tissues, posing an extreme challenge to disinfection technology.

[0005] Therefore, there is an urgent need to overcome the challenges of in vitro culture, particularly for flower buds, and to develop innovative technical solutions that can simultaneously achieve an ultra-long viewing period for cut flowers and high technical stability. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides a method for the integrated cultivation of detached rose buds for long-term sterile viewing and regeneration.

[0007] In a first aspect, the present invention provides a culture medium for in vitro culture of roses, with MS medium as the base medium, wherein 1.5 mg / L to 2.5 mg / L naphthaleneacetic acid, 5 g / L to 8 g / L plant gel, 1.5 g / L to 3.0 g / L sucrose and 0.10 g / L to 0.20 g / L antibacterial agent are added to the base medium; wherein the content of potassium dihydrogen phosphate in the culture medium for in vitro culture of roses is 300 mg / L to 400 mg / L.

[0008] According to the present invention, a culture medium for in vitro culture of roses is preferably provided in which the mass ratio of naphthaleneacetic acid to sucrose is 1:(0.75-3), and / or the mass ratio of plant gel to sucrose is (5-16):3, and / or the mass ratio of sucrose to antibacterial agent is (10-30):1.

[0009] More preferably, the mass ratio of naphthaleneacetic acid to sucrose is 1:(0.75-3), the mass ratio of plant gel to sucrose is (5-16):3, and the mass ratio of sucrose to antibacterial agent is (10-30):1.

[0010] More preferably, the potassium dihydrogen phosphate content in the culture medium for rose in vitro culture is 350 mg / L.

[0011] More preferably, the culture medium used for in vitro rose culture is adjusted to a pH of 5.8 to 6.0 before sterilization.

[0012] In some specific embodiments, the culture medium used for in vitro culture of roses is MS medium containing 1.5 mg / L naphthaleneacetic acid, 8 g / L plant gel, 3.0 g / L sucrose and 0.10 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 350 mg / L.

[0013] In some specific embodiments, the culture medium used for in vitro rose culture is MS medium containing 1.5 mg / L naphthaleneacetic acid, 8 g / L plant gel, 1.5 g / L sucrose and 0.15 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 350 mg / L.

[0014] In some specific embodiments, the culture medium used for in vitro culture of roses is MS medium containing 1.5 mg / L naphthaleneacetic acid, 8 g / L plant gel, 2.0 g / L sucrose and 0.20 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 350 mg / L.

[0015] In some specific embodiments, the culture medium used for in vitro culture of roses is MS medium containing 1.0 mg / L naphthaleneacetic acid, 5 g / L plant gel, 3.0 g / L sucrose and 0.10 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 350 mg / L.

[0016] In some specific embodiments, the culture medium used for in vitro culture of roses is MS medium containing 2.0 mg / L naphthaleneacetic acid, 6 g / L plant gel, 1.5 g / L sucrose and 0.15 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 300 mg / L.

[0017] In some specific embodiments, the culture medium used for in vitro culture of roses is MS medium containing 2.5 mg / L naphthaleneacetic acid, 7 g / L plant gel, 4.0 g / L sucrose and 0.20 g / L plant tissue culture antibacterial agent, wherein the content of potassium dihydrogen phosphate is 400 mg / L.

[0018] More preferably, the culture medium for in vitro culture of roses provided by the present invention uses MS medium as the base medium, and the base medium also contains 1.5 mg / L naphthaleneacetic acid, 8 g / L plant gel, 3.0 g / L sucrose and 0.10 g / L antibacterial agent, and the content of potassium dihydrogen phosphate is 350 mg / L.

[0019] According to the present invention, a culture medium for in vitro culture of roses is provided, preferably wherein the antibacterial agent is a plant tissue culture antibacterial agent.

[0020] More preferably, the plant tissue culture antibacterial agent is a Plant Preservative Mixture (PPM) plant tissue culture antibacterial agent.

[0021] The application of the culture medium for in vitro rose culture provided by this invention in in vitro rose culture and / or in extending the flowering cycle of in vitro roses should also be within the protection scope of this invention.

[0022] The application of the culture medium for in vitro culture of roses provided by this invention in any of the following should also be within the protection of this invention: (1) culturing rose explants; (2) culturing rose cut flowers; (3) prolonging the flowering cycle of rose explants and / or rose cut flowers; (4) inhibiting bacterial contamination of rose explants and / or rose cut flowers; (5) improving the environmental adaptability of rose explants and / or rose cut flowers; (6) preparing products for achieving any of the purposes of (1) to (5).

[0023] Secondly, the present invention provides a method for cultivating cut rose flowers, wherein rose explants are cultivated using the culture medium for rose in vitro culture.

[0024] According to a method for cultivating cut roses provided by the present invention, preferably, the rose explant contains at least one flower bud in the color-revealing stage.

[0025] More preferably, the rose explant contains one or two flower buds at the color-showing stage before cultivation.

[0026] More preferably, the rose explant contains a flower bud at the color-showing stage before cultivation.

[0027] More preferably, before cultivation, the rose explant contains a 7cm to 9cm pedicel. More preferably, the rose explant contains a 7.5cm to 8.5cm pedicel.

[0028] More preferably, prior to cultivation, the rose explant also contains at least one pinnate compound leaf.

[0029] More preferably, before cultivation, the rose explant has at least one pinnate compound leaf on each side.

[0030] More preferably, before cultivation, the rose explant has a pinnate compound leaf on each side.

[0031] More preferably, each pinnate compound leaf has 1 to 5 leaflets.

[0032] More preferably, each pinnate compound leaf has 2 to 3 leaflets.

[0033] According to the present invention, a method for cultivating cut roses is provided, wherein the rose explants are preferably fresh and / or refrigerated rose explants.

[0034] More preferably, the refrigeration and preservation conditions include: a temperature of 3°C to 5°C, and / or, preservation for 1 day to 30 days.

[0035] More preferably, the cold storage preservation method includes: sterilizing the rose explants with a 0.70 mg / L to 0.80 mg / L copper sulfate solution during cold storage.

[0036] More preferably, the cold storage preservation method includes: sterilizing the rose explants with a 0.75 mg / L copper sulfate solution during cold storage.

[0037] In some specific implementations, during refrigeration, the rose explants are wrapped with filter paper soaked in a 0.75 mg / L copper sulfate solution.

[0038] According to the method for preparing cut roses provided by the present invention, preferably, the rose explant is obtained by cutting it with scissors at a 40° to 50° angle at the distal end from the base of the flower bud. More preferably, it is obtained by cutting it with scissors at a 45° angle at the distal end from the base of the flower bud.

[0039] According to a method for cultivating cut roses provided by the present invention, preferably, the rose explants are treated with a disinfectant before cultivation; the disinfectant used includes 0.08% m / v to 0.12% m / v HgCl2.

[0040] More preferably, the disinfectant used includes 0.10% m / v HgCl2.

[0041] More preferably, the disinfectant used further includes a 0.008% m / v to 0.012% m / v antibacterial agent, wherein the effective disinfectant components of the antibacterial agent include 48 mg / L to 52 mg / L ketoconazole, 48 mg / L to 52 mg / L nystatin, and 0.08% m / v to 0.12% m / v sodium hypochlorite.

[0042] More preferably, the disinfectant used also includes a 0.008% m / v to 0.012% m / v antibacterial agent, wherein the effective disinfectant components of the antibacterial agent include 50 mg / L ketoconazole, 50 mg / L nystatin and 0.1% m / v sodium hypochlorite.

[0043] More preferably, the disinfectant used also includes a 0.01% m / v antibacterial agent, wherein the effective disinfectant components of the antibacterial agent include 48 mg / L to 52 mg / L ketoconazole, 48 mg / L to 52 mg / L nystatin, and 0.08% m / v to 0.12% m / v sodium hypochlorite.

[0044] More preferably, the disinfectant used also includes a 0.01% m / v antibacterial agent, wherein the effective disinfectant components of the antibacterial agent include 50 mg / L ketoconazole, 50 mg / L nystatin and 0.1% m / v sodium hypochlorite.

[0045] More preferably, the disinfectant treatment time is 3 to 9 minutes.

[0046] In some specific implementations, the disinfectant treatment time is 3 min, 5 min, 7 min, or 9 min.

[0047] More preferably, the disinfectant treatment time is 5 minutes.

[0048] According to a method for cultivating cut roses provided by the present invention, preferably, the entire rose explant is immersed in the disinfectant for treatment.

[0049] According to the present invention, a method for cultivating cut roses is provided, wherein the rose is preferably a miniature rose.

[0050] More preferably, the variety of the miniature rose is Little Red Riding Hood, Rouge Red, or Hangmei No. 1.

[0051] More preferably, the variety of the miniature rose is Carmine Red.

[0052] Thirdly, the cut roses obtained by the method for cultivating cut roses provided by the present invention.

[0053] The application of the cut roses described in this invention in the preparation of floral decorations should also be within the scope of protection of this invention.

[0054] Fourthly, the present invention provides a rose flower decoration, comprising a container, the culture medium for in vitro culture of roses provided by the present invention, and cut roses cultured according to a method for culturing cut roses provided by the present invention.

[0055] According to the present invention, a rose flower decoration is preferably provided in a container that is openable, closable and sealable, wherein the culture medium for in vitro rose culture and the cut rose flowers are both disposed inside the container, and the interior of the container is a sterile environment.

[0056] According to the present invention, a rose flower decoration is preferably provided in which the cut rose is planted in the culture medium for in vitro culture of roses.

[0057] According to the present invention, in a rose flower decoration, preferably, the culture medium for in vitro rose culture accounts for 25% to 35% of the container volume.

[0058] More preferably, the culture medium for in vitro rose culture accounts for 30% of the container.

[0059] According to the present invention, the container for a rose flower decoration is preferably a test tube with a volume of 50 mL to 200 mL.

[0060] More preferably, the container is a flat-bottomed test tube.

[0061] More preferably, each container holds at least one of the cut rose flowers.

[0062] According to the present invention, a rose flower decoration is preferably provided in a visible container, the main body of which is made of a transparent material.

[0063] More preferably, the transparent material includes glass and plastic.

[0064] More preferably, the container is made of a material resistant to high temperature and high pressure.

[0065] According to the present invention, a rose flower decoration is preferably placed in an environment with light and / or without light, and / or the ambient temperature is 0℃~38℃.

[0066] More preferably, the rose flower decoration is intended to be placed in an environment with and / or without light, and / or at an ambient temperature of 3°C to 35°C.

[0067] More preferably, the rose flower decoration is placed in an environment without direct sunlight, and / or at an ambient temperature of 15℃~25℃.

[0068] More preferably, the rose flower decoration is placed in an indoor environment without direct sunlight, where the indoor temperature is 20℃~25℃.

[0069] The method for preparing the rose flower decorations provided by the present invention should also be within the scope of protection of the present invention, including the following steps: assembling the container, the culture medium for in vitro culture of roses, and the cut roses using aseptic operation.

[0070] According to the method for preparing the rose flower decoration provided by the present invention, preferably, the container is sterilized and / or defogging before assembly.

[0071] Terminology Explanation In this invention, the term "explant" refers to an organ, tissue, or cell fragment excised from a living plant and used for in vitro aseptic culture; in subculture, a tissue segment excised from a culture and transferred to a new culture medium can also be called an explant.

[0072] In this invention, the term "culture medium" refers to an artificially prepared nutrient substrate used in plant tissue culture to maintain the survival and development of explants. Its core function is to provide the explants with the necessary living conditions, such as water, inorganic nutrients, carbon sources, vitamins, and growth regulators. Common basal culture media for plants include MS and B5.

[0073] In this invention, the term "cut flower" refers to fresh-cut flower branches cut from plants primarily for the purpose of viewing flowers or inflorescences, which is one of the main forms of flower commodity circulation. In the context of tissue culture, explants can be used as cut flowers after in vitro culture, and the cut flowers themselves or specific parts (such as pedicels and lateral buds) can serve as a source of materials for obtaining explants.

[0074] In this invention, the term "disinfection" refers to killing or removing some microorganisms so that they no longer pose a harmful effect, but does not necessarily kill all microorganisms (such as spores).

[0075] In this invention, the term "sterilization" refers to the complete killing or elimination of all microorganisms (including bacteria, fungi and their spores) on the surface of a material and in the environment by means of physical or chemical methods.

[0076] In this invention, the term "aseptic operation" refers to an operation performed in a sterilized environment, utensils, and tools to prevent microbial contamination.

[0077] In this invention, the term "visual container" refers to a specific part of a container that allows a user to observe its internal condition from the outside.

[0078] In this invention, the term "transparent material" refers to a material that allows visible light to pass through effectively, thereby allowing the user to clearly see the interior of the container.

[0079] The present invention has the following beneficial effects: This invention innovatively integrates plant tissue culture and cut flower preservation technologies, and optimizes a specific solid culture medium formula. It overcomes the core contradiction in existing technologies where portability, sterility, long-lasting ornamental value, and plant regeneration ability cannot coexist, reducing the risk of contamination. Its broad-spectrum and long-lasting antibacterial effect provides a fundamental guarantee for the long-term sterile culture of rose bud explants. Under this system, rose bud explants complete the entire cycle from flowering to decay using slow-release nutrients, significantly extending the ornamental period of cut roses. Furthermore, after the ornamental period, the cut roses possess complete transplant survival ability, realizing a shift from one-time consumption to sustainable cultivation. This solution has the industrialization advantages of short production cycle, high stability, and low maintenance costs, making it suitable for all-season and regional applications. It significantly enhances the ornamental value and practicality of products, providing innovative products for home decoration, high-end gifts, medical and health care, and education and popular science fields. Attached Figure Description

[0080] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0081] Figure 1 This is a photograph of the morphology of a rosebud explant in the blooming stage, provided in Embodiment 2 of the present invention.

[0082] Figure 2 This is a photograph of the morphology of the rose bud explant in the early flowering stage provided in Embodiment 2 of the present invention.

[0083] Figure 3 This is a photograph of the morphology of a rosebud explant during its full bloom period, provided in Embodiment 2 of the present invention. Detailed Implementation

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0085] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0086] The experimental materials and reagents involved in the following examples are as follows: The rose used is a miniature rose, the variety is Rouge Red (also known as Crimson Princess Rose); Plant Preservative Mixture (PPM) plant tissue culture antibacterial agent (i.e. PPM antibacterial agent) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0087] Unless otherwise specified, all other experimental materials and reagents are commercially available.

[0088] Example 1: Establishment of a disinfection method for rose bud explants 1. Pretreatment of rose bud explants To grow roses in indoor pots, select a flower bud that is showing signs of color. Cut the bud at a 45° angle from the base, leaving an 8±0.5 cm peduncle. Remove any yellowed, withered, or deformed leaves. Retain one pinnate compound leaf on each side; 2-3 leaves are ideal, but leaves on only one side are also acceptable. This will give you a fresh rose bud explant. Rinse the rose bud explant under running water for 5 minutes to ensure it is thoroughly cleaned.

[0089] If subsequent operations cannot be carried out in time, the rose bud explants can be wrapped with filter paper soaked in 0.75 mg / L copper sulfate solution (to achieve a sterilization effect), and then stored in a refrigerator at 4±1℃ for preservation. The maximum storage period is 30 days. This method can alleviate the problem that the flowering period of potted roses cannot meet the current demand.

[0090] 2. Disinfection of rose bud explants Disinfection procedures were performed in a clean bench. Fresh rosebud explants were placed in sterile beakers and disinfected according to schemes 1-5 in Table 1. Rosebud explants that had been refrigerated for 15 days were placed in sterile beakers and disinfected according to schemes 6-10 in Table 1. Rosebud explants that had been refrigerated for 15 days and wrapped in filter paper soaked in 0.75 mg / L copper sulfate solution were removed from the filter paper and placed in sterile beakers and disinfected according to schemes 11-15 in Table 1.

[0091] During the disinfection process, 20 rosebud explants were sterilized at a time for each method, and each method was set up in 3 replicates. During each sterilization, each rosebud explant was fully immersed in the disinfectant, and the beaker was gently shaken continuously to ensure complete disinfection.

[0092] Table 1 Disinfection Treatment Plan

[0093] Note: The effective disinfectant components of the antibacterial agent used are: 50 mg / L ketoconazole, 50 mg / L nystatin and 0.1% m / v sodium hypochlorite, and the solvent is sterile distilled water.

[0094] Afterwards, the rose bud explants, sterilized under high temperature and pressure, were rinsed three times with distilled water and left to stand at 23±1℃ for 7 days. The contamination, petal damage, and growth of the rose bud explants were observed and recorded. Petals that discolored or fell off were considered damaged. The presence of fungal mycelium at the base of the explant, or visible mold spots or colonies on the surface of the culture medium, indicated contamination.

[0095] Table 2. Effects of different disinfection treatments on petals and growth of rose bud explants. (mean ± standard deviation)

[0096] Note: Sterilization rate (%) = [(Total number of rose bud explants (strips) - Number of contaminated rose bud explants (strips)) ÷ Total number of rose bud explants (strips)] × 100%; The degree of petal damage is indicated by the number of "+" signs: "+" indicates that less than 2 petals have slightly fallen off and there is no discoloration; "++" indicates that 3 to 5 petals have fallen off and less than 1 petal has discoloration; "+++" indicates that 3 to 5 petals have fallen off or more than 2 petals have discoloration; "++++" indicates that 3 to 5 petals have fallen off and more than 2 petals have discoloration; "+++++" indicates that 6 or more petals have fallen off and more than 2 petals have discoloration, indicating that the petal damage is serious and affects subsequent growth.

[0097] As shown in Table 2, from the perspective of material selection, regardless of the disinfection method used, the sterilization rate of fresh flower branches is higher than that of flower branches treated with cold storage and copper sulfate, which is higher than that of flower branches treated with cold storage. Regarding the choice of disinfectant, while short-term surface disinfection with 75% v / v alcohol (30s–1min) can achieve some disinfection, it severely damages the petals of rose bud explants, causing color fading and structural damage, making it difficult to maintain their ornamental value. In contrast, short-term surface disinfection with 0.1% m / v HgCl2 (5min) effectively sterilizes while protecting the petals of rose bud explants from discoloration, ensuring their continued normal growth and development and maintaining their ornamental value. Disinfection with 0.1% m / v HgCl2 for more than 10min causes some irreversible damage to the petals, preventing some buds from blooming properly and compromising their ornamental value. Among them, disinfection with 0.1% m / v HgCl2 solution containing 0.01% m / v antibacterial agent for 5 minutes was determined to be the optimal disinfection scheme, with the highest sterilization rate and the lowest degree of petal damage.

[0098] Example 2: Development of a solid culture medium for planting rose bud explants 1. Solid culture medium formulation design This invention uses MS medium as the basal medium to investigate the effects of adding plant growth regulators, gelling agents, sugar sources, and antibacterial agents, as well as adjusting the content of potassium dihydrogen phosphate (KH2PO4), on the planting effect of rose bud explants.

[0099] Commonly available MS culture media can be used as the basic culture medium for this invention. The following is a specific MS culture medium (powder) composition formula: Potassium nitrate (KNO3) 1900 mg / L, ammonium nitrate (NH4NO3) 1650 mg / L, potassium dihydrogen phosphate (KH2PO4) 150 mg / L, magnesium sulfate (MgSO4) 370 mg / L, calcium chloride (CaCl2) 440 mg / L, potassium iodide (KI) 0.83 mg / L, boric acid (H3BO3) 6.2 mg / L, manganese sulfate (MnSO4) 22.3 mg / L, zinc sulfate (ZnSO4) 8.6 mg / L, sodium molybdate (Na2MoO4) 0.25 mg / L, copper sulfate (CuSO4) 0.025 mg / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.025 mg / L, disodium ethylenediaminetetraacetate (C 10 H 14 N2Na2O8·2H2O) 37.3 mg / L, ferrous sulfate (FeSO4) 27.8 mg / L, inositol (C6H) 12 O6) 100mg / L, glycine (C2H5NO2) 2mg / L, thiamine hydrochloride (C12 H 17 ClN4OS) 0.1 mg / L, pyridoxine hydrochloride (C8H) 11 NO3) 0.5 mg / L, nicotinic acid (C6H5NO2) 0.5 mg / L.

[0100] Fifteen solid culture medium formulations were designed, as shown in Table 3.

[0101] Table 3 Formulation of solid culture medium

[0102] 2. Preparation of solid culture medium According to the information shown in Table 3, weigh each raw material, then dissolve it completely in deionized water, adjust the pH value to 5.8-6.0 with 1M KOH or 1M HCl solution, autoclave (121℃, 0.1 MPa, 20 min), and dispense 12±0.5mL into sterile test tubes (transparent glass test tubes that have been defogging and sterilized with an anti-fogging agent, flat bottom, volume 86mL) that can hold rose bud explants, cool to room temperature, and seal for later use.

[0103] To simulate the mass production mode of actual products, 100 replicates of solid culture medium for each formulation were prepared in parallel.

[0104] 3. Antibacterial effect of solid culture media All samples of the above 15 solid culture media were left to stand at 23±1℃ for 7 days. Then, referring to "Experimental Guide to Plant Tissue Culture" (edited by Li Yun, published by China Forestry Publishing House in 2021), the bacterial and fungal contamination of each solid culture medium was statistically analyzed using conventional methods. The contamination rate of each solid culture medium was calculated using the following formula: Contamination rate (%) = Number of contaminated items (items) ÷ Total number (100 items) × 100%.

[0105] Table 4. Microbial contamination of different solid culture media (average values)

[0106] As shown in Table 4, none of the solid culture media formulated 1-6 showed bacterial contamination, and the mold contamination rate was also below 5%. Formula 2, due to its low sucrose content and the addition of a 0.15 g / L PPM antibacterial agent, exhibited the lowest contamination rate. When no sucrose was added to the solid culture medium in Formula 7, the lack of necessary sugar sources for microbial growth resulted in virtually no mold and bacterial growth, with a contamination rate of only 1%. In Formula 8, with a sucrose concentration of 3.0 g / L, the absence of a PPM antibacterial agent failed to completely inhibit bacterial and fungal growth. In Formula 9, with a sucrose concentration >3.0 g / L... At a sugar content of 1.0 g / L, the culture medium is rich in sugar sources, and even with the addition of antibacterial agents, the growth of molds and bacteria cannot be inhibited. Although the sucrose content in the culture medium of Formula 10 is as low as 1.0 g / L, even with the addition of PPM antibacterial agents at a concentration equivalent to one-tenth of the sucrose, the growth of bacteria and fungi cannot be effectively inhibited. The fungal infection rate of Formulas 11-15 is slightly higher than that of Formulas 1-6, and the bacterial infection rate is 0. This indicates that reasonable control of the sucrose content and the content of PPM antibacterial agents in the solid culture medium of this invention can effectively ensure antibacterial performance. The concentration changes of potassium dihydrogen phosphate (KH2PO4) and naphthaleneacetic acid (NAA) have no significant effect on this, providing the necessary sterile environment support for maintaining the flowering and growth of rose bud explants.

[0107] 4. Flowering of rose bud explants In a clean bench, 300 fresh rose bud explants were obtained by following the method in Example 1 and pretreated with disinfection scheme 5. The surface moisture of the explants was dried with sterilized filter paper, and the explants were randomly divided into 15 groups of 20 each. The rose bud explants of each group were vertically inserted into the test tubes containing different solid culture media (formulas 1 to 15) to fix them upright and prevent them from tipping over.

[0108] After sealing the test tubes, remove them from the clean bench and place them in a cool, shaded environment (20℃~25℃) without direct sunlight. Do not treat them further. Observe the flowering status of the rose bud explants regularly and record the time of each stage of the flowering process. After the flowering period ends, observe the axillary bud differentiation and root system of each rose bud explant and count the number of axillary buds and roots.

[0109] Before being inserted into the solid culture medium, the rose buds of the explant were firm, with tightly closed sepals, but the color of the petals was still visible. By the time they opened, as... Figure 1 As shown, the sepals of the rosebud begin to separate, and the tips of the petals slightly peek out, but the petals are not yet fully open. At this time, the bud begins to bloom. By the initial flowering stage, as... Figure 2 As shown, the rose petals begin to unfold outwards from the tip, indicating the flower is beginning to open, but not yet fully open. When it reaches its peak bloom, as... Figure 3As shown, the rose petals are fully open. After blooming, the flower begins to age and enters its decline stage. At this time, the rose petals darken in color, lose their elasticity, and the edges of the petals wilt, curl, or fade. The stamens wither or turn brown. Finally, the rose petals wilt severely and gradually fall off, and the flower stalk dries up.

[0110] Table 5. Effects of different solid culture media on the flowering cycle of rose bud explants (average values)

[0111] Table 6. Effects of different solid culture media on the long-term ornamental value of rose bud explants (average values)

[0112] As shown in Tables 5 and 6, formulations 1-6 exhibit significant advantages: the average opening period of rose bud explants cultured with these formulations is less than 6 days, the average total flowering period is greater than 56 days, the optimal viewing time (initial flowering period + full bloom period + end of flowering period) averages greater than 42 days (accounting for more than 74% of the total flowering period), and the average full bloom period is greater than 20 days (accounting for more than 35% of the total flowering period). This indicates that the solid culture medium of these formulations can not only promote rapid bud opening but also effectively extend the flowering period, maximizing the maintenance of ornamental value. In contrast, the total flowering period of rose bud explants cultured with formulations 7-15 is less than 51 days on average, showing significantly poorer overall performance. Among them, although the average optimal viewing time of rose bud explants cultured with formulations 9 and 15 is 40 days and the average full bloom period is 18 days, the total flowering period is relatively short. Furthermore, the average optimal viewing time of rose bud explants cultured with formulations 7, 8, and 10-14 is less than 40 days, failing to achieve long-term flowering and ornamental value. Formula 1 is the optimal solid culture medium formula, with the longest total flowering period and the best viewing time reaching 54.75 days (accounting for more than 84% of the total flowering period). The average peak flowering period lasts for 28.95 days (accounting for more than 44% of the average total flowering period), and the stability at each stage is significant.

[0113] 5. Transplanting of rose bud explants After the flowering period, 300 rosebud explants cultured on 15 different solid culture media were transplanted into a cultivation substrate (peat and perlite in a 3:1 ratio). One rosebud explant was transplanted into each pot. The pots were then moved to a cool, well-ventilated area, and thoroughly watered. The tops of the pots were then covered with plastic wrap to maintain warmth and moisture, with several holes evenly punched in the wrap for ventilation. During the maintenance period, the plastic wrap was opened twice daily, once in the morning and once in the afternoon, for ventilation. Water was sprayed as needed based on the surface dryness of the substrate and the ambient humidity for 15 days. After this period, the plastic wrap was completely removed, and the survival rate of each rose was recorded.

[0114] Table 7. Effects of different solid culture media on axillary bud differentiation and rooting of rose bud explants (average values)

[0115] Note: Transplant survival rate (%) = (Number of surviving plants ÷ Total number of transplanted plants) × 100%.

[0116] As shown in Table 7, the solid culture media of formulas 1-6 and formula 11 can meet the potential and vitality of rose bud explants to continue growing after the ornamental period, with a transplant survival rate of over 90%. The transplant survival rates of formulas 7, 8, 9 and 10 are all below 85%, indicating that when sucrose is not added or the antibacterial properties of the solid culture medium are weak, the root development of rose bud explants is poor, making them susceptible to disease and resulting in a low transplant survival rate. The transplant survival rates of formulas 12, 13, 14 and 15 are all below 85%, indicating that although the solid culture medium has certain antibacterial properties and reduces the disease rate of rose bud explants, the content of potassium dihydrogen phosphate is too low (<300 mg / L) or too high (>400 mg / L), and the content of NAA is too low (<1 mg / L), which are all detrimental to the root development of rose bud explants and result in a low transplant survival rate. Among them, the transplant survival rate of rose bud explants cultured in the solid culture medium of Formula 1 was the best, reaching 100%.

[0117] Example 3: The Influence of the Placement Environment on the Ornamental Value of Cut Roses One hundred fresh rose bud explants were prepared according to the method of Example 1 and pretreated with disinfection scheme 5. After they were naturally dried at room temperature, they were vertically inserted into a test tube containing solid culture medium of formula 1 prepared according to the method of Example 2 in a clean bench, so that it was fixed upright and not tipped over. After sealing the test tube, it was taken out of the clean bench to obtain the finished cut rose flowers.

[0118] One hundred cut roses were randomly divided into five groups of 20 each. The groups were placed in five environments: an indoor environment with no direct sunlight (20℃~25℃) (Environment 1), a refrigerator at 4±1℃ (Environment 2), an indoor environment with direct sunlight (20℃~35℃) (Environment 3), an outdoor environment with no direct sunlight (15℃~25℃) (Environment 4), and an outdoor environment with direct sunlight (15℃~35℃) (Environment 5). No further treatment was given. The flowering status of the cut roses was observed regularly, and the time of each flowering stage was recorded.

[0119] Table 8. Effects of different placement environments on the flowering cycle of cut roses (average values)

[0120] As shown in Table 8, the total flowering period of cut roses cultivated with Formula 1 can be up to 4 months under different placement environments. Even in outdoor environments with significant changes in light and temperature, the total flowering period can be maintained for more than 1 month.

[0121] Rose cut flowers were prepared using solid culture media with formulas 2-6 in the same manner and observed in different environments. It was found that all rose cut flowers showed similar characteristics, with the longest total flowering period being 4 months. Even in outdoor environments with light, the total flowering period could be maintained for more than 1 month.

[0122] The above results indicate that the solid culture medium (formulas 1-6) provided by the present invention can not only meet the normal flowering cycle maintenance of cut roses under different environments, but also better meet the viewing needs of different consumer groups.

[0123] In addition, temperature is a key factor in regulating the flowering period. Compared with environment 1, low temperature (4℃) can extend the total flowering period to 123 days (1.90 times that under normal temperature conditions), with no significant difference in the proportion of peak bloom, but the refrigerated environment is inconvenient for daily viewing. In contrast, a cool indoor environment of 20℃~25℃ can better balance the advantages of flowering period length and viewing quality. Light intensity significantly affects the flowering rate. Although direct sunlight accelerates bud opening, it can induce photooxidative damage, causing the flowers to enter the decay period prematurely. Therefore, considering production feasibility, viewing continuity, and consumer convenience, a cool indoor environment is the preferred solution for commercial application. It is recommended to place the cut rose products provided by this invention in an indoor area without direct sunlight to extend the optimal viewing period to more than 54 days.

[0124] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for in vitro culture of roses, using MS medium as the base medium, characterized in that, The basic culture medium also contains 1.5 mg / L to 2.5 mg / L naphthaleneacetic acid, 5 g / L to 8 g / L plant gel, 1.5 g / L to 3.0 g / L sucrose, and 0.10 g / L to 0.20 g / L antibacterial agent. The potassium dihydrogen phosphate content in the culture medium used for in vitro rose culture is 300 mg / L to 400 mg / L.

2. The culture medium according to claim 1, characterized in that, The mass ratio of naphthaleneacetic acid to sucrose is 1:(0.75-3), and / or, The mass ratio of the plant gel to the sucrose is (5-16):3, and / or, The mass ratio of sucrose to antibacterial agent is (10-30):

1.

3. A method for cultivating cut roses, characterized in that, Rose explants were cultured using the culture medium for rose in vitro culture as described in claim 1 or 2.

4. The method according to claim 3, characterized in that, The rose explant contains at least one flower bud at the color-revealing stage.

5. The method according to claim 3, characterized in that, Before cultivation, the rose explants were treated with a disinfectant, including 0.08% m / v to 0.12% m / v HgCl2.

6. Cut roses cultivated according to any one of claims 3 to 5.

7. A rose-themed floral decoration, characterized in that, It includes a container, the culture medium as described in claim 1 or 2, and the cut rose as described in claim 6.

8. The rose flower decoration according to claim 7, characterized in that, The container is optionally sealed, and both the culture medium and the cut roses are placed inside the container, which is a sterile environment.

9. The rose flower decoration according to claim 8, characterized in that, The culture medium used for in vitro rose culture occupies 25% to 35% of the container volume.

10. The rose flower decoration according to claim 8 or 9, characterized in that, The container is a visible container, and its main body is made of transparent material.

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