Application of compound enzyme in preparation of rosa chinensis protoplast

Abstract

The invention provides an application of a compound enzyme in preparation of a Chinese rose protoplast, and belongs to the technical field of plant protoplast preparation. According to the compound enzyme disclosed by the invention, the cellulase R-10 and the macerozyme R-10 are compounded for use, and the cellulase R-10 mainly acts on a cellulose component in a cell wall of a Chinese rose leaf, so that a cell wall structure begins to collapse; the macerozyme R-10 can act on components such as pectin in cell walls, and has a synergistic effect with cellulase R-10, so that the cell wall structure is further destroyed, and the release of protoplast is promoted. When the compound enzyme is used for preparing the rosa chinensis protoplast, the high activity of the protoplast is kept, and the number of the obtained protoplast is large. The method disclosed by the invention has outstanding beneficial effects in the aspects of keeping the high activity of the rosa chinensis protoplast and increasing the number of the rosa chinensis protoplast, and opens up a broader way for related research and application of the rosa chinensis protoplast.

Description

Technical Field

[0001] This invention belongs to the field of plant protoplast preparation technology, specifically relating to the application of a complex enzyme in the preparation of rose protoplasts. Background Technology

[0002] In the field of plant biotechnology, protoplast preparation is a crucial fundamental operation, playing a key role in many aspects such as plant cell fusion, genetic transformation, and gene function research. Roses, as world-renowned ornamental flowers, occupy a pivotal position in the floriculture industry. However, there are currently many unresolved issues in rose protoplast extraction.

[0003] Existing methods for extracting protoplasts from roses mostly draw upon techniques from other plants, with adaptive adjustments. However, due to the unique physiological characteristics and cellular structure of roses, these methods often fail to achieve ideal extraction results. One prominent issue is low extraction efficiency. Many traditional methods suffer from incomplete cell wall disruption due to improper control of enzymatic hydrolysis conditions, resulting in a large number of protoplasts remaining unreleased and yielding a significantly lower number of protoplasts than expected. This severely limits subsequent experiments. For example, some conventional methods use a single cellulase for enzymatic hydrolysis, which can partially disrupt the cell wall. However, a single enzyme cannot fully decompose the complex structural components of rose cell walls, thus affecting protoplast release efficiency.

[0004] On the other hand, low protoplast activity is also a major challenge for researchers. During the extraction process, some methods, due to inaccurate control of conditions such as temperature and osmotic pressure, cause a rapid decline in protoplast activity after separation. Reduced protoplast activity leads to lower survival rates in subsequent cell fusion and culture operations, severely affecting the reliability and reproducibility of experimental results. For example, enzymatic hydrolysis at excessively high temperatures, while potentially accelerating cell wall decomposition, can also damage protoplast structures such as the cell membrane, thereby reducing their activity.

[0005] Furthermore, existing methods also have shortcomings in cost control. Some extraction methods require the use of expensive special enzymes or reagents, or complex equipment and operating procedures during the extraction process, which undoubtedly increases experimental costs and limits the widespread application of related technologies in actual production and scientific research.

[0006] In summary, existing rose protoplast extraction technologies have significant drawbacks in terms of extraction efficiency, protoplast activity, and cost control, and there is an urgent need for a more efficient, economical extraction method that can guarantee high protoplast activity. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application of a compound enzyme in the preparation of rose protoplasts. The compound enzyme can significantly improve the yield and activity of rose protoplasts while reducing costs.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides an application of a compound enzyme in the preparation of rose protoplasts. The working concentration of the compound enzyme, based on the volume of the enzymatic hydrolysis working system, is: cellulase R-10 2~4 g / 100mL and dissociative enzyme R-10 1~2 g / 100mL.

[0009] Preferably, the roses include 'Monthly Pink' roses and / or 'Blue Rhapsody' roses.

[0010] Preferably, the cellulase R-10 includes Yakult cellulase R-10; the dissociation enzyme R-10 includes Yakult dissociation enzyme R-10.

[0011] Preferably, based on the volume of the enzymatic hydrolysis working system, the working concentration of the composite enzyme is: cellulase R-10 3 g / 100 mL and dissociative enzyme R-10 1.5 g / 100 mL.

[0012] This invention provides an enzymatic hydrolysate for preparing rose protoplasts, comprising, by total volume of the hydrolysate: 2~4 g / 100mL cellulase R-10, 1~2 g / 100mL anabolase R-10, mannitol 0.3~0.8 mol / L, KCl 5~20mmol / L, MES dosage to adjust the pH of the enzyme hydrolysate to 5.5~6.0, CaCl2 5~20mmol / L, 0.01~0.02 g / 100mL PVP and 0.1~0.5 g / 100mL BSA.

[0013] This invention provides the application of the enzymatic hydrolysate described in the above-mentioned technical solution in any one or more of the following: preparing rose protoplasts, increasing the yield of rose protoplasts, and improving the activity of rose protoplasts; wherein the rose includes 'Monthly Powder' rose (…). Rosa chinensis 'Old Blush' and / or 'Blue Rhapsody' roses Rosa 'Rhapsody in Blue').

[0014] This invention provides a method for preparing rose protoplasts, comprising: Rose leaves were enzymatically hydrolyzed using the enzymatic hydrolysate described in the above technical solution to obtain rose protoplasts.

[0015] Preferably, the rose leaves include leaves from rose cuttings and / or leaves from rose tissue culture seedlings.

[0016] Preferably, the enzymatic hydrolysis temperature is 25~28℃; and the enzymatic hydrolysis time is 14~16h.

[0017] Preferably, the rose leaves are pretreated before enzymatic hydrolysis; the pretreatment includes the following steps: Rose leaves were cut into thin strips and soaked in mannitol solution to obtain tissue material treated with mannitol solution. Tissue materials treated with mannitol solution were mixed with enzymatic hydrolysate and then subjected to vacuum treatment.

[0018] This invention provides a reagent for preparing rose protoplasts, comprising: the enzymatic hydrolysate described in the above technical solution, reagent B, and reagent C; Reagent B includes W5 solution; The reagent C includes FDA working solution.

[0019] The beneficial effects of this invention are: This invention provides an application of a compound enzyme in the preparation of rose protoplasts. Based on the volume of the enzymatic hydrolysis system, the working concentration of the compound enzyme is: cellulase R-10 2~4 g / 100mL and cleavage enzyme R-10 1~2 g / 100mL. The compound enzyme of this invention utilizes the combined use of cellulase R-10 and cleavage enzyme R-10. Cellulase R-10 primarily acts on the cellulose components in the cell wall of rose leaves, causing the cell wall structure to begin to disintegrate; cleavage enzyme R-10 can act on components such as pectin in the cell wall, working synergistically with cellulase R-10 to further destroy the cell wall structure and promote the release of protoplasts. Using this compound enzyme for the preparation of rose protoplasts maintains high protoplast activity and yields a large number of protoplasts. The results of the embodiments of this invention show that using the compound enzyme to prepare 'Yueyuefen' rose protoplasts achieves a protoplast yield of 46 × 10⁻⁶. 5 The compound enzyme was used to prepare protoplasts of the 'Blue Rhapsody' rose variety, achieving a protoplast yield of 31 × 10⁶ cells / g. 5The protoplasts obtained from this invention, specifically the 'Monthly Powder' and 'Blue Rhapsody' roses, exhibit high activity. Furthermore, the method for preparing rose protoplasts provided by this invention demonstrates significant advantages in cost control. On one hand, the use of common and relatively inexpensive cellulase R-10 and anabolase R-10 reduces enzyme reagent costs at the source. On the other hand, by optimizing enzymatic hydrolysis conditions, such as determining the optimal enzyme ratio and hydrolysis time, the amount of enzyme used and reaction time are reduced, further lowering costs. In addition, this invention can use cuttings as experimental materials; compared to conventional tissue culture materials, cuttings are readily available, greatly reducing material costs. These combined measures significantly reduce the cost of the entire protoplast preparation process, making this technology more economically feasible in scientific research and actual production. This invention achieves outstanding beneficial effects in maintaining high protoplast activity, increasing protoplast quantity, and reducing costs, opening up broader avenues for rose protoplast-related research and applications. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 Image of 'Monthly Pink' cuttings; Figure 2 The diagram shows the preparation process of 'Yueyuefen' protoplasts; A shows the observation results of immersing 'Yueyuefen' leaves into 1mm leaf strips in a 0.4mol / L mannitol solution; B shows the observation results of terminating the reaction by adding W5 solution after enzymatic hydrolysis under dark conditions; C shows the observation results of centrifugation after gentle resuspending with reagent B. Figure 3 This is a photograph of the protoplast suspension prepared in Example 4 under an optical microscope; Figure 4 Photograph of the protoplast suspension prepared in Example 6 under an inverted fluorescence microscope; Figure 5 Photograph of the protoplast suspension prepared for Comparative Example 1 under an optical microscope; Figure 6 Photograph of the protoplast suspension prepared for Comparative Example 2 under an optical microscope; Figure 7 Photograph of the protoplast suspension prepared in Example 5 under an optical microscope; Figure 8 This is a photograph of the protoplast suspension prepared in Example 4 under an optical microscope; Figure 9 The effects of different cultivation conditions on rose protoplast yield and vigor. Detailed Implementation

[0022] This invention provides an application of a compound enzyme in the preparation of rose protoplasts. The working concentration of the compound enzyme, based on the volume of the enzymatic hydrolysis working system, is: cellulase R-10 2~4 g / 100mL and dissociation enzyme R-10 1~2 g / 100mL.

[0023] Unless otherwise specified, the present invention does not have any special limitations on the source of each raw material in the following technical solutions, and any commercially available products in the field can be used.

[0024] As an optional embodiment of the present invention, the rose includes the 'Monthly Pink' rose and / or the 'Blue Rhapsody' rose. The leaves can be leaves from rose cuttings and / or leaves from rose tissue culture seedlings. The present invention does not specifically limit the source or cultivation method of the rose cuttings and rose tissue culture seedlings; conventional sources or cultivation methods in the art are acceptable. As an optional embodiment of the present invention, the rose cuttings can be cuttings cultured for 60-90 days. The results of the embodiments of the present invention show that: using an enzymatic hydrolysate containing the aforementioned complex enzyme to prepare protoplasts from rose cutting leaves or rose tissue culture seedling leaves can significantly improve the yield of rose protoplasts compared to using leaves from ground-grown rose seedlings, and also improve the activity of the obtained protoplasts.

[0025] As an optional embodiment of the present invention, the working concentration of cellulase R-10 in the composite enzyme is 2-4 g / 100 mL, based on the volume of the enzymatic hydrolysis working system; it can be 2, 3, or 4 g / 100 mL. In this invention, the cellulase R-10 can be Yakult cellulase R-10. In this invention, the cellulase R-10 mainly acts on the cellulose component in the cell wall of rose leaves, breaking its β-1,4-glycosidic bonds, thus causing preliminary degradation of the cell wall.

[0026] As an optional embodiment of the present invention, the working concentration of the dissociative enzyme R-10 in the complex enzyme is 1~2 g / 100mL, based on the volume of the enzymatic hydrolysis working system, and can be 1, 1.5, or 2 g / 100mL. In the present invention, the dissociative enzyme R-10 can be Yakult dissociative enzyme R-10. In the present invention, the dissociative enzyme R-10 can act on components such as pectin in the cell wall, assisting cellulase R-10 to more comprehensively decompose the cell wall and promote the release of protoplasts.

[0027] This invention innovatively employs a specific ratio of cellulase R-10 and dissociative enzyme R-10 to enzymatically hydrolyze rose leaves, significantly increasing protoplast yield compared to traditional methods. The optimal enzyme ratio, determined through extensive experimentation, allows the two enzymes to work synergistically, fully and efficiently breaking down cell walls and releasing more protoplasts from the cells. This solves the problem of protoplasts being trapped within incompletely decomposed cell walls due to insufficient enzymatic hydrolysis in traditional methods.

[0028] The present invention uses the aforementioned complex enzyme to prepare rose protoplasts, which maintains the high activity of the protoplasts and yields a large number of protoplasts.

[0029] This invention provides an enzymatic hydrolysate for preparing rose protoplasts, comprising, by total volume of the hydrolysate: 2~4 g / 100mL cellulase R-10, 1~2 g / 100mL analyte R-10, 0.3~0.8 mol / L mannitol, 5~20mmol / L KCl, MES (2-(N-morpholino)ethanesulfonic acid) dosage to adjust the pH of the enzyme hydrolysate to 5.5~6.0, 25~20mmol / L CaCl2, 0.01~0.02 g / 100mL PVP (polyvinylpyrrolidone), and 0.1~0.5 g / 100mL BSA (bovine serum albumin).

[0030] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 2-4 g / 100 mL of cellulase R-10, or 2, 3, or 4 g / 100 mL, based on the total volume of the hydrolysate. In the present invention, the cellulase R-10 may be Yakult cellulase R-10.

[0031] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 1-2 g / 100 mL of analyte R-10, or 1, 1.5, or 2 g / 100 mL, based on the total volume of the hydrolysate. In the present invention, the analyte R-10 may be Yakult analyte R-10.

[0032] As an optional embodiment of the present invention, the enzymatic hydrolysate includes 0.3~0.8 mol / L mannitol, which can be 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mol / L, based on the total volume of the hydrolysate. In the present invention, the mannitol can be D-mannitol. In the present invention, the role of the mannitol is to maintain the osmotic pressure balance of the protoplasts during enzymatic hydrolysis, making it consistent with the osmotic pressure inside the rose leaf cells, and preventing the protoplasts from rupturing or shrinking due to osmotic pressure imbalance.

[0033] As an optional embodiment of the present invention, the enzymatic hydrolysate, based on its total volume, comprises KCl at a concentration of 5-20 mmol / L, which can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mmol / L. The potassium chloride plays a positive role in maintaining enzyme activity and cell membrane stability by regulating the ionic strength of the enzymatic hydrolysate. In protoplast extraction, potassium chloride ensures the smooth progress of the protoplast extraction process and the activity and integrity of the protoplasts by maintaining ion balance and osmotic pressure, influencing cell membrane properties (stabilizing structure and regulating permeability), and affecting enzyme activity (activating related enzymes and maintaining enzyme stability).

[0034] In an optional embodiment of the present invention, the enzymatic hydrolysate includes MES, and the amount of MES used is sufficient to adjust the pH of the enzymatic hydrolysate to 5.5-6.0. The pH value can also be 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In another optional embodiment of the present invention, when adjusting the pH of the enzymatic hydrolysate, the molar concentration of the MES mother liquor can be 0.2 mol / L. The MES mainly stabilizes the pH of the enzymatic hydrolysate, providing a suitable acid-base environment for cellulase R-10 and ionase R-10 to exert their optimal activity.

[0035] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 5-20 mmol / L CaCl2, or may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mmol / L, based on the total volume of the hydrolysate. The calcium chloride enhances the stability of the cell membrane, helping to protect the integrity of the protoplast during the enzymatic hydrolysis process.

[0036] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 0.01~0.02 g / 100 mL PVP, based on the total volume of the hydrolysate. The PVP has strong hydrophilicity and complexing ability, and can bind to phenolic substances released from rose leaves such as 'Yuehuefen' during enzymatic hydrolysis, preventing oxidative browning of the phenolic substances, thereby protecting the protoplasts from oxidative damage and further improving protoplast activity and yield.

[0037] As an optional embodiment of the present invention, the enzymatic hydrolysate comprises 0.1~0.5 g / 100mL BSA, or 0.1, 0.2, 0.3, 0.4 or 0.5 g / 100mL, based on the total volume of the hydrolysate. The BSA can reduce non-specific protein adsorption during enzymatic hydrolysis, protect enzyme activity, and also help maintain protoplast stability.

[0038] The present invention does not specifically limit the preparation method of the enzymatic hydrolysate; any conventional preparation method in the art can be used. As an optional embodiment of the present invention, the preparation of the enzymatic hydrolysate includes the following steps: According to the concentration range of each component mentioned above, accurately weigh cellulase R-10 and ionase R-10, and dissolve them in a solution containing the corresponding concentrations of D-mannitol, MES buffer, and potassium chloride to obtain an enzyme solution; incubate the enzyme solution in a 55°C water bath for 10 min, and then place it on ice to cool to room temperature. Once the enzyme solution is clear, add BSA, PVP, and calcium chloride solution, and stir thoroughly to ensure that all components are completely dissolved, to obtain a homogeneous and stable enzymatic hydrolysate.

[0039] The enzymatic hydrolysate provided by this invention has a density range of 1.02~1.05 g / cm³ at 25°C. 3 This density range facilitates sufficient contact between the enzymatic hydrolysate and the leaf tissue, ensuring uniform enzymatic hydrolysis throughout the system. For example, when the density is 1.03 g / cm³... 3 At this time, the enzyme hydrolysate can better penetrate into the intercellular spaces of the leaf cells, increasing the chances of contact between the enzyme and the substrate.

[0040] The enzymatic hydrolysate has a viscosity range of 1.0–1.2 mPa·s at 25°C. This suitable viscosity allows the hydrolysate to effectively coat the leaf strips, ensuring sufficient contact between the enzyme and the leaf, while also preventing excessive viscosity from hindering the relative movement of the hydrolysate and the leaf when the culture dish is shaken, thus ensuring the smooth progress of the enzymatic reaction. For example, a viscosity of 1.1 mPa·s achieves a good balance between the fluidity and adhesion of the hydrolysate.

[0041] The enzymatic hydrolysate, when placed at 25°C for 14-16 hours, exhibits a pH fluctuation range within ±0.1, demonstrating good pH stability. This provides a stable acid-base environment for cellulase R-10 and ionase R-10 over a relatively long period, ensuring the continuous and stable progress of the enzymatic hydrolysis reaction.

[0042] The enzymatic hydrolysate is transparent and colorless in the visible light range, which is beneficial for observing leaf tissues during enzymatic hydrolysis and also indicates that there are no large molecular particles or suspended matter in the solution that could interfere with the enzymatic hydrolysis reaction. Its specific absorption peaks in the ultraviolet-visible spectrum can be used for qualitative and quantitative analysis of the components in the solution; for example, by detecting the absorbance at a specific wavelength, the concentration changes of cellulase R-10 or analyte R-10 can be determined.

[0043] The appropriate osmotic pressure regulator and precisely controlled concentration in the enzymatic hydrolysate provided by this invention ensure that the protoplasts are always in a suitable osmotic pressure environment during the separation process, preventing protoplast rupture or dehydration and shrinkage due to improper osmotic pressure, thereby ensuring the activity of the protoplasts.

[0044] The enzymatic hydrolysate provided by this invention is a homogeneous and stable mixed solution system in which all components are evenly distributed. These components work synergistically to the leaf cells of roses such as 'Yuehuefen' from different aspects. For example, cellulase R-10 and ionase R-10 are responsible for degrading the cell wall, osmotic pressure regulators maintain osmotic pressure balance, buffers stabilize pH, and calcium chloride, potassium chloride, BSA, and PVP enhance membrane stability, regulate ionic strength, prevent protein adsorption, and inhibit phenolic oxidation, respectively, thus jointly ensuring the effective decomposition of the cell wall and the efficient release and stable existence of protoplasts.

[0045] This invention provides the application of the enzymatic hydrolysate described in the above technical solution in any one or more of the following: preparing rose protoplasts, increasing the yield of rose protoplasts, and improving the activity of rose protoplasts; wherein the roses include 'Monthly Pink' roses and / or 'Blue Rhapsody' roses.

[0046] This invention provides a method for preparing rose protoplasts, comprising: Rose leaves were enzymatically hydrolyzed using the enzymatic hydrolysate described in the above technical solution to obtain rose protoplasts.

[0047] As an optional embodiment of the present invention, the rose leaves include leaves from rose cuttings and / or leaves from rose tissue culture seedlings. The present invention does not specifically limit the source or cultivation method of the rose cuttings; cuttings obtained using conventional cuttings or conventional cultivation methods in the art are acceptable. The present invention also does not specifically limit the source or cultivation method of the rose tissue culture seedlings; tissue culture seedlings obtained using conventional tissue culture methods in the art are acceptable.

[0048] In an optional embodiment of the present invention, the cuttings can be cuttings cultured for 60-90 days. In an optional embodiment of the present invention, the preparation method of the cuttings can be: rooting rose cuttings in a rooting substrate to obtain rooted seedlings; and then culturing the rooted seedlings in a transplanting substrate to obtain cuttings. In an optional embodiment of the present invention, the rose cuttings are preferably selected from plump, disease-free branches of the current year's axillary buds before spring budding or after autumn flowering; the cuttings preferably retain two buds and two small leaves, and are morphologically cut flat at both ends. In the present invention, the rooting substrate can be a mixture of vermiculite and perlite in a volume ratio of 3:1. In the present invention, the cuttings are preferably propagated using a thickened 230-size plastic two-color flowerpot as the propagation container. The rooting culture temperature can be 20-25℃, or 20, 21, 22, 23, 24, or 25℃. During the rooting culture process, diffused light is preferred to avoid direct sunlight and leaf burn. Water every 3 days, and cover the bottom of the pot with a plastic wrap to prevent rapid water loss. In this invention, the rooting culture time can be 20-35 days, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days. During the rooting culture process, new buds sprout from the tip of the cutting, and white new roots grow from the base. After the rooting culture is completed, this invention yields rooted seedlings with new roots reaching 3-5 cm in length and 2-3 new leaves unfolding. After obtaining the rooted seedlings, this invention cultivates them in a transplanting substrate to obtain cuttings. As an optional embodiment of the present invention, the transplanting substrate can be a mixed substrate of vermiculite, peat moss, and perlite in a volume ratio of 2:3:1; the cultivation temperature can be 20-25℃, or 20, 21, 22, 23, 24, or 25℃; the cultivation process preferably uses diffused light, keeping the substrate moist, watering every 5-8 days, following the principle of watering only when the soil is dry; the cultivation time can be 60-90 days, or 60, 65, 70, 75, 80, 85, or 90 days. After cultivation, cuttings are obtained. The leaves of the obtained cuttings can be directly used for subsequent protoplast preparation.

[0049] As an optional embodiment of the present invention, the tissue culture seedling can be a tissue culture seedling cultured for 50-60 days. Further, the tissue culture seedling can be a tissue culture seedling cultured for 50-60 days after the rose stem segments have been disinfected and induced to sprout axillary buds.

[0050] As an optional embodiment of the present invention, the rose cuttings can be selected from those with good growth and free from pests and diseases. The rose tissue culture seedlings can be selected from those with good growth. The present invention uses cuttings as the material for collecting rose leaves, which is easier to obtain than conventional tissue culture materials, thus reducing material costs. After obtaining the cuttings and / or tissue culture seedlings, the present invention preferably takes tender and fully unfolded rose leaves from the cuttings and / or tissue culture seedlings for enzymatic hydrolysis. After obtaining the rose leaves, the present invention preferably washes the rose leaves 3-5 times, or even 4 times; the washing is preferably done with distilled water. The present invention washes the rose leaves to thoroughly remove surface dust and impurities. After washing, the present invention preferably uses filter paper to absorb the surface moisture of the leaves and then performs subsequent processing on the obtained rose leaves.

[0051] After obtaining the aforementioned leaves, the present invention preferably removes the main vein and the tissue portion of the leaf edge to obtain leaves with full color, a radius of 0.5~1.5cm, and no wrinkles. The present invention may also refer to leaves with full color, a radius of 0.5~1.5cm, and no wrinkles as pre-treated leaves. The present invention preferably uses a sharp single-edged safety blade to cut the leaves, removing the main vein and the tissue portion of the leaf edge. After obtaining the pre-treated leaves, the present invention preferably cuts the pre-treated leaves into thin strips. As an optional embodiment of the present invention, the thin strips can be cut along the direction of the main vein of the leaf tissue. In the present invention, the width of the thin strips can be 0.5~2mm, or 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0mm. After obtaining the thin strips, the present invention preferably soaks the thin strips in a mannitol solution to obtain mannitol-treated tissue material. In the present invention, the mannitol solution can be a 0.3~0.8 mol / L mannitol solution; the molar concentration of the mannitol solution can also be 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mol / L. The present invention does not have a particular limitation on the amount of mannitol solution used, as long as it is sufficient to immerse the thin strips in the mannitol solution. In the present invention, the soaking temperature can be 25~28℃, or 25, 26, 27, or 28℃, or room temperature; the soaking time can be 0.25~0.5 h, or 0.5 h. After soaking, the present invention obtains mannitol-treated tissue material. Before enzymatic hydrolysis, the present invention treats the leaf strips with mannitol solution, which can fully release the secondary metabolites in the leaf strips, further improve the hydrolysis efficiency, and also keep the leaf strips fresh before enzymatic hydrolysis, thereby improving the activity of protoplasts.

[0052] After obtaining the tissue material treated with mannitol solution, the present invention mixes the obtained tissue material with an enzymatic hydrolysate and performs vacuum treatment. As an optional embodiment of the present invention, the mass ratio of the tissue material to the enzymatic hydrolysate can be 1 g:(10~20) mL or 1 g:10 mL. In the present invention, the vacuum degree of the vacuum treatment can be 0.04~0.06 MPa or 0.05 MPa; the vacuum treatment time can be 0.5~1 h or 1 h. After vacuum treatment, the present invention performs enzymatic hydrolysis on the mixture of tissue material and enzymatic hydrolysate. As an optional embodiment of the present invention, the enzymatic hydrolysis temperature can be 25-28℃, or 25, 26, 27, or 28℃; the enzymatic hydrolysis time can be 14-16 hours, or 14, 15, or 16 hours; the enzymatic hydrolysis is preferably carried out in a shaker; the shaking speed can be 40-50 rpm, or 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 rpm. After enzymatic hydrolysis, an enzymatically hydrolyzed solution is obtained. In the present invention, the enzymatic hydrolysis temperature avoids thermal damage to the rose protoplasts caused by high temperatures, ensuring the integrity of cell membranes, organelles, and other structures, thereby maintaining the high activity of the protoplasts.

[0053] After obtaining the enzymatically hydrolyzed solution, the present invention preferably terminates the reaction by mixing the enzymatically hydrolyzed solution with W5 solution, resulting in a turbid mixture of enzymatic hydrolysate and protoplasts. As an optional embodiment of the present invention, the W5 solution and the enzymatically hydrolyzed solution can be mixed in equal volumes. When adding W5 solution to the enzymatically hydrolyzed solution, the W5 solution is preferably added slowly, and then allowed to stand for 5 minutes after addition.

[0054] After obtaining a turbid mixture of enzymatic hydrolysate and protoplasts, this invention separates and purifies the protoplasts. The steps of protoplast separation and purification in this invention include: filtering the turbid mixture of enzymatic hydrolysate and protoplasts, collecting the protoplasts, and washing the protoplasts. As an optional embodiment of this invention, the filtration can be performed using a 70 μm cell sieve. This invention uses filtration through a 70 μm cell sieve to remove impurities such as incompletely hydrolyzed leaf tissue fragments, obtaining a filtrate containing protoplasts. Preferably, the filtration is performed into pre-cooled centrifuge tubes, and then the centrifuge tubes containing the filtrate are placed on ice. The method for separating protoplasts in this invention can be centrifugation; the centrifugation speed can be 600-1000 rpm, or 600, 700, 800, 900, or 1000 rpm; the centrifugation time can be 5 min. After centrifugation, this invention removes the supernatant, and the resulting precipitate is the protoplast. This invention preferably uses W5 solution to wash the protoplasts; the washing can be performed twice. When washing protoplasts with the W5 solution in this invention, the amount of W5 solution used is determined by the amount of enzymatic hydrolysis product. Approximately 5 mL less than the amount of enzymatic hydrolysis product is added, or an equal volume of W5 solution is added, to avoid adding too much W5 solution and submerging the precipitate. The precipitate is gently suspended during the washing process. After washing, this invention preferably resuspends the obtained protoplasts with the W5 solution to obtain a rose protoplast suspension. When resuspending, an equal volume of W5 solution as the enzymatic hydrolysis solution can be used.

[0055] The rose protoplasts prepared according to this invention can be used for genetic transformation research. Take an appropriate amount of prepared rose leaf protoplasts, such as 'Yueyuefen' protoplasts, and mix them with the constructed expression vector in a transformation solution (containing mannitol, calcium chloride, MES, etc.), and mix gently. Add an appropriate amount of 30% (w / v) PEG solution and let stand at room temperature for 20-30 minutes to promote the expression vector to enter the protoplasts. Incubate the transfected protoplasts at 25℃ in the dark for 12-16 h. Observe the expression of green fluorescent protein in the protoplasts using a laser confocal scanning microscope, providing an effective means for studying rose gene function and genetic improvement.

[0056] This invention provides a unique enzymatic hydrolysate formulation and a complete preparation method for rose leaf protoplasts. Compared with existing rose protoplast preparation technologies, the enzyme combination, component concentrations, and parameter control during the preparation process are significantly different. Existing technologies have shortcomings in terms of rose protoplast extraction efficiency, activity, and cost control. This invention effectively solves the above problems by innovatively combining cellulase R-10 and ionizing enzyme R-10, and precisely optimizing the concentration of each component in the hydrolysate and the hydrolysis conditions. The composite enzyme, hydrolysate, and method provided by this invention can efficiently prepare highly active protoplasts, and have wide application value in various plant biotechnology fields such as plant cell fusion, genetic transformation, and gene function research.

[0057] Furthermore, the method for preparing rose protoplasts provided by this invention also brings several significant benefits. Firstly, it demonstrates excellent performance in maintaining protoplast activity. This invention precisely controls key conditions such as enzymatic hydrolysis temperature and osmotic pressure, minimizing damage to the protoplasts during separation. For example, the precise setting of the enzymatic hydrolysis temperature between 25 and 28°C satisfies the activity requirements of cellulase R-10 and dissociative enzyme R-10 while avoiding thermal damage to the protoplasts from high temperatures, ensuring the integrity of cell membranes, organelles, and other structures, thereby maintaining high protoplast activity. FDA staining analysis shows that the 'Yueyuefen' leaf protoplasts prepared by this invention exhibit higher activity compared to traditional methods. This provides a reliable material basis for subsequent experiments such as cell fusion, genetic transformation, and gene function research, greatly increasing the probability of experimental success.

[0058] Secondly, the number of protoplasts obtained was significantly increased. By innovatively employing a specific ratio of cellulase R-10 and dissociation enzyme R-10 to enzymatically hydrolyze 'Yueyuefen' leaves, the cell wall was fully and efficiently broken down, prompting the release of more protoplasts from the cells. This increased number of protoplasts not only meets the needs of large-scale experiments but also provides ample sample resources for further exploration of the genetic characteristics and biotechnological applications of roses.

[0059] This invention provides a reagent for preparing rose protoplasts, comprising: the enzymatic hydrolysate described in the above technical solution, reagent B, and reagent C; reagent B includes a W5 solution; and reagent C includes an FDA working solution. In this invention, the W5 solution comprises 2 mmol / L 2-(N-morpholino)ethanesulfonic acid monohydrate, 154 mmol / L NaCl, 125 mmol / L CaCl2·2H2O, and 5 mmol / L KCl.

[0060] As an optional embodiment of the present invention, the reagent further includes a mannitol solution; the mannitol solution may be a 0.3~0.8 mol / L mannitol solution; the molar concentration of the mannitol solution may also be 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mol / L.

[0061] Rose leaves, rich in polyphenols and various secondary metabolites, are highly susceptible to severe browning during protoplast preparation, leading to protoplast inactivation and making preparation extremely difficult. This invention addresses the characteristics of rose materials by combining specific types and ratios of cellulase R-10 and analyte R-10, along with a specific concentration of PVP for browning prevention, mannitol soaking of leaf strips to pre-release secondary metabolites, and vacuum treatment. This multi-step synergistic approach solves the rose browning problem and successfully prepares highly viable protoplasts. For example, during the research process, this invention used an enzymatic hydrolysate without added PVP for protoplast preparation; however, regardless of adjustments to the content of other components in the hydrolysate, the yield of rose protoplasts remained extremely low.

[0062] This invention further reveals that the yield and viability of protoplasts prepared from cuttings are not only significantly higher than those from field-grown seedlings, but also reach a level comparable to tissue-cultured seedlings (with no significant difference). Given the significant advantages of cuttings, such as short acquisition cycle, low cost, and resistance to vitrification, they are more economically valuable and feasible in practical applications. This invention utilizes cuttings at a vigorous vegetative growth stage, with moderately structured leaf cell walls and low levels of cellulose and lignification, making them more easily degraded by enzymatic hydrolysate. Simultaneously, the leaves exhibit stable physiological states and active metabolism, with moderate accumulation of secondary metabolites such as polyphenols and tannins. This avoids the defects of tissue-cultured seedlings, such as easy vitrification and cell fragility, and also solves the problem of severe browning caused by environmental stress leading to leathery leaves, thick cell walls, and excessive accumulation of secondary metabolites in field-grown seedlings. Furthermore, the uniform leaf tissue structure and good cell synchronization of cuttings, combined with the enzymatic hydrolysis system and anti-browning treatment of this invention, can minimize browning and cell membrane damage, thereby significantly improving protoplast release and survival rate.

[0063] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0064] The cultivation method for the 'Monthly Powder' cuttings used in this embodiment is as follows: The leaves of 'Yueyuefen' were taken from 'Yueyuefen' cuttings that had been cultured for 60-90 days.

[0065] As an ancient rose variety, the survival rate of 'Yueyuefen' cuttings is significantly affected by time, substrate, cutting quality, and temperature and humidity management. The following are the cultivation methods for 'Yueyuefen' cuttings, which can steadily improve the survival rate.

[0066] 1. Cutting time and cutting preparation In spring (March-April, before budding) or autumn (September-October, after flowering), take cuttings from healthy, disease-free branches with plump axillary buds from the current year. Each cutting should retain two buds and two small leaves, and be morphologically cut flat at both ends.

[0067] The cuttings should be taken in an environment with a temperature of 20-25℃ and a humidity of 70-80%. These conditions are conducive to root development.

[0068] The cutting substrate used is pure vermiculite:perlite = 3:1 (by volume). This substrate is breathable and retains water, preventing root rot.

[0069] Thickened 230-size plastic two-tone flowerpots were used as the cutting containers. This container choice facilitates management and transplanting. The substrate was pre-watered until moist. Fifteen cuttings were inserted into each pot, and the surrounding substrate was gently compacted to ensure close contact between the cut and the medium. The cuttings were thoroughly watered immediately after insertion.

[0070] After propagation by cuttings, provide diffused light to prevent direct sunlight from scorching the leaves. Water every 3 days and cover the bottom of the pot with plastic wrap to prevent rapid water loss.

[0071] 20-35 days after cutting, new buds will sprout at the top of the cutting and white new roots will grow at the base (there will be resistance when you gently lift it), indicating that the rooting is successful. Transplant when the new roots grow to 3-5cm and 2-3 new leaves unfold, to avoid damaging the roots too early.

[0072] 6. The transplanting substrate should consist of vermiculite, peat moss, and perlite in a 2:3:1 volume ratio. After transplanting, place the plants in a location with diffused light. Do not fertilize for the first 7 days. Keep the substrate moist, watering every 5-8 days, following the principle of watering only when the soil is dry to the touch. After 60-90 days of cultivation, cuttings will be obtained.

[0073] The 'Blue Rhapsody' rose greenhouse cuttings were taken from 'Blue Rhapsody' cuttings that had been cultivated for 60-90 days.

[0074] The preparation method for 'Blue Rhapsody' cuttings is the same as that for 'Monthly Powder' cuttings.

[0075] The culture method for tissue culture seedlings in this embodiment is as follows: 'Monthly Powder' stem segments were inoculated as explants. These segments should be healthy, one-year-old branches in a semi-lignified growth stage, with well-developed axillary buds, free from disease and pests. After rinsing with running water for 1 hour, the segments were disinfected in a clean bench with 75% alcohol for 45 seconds and 84 disinfectant for 7 minutes. Rotten parts were removed, and the segments were rinsed with sterile water. They were then inoculated onto a culture medium (1 / 2 MS + 6-BA 3.0 mg / L + NAA 0.05 mg / L + 3% sucrose + 0.62% agar powder, pH 5.8). Axillary bud germination was induced under suitable light and temperature conditions. After germination, the segments were subcultured every 25-30 days to ensure sufficient experimental material. In subsequent experiments, when preparing protoplasts from tissue-cultured seedling leaves, leaves from seedlings with germinating axillary buds and cultured for 50-60 days were used for protoplast preparation.

[0076] The chemical reagents involved in the following technical solutions of this invention are of chemical purity and are commercially available.

[0077] Example 1 A composite enzyme for preparing protoplasts of "Yueyuefen" (a type of powder) has the following working concentrations based on the volume of the enzymatic hydrolysis working system: cellulase R-10 3 g / 100 mL and macerozyme R-10 2 g / 100 mL. Both cellulase R-10 and macerozyme R-10 were purchased from Beijing Beicheng Hongye Co., Ltd., specifically: cellulase R-10 is Yakult cellulase R-10, and macerozyme R-10 is Yakult macerozyme R-10. The enzyme activity of cellulase R-10 is 10000 U / g, and the enzyme activity of macerozyme R-10 is 3000 U / g.

[0078] Example 2 An enzymatic hydrolysate for preparing 'Yueyuefen' protoplasts comprises the following components at the following concentrations: cellulase R-10 3 g / 100 mL, analyte R-10 2 g / 100 mL, mannitol 0.4 mol / L, KCl 20 mmol / L, 2-(N-morpholino)ethanesulfonic acid (MES) 20 mmol / L, CaCl2 10 mmol / L, 0.01 g / 100 mL PVP, and 0.1 g / 100 mL BSA.

[0079] The method for preparing the enzymatic hydrolysate of 'Monthly Powder' protoplasts is as follows: 10mL composition system (a) Add 0.3g cellulase R-10, 0.2g analyte R-10, 5mL 0.8mol / L D-mannitol, 1mL 0.2mol / L KCl, and 1mL 0.2mol / L MES (pH=5.7, i.e., the pH value of 0.2mol / L MES is 5.7) sequentially to a 50mL round-bottom centrifuge tube. Use distilled water for all solutions. After thoroughly shaking and mixing the above solutions, incubate them in a water bath at 55℃ for 10min, and then cool to room temperature. The water bath allows the enzymes to fully dissolve and mix evenly at a suitable temperature without destroying the enzyme activity, thus improving the efficiency of subsequent enzymatic hydrolysis.

[0080] (b) Add 0.1 mL of 1 mol / L CaCl2·2H2O, 0.1 mL of 10 g / 100 mL BSA, 0.1 mL of 1 g / 100 mL PVP, and 2.7 mL of ddH2O to the solution after it has been cooled to room temperature, and mix thoroughly to complete the preparation.

[0081] The enzyme used in the enzymatic hydrolysate in this embodiment is the composite enzyme from Example 1.

[0082] Example 3 A reagent for preparing leaf protoplasts of 'Monthly Powder', comprising reagent A, reagent B and reagent C; Reagent A is the enzymatic hydrolysate from Example 2; Reagent B is solution W5, which is composed of the following components in the following amounts: 2-(N-morpholino)ethanesulfonic acid monohydrate 2 mmol / L, NaCl 154 mmol / L, CaCl2·2H2O 125 mmol / L, KCl 5 mmol / L; Reagent C is the FDA working solution, a homogeneous solution prepared by mixing FDA stock solution and 0.8 mol / L D-mannitol solution at a volume ratio of 1:100; wherein the FDA stock solution is a 2% (w / v) FDA acetone solution.

[0083] Example 4 A method for preparing leaf protoplasts of 'Monthly Powder' includes the following steps: 1) Select 'Monthly Pink' cuttings that are 2-3 months old and growing in the same manner. Figure 1 Using a sharp, single-edged blade, remove the main vein and leaf margin tissue from the leaf. Select leaves with rich color, a radius of approximately 1 cm, and no wrinkles. Divide the leaf tissue along the main vein, making 1 mm wide cuts. Immerse the cut leaf tissue in 10 mL of 0.4 mol / L mannitol solution for 0.5 h to obtain the mannitol-treated tissue material. Figure 2 China A; 2) The mannitol-treated tissue material was mixed with the enzymatic hydrolysate from Example 2 at a mass-to-volume ratio of 1 g:10 mL. The mixture was then subjected to vacuum treatment at 25–28°C in the dark, with the vacuum pressure reduced to 0.05 MPa for 1 hour. After vacuum treatment, the tissue was subjected to enzymatic hydrolysis at 25–28°C and a shaking speed of 40–50 rpm for 16 hours. During the enzymatic hydrolysis process, the tissue needed to be completely immersed in the enzymatic hydrolysate.

[0084] 3) After enzymatic hydrolysis, add an equal volume of W5 solution to the hydrolyzed solution to terminate the reaction, resulting in a turbid mixture of enzymatic hydrolysate and protoplasts. Figure 2 For cell culture B, filter the filtrate through a 70 μm cell sieve into a pre-chilled 50 mL round-bottom centrifuge tube and place the centrifuge tube containing the filtrate on ice. Then, place the centrifuge tube containing the filtrate in a centrifuge at 4 °C and centrifuge at 1000 rpm for 5 min.

[0085] 4) Use a Pasteur pipette to remove the supernatant, and the precipitate should be removed as shown in the image. Figure 2 C; Add 10 mL of reagent B from Example 3 to the precipitate to resuspend the protoplasts. After resuspending, continue centrifugation. Repeat this step twice to wash the protoplasts.

[0086] 5) Remove the supernatant with a Pasteur pipette and gently resuspend the protoplasts in 10 mL of reagent B. Place the centrifuge tube containing the resuspended solution on ice for 30 min to allow the protoplasts to precipitate to the bottom of the 50 mL centrifuge tube, thus obtaining the 'Yueyuefen' protoplasts.

[0087] Comparative Example 1 A method for preparing leaf protoplasts of 'Yueyuefen' is provided, with the same steps as in Example 4, except that the Yakult cellulase R-10 and Yakult dissociative enzyme R-10 in the enzymatic hydrolysate of Example 2 are replaced with Coolaber cellulase R-10 and Coolaber dissociative enzyme R-10, with Coolaber cellulase R-10 having an activity of approximately ≥10 U / mg and Coolaber dissociative enzyme R-10 having an activity of >3000 U / g.

[0088] Comparative Example 2 A method for preparing leaf protoplasts of 'Yueyuefen' is the same as in Example 4, except that the 'Yueyuefen' leaves are derived from field-grown seedlings, and the enzymatic hydrolysate contains 3 g / 100 mL of cellulase R-10 and 1.5 g / 100 mL of dissociative enzyme R-10.

[0089] The seedlings planted in the field are 50-60cm tall, have been planted for more than 3 years, and require relatively extensive management, with infrequent watering. In winter, they are pruned and covered with soil for protection against the cold.

[0090] Example 5 A method for preparing protoplasts of the 'Blue Rhapsody' rose, with the same steps as in Example 4, except that leaves from 'Blue Rhapsody' rose greenhouse cuttings are used instead of leaves from 'Monthly Pink' rose greenhouse cuttings.

[0091] Example 6 20 μL of protoplast suspension (resuspended after treatment with the reaction solution in step 5) of Examples 4-5 and Comparative Examples 1-2) and 20 μL of reagent C from Example 3 were respectively dropped into the groove of a glass slide with grooves, gently mixed, covered with a coverslip, and observed after 5-10 minutes. The effective observation time is within 30 minutes. Select a field of view where the protoplasts are completely and evenly distributed, take a picture, and observe that the protoplasts that emit a pale green fluorescence under a fluorescence microscope are live protoplasts, while those without fluorescence are dead cells.

[0092] Application Example 1 Observing protoplasts under a microscope The protoplasts prepared in Examples 4-5 and Comparative Examples 1-2 were observed under a microscope to examine their yield and viability. The results are as follows: Figures 3-8 ,in Figure 3 and Figure 8 These are photographs of the protoplast suspension prepared in Example 4 under an optical microscope, showing different fields of view. Figure 4 This is a photograph of the protoplast suspension from Example 6 under an inverted fluorescence microscope. Figure 5 This is a photograph of the protoplast suspension prepared in Comparative Example 1 under an optical microscope. Figure 6 A photograph of the protoplast suspension prepared for Comparative Example 2 under an optical microscope. Figure 7 The image shows a photograph of the protoplast suspension prepared in Example 5 under an optical microscope. The number and activity of protoplasts were counted using a hemocytometer, and the results are shown in Table 1.

[0093] Table 1. Statistics on the number of protoplasts prepared in Examples 4, 5 and Comparative Examples 1-2 (mean ± standard deviation)

[0094] Note: Protoplast count / g means the number of protoplasts obtained per gram of leaf.

[0095] Depend on Figure 3 It can be seen that the morphology of the leaf protoplasts of 'Monthly Powder' prepared by this invention is complete, and it is composed of... Figure 4 It can be seen that protoplasts under an inverted fluorescence microscope emit green fluorescence, indicating high activity; Figure 5 It can be seen that treatment with Coolaber cellulase R-10 and dissociation enzyme R-10 yields intact protoplasts, but the protoplast extraction efficiency is low; Figure 6 It can be seen that the leaves of 'Yueyue Pink' are from field-grown seedlings, and a relatively small amount of protoplasts are obtained. From Figure 7 it can be seen that the number of protoplasts of the rose 'Blue Rhapsody' is significantly lower than that of 'Yueyue Pink', but it can also reach a relatively high number. As shown in Table 1, the number of protoplasts obtained in Example 4 of the present invention can reach 46×10 5 per gram, which can meet the needs of subsequent scientific research experiments; while the number of protoplasts obtained in Comparative Example 1 and Comparative Example 2 has a difference of 2 to 3 orders of magnitude from the number of protoplasts of the present invention, which is significantly worse than the present invention. The number of protoplasts of the rose 'Blue Rhapsody' is significantly lower than that of 'Yueyue Pink'.

[0096] As shown in Table 1, the number of protoplasts obtained in Example 4 of the present invention can reach 46×10 5 per gram, which can meet the needs of subsequent scientific research experiments; while the number of protoplasts obtained in Comparative Example 1 and Comparative Example 2 has a difference of 2 to 3 orders of magnitude from the number of protoplasts of the present invention, which is significantly worse than the present invention. The number of protoplasts of the rose 'Blue Rhapsody' is significantly lower than that of 'Yueyue Pink', but it can also reach a relatively high level.

[0097] From the above results, it can be seen that the enzyme solution and preparation method provided by the present invention not only have good protoplast separation effect, but also have good activity and a large number of protoplasts prepared.

[0098] Examples 7 - 15 A method for preparing protoplasts from the leaves of 'Yueyue Pink', the steps are the same as in Example 4, and the only difference is that the concentrations of cellulase R-10 and macerozyme R-10 in the enzyme solution are different. The concentrations of cellulase R-10 and macerozyme R-10 are shown in Table 2. The extraction efficiency and activity of protoplasts are shown in Table 2.

[0099] Table 2 Effects of different enzyme combinations in Examples 7 - 15 on the extraction of protoplasts from the leaves of greenhouse cuttings of 'Yueyue Pink'

[0100] Note: The yield is the number of protoplasts; the activity is the protoplast viability, the same below.

[0101] Comparative Examples 3 - 29 A method for preparing protoplasts from the leaves of 'Yueyue Pink', the steps are the same as in Example 4, and the only difference is that the enzyme composition in the enzyme solution is: Coolaber cellulase R-10, pectinase Pectolyase Y-23 and Coolaber macerozyme R-10. The specific concentrations are shown in Table 3. The extraction efficiency and activity of protoplasts are shown in Table 3.

[0102] Table 3 Effects of Different Concentrations of Enzyme Combinations in Comparative Examples 3 - 29 on the Extraction of Protoplasts from Leaves of 'Yueyuefen' Greenhouse Cuttings

[0103] Examples 16 - 21 A method for preparing protoplasts from 'Yueyuefen' leaves, the steps are the same as in Example 4, the only difference is: in the enzyme solution, cellulose enzyme R - 10 with a concentration of 3.0 g / 100 mL and macerozyme R - 10 with a concentration of 2.0 g / 100 mL are used, and the concentration of mannitol in the enzyme solution is shown in Table 4. The extraction efficiency and activity of protoplasts are shown in Table 4.

[0104] Table 4 Results of Protoplast Extraction from Leaves of 'Yueyuefen' Greenhouse Cuttings in Examples 16 - 21

[0105] Examples 22 - 31 A method for preparing protoplasts from 'Yueyuefen' leaves, the steps are the same as in Example 4, the only difference is: in the enzyme solution, cellulose enzyme R - 10 with a concentration of 3.0 g / 100 mL and macerozyme R - 10 with a concentration of 2.0 g / 100 mL are used, and the enzyme digestion time is shown in Table 5. The extraction efficiency and activity of protoplasts are shown in Table 5.

[0106] Table 5 Results of Protoplast Extraction from Leaves of 'Yueyuefen' Greenhouse Cuttings in Examples 22 - 31

[0107] Comparative Example 30 A method for preparing protoplasts from 'Yueyuefen' leaves, the steps are the same as in Example 4, the only difference is: the enzyme solution does not contain PVP.

[0108] After treatment with the enzyme solution without PVP, the leaves showed severe browning during the enzyme digestion process, the activity of protoplasts decreased significantly, and the obtained protoplasts were completely inactivated and had no practical application value. Therefore, their yields and activities were not statistically analyzed. Browning of leaves during the enzyme digestion process is a key inhibitory factor for protoplast extraction. Its essence is that after the leaf cells are damaged, polyphenolic substances are oxidized to quinone substances under the action of polyphenol oxidase, which then causes multiple damages to the protoplasts and directly affects the yield, vitality and success rate of subsequent experiments.

[0109] Application Example 2 Using the method of Example 4, protoplasts were prepared from the leaves of 'Yueyuefen' tissue - cultured seedlings, cuttings and field - grown seedlings as raw materials respectively. The difference from Example 4 is that the dosage of enzymes in the enzyme solution is shown in Table 6.

[0110] The methods for obtaining cuttings, field - grown seedlings and tissue - cultured seedlings are the same as described above.

[0111] A comparison of the effects of tissue culture seedlings, cuttings, and ground-grown seedlings on protoplast yield and activity is shown in Table 6. Figure 9 As shown.

[0112] Table 6. Comparison of the effects of tissue culture seedlings, cuttings, and ground-grown seedlings on protoplast yield and activity of 'Yueyuefen'.

[0113] Note: The same letter indicates that the difference is not significant according to Duncan's new compound grade test (P<0.05).

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a compound enzyme in the preparation of rose protoplasts, wherein the working concentration of the compound enzyme is: cellulase R-10 2~4 g / 100mL and dissociation enzyme R-10 1~2 g / 100mL, based on the volume of the enzymatic hydrolysis working system.

2. The application according to claim 1, characterized in that, The roses include 'Monthly Pink' roses and / or 'Blue Rhapsody' roses.

3. The application according to claim 1, characterized in that, The cellulase R-10 includes Yakult cellulase R-10; the dissociative enzyme R-10 includes Yakult dissociative enzyme R-10.

4. An enzymatic hydrolysate for preparing rose protoplasts, characterized in that, Based on the total volume of the enzyme hydrolysate, it includes: 2~4 g / 100mL cellulase R-10, 1~2 g / 100mL analyte R-10, mannitol 0.3~0.8 mol / L, KCl 5~20mmol / L, MES dosage to adjust the pH of the enzyme hydrolysate to 5.5~6.0, CaCl2 5~20mmol / L, 0.01~0.02 g / 100mL PVP and 0.1~0.5 g / 100mL BSA.

5. The use of the enzymatic hydrolysate of claim 4 in any one or more of the following: preparing rose protoplasts, increasing the yield of rose protoplasts, and increasing the activity of rose protoplasts; wherein the rose includes 'Monthly Pink' rose and / or 'Blue Rhapsody' rose.

6. A method for preparing rose protoplasts, characterized in that, include: Rose protoplasts were obtained by enzymatically hydrolyzing rose leaves using the enzymatic hydrolysate described in claim 4.

7. The method according to claim 6, characterized in that, The rose leaves include the leaves of rose cuttings and / or the leaves of rose tissue culture seedlings.

8. The method according to claim 6, characterized in that, The enzymatic hydrolysis temperature is 25~28℃; the enzymatic hydrolysis time is 14~16h.

9. The method according to any one of claims 6 to 8, characterized in that, Before enzymatic hydrolysis of rose leaves, the rose leaves undergo pretreatment; the pretreatment includes the following steps: Rose leaves were cut into thin strips and soaked in mannitol solution to obtain tissue material treated with mannitol solution. Tissue materials treated with mannitol solution were mixed with enzymatic hydrolysate and then subjected to vacuum treatment.

10. A reagent for preparing rose protoplasts, characterized in that, include: The enzymatic hydrolysate, reagent B, and reagent C as described in claim 4; Reagent B includes W5 solution; The reagent C includes FDA working solution.

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