A tissue culture method of short calyx instrument flower

Abstract

This invention discloses a tissue culture method for *Aureobasidium brevicornum*, belonging to the field of plant culture technology, comprising the following steps: (1) sterilizing *Aureobasidium brevicornum* seeds; (2) soaking *Aureobasidium brevicornum* seeds in mercuric chloride solution and then inoculating them in MS medium to induce germination into sterile seedlings; (3) inoculating the budded stem segments of the sterile seedlings into germination medium for germination and proliferation; (4) inoculating the germinating and proliferating leaves into induction medium for callus induction culture; (5) using proliferation medium to proliferate the induced callus; (6) using differentiation medium to differentiate the proliferated callus to obtain rootless seedlings; (7) using root induction medium to induce roots in the rootless seedlings. This invention explores the tissue culture technology of *Aureobasidium brevicornum* to fill the gaps and deficiencies in the seedling cultivation technology of *Aureobasidium brevicornum*, bringing a new approach to the propagation of *Aureobasidium brevicornum*.

Description

Technical Field

[0001] This invention relates to the field of plant culture technology, specifically to a tissue culture method for *Aureobasidium brevicornum*. Background Technology

[0002] *Lysidice brevicalyx*, an evergreen tree belonging to the genus *Lysidice* in the family Caesalpinioideae, is native to southern and southwestern my country, mainly distributed in Guangdong, Guangxi, Guizhou, and Yunnan provinces. It grows in sparse or dense forests at altitudes of 500–1000 m, commonly found in valleys and along streams. It prefers sunny, warm, and humid climates, has relatively strong cold resistance, and can tolerate light frost. It is not particular about soil pH. This species has abundant and vibrant flowers, hard wood, and its roots, stems, and leaves can be used medicinally. It is a native tree species with great development potential in South China.

[0003] Current research on *Aureobasidium brevicornum* mainly focuses on morphological studies, medicinal applications, and biological community diversity, with relatively few reports on propagation and seedling cultivation. Production primarily relies on seed propagation, resulting in slow propagation speed, low seedling efficiency, weak selection of superior varieties, and a lack of systematic development and utilization. Consequently, the market supply of seedlings is limited, and their quality varies greatly, failing to fully realize the value of this species.

[0004] Short-calyxed flowers primarily reproduce by seeds, but their seeds are large, leathery, and covered with a waxy outer layer that makes them impermeable to water, resulting in difficulties in water absorption and low germination rates. Furthermore, seed propagation is slow and inefficient, and the genetic stability of desirable traits in offspring cannot be guaranteed.

[0005] Propagation using tissue culture can provide a large number of high-quality seedlings in a short period of time, providing scientific guidance for its industrialized seedling production and large-scale development and utilization, and providing technical support for scientific research such as genetic improvement and accelerating the breeding process. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a tissue culture method for *Aureocarpus brevicornu*. This invention explores tissue culture technology for *Aureocarpus brevicornu* in order to fill the gaps and deficiencies in the seedling technology of *Aureocarpus brevicornu*, bring new ways to the propagation of *Aureocarpus brevicornu*, and provide technical support and reference for the collection, preservation, large-scale production and further genetic transformation research of its germplasm resources.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for tissue culture of *Aureobasidium brevicornu*, comprising the following steps:

[0009] (1) Disinfect the seeds of the short-calyxed flower;

[0010] (2) The seeds of the short-calyxed flower were soaked in mercuric chloride solution and then inoculated into MS medium to induce the germination of sterile seedlings;

[0011] (3) Inoculate the budded stem segments of sterile seedlings into germination medium for germination and proliferation;

[0012] (4) The germinating and proliferating leaves were inoculated into an induction medium for callus induction culture;

[0013] (5) The induced callus tissue was cultured in a proliferation medium;

[0014] (6) The callus tissue after proliferation culture was cultured in differentiation medium to obtain rootless seedlings;

[0015] (7) Roots were induced in the rootless seedlings using root induction medium.

[0016] In a preferred embodiment of the present invention, the seeds of the short-calyxed flower are plump and free from pests and diseases.

[0017] As a preferred embodiment of the present invention, the disinfection of the seeds of *Symplocos brevicornu* is specifically carried out as follows: cut the seeds along the edge, wash and soak them in detergent for 8-12 minutes after cutting, then rinse them with water for 20-40 minutes. After washing, soak the seeds in 60-80% ethanol solution for 0.5-2 minutes, rinse them with water 2-4 times, then soak them in 0.05-0.2% mercuric chloride solution for 8-12 minutes, rinse them with water 4-6 times, then soak them in water for 20-30 hours. After soaking, soak them in 60-80% ethanol solution for 20-40 seconds, rinse them with water 2-4 times, then soak them in 0.05-0.2% mercuric chloride solution for 5-15 minutes, and rinse them with water 4-6 times.

[0018] The germination induction conditions are: temperature (25±2)℃, light intensity 1500~2000Lx, and light intensity 12h / day. As a preferred embodiment of the present invention, the germination medium comprises: MS medium, 0.5~1.5mg / L 6-BA, 0~0.3mg / L NAA, and 0~0.4mg / L IBA.

[0019] The conditions for germination and proliferation are: temperature (25±2)℃, light intensity 1500~2000Lx, and light exposure 12h / day.

[0020] As a preferred embodiment of the present invention, the induction medium comprises: MS medium, 0.5-2 mg / L 6-BA, 0.01-0.1 mg / L IBA, and 0.05-0.5 mg / L 2,4-D.

[0021] The conditions for induction culture were: temperature (25±2)℃, light intensity 1500~2000Lx, and light exposure 12h / day.

[0022] As a preferred embodiment of the present invention, the proliferation medium comprises: MS medium, 1-3 mg / L 6-BA, 0.05-0.1 mg / L IBA, 0.05-0.1 mg / L 2,4-D, 0-0.1 g / L citric acid, 0-1 g / L PVP, and 0-1 g / L activated carbon.

[0023] The conditions for proliferation culture were: temperature (25±2)℃, light intensity 1500~2000Lx, and light exposure 12h / day.

[0024] As a preferred embodiment of the present invention, the proliferation medium comprises: MS medium, 2 mg / L 6-BA, 0.1 mg / L IBA, 0.1 mg / L 2,4-D, and 0.1 g / L citric acid.

[0025] As a preferred embodiment of the present invention, the differentiation medium comprises: MS medium, 0.5-2 mg / L 6-BA, 0-0.5 mg / L TDZ, 0.1-0.3 mg / L IBA, and 0.2 g / L acid-hydrolyzed complexin.

[0026] The conditions for differentiation culture were: temperature (25±2)℃, light intensity 1500~2000Lx, and light exposure 12h / day.

[0027] As a preferred embodiment of the present invention, the differentiation medium comprises: MS medium, 2 mg / L 6-BA, 0.5 mg / L TDZ, 0.2 mg / L IBA, and 0.2 g / L acid-hydrolyzed complexin.

[0028] As a preferred embodiment of the present invention, the root induction medium comprises: 1 / 2 MS medium, 0.1-0.5 mg / L NAA, and 0-0.5 mg / L IBA.

[0029] The conditions for root induction are: temperature (25±2)℃, light intensity 1500~2000Lx, and light exposure 12h / day.

[0030] In this application, 6-BA refers to 6-benzylaminopurine, TDZ refers to cytokinin thiamethoxam, NAA refers to naphthaleneacetic acid, IBA refers to indoleacetic acid, 2,4-D refers to dichlorophenoxyacetic acid, and PVP refers to polyvinylpyrrolidone. These substances are all conventional commercially available products.

[0031] In this invention, the term "culture medium" refers to the addition of a substance of a corresponding concentration to the culture medium.

[0032] For example, differentiation media include: MS medium, 0.5–2 mg / L 6-BA, 0–0.5 mg / L TDZ, 0.1–0.3 mg / L IBA, and 0.1–0.3 g / L acid-hydrolyzed complexin. In other words, differentiation medium refers to MS medium supplemented with 0.5–2 mg / L 6-BA, 0–0.5 mg / L TDZ, 0.1–0.3 mg / L IBA, and 0.1–0.3 g / L acid-hydrolyzed complexin.

[0033] Root induction medium refers to 1 / 2 MS medium supplemented with 0.1–0.5 mg / L NAA, 0–0.5 mg / L IBA, and 10–30 g / L sucrose.

[0034] The beneficial effects of this invention are as follows: (1) This invention explores the tissue culture technology of *Aureocarpus brevicornus* to fill the gaps and deficiencies in the seedling technology of *Aureocarpus brevicornus*, bringing new ways to the propagation of *Aureocarpus brevicornus*, and also providing technical support and reference for the collection, preservation, large-scale production and further genetic transformation research of its germplasm resources; (2) This invention uses alcohol and mercuric chloride in combination to disinfect the seeds, which can effectively sterilize them, reduce the browning rate, and promote seed germination to a certain extent. By using the budded stem segments of sterile seedlings of *Aureocarpus brevicornus* seeds, bud proliferation is carried out through the bud-to-bud propagation method. Germination of *Aureocarpus brevicornus* seeds is promoted by removing the cotyledons, which can effectively improve the seed germination rate and germination speed. Plant regeneration is achieved through callus redifferentiation, which can provide technical support for genetic transformation research. Rooting is induced by hormone ratio. By exploring the appropriate explant, culture medium type and hormone ratio for the induction and regeneration process of callus in *Aureocarpus brevicornus*, the foundation for the establishment of its genetic transformation system is laid, which has broad application prospects. Attached Figure Description

[0035] Figure 1 This diagram illustrates the process of lateral bud germination and proliferation.

[0036] Figure 2 This image shows the callus formation after 30 days of induction on different culture media from different explants of *Echeveria brevicornu*.

[0037] Figure 3 Figures showing leaf callus tissue induced by different hormone ratios for 30 days.

[0038] Figure 4 Diagram of adventitious bud differentiation of callus tissue.

[0039] Figure 5 Images showing the rooting effect after 30 days under different hormone ratios. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0041] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0042] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0043] In this application, there are no particular restrictions on the specific dispersion and mixing methods.

[0044] Unless otherwise specified, all components, raw materials, or instruments used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0045] Example

[0046] 1. Materials and Methods

[0047] 1.1 Experimental Materials

[0048] 1.1.1 Source of experimental materials

[0049] A number of mature seeds of *Echeveria pulvinata* were collected from the campus of Guangxi University. After removing the pods and extracting the seeds, they were stored in a cool, shady place for later use. For the experiment, plump, disease-free, and healthy seeds were selected to induce sterile seedlings. After 30 days, leaves, stem segments, and hypocotyls of the sterile seedlings were harvested as initial materials for the culture. Cotyledons were taken from seeds after they absorbed water, swelled, and were sterilized. All materials for subsequent experiments were taken from callus tissue and regenerated seedlings produced in the sterile system.

[0050] 1.1.2 Overview of the source of experimental materials

[0051] Guangxi University is located at 108°16′~108°18′E, 22°50′~23°51′N, in Nanning City, Guangxi Province. It belongs to the humid subtropical region with an average annual temperature of 21.6℃, an average annual precipitation of 1304.2mm, and an average annual relative humidity of 79%. It has abundant rainfall, little frost and no snow, and a mild and suitable climate.

[0052] 1.2 Cultivation Conditions

[0053] Unless otherwise specified, the basic culture medium (MS medium) is supplemented with 7 g / L agar, 30 g / L sucrose, pH 5.8-6.0. The prepared culture media are all autoclaved at 121℃ for 20 min, cultured at (25±2)℃, with a light intensity of 1500-2000 Lx for 12 h / d.

[0054] 1.3 Experimental Methods

[0055] 1.3.1 Establishment of a sterile system

[0056] 1.3.1.1 Seed disinfection and sterilization treatment

[0057] Cut the collected seeds along the edges, then wash and soak them in detergent for 10 minutes, followed by rinsing with tap water for 30 minutes. After washing, transfer them to a laminar flow hood for pre-sterilization. First, soak the seeds in 75% alcohol for 1 minute on the laminar flow hood, then rinse three times with sterile water, followed by soaking in 0.1% HgCl2 for 10 minutes, then rinsing five times with sterile water. Finally, soak them in a culture bottle containing sterile water for 24 hours. The next day, after removing the seed coat, soak the seeds in 75% alcohol for 30 seconds on the laminar flow hood, rinse three times with sterile water, disinfect with 0.1% HgCl2 for 5, 10, and 15 minutes, rinse five times with sterile water, retaining 1 / 4 of the cotyledon, and finally inoculate them onto MS blank medium. Each treatment was inoculated with 30 explants, replicated three times. After 30 days, the average germination time, germination rate (based on 1 cm shoot elongation), contamination rate, and browning rate were recorded.

[0058] 1.3.1.1 Seed germination promotion treatment

[0059] Pre-sterilization treatment was the same as in 2.3.1.1. After seed swelling and removal of the seed coat, the seeds were soaked in 75% alcohol for 30 seconds in a clean bench, rinsed three times with sterile water, sterilized with 0.1% HgCl2 for 10 minutes, and rinsed five times with sterile water. Seeds with different cotyledon sizes (intact, 1 / 2, 1 / 4) were inoculated onto MS blank medium. Each treatment was inoculated with 30 explants, replicated three times. After 30 days, the average germination time, germination rate, contamination rate, and browning injury rate were recorded.

[0060] 1.3.2 Initiating Culture

[0061] 1.3.2.1 Investigating the effects of hormone combinations on the induction of lateral buds in stem segments with buds.

[0062] Sterile seedling stem segments with buds were inoculated into MS medium supplemented with (0.5, 1.0, 1.5) mg / L 6-BA, (0, 0.1, 0.3) mg / L NAA, and (0, 0.2, 0.4) mg / L IBA (Table 1). An L9(33) orthogonal design was used for the combined experiment (Table 2). Each treatment was inoculated with 30 stem segments with buds, and the results were repeated 3 times. After 30 days, the induction rate, bud proliferation coefficient, budding index, and effective bud rate were counted. The growth status of lateral buds was recorded. The optimal hormone ratio for lateral bud induction was screened based on the comprehensive experimental results.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] 1.3.2.2 Investigating the effects of explant and culture medium type on callus induction

[0068] 1.3.2.3 Select sterile seedling leaves, stem segments without buds, hypocotyls, and cotyledons after seeds have absorbed water, swelled, and been sterilized. Cut the leaves and cotyledons into 1cm pieces. 2 The stem segments and hypocotyls were cut into pieces about 1 cm long and were of approximately the same size. Each type of explant was inoculated on MS, WPM, or B5 medium supplemented with 2.0 mg / L 6-BA, 0.05 mg / L IBA, and 0.05 mg / L 2,4-D. 30 explants were inoculated for each treatment, and the treatment was repeated 3 times. The induction time was recorded every 7 days. After 30 days, the number of induced and browned calluses was counted, and the color and texture of the calluses were recorded. After comprehensive analysis, the optimal explants and medium for inducing callus tissue were determined.

[0069] 1.3.2.3 Investigating the effects of exogenous hormone combinations on leaf-induced callus tissue

[0070] Cut the sterile seedling leaves into 1cm pieces. 2 Plants of approximately the same size, with the abaxial surface facing upwards, were inoculated on MS medium supplemented with (0.5, 1.0, 2.0) mg / L 6-BA, (0.01, 0.05, 0.1) mg / L IBA, and (0.05, 0.1, 0.5) mg / L 2,4-D (Table 3). An L9(33) orthogonal design was used for combined experiments (Table 4). Each treatment was inoculated with 30 explants, and the results were repeated 3 times. After 30 days, the number of induced calluses and the number of brownings were counted, and the color and texture of the calluses were recorded. Based on the combined experimental results, the optimal hormone ratio for inducing leaf calluses was selected.

[0071] Table 3

[0072]

[0073] Table 4

[0074]

[0075] 1.3.3 Subculture

[0076] New shoots were induced by initiating culture and then propagated using the optimal lateral bud induction medium.

[0077] 1.3.3.1 Investigating the effects of exogenous hormones on callus proliferation

[0078] The induced callus tissue was first transferred to the optimal induction medium for the first subculture. Dense, green callus tissue was then selected and transferred to MS medium containing 2.0 mg / L 6-BA, (0.05, 0.1) mg / L IBA, and (0.05, 0.1) mg / L 2,4-D for proliferation culture. Different hormones at different concentrations were combined in pairs, and 0.5 g / L PVP (polyvinylpyrrolidone) was added to the medium. Each treatment was inoculated with 30 explants, and the results were repeated three times. After 30 days, the callus proliferation rate was counted, and the callus growth status was recorded. The optimal proliferation medium was selected based on comprehensive analysis.

[0079] 1.3.3.2 Effects of the type and concentration of anti-browning agents on callus proliferation

[0080] Dense, green callus tissue was transferred to optimal proliferation media containing (0.5, 1.0) g / L AC (activated charcoal), (0.05, 0.1) g / L CA (citric acid), and (0.5, 1.0) g / L PVP. Each treatment was inoculated with 30 explants, and the treatment was repeated three times. After 30 days, the callus proliferation rate was recorded, and the callus growth status was analyzed to identify the types and concentrations of substances that could effectively inhibit callus browning.

[0081] 1.3.4 Callus Differentiation and Culture

[0082] The induced embryogenic callus was cut into small pieces of about 1 cm² and inoculated into MS medium supplemented with (0.5, 1.0, 2.0) mg / L 6-BA, (0, 0.2, 0.5) mg / L TDZ, (0.1, 0.2, 0.3) mg / L IBA, and 0.2 g / L acid-hydrolyzed casein for adventitious shoot induction (Table 5). An L9(33) orthogonal design was used for combination experiments (Table 6). Each treatment was inoculated with 30 explants, and the results were repeated 3 times. After 40 days, the differentiation of adventitious shoots in the callus was observed and the differentiation rate was calculated. The best combination of exogenous hormones that could induce adventitious shoots was selected through comprehensive analysis.

[0083] Table 5

[0084]

[0085] Table 6

[0086]

[0087] 1.3.5 Rooting Culture

[0088] Healthy, rootless seedlings with a height ≥3cm were selected for root induction. They were inoculated into 1 / 2 MS medium supplemented with (0.1, 0.2, 0.5) mg / L NAA, (0, 0.1, 0.5) mg / L IBA, and 20 g / L sucrose (Table 7). An L9(32) orthogonal design combination experiment was used (Table 8). Each treatment was inoculated with 30 seedlings, and the results were repeated 3 times. After 30 days, the rooting rate, average number of roots, and average root length of the test-tube seedlings in each experimental group were counted to screen out the optimal combination of exogenous hormones for root induction.

[0089] Table 7

[0090]

[0091] Table 8

[0092]

[0093]

[0094] 1.3.6 Observation of tissue sections during callus differentiation process

[0095] 1.3.6.1 Sampling and Fixation

[0096] (1) Cytological observation of callus formation induced by different explants.

[0097] (2) Cytological observation of different callus types to determine embryogenic and non-embryonic callus.

[0098] (3) Cytological observation of the process of callus differentiation into adventitious shoots.

[0099] (4) Cytological observation of the rooting process

[0100] All induction and culture processes were conducted using the optimal culture medium selected at each stage. Ten healthy explants with distinct and representative characteristics were randomly selected from each stage of each treatment. Sampling for differentiation and rooting was performed as follows: the first sampling was conducted before inoculation, followed by sampling every 7 days after inoculation, and a final sampling was conducted at the end of the culture. Callus tissue sampling was conducted after 30 days of proliferation culture. All samples were fixed with FAA prepared in 70% ethanol for 24 hours and stored at 4°C until use.

[0101] 1.3.6.2 Preparation of Paraffin Sections

[0102] The specific steps are as follows:

[0103] ① Dehydration: The material fixed with FAA for 24 hours was placed in (70%, 85%, 90%, 95%, 100%, 100%) alcohol for dehydration, and treated for 30 minutes in sequence.

[0104] ② Transparency: The dehydrated material is sequentially transferred into 1 / 2 anhydrous ethanol + 1 / 2 TO (2h) → pure TO (2h, twice) to achieve transparency.

[0105] ③ Wax impregnation: The transparent material is sequentially transferred into 1 / 2 TO + 1 / 2 paraffin (2h) → pure paraffin (4h) → pure paraffin (6h) for wax impregnation.

[0106] ④ Embedding and wax trimming: Place the wax-impregnated material into a cardboard box and embed it at the wax outlet of the embedding machine. After adjusting the position, quickly place it on the freezing stage to solidify it. Use a knife to trim the wax block to a suitable size and attach it to the bottom of the embedding box specifically for hand-cranked microtome.

[0107] ⑤ Slicing, spreading and baking: Set the slice thickness to 8μm, cut complete wax strips of appropriate length according to the length of the glass slide, place them in 43℃ warm water to flatten them, attach the flattened wax strips to a clean glass slide, and place them on a 42℃ baking table to dry. Pay attention to controlling the time to avoid over-drying and causing air bubbles.

[0108] ⑥ Dewaxing, rehydration and staining: Before dewaxing, place the completely dried sections in a 65℃ oven and heat for 30 minutes. Then, quickly place them in pure TO (twice) for dewaxing for 10 minutes each time. After that, place the sections in 1 / 2 TO + 1 / 2 anhydrous ethanol, (100%, 95%, 85%, 70%) alcohol, 1% safranin, (70%, 85%, 95%, 100%) alcohol, 1 / 2 TO + 1 / 2 anhydrous ethanol, and pure TO (twice) for rehydration and staining. Each treatment is soaked for 5 minutes.

[0109] ⑦ Observation: Place the slide under a Nikon E200 optical microscope for observation and take pictures.

[0110] 1.3.7 Determination of physiological indicators during callus differentiation

[0111] 1.3.7.1 Sampling Method

[0112] (1) Determination of physiological indicators of callus induction process of different explants.

[0113] (2) Determination of physiological indicators of different callus types.

[0114] (3) Determination of physiological indicators of callus differentiation into adventitious shoots.

[0115] All the above induction and culture processes used the optimal culture medium selected at each stage. The sampling method for the differentiation process was to take a first sample before inoculation, take a sample once every 7 days after inoculation, and take a final sample at the end of the culture. For callus tissue, it was collected after 30 days of proliferation culture. 0.2g of fresh sample was randomly weighed and repeated 3 times. An appropriate amount of pH 7.0 phosphate buffer solution was added, and the sample was ground in an ice bath. After thorough grinding, it was transferred to a centrifuge tube and brought to a volume of 20ml. The sample was centrifuged at 5000r / min for 20min using a high-speed refrigerated centrifuge. The supernatant, which is the enzyme solution, was stored at -80℃ for later use. The experiment was carried out after all the materials were collected.

[0116] 1.3.7.2 Determination Method

[0117] Superoxide dismutase (SOD) activity was measured using nitro blue tetrahydroquinone. The peroxidase (POD) activity was determined using the guaiacol method, the polyphenol oxidase (PPO) activity was determined using the catechol method, the indoleacetic acid oxidase (IAAO) activity was determined using the indoleacetic acid colorimetric method, the soluble sugar content was determined using the anthrone colorimetric method, and the soluble protein content was determined using the Coomassie Brilliant Blue (G-250) method.

[0118] 1.4 Data Statistics and Analysis

[0119] Preliminary statistical analysis of the experimental data was performed using Excel 2010, and one-way ANOVA and Duncan's multiple comparisons were conducted using SPSS 22.0. The calculation methods for each indicator in this experiment are as follows:

[0120] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) × 100%.

[0121] Brown bruising rate = (number of sterile seedlings with brown bruising / total number of seeds inoculated) × 100%.

[0122] Germination rate = (Number of germinated seeds / Total number of seeds inoculated) × 100%.

[0123] Lateral bud induction rate = (number of axillary buds sprouting / total number of inoculated axillary buds) × 100%.

[0124] Bud proliferation coefficient = (number of axillary buds proliferated / total number of inoculated axillary buds) × 100%.

[0125] The budding index is the average number of buds that emerge from an explant that has produced buds.

[0126] Effective budding rate = (number of new shoots ≥ 1 cm in length / total number of inoculated axillary shoots) × 100%.

[0127] Callus induction rate = (number of callus induced / total number of explants inoculated) × 100%.

[0128] Browning rate = (Number of browned explants / Total number of inoculated explants) × 100%.

[0129] Callus proliferation coefficient = Fresh weight of proliferating callus / Fresh weight of callus before inoculation.

[0130] Bud differentiation rate = (number of callus tissues that produce adventitious buds / total number of inoculated callus tissues) × 100%.

[0131] Rooting rate = (Number of rooted explants / Total number of inoculated explants) × 100%.

[0132] Average number of roots = total number of roots / number of rooted explants.

[0133] Average root length = sum of all root lengths / total number of roots.

[0134] 2. Results and Analysis

[0135] 2.1 Tissue culture of short-calyxed flowers

[0136] 2.1.1 Establishment of a sterile system

[0137] To screen the optimal sterilization scheme for the rapid induction of aseptic seedlings from *Aureobasidium brevicornu* seeds and lay the foundation for providing healthy, sterile, and efficient culture materials for subsequent experiments, the seeds were dehulled and then sterilized with 0.1% HgCl2. After being transversely cut and retaining 1 / 4 of the cotyledons, they were inoculated into MS blank medium for germination culture to establish a sterile system. Table 9 shows that the contamination rate decreased with prolonged sterilization time, but the browning injury rate increased. The lowest contamination rate (6.67%) was observed after 15 min of immersion sterilization, which was not significantly different from that after 10 min (P>0.05). The lowest browning injury rate (6.67%) was observed after 5 min of immersion sterilization, which was not significantly different from that after 10 min. The highest germination rate (94.44%) was observed after 10 min of immersion sterilization, which was significantly different from that after 5 min (P<0.05). Treatment time with 0.1% HgCl2 had no significant effect on seed germination time, mainly concentrated around 7 days after inoculation. In summary, treatment with 0.1% HgCl2 for 10 minutes on dehulled seeds showed no significant difference in sterilization effect, minimum contamination rate, or browning damage rate, and exhibited the highest germination rate, making it the optimal disinfection treatment.

[0138] Table 9

[0139]

[0140] The sterilization process revealed that the amount of cotyledons retained affected sterilization effectiveness and seed germination. Therefore, the optimal disinfection treatment of soaking peeled seeds in 0.1% HgCl2 for 10 minutes was used to study the influencing factor of cotyledon integrity. The sterilization effect and seed germination results are shown in Table 10. All indicators for retaining intact cotyledons were significantly lower than those for retaining 1 / 4 cotyledons. The contamination rate increased from 8.89% to 17.78%, the brown damage rate decreased from 13.33% to 1.11%, the germination rate decreased from 94.44% to 74.44%, and the germination time was prolonged to 14 days. Compared to retaining 1 / 4 cotyledons, retaining 1 / 2 cotyledons significantly reduced the brown damage rate, while there was no significant difference in the contamination rate and germination rate, and the germination time was slightly prolonged.

[0141] Comprehensive analysis shows that the optimal scheme for establishing a sterile system is as follows: after the seeds have been peeled, they are disinfected with 0.1% HgCl2 for 10 minutes, and then half of the cotyledons are retained for germination culture.

[0142] Table 10

[0143]

[0144] 2.1.2 Initiating Culture

[0145] 2.1.2.1 Effects of Hormone Combinations on the Induction of Lateral Buds in Short-calyxed Flowering Stem Segments

[0146] To achieve bud propagation and improve proliferation efficiency, a combination of three exogenous hormones—6-BA, NAA, and IBA—was used to induce the germination and proliferation of lateral buds from stem segments with buds. During observation of the lateral bud induction, it was found that after 7 days of induction culture, the lateral buds swelled, forming protrusions in the leaf axils. After 12 days, the swollen lateral buds split open and began to sprout and produce shoots. After the new shoots emerged, they underwent elongation growth, and within 30 days, clusters of buds could form at the lateral bud sites. Figure 1 The hormones were induced to: A. swell lateral buds after 7 days; B. germinate lateral buds after 12 days; C. elongate new shoots after 16 days; D. germinate multiple buds in leaf axils after 30 days; and E. form cluster buds. Table 11 shows that different hormone ratios had significant differences in the effects on lateral bud germination and proliferation. Treatment B8, with the highest concentration of 6-BA and a moderate concentration of NAA, had the highest lateral bud induction rate (92.57%), followed by treatments B4 and B1, with induction rates of 92.22% and 83.33%, respectively. There was no significant difference in induction rates among these three treatments. Treatment B3 had a significantly lower induction rate than the other treatments (27.39%), a decrease of 70.42% compared to the maximum value. Regarding the bud proliferation coefficient, treatments B4 and B8 were significantly higher than the other treatments, differing by only 0.06 (3.57 and 3.51, respectively). Treatment B3 had a significantly lower bud proliferation coefficient than the other hormone combinations (0.82), a decrease of 77.03% compared to the maximum value. In terms of germination index, only treatments B7 and B8 had values ​​greater than 1, at 1.15 and 1.1 respectively. Treatments B4, B5, and B1 followed, with germination indices above 0.9. Treatment B3 had the lowest germination index at 0.58, only half the maximum value. Regarding effective germination rate, treatments B4, B8, B1, and B7 had the highest values, ranging from 71.11% to 62.22%. There were no significant differences among these four treatments, but all were significantly higher than the others. Treatment B3 had the lowest effective germination rate, at only 11.56%, a decrease of 83.74% compared to the maximum value.

[0147] In summary, the differences in these four indicators between treatments B8 and B4 were not significant, and treatment B8 showed significantly higher values ​​for all four indicators than treatments other than B4. Treatment B3, with the highest concentrations of NAA and IBA, exhibited the worst induction effect, with all four indicators reaching their minimum values. Within the same 6-BA concentration, the ability to induce lateral bud germination and proliferation decreased with increasing NAA and IBA concentrations, while the effect was best when no NAA or IBA was added. Therefore, NAA and IBA inhibit lateral bud growth, while the ability to induce lateral bud germination and proliferation improves with increasing 6-BA concentration.

[0148] Regarding the order of influence on lateral bud proliferation, the ANOVA of the factors in Table 12 showed that 6-BA and NAA had extremely significant effects on all four indicators (P<0.01, the same below); IBA had extremely significant effects on both the induction rate and the bud proliferation coefficient, and showed significant differences in its effect on the effective bud rate, but no significant effect on the budding index. By comparing the R values ​​of each factor in the range analysis (Table 13), it can be seen that, except for the lateral bud induction rate, where IBA's effect was slightly greater than 6-BA, the order of influence of the three hormones on lateral bud proliferation was: NAA>6-BA>IBA. Furthermore, the range analysis showed that the optimal level for 6-BA was 1.5 mg / L, while the optimal levels for both NAA and IBA were 0 mg / L.

[0149] Comprehensive analysis shows that the optimal hormone combination for inducing lateral bud proliferation in this experiment is 1.5 mg / L 6-BA + 0.1 mg / L NAA. Since the orthogonal design cannot cover all treatment combinations, the optimal level combination of 1.5 mg / L 6-BA determined by range analysis was not included in this orthogonal experiment and can be optimized in subsequent studies.

[0150] Table 11

[0151]

[0152] Table 12

[0153]

[0154]

[0155] Table 13

[0156]

[0157] 2.1.2.2 Effects of explant and culture medium type on callus induction in *Euphorbia tirucalli*

[0158] Four types of explants—hypocotyls, stem segments, leaves, and sterile cotyledons—from aseptic seedlings of *Aureobasidium brevicornu* were placed in MS, WPM, and B5 culture media for callus induction to investigate the differences in callus induction caused by explant type and culture medium type. Table 14 shows that different types of explants could induce various callus tissues, and the induction rates differed significantly between different basic culture media. Hypocotyls and cotyledons could both induce callus tissue in B5 medium. The induction rate of leaves on WPM medium was significantly lower than other treatments, at only 58.89%, followed by treatment C6 with an induction rate of 84.62%. The callus induction rates of the remaining treatments were all above 90%. Regarding the browning rate of callus tissue, cotyledons, hypocotyls, and stem segments showed the most severe browning on WPM medium, with browning rates of 88.92% and 82.22%, respectively. Leaves showed the most severe browning on B5 medium, with a browning rate of 70%. Leaves inoculated on MS medium exhibited the lowest browning rate for callus induction, at only 27.78%, a difference of 61.14% from the highest browning rate, significantly lower than other treatments. The induction results of the same explants on different basal media revealed that the induction rate of stem segments and leaves was significantly affected by the medium. All four explant types showed the lowest browning rate on MS medium.

[0159] Regarding the order of influence on callus induction, the analysis of variance in Table 15 shows that explant type has a significant impact on the induction rate of *Hylocereus brevicornu* callus, while its impact on the browning rate is extremely significant; culture medium type has no significant impact on the callus induction rate, but its impact on the browning rate is extremely significant. By comparing the F-values, it can be concluded that explant type is the main factor affecting the callus induction rate, and culture medium type is the main factor affecting the browning rate.

[0160] from Figure 2 It is known that soft, moist callus tissue is prone to browning during induction. Although hypocotyls and cotyledons have high induction rates, their induction degree is low, resulting in fewer callus formations. Callus induced from stem segments is mostly water-soaked and easily browns and loses its activity during subsequent subcultures. Callus induced from leaves on MS medium is yellowish-green, brittle, and grows vigorously with a low browning rate. Its induction degree on WPM medium is low, and browning is severe on B5 medium. Therefore, using leaves as explants for inoculation on MS medium is optimal.

[0161] Table 14

[0162]

[0163] Table 15

[0164]

[0165] 2.1.2.3 Effects of exogenous hormone combinations on callus induction in leaves of *Acer brevicornu*

[0166] Leaves of sterile seedlings of *Aeonium brevicornu* were inoculated with their abaxial surface facing upwards on MS medium containing 6-BA, IBA, and 2,4-D to investigate the effects of exogenous hormones on callus induction. The results are shown in Table 16. Different combinations of the three hormones at different concentrations showed significant differences in callus induction rate and browning rate, and the color and texture of the callus also differed. Regarding induction rate, all nine treatments had high induction rates. The highest induction rate was achieved by treatment D9, which contained the highest concentrations of 6-BA and IBA, as well as a moderate concentration of 2,4-D, reaching 97.85%. Treatments D8 and D6 followed. Treatment D3 had the lowest induction rate at 80.25%, containing the highest concentrations of IBA and 2,4-D, as well as the lowest concentration of 6-BA. The browning rates varied greatly among the nine treatments, falling into three ranges: 4.44%–8.82%, 12.22%–27.78%, and 84.59%–92.29%. Treatment D2 had the lowest browning rate, with no significant difference from treatments D1, D9, and D6. Treatment D3 had the highest browning rate, with no significant difference from treatments D5 and D7.

[0167] Regarding the order of influence on callus induction in leaves, the ANOVA in Table 17 shows that 6-BA had a highly significant effect on callus induction rate, but no significant effect on browning rate; IBA had significant effects on both callus induction rate and browning rate; 2,4-D had highly significant effects on both callus induction rate and browning rate. Combined with the range analysis results in Table 18, the order of influence of factors affecting callus induction rate in leaves is 2,4-D > 6-BA > IBA. The induction rate first increases and then decreases with increasing 2,4-D concentration, and increases with increasing 6-BA concentration. IBA has a relatively small effect on the induction rate, showing a slight decrease followed by an increase with increasing IBA concentration. Therefore, the optimal combination of concentrations for induction rate is 2.0 mg / L 6-BA + 0.1 mg / L IBA + 0.1 mg / L 2,4-D. The order of hormone effects influencing the browning rate of leaf callus is 2,4-D >> 6-BA > IBA. The browning rate is greatly affected by the concentration of 2,4-D; it initially decreases and then increases with increasing 2,4-D concentration, increases with increasing 6-BA concentration, and initially increases and then decreases with increasing IBA concentration. The trends of 2,4-D and IBA with concentration are opposite to those of the induction rate. In summary, 2,4-D mainly affects the callus induction effect; an appropriate concentration of 2,4-D is beneficial for callus induction, while excessively high concentrations easily induce browning.

[0168] from Figure 3It is evident that the callus induced from the leaves mainly falls into three types, and all nine treatments can induce them to varying degrees. Type 1 is soft and moist, with a creamy white or pale yellow color, primarily appearing in treatments D1 and D2. Type 2 is firm and crisp, with a green or yellowish-green color, primarily appearing in treatments D6, D8, and D9. Type 3 is firm and dry, with a pale green color, primarily appearing in treatment D9. Comprehensive analysis shows that although treatment D2 has a high induction rate, the induced callus is soft and moist, making it prone to browning in subsequent subculturing. Treatments D6 and D9 have high induction rates and low browning rates, producing firmer callus with vigorous growth. Furthermore, callus induced by treatment D9 is more likely to form dense embryogenic callus during subsequent subculturing. Therefore, the optimal combination is treatment D9: 2.0 mg / L 6-BA + 0.1 mg / L IBA + 0.1 mg / L 2,4-D, consistent with the optimal level combination in the range analysis.

[0169] Table 16

[0170]

[0171] Table 17

[0172]

[0173] Table 18

[0174]

[0175] 2.1.3 Subculture 2.1.3.1

[0177] Effects of exogenous hormones on callus proliferation in *Aglaonema brevicornu*

[0178] Before inducing adventitious shoot differentiation in callus tissue, it is necessary to conduct proliferation culture to obtain sufficient and high-quality dense callus tissue. Table 19 shows that, under the same addition of 2 mg / L 6-BA, different concentration ratios of IBA and 2,4-D significantly affected the proliferation culture of light green, dense callus tissue. Treatment E4 showed the best proliferation effect, with a proliferation coefficient of 4.77, significantly higher than other treatments. Treatment E3 had the lowest proliferation coefficient, only 2.83, significantly lower than other treatments. With a constant IBA concentration, the proliferation coefficient increased with increasing 2,4-D concentration; however, at the same 0.05 mg / L 2,4-D concentration, the proliferation coefficient decreased with increasing IBA concentration. Looking at the 2,4-D to IBA concentration ratio, only treatment E3, with the lowest proliferation coefficient (0.5, less than 1), showed a ratio of 0.5. Combined with the range analysis results in Table 20, it can be concluded that 2,4-D plays a major role in promoting callus proliferation, while IBA has a relatively small impact.

[0179] Table 19

[0180]

[0181] Table 20

[0182]

[0183] 2.1.3.3 Effects of the type and concentration of anti-browning agents on the proliferation of callus tissue in *Bretschneidera sinensis*

[0184] 2.1.4 Callus Differentiation Culture

[0185] To achieve plant regeneration, adventitious bud differentiation was induced in green, dense, and compact callus tissue with differentiation capacity. Figure 4 It is evident that callus tissue can differentiate into single buds and clusters of buds. After 30 days of continued culture, adventitious buds elongate and grow into robust seedlings. According to the experimental results in Table 21, different concentrations of 6-BA, TDZ, and IBA showed significant differences in callus differentiation rates. When the 6-BA concentration was 0.5 mg / L, no adventitious buds were observed to differentiate from the callus tissue. Furthermore, at the same 6-BA concentration, the differentiation rate increased with increasing TDZ concentration. Among the nine treatments, treatment G9 had the highest differentiation rate at 30.82%, significantly higher than all treatments except G6. Treatment G9 also had the highest budding index, but the difference between it and treatments G6 and G8 was not significant.

[0186] Regarding the order of influence on callus differentiation into adventitious shoots, the ANOVA in Table 22 shows that 6-BA, TDZ, and IBA all had highly significant effects on callus differentiation rate and shoot emergence index. Comparing the F-values ​​indicates that 6-BA is the main influencing factor. Combined with the range analysis results in Table 23, the order of influence of these three hormones on callus differentiation into adventitious shoots is 6-BA > TDZ > IBA. Both differentiation rate and shoot emergence index increase with increasing 6-BA and TDZ concentrations, and decrease with increasing IBA concentrations. The optimal combination of these three hormones for differentiation rate and shoot emergence index is 2.0 mg / L 6-BA + 0.5 mg / L TDZ + 0.1 mg / L IBA, which slightly differs from the optimal result of this experiment (2.0 mg / L 6-BA + 0.5 mg / L TDZ + 0.2 mg / L IBA) and can be optimized in future studies.

[0187] Table 21

[0188]

[0189] Table 22

[0190]

[0191] Table 23

[0192]

[0193] 2.1.5 Peanut root culture using the short-calyx apparatus

[0194] Rooting of short-calyx flower bud seedlings was induced using a combination of NAA and IBA growth hormones, as shown in Table 24. Figure 5 It can be seen that different combinations of hormone concentrations have significant effects on induced rooting. NAA and IBA synergistically promote the rooting of *Peanuts spicata*, and the rooting rate increases with increasing NAA and IBA concentrations. The H9 treatment has the highest rooting rate at 42.22%, which is not significantly different from the H6 treatment. The H1 treatment has the lowest rooting rate at 18.89%, which is not significantly different from H4 and H7, but is 55.26% lower than the maximum value. Regarding the average number of roots, while keeping the IBA concentration constant, the average number of roots increases with increasing NAA concentration. The H9 treatment has the highest number at 2.81, significantly higher than other treatments, while the H1 treatment has the lowest number at 1.0, significantly lower than other treatments, and is 64.41% lower than the maximum value. In terms of average root length, treatment H1 had the highest at 1.62, significantly greater than other treatments, while treatment H4 had the lowest at 0.37, which was not significantly different from H9 and decreased by 77.16% compared to the maximum value. Root length decreased with increasing NAA concentration and showed a trend of first increasing and then decreasing with increasing IBA concentration.

[0195] Regarding the order of influence on the rooting induction of seedlings, the analysis of variance of each factor in Table 25 showed that NAA had a highly significant effect on the rooting rate and number of roots of *Aureocarpus septemlobus*, while having a significant effect on its average root length; IBA had a highly significant effect on the rooting rate, but no significant effect on the average number of roots and average root length. Based on the range analysis results in Table 26, the ranges of the two auxins affecting the rooting rate of seedlings are: RNAA = 5.926 and RIBA = 16.750, indicating that the order of influence is IBA > NAA. The ranges affecting the average number of roots are: RNAA = 1.066 and RIBA = 0.212, and the ranges affecting the average root length are: RNAA = 0.510 and RIBA = 0.092, indicating that the order of influence on the average number of roots and the average root length is NAA > IBA. The optimal combination of levels for both rooting rate and average number of roots is 0.5 mg / L NAA + 0.5 mg / L IBA, which is consistent with the optimal hormone combination for inducing rooting in this experiment.

[0196] Table 24

[0197]

[0198] Table 25

[0199]

[0200]

[0201] Table 26

[0202]

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A tissue culture method of short calyx instrument flower, characterized by, Includes the following steps: (1) Disinfect the seeds of the short-calyxed flower; (2) The seeds of the short-calyxed flower were soaked in mercuric chloride solution and then inoculated into MS medium to induce the germination of sterile seedlings; (3) Inoculate the budded stem segments of sterile seedlings into the germination medium for germination and proliferation; (4) The germinating and proliferating leaves were inoculated into an induction medium for callus induction culture; (5) The induced callus tissue was cultured in a proliferation medium; (6) The callus tissue after proliferation culture was cultured in differentiation medium to obtain rootless seedlings; (7) Roots were induced in the rootless seedlings using root induction medium; The germination medium is: MS medium, 0.5~1.5 mg / L 6-BA, 0~0.3 mg / L NAA, 0~0.4 mg / L IBA; The induction medium was: MS medium, 0.5~2 mg / L 6-BA, 0.01~0.1 mg / L IBA, 0.05~0.5 mg / L 2,4-D; The proliferation medium consisted of: MS medium, 1-3 mg / L 6-BA, 0.05-0.1 mg / L IBA, 0.05-0.1 mg / L 2,4-D, 0-0.1 g / L citric acid, 0-1 g / L PVP, and 0-1 g / L activated carbon. The differentiation medium consisted of MS medium, 1-2 mg / L 6-BA, 0.2-0.5 mg / L TTDZ, 0.1-0.3 mg / L IBA, and 0.2 g / L acid-hydrolyzed casein. The root induction medium consisted of 1 / 2 MS medium, 0.1–0.5 mg / L NAA, and 0–0.5 mg / L IBA. All MS media were supplemented with 7 g / L agar and 30 g / L sucrose.

2. The method of tissue culture of short calyx instrument flower according to claim 1, characterized in that, The seeds of the short-calyxed flower are plump and free from pests and diseases.

3. The method of tissue culture of short calyx instrument flower as claimed in claim 1, wherein, The specific steps for disinfecting the seeds of *Echeveria pulvinata* are as follows: Cut the seeds along the edge, then wash and soak them in detergent for 8-12 minutes, followed by rinsing with water for 20-40 minutes. After washing, soak the seeds in a 60-80% ethanol solution for 0.5-2 minutes, rinse with water 2-4 times, then soak them in a 0.05-0.2% mercuric chloride solution for 8-12 minutes, rinse with water 4-6 times, and then soak them in water for 20-30 hours. After soaking, soak them in a 60-80% ethanol solution for 20-40 seconds, rinse with water 2-4 times, and then soak them in a 0.05-0.2% mercuric chloride solution for 5-15 minutes, rinsing with water 4-6 times.

4. The tissue culture method for *Bructus brevicornu* according to claim 1, characterized in that, The proliferation medium consisted of MS medium, 2 mg / L 6-BA, 0.1 mg / L IBA, 0.1 mg / L 2,4-D, and 0.1 g / L citric acid.

5. The method for tissue culture of *Bructus brevicornu* according to claim 1, characterized in that, The differentiation medium consisted of MS medium, 2 mg / L 6-BA, 0.5 mg / L TTDZ, 0.2 mg / L IBA, and 0.2 g / L acid-hydrolyzed casein.

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