A culture medium and culture method for culturing leaf callus of leatherleaf mahonia

Abstract

The present application belongs to the technical field of tissue culture, and particularly relates to a culture medium and a culture method for culturing leaf callus of Leucadendron. The present application provides a culture medium for culturing leaf callus of Leucadendron, wherein the culture medium comprises an induction culture medium and a proliferation culture medium; 6-BA in the induction culture medium and the proliferation culture medium promotes cell division, 2,4-D induces the generation of callus and promotes the growth of callus, and NAA can promote the generation and proliferation of callus; under the specific 6-BA, 2,4-D, NAA, suitable sucrose concentration and specific basic culture medium, the hormone balance of Leucadendron in vitro leaf can be maintained, and the growth and proliferation of callus are promoted. The present application provides a method for culturing callus of Leucadendron by taking Leucadendron leaf as an explant, inducing leaf dedifferentiation to form callus and proliferating culture, wherein the proliferation coefficient of callus is high, and the texture is good.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a culture medium and culture method for culturing callus tissue from silver thread plum leaves. Background Technology

[0002] Silver-thread plum (scientific name: Shaniodendron subaequale (HT Chang) MB Deng&al.) is a deciduous small tree belonging to the genus *Hamamelis* in the family Hamamelidaceae. The *Hamamelis* can grow to 4-5 meters tall and blooms in May. It is distributed in Anhui, Jiangsu, and Zhejiang provinces of China, preferring warm and humid climates and exhibiting strong cold resistance. It can adapt to acidic, neutral, slightly alkaline, and calcareous loam soils, but deep, fertile, moist, and well-drained loam is preferred.

[0003] Silver-thread plum (Prunus armeniaca) has a dignified shape, large crown, and provides excellent shade, making it suitable as a garden tree, shade tree, and street tree. It is a Class I protected wild plant in my country. Due to its rarity, it has been listed as a Class I key protected wild plant. Because its leaves contain a large amount of active substances, making it an excellent species for producing secondary metabolites, there are currently no reports of tissue culture of Silver-thread plum using leaves as explants. Summary of the Invention

[0004] The purpose of this invention is to provide a culture medium and a culture method for cultivating callus tissue from silver thread plum leaves, using newly grown leaves from the current year's branches of silver thread plum as explants, resulting in a high proliferation coefficient of the callus tissue.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0006] This invention provides a culture medium for culturing callus tissue from *Haloxylon ammodendron* leaves, the culture medium comprising an induction medium and a proliferation medium;

[0007] The induction medium uses WPM as the basic medium and also includes 0.2~1.0 mg / L 6-BA, 0.5~1.5 mg / L 2,4-D, 0.1~1.0 mg / L NAA, 29~30 g / L sucrose and 7~8 g / L agar;

[0008] The proliferation medium uses WPM as the basic medium and also includes 0.5~1.0 mg / L 6-BA, 0.5~1.0 mg / L NAA, 25~30 g / L sucrose and 7~8 g / L agar.

[0009] As a preferred embodiment, the initial pH of the induction medium is 5.7-6.0; the initial pH of the proliferation medium is 5.7-6.0.

[0010] This invention provides a method for culturing callus tissue from *Haloxylon ammodendron* leaves, using the culture medium described in the above technical solution, and comprising the following steps:

[0011] First, the leaves of *Silver Witch Hazel* were inoculated sequentially into the induction medium to induce callus tissue. Then, the callus tissue was placed in the proliferation medium for proliferation culture to obtain callus tissue.

[0012] As a preferred embodiment, the leaves are further disinfected before inoculation. The disinfection includes soaking in 75% ethanol for 25-35 seconds, rinsing with sterile water 3-4 times, soaking in sodium hypochlorite solution with a mass concentration of 0.21%-0.30% for 6-10 minutes, and rinsing with sterile water 5-6 times.

[0013] As a preferred embodiment, the induction culture and proliferation culture temperatures are 23~27℃, respectively.

[0014] As a preferred embodiment, the induction culture and proliferation culture are respectively light-dark alternating cultures.

[0015] As a preferred embodiment, the light duration in the alternating light and dark culture is 14-16 h / d; the light intensity is 2100-2600 lx; ​​and the darkness duration in the alternating light and dark culture is 8-10 h / d.

[0016] As a preferred embodiment, the induction culture time is 28-35 days; the proliferation culture time is 28-45 days.

[0017] As a preferred embodiment, before proliferation culture, the method further includes: subculturing the callus tissue obtained from the induced culture.

[0018] As a preferred option, the leaves need to be incised before induction culture.

[0019] The beneficial effects of this invention are as follows: This invention provides a culture medium for culturing callus tissue from *Hypericum esculentum* leaves. The culture medium includes an induction medium and a proliferation medium. The basic culture medium provides the essential nutrients required for plant cell growth, including inorganic salts, carbon sources, vitamins, etc. 6-BA (6-benzylaminopurine) in the induction and proliferation media promotes cell division, 2,4-D induces callus formation and promotes its growth, and NAA can promote the production and proliferation of callus. Under specific conditions of 6-BA, 2,4-D, NAA, suitable sucrose concentration, and a specific basic culture medium, a new hormonal balance in detached *Hypericum esculentum* leaves can be maintained, promoting the growth and proliferation of callus tissue.

[0020] This invention provides a method for culturing callus tissue from *Hypericum esculentum* leaves, comprising the following steps: first, sequentially inoculating *Hypericum esculentum* leaves into an induction medium to induce callus formation; then, placing the callus tissue into a proliferation medium for proliferation culture to obtain callus tissue. This invention provides a method for culturing *Hypericum esculentum* callus tissue using *Hypericum esculentum* leaves as explants, inducing leaf dedifferentiation to form callus tissue, and then proliferating and culturing the resulting callus tissue. The callus tissue exhibits a high proliferation coefficient and excellent quality. Existing technologies have not reported methods for culturing *Hypericum esculentum* callus tissue using leaves as explants. This invention enriches the types of explants for inducing *Hypericum esculentum* callus tissue, enabling the callus tissue to proliferate in large quantities, laying a foundation for subsequent *Hypericum esculentum* callus tissue differentiation and the production of active metabolites. Attached Figure Description

[0021] Figure 1 A photograph of a new leaf used as an explant;

[0022] Figure 2 Figure showing the effect of different concentrations of 6-BA on callus proliferation in leaves of *Hypericum esculentum*.

[0023] Figure 3 Figure showing the effect of different concentrations of 2,4-D on callus proliferation in leaves of *Hypericum silveripes*.

[0024] Figure 4 Figure showing the effect of different concentrations of NAA on callus proliferation in Silver Witch Hazel leaves;

[0025] Figure 5 Figure showing the effect of different concentrations of KT on callus proliferation in Silver Witch Hazel leaves;

[0026] Figure 6 Figure showing the effect of high concentrations of 6-BA and NAA on callus proliferation in leaves of *Hypericum esculentum*.

[0027] Figure 7 Figure showing the effect of low concentrations of 6-BA and NAA on callus proliferation in *Hypericum esculentum* leaves. Detailed Implementation

[0028] This invention provides a culture medium for culturing callus tissue from silverwort leaves, the culture medium comprising an induction medium and a proliferation medium.

[0029] The induction medium of the present invention uses WPM as the basic medium and also includes 1.0 mg / L 6-BA, 1.0 mg / L 2,4-D, 0.1 mg / L NAA, 29~30 g / L sucrose and 7~8 g / L agar.

[0030] As an optional embodiment, the concentration of 6-BA in the induction medium provided by the present invention is 0.2~1.0 mg / L; in specific embodiments of the present invention, the concentration of 6-BA in the induction medium can be 0.2, 0.4, 0.6, 0.8, or 1.0 mg / L. 6-BA at an appropriate concentration helps induce callus formation in leaves. As an optional embodiment, the concentration of 2,4-D in the induction medium provided by the present invention is 0.5~1.5 mg / L; in specific embodiments of the present invention, the concentration of 2,4-D in the induction medium can be 0.5, 0.8, 1.0, 1.2, or 1.5 mg / L. 2,4-D at an appropriate concentration helps induce callus formation in leaves and promotes callus growth. As an optional embodiment, the concentration of NAA in the induction medium provided by the present invention is 0.1~1.0 mg / L; in specific embodiments of the present invention, the concentration of NAA in the induction medium can be 0.1, 0.3, 0.5, 0.8, or 1.0 mg / L. NAA at appropriate concentrations helps induce callus formation in leaves. The sucrose concentration in the induction medium provided by this invention is 29-30 g / L. In specific embodiments of this invention, the sucrose concentration in the induction medium can be 29, 29.5, or 30 g / L. The agar concentration in the induction medium provided by this invention is 7-8 g / L. In specific embodiments of this invention, the agar concentration in the induction medium can be 7, 7.5, or 8 g / L. In this invention, the pH value of the induction medium can be 5.7-6.0, or 5.7, 5.8, 5.9, or 6.0.

[0031] The proliferation medium of the present invention uses WPM as the basic medium and also includes 0.5~1.0 mg / L 6-BA, 0.5~1.0 mg / L NAA, 29~30 g / L sucrose and 7~8 g / L agar.

[0032] As an optional implementation, the concentration of 6-BA in the proliferation medium provided by the present invention is 0.5~1.0 mg / L; in specific embodiments of the present invention, the concentration of 6-BA in the proliferation medium can be 0.5, 0.6, 0.8, 0.9, or 1.0 mg / L. 6-BA helps to accelerate the proliferation rate of callus tissue. The concentration of NAA in the proliferation medium provided by the present invention is 0.5~1.0 mg / L; in specific embodiments of the present invention, the concentration of NAA in the proliferation medium can be 0.5, 0.6, 0.8, 0.9, or 1.0 mg / L. The concentration of sucrose in the proliferation medium provided by the present invention is 25~30 g / L. In specific embodiments of the present invention, the concentration of sucrose in the proliferation medium can be 25, 28, or 30 g / L. The concentration of agar in the proliferation medium provided by the present invention is 7~8 g / L. In specific embodiments of the present invention, the concentration of agar in the proliferation medium can be 7, 7.5, or 8 g / L. In this invention, the pH value of the proliferation medium can be 5.7~6.0, or it can be 5.7, 5.8, 5.9 or 6.0.

[0033] The induction and proliferation media provided by this invention both contain sucrose. Sucrose can provide the carbon source required for the growth of silver witch hazel leaves, maintain the hormone balance of silver witch hazel leaves, and promote the growth and proliferation of callus tissue.

[0034] The culture medium provided by this invention has good application effects, which can improve the proliferation coefficient of callus induced from leaves of *Hypericum esculentum*, and the callus texture is excellent. This invention does not have special limitations on the source of each component in the induction and proliferation culture media; conventional commercially available products can be used.

[0035] This invention provides a method for culturing callus tissue from *Haloxylon ammodendron* leaves, using the culture medium described in the above technical solution, and comprising the following steps:

[0036] First, the leaves of *Hypericum esculentum* were sequentially inoculated into the induction medium to induce callus tissue. Then, the callus tissue was placed in the proliferation medium for proliferation culture to obtain callus tissue.

[0037] As an optional implementation, the leaves of this invention further include cleaning and disinfection before inoculation. As an optional implementation, the cleaning method of this invention includes rinsing with running water. As an optional implementation, the disinfection method of this invention includes first soaking in 75% ethanol for 25-35 seconds (or 30 seconds); then rinsing 3-4 times with sterile water; then soaking in a 0.21%-0.30% sodium hypochlorite solution for 6-10 minutes (or 8 minutes); the sodium hypochlorite solution used is 0.21%-0.30% (or 0.26%); finally rinsing 5-6 times with sterile water. This disinfection method can reduce the browning rate of callus tissue, resulting in a high aseptic survival rate of explants and better callus quality. In a specific embodiment of this invention, leaf disinfection involves first soaking in 75% ethanol for 30 seconds, followed by treatment with 0.26% NaClO for 8 minutes, achieving a callus induction rate of up to 81.67%.

[0038] As an optional implementation method, the present invention involves cutting the disinfected leaves. The leaves are preferably new leaves from the current year's growth branches of *Hymenochloa chinensis*. New leaves from the current year's growth branches are chosen because they are tender, have high vitality, and are clean and not easily contaminated. *Hymenochloa chinensis* leaves have fine folds, making them more prone to accumulating dirt and making disinfection more difficult. The size of the cut leaves in this invention is 0.5~1cm. 2 The leaf is rectangular. In a specific embodiment of the present invention, the shape of the segmented leaf is preferably rectangular. The present invention preferably involves making incisions on the segmented leaf. The incisions are preferably located on the back of the leaf, perpendicular to the main vein, ensuring that the leaf is not severed. Making incisions increases callus induction efficiency; callus tissue is more easily formed at wound sites. The present invention does not limit the number of incisions; in a specific embodiment, the number of incisions is three.

[0039] As an optional implementation, the present invention involves inoculating the divided and wounded leaves into an induction medium for induction culture to obtain callus tissue. As an optional implementation, the induction culture temperature is 23-27℃, or 25℃; the induction culture time is 28-35 days, preferably 30 days. As an optional implementation, the induction culture is a light-dark alternating culture. The light culture time in the light-dark alternating culture of the present invention is 14-16 h / d, more preferably 16 h / d; the light intensity can be 2200-2500 lx, or 2300-2450 lx, more preferably 2400 lx. The dark time in the light-dark alternating culture of the present invention is 8-10 h / d, more preferably 8 h / d.

[0040] The induction culture time described in this invention is calculated from the time the leaf is inoculated onto the induction medium until callus tissue grows. Once the callus tissue clumps reach a diameter of 1-5 mm, the leaf is transferred to the proliferation medium. Alternating light and dark culture in darkness helps promote callus formation, maintains good growth, and reduces browning.

[0041] As an optional implementation, the present invention performs subculture followed by proliferation culture on the callus tissue. As an optional implementation, the culture medium used for subculture in the present invention has the same composition as the induction culture medium. Subculture is used to stabilize the newly induced callus tissue, especially since callus tissue at leaf cuts may still have leaf remnants after cutting, and will continue to dedifferentiate to form more callus tissue until the leaf completely dedifferentiates to form callus tissue, and secondary callus tissue begins to form. The subculture time is 30-60 days. As an optional implementation, the subculture temperature is 23-27℃, or 25℃. As an optional implementation, the subculture is a light-dark alternating culture. The light culture time for the light-dark alternating culture in the present invention is 14-16 h / d, more preferably 16 h / d; the light intensity can be 2200-2500 lx, or 2300-2450 lx, more preferably 2400 lx. The dark time in the light-dark alternating culture of the present invention is 8-10 h / d, more preferably 8 h / d.

[0042] As an optional implementation, the callus tissue obtained after subculture is divided into clumps and then inoculated into proliferation medium for proliferation culture. The size of the clumps can be 1-5 mm in diameter. The proliferation culture time of the present invention can be 28-45 days, more preferably 30 days. As an optional implementation, the proliferation culture temperature is 23-27℃, or 25℃. As an optional implementation, the proliferation culture is a light-dark alternating culture. The light culture time of the light-dark alternating culture of the present invention is 14-16 h / d, more preferably 16 h / d; the light culture intensity can be 2200-2500 lx, or 2300-2450 lx, more preferably 2400 lx. The dark time in the light-dark alternating culture of the present invention is 8-10 h / d, more preferably 8 h / d. The callus tissue obtained by the proliferation culture of the present invention has a high proliferation coefficient, the callus tissue is yellowish-white, has a strong granular feel, is loose and brittle, and has excellent texture.

[0043] This invention uses newly grown leaves from current-year branches of *Hypericum esculentum* as explants to investigate the effects of different disinfection methods, basic culture media, and plant growth regulators on callus induction and proliferation, laying a foundation for subsequent callus differentiation from *Hypericum esculentum* leaves.

[0044] The optimal culture medium for inducing callus from leaves is: WPM + 1.0 mg / L 6-BA + 1.0 mg / L 2,4-D + 0.1 mg / L NAA + 30 g / L sucrose + 8 g / L Agar, pH 5.7.

[0045] In callus proliferation culture, low concentrations of 6-BA and NAA are significantly more effective than high concentrations. The optimal formulation is 1.0 mg / L 6-BA + 0.5 mg / L NAA, resulting in a high callus proliferation coefficient and excellent texture.

[0046] 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.

[0047] This invention uses Excel and SPSS software for data statistics and analysis. Duncan's method is used to test for significant differences.

[0048] The calculation method for the survey indicators is as follows:

[0049] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) × 100%;

[0050] Browning rate = (Number of browned explants / Total number of inoculated explants) × 100%;

[0051] Survival rate = (Number of surviving explants / Total number of inoculated explants) × 100%;

[0052] Callus induction rate (%) = (Number of explants producing callus / Total number of inoculated explants) × 100%;

[0053] Callus proliferation coefficient = (mass of the callus after proliferation - mass of the callus immediately after inoculation) / mass of the callus immediately after inoculation.

[0054] The culture conditions were as follows: all culture media were supplemented with 30 g / L sucrose and 8 g / L agar, and the pH value was 5.7. The culture temperature was (25±2)℃, the photoperiod was 16 h light / 8 h dark, and the light intensity was 2400 lx.

[0055] KT: Furanoaminopurine;

[0056] 6-BA: 6-Benzylaminopurine;

[0057] 2,4-D: 2,4-Dichlorophenoxyacetic acid;

[0058] NAA: Naphthaleneacetic acid.

[0059] Example 1

[0060] 1. Materials and Methods

[0061] 1.1 Test Materials

[0062] The experimental material was obtained from three-year-old juvenile *Prunus cerasifera* trees in the intelligent greenhouse of Zhejiang Agriculture and Forestry University. The plants were robust and free from pests and diseases, and the seed source was wild *Prunus cerasifera* plants from Longwang Mountain in Anji County. New leaves from the current year's new shoots were used as explants (see...). Figure 1 ).

[0063] 1.2 Screening of Leaf Disinfection Methods

[0064] Take new leaves from 3-year-old silver thread plum trees, rinse them under running water for 2 hours, and then transfer them to a clean bench for later use. First, treat the leaves with 75% ethanol for 30 seconds, rinse them with sterile water 3-4 times, and then vacuum immerse them in NaClO (sodium hypochlorite) solutions of different mass concentrations (0.26%, 0.35%, 0.52%, and 1.05%) for 8 minutes. After disinfection, rinse them with sterile water 5-6 times and blot dry with sterile filter paper.

[0065] Disinfected leaves were inoculated into A2 medium (MS + 1.0 mg / L 6-BA + 0.5 mg / L 2,4-D + 0.1 mg / L NAA); 20 leaves were inoculated for each treatment, and the experiment was repeated 3 times. The contamination rate was counted 14 days after inoculation, and the browning rate and sterile survival rate were counted 30 days after inoculation.

[0066] The composition of A2 medium is: MS as the basic medium, supplemented with 1.0 mg / L 6-BA, 0.5 mg / L 2,4-D and 0.1 mg / L NAA.

[0067] 1.3 Effect of different NaClO concentrations on leaf disinfection

[0068] This experiment mainly studied the effect of different NaClO concentrations on the disinfection effect of *Hypericum esculentum* leaves. As shown in the table, with an average disinfection time of 8 minutes, the browning rate of the leaves increased with increasing NaClO concentration, reaching a maximum of 40% at 1.05% NaClO. The contamination rate was lowest at 0.52% NaClO, reaching 3.33%. The survival rate was highest at 0.26% NaClO, reaching 81.67%. In conclusion, 0.26% NaClO treatment for 8 minutes is the most suitable for the disinfection of newly sprouted *Hypericum esculentum* leaves (Table 1).

[0069] Table 1. Effects of different NaClO concentrations on the disinfection effect of *Gynostemma pentaphyllum* leaves.

[0070]

[0071] Note: The contamination rate, survival rate, and browning rate in the table represent the mean ± standard deviation; Duncan's multiple comparison method was used; different letters in the same column represent significant differences ( P <0.05), the same applies below.

[0072] Example 2: Induction of leaf callus

[0073] After disinfecting the leaves of the silver wisteria, cut off the leaf edges and cut them into 0.5-1cm pieces. 2 Rectangular-sized explants were incised on the underside of the leaves, perpendicular to the main vein, without cutting the leaves. The leaves were then inoculated onto different culture media M1-M9, with the underside facing upwards. Twenty leaves were inoculated into each of the M1-M9 media, with one leaf per media. Three replicates were set up for orthogonal treatment. The callus induction rate and explant status were recorded after 30 days. The results are shown in Tables 2 and 3.

[0074] M1 consists of MS as the basic medium, supplemented with 0.2 mg / L 6-BA, 0.5 mg / L 2,4-D, 0.1 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0075] M2 consists of MS as the basic medium, supplemented with 0.5 mg / L 6-BA, 1.0 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0076] M3 consists of MS as the basic medium, supplemented with 1.0 mg / L 6-BA, 1.5 mg / L 2,4-D, 1.0 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0077] M4 consists of 1 / 2 MS as the basic medium, supplemented with 0.2 mg / L 6-BA, 1.0 mg / L 2,4-D, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0078] M5 consists of 1 / 2 MS as the basal medium, supplemented with 0.5 mg / L 6-BA, 1.5 mg / L 2,4-D, 0.1 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0079] M6 consists of 1 / 2 MS as the basal medium, supplemented with 1.0 mg / L 6-BA, 0.5 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0080] M7 consists of WPM as the basic culture medium, supplemented with 0.2 mg / L 6-BA, 1.5 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0081] M8 consists of WPM as the basic culture medium, supplemented with 0.5 mg / L 6-BA, 0.5 mg / L 2,4-D, 1.0 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0082] M9 consists of WPM as the basic culture medium, supplemented with 1.0 mg / L 6-BA, 1.0 mg / L 2,4-D, 0.1 mg / L NAA, 30 g / L sucrose, and 8 g / L agar.

[0083] Table 2 Results and range analysis of callus induction experiment on Witch hazel leaves

[0084]

[0085] Table 3. Analysis of variance of orthogonal experiments on callus induction from *Silver Witch Hazel* leaves.

[0086]

[0087] It can be seen that the leaf induction rate in the M9 induction medium was the highest, reaching 67.31%, with swollen leaf veins and good callus growth; followed by the M1 induction medium.

[0088] Range analysis of the callus induction rate revealed that the basal culture medium had the greatest impact on callus induction from *Hypericum esculentum* leaves, with a range R of 20.77, followed by 6-BA, while NAA had the least impact. The order of influence of the four factors on the callus induction rate of *Hypericum esculentum* leaves was: basal culture medium > 6-BA > 2,4-D > NAA (Table 3). According to the ANOVA results in the table, the basal culture medium had a highly significant effect on callus induction from *Hypericum esculentum* leaves, 6-BA and 2,4-D had significant effects on callus induction, while NAA had no significant effect on callus induction (Table 3). Combining the range and ANOVA results of the orthogonal experiments on *Hypericum esculentum* leaves, M9 showed the highest induction rate. The optimal culture medium for callus induction from *Hypericum esculentum* leaves was determined to be: WPM + 1.0 mg / L 6-BA + 1 mg / L 2,4-D + 0.1 mg / L NAA.

[0089] Basic culture media provide essential nutrients for plant cell growth, including inorganic salts, carbon sources, and vitamins. Different basic culture media vary in composition and content, thus affecting plant cell growth and differentiation. In the induction of callus from *Hypericum esculentum* leaves, callus induction failed under simple basic culture media conditions. Therefore, an orthogonal design was used to explore the effects of basic culture media and plant growth regulators on callus induction. The experiment revealed that basic culture media had the greatest impact on callus induction, followed by 6-BA, 2,4-D, and NAA. The study indicates that selecting a suitable basic culture medium is crucial for improving callus induction rates, but the combination with different plant growth regulators is also necessary for successful callus induction.

[0090] Example 3: Effects of single-factor treatments with different concentrations of plant growth regulators on the subculture proliferation of callus tissue in *Hypericum esculentum* leaves.

[0091] Callus induced from silverwort leaves was transferred whole to proliferation medium for proliferation experiments. The proliferation medium consisted of C1-C4, H1-H4, I1-I4, and G1-G4.

[0092] C1 consists of WPM as the basic culture medium, supplemented with 0.5 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0093] C2 consists of WPM as the basic culture medium, supplemented with 1.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0094] C3 consists of WPM as the basic culture medium, supplemented with 2.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0095] C4 consists of WPM as the basic culture medium, supplemented with 4.0 mg / L 6-BA, 30 g / L sucrose and 8 g / L agar;

[0096] The composition of H1 is: WPM as the basic culture medium, with the addition of 0.5 mg / L 2,4-D, 30 g / L sucrose and 8 g / L agar;

[0097] The composition of H2 is: WPM as the basic culture medium, with the addition of 1.0 mg / L 2,4-D, 30 g / L sucrose and 8 g / L agar;

[0098] The composition of H3 is: WPM as the basic culture medium, with the addition of 2.0 mg / L 2,4-D, 30 g / L sucrose and 8 g / L agar;

[0099] The composition of H4 is: WPM as the basic culture medium, with the addition of 4.0 mg / L 2,4-D, 30 g / L sucrose and 8 g / L agar;

[0100] The composition of I1 is: WPM as the basic medium, with the addition of 0.1 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0101] The composition of I2 is: WPM as the basic medium, with the addition of 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0102] The composition of I3 is: WPM as the basic medium, with the addition of 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0103] The composition of I4 is: WPM as the basic medium, with the addition of 2.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0104] The composition of G1 is: WPM as the basic medium, with the addition of 0.5 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0105] The composition of G2 is: WPM as the basic medium, with the addition of 1.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0106] The composition of G3 is: WPM as the basic culture medium, with the addition of 2.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0107] The composition of G4 is: WPM as the basic medium, with the addition of 4.0 mg / L KT, 30 g / L sucrose and 8 g / L agar;

[0108] Each treatment was inoculated into 20 culture dishes, with one callus mass inoculated into each dish. Three replicates were performed. After 30 days of culture, the callus proliferation coefficient and texture were analyzed. Results are as follows:

[0109] The effects of different concentrations of 6-BA on callus proliferation are shown in Table 4 and Figure 2 .

[0110] 6-BA significantly affected callus proliferation in *Haloxylon ammodendron* leaves. With increasing 6-BA concentration, the callus proliferation coefficient first increased and then decreased (Table 4). The highest coefficient (3.70) was observed at a concentration of 1.0 mg / L, with the callus exhibiting a granular texture and a yellowish-green color. Further concentration increase, reaching 4.0 mg / L, led to vitrification of the callus. Figure 2 ).

[0111] Table 4. Effects of different concentrations of 6-BA on callus proliferation.

[0112]

[0113] The effects of different concentrations of 2,4-D on callus proliferation are shown in Table 5 and Figure 3 .

[0114] During the induction of callus tissue from *Hypericum esculentum* leaves using 2,4-D, the callus proliferation coefficient was generally high, but the callus tissue was in a vitrified and hydrated state, resulting in low callus quality. Figure 3 As the concentration of 2,4-D increased, the callus proliferation coefficient showed a trend of first increasing and then decreasing. The highest proliferation coefficient reached 3.91 at 2.0 mg / L and the lowest was 2.24 at 4.0 mg / L (Table 5).

[0115] Table 5. Effects of different concentrations of 2,4-D on callus proliferation.

[0116]

[0117] The effects of different concentrations of NAA on callus proliferation are shown in Table 6 and Figure 4 .

[0118] NAA showed a significant effect on callus proliferation from *Hypericum esculentum* leaves. The callus proliferation coefficient increased continuously with increasing concentration (Table 6). The lowest proliferation coefficient (0.67) was observed at a concentration of 0.1 mg / L, with the callus becoming brown and hard. The best proliferation effect was observed at a concentration of 2.0 mg / L, with a callus proliferation coefficient reaching 2.20; the callus was dense and whitish in color. Figure 4 ).

[0119] Table 6. Effects of different concentrations of NAA on callus proliferation

[0120]

[0121] The effects of different concentrations of KT on callus proliferation are shown in Table 7 and Figure 5 .

[0122] KT had no significant effect on callus proliferation from *Hypericum sylvestris* leaves (Table 7). After 30 days of culture, with increasing KT concentration, the callus gradually vitrified and died. The callus proliferation coefficient was lowest at a concentration of 4.0 mg / L, and extensive browning occurred. At a low concentration of 0.5 mg / L, the callus proliferation coefficient was the highest at 1.59, and the callus had a strong granular texture and appeared yellowish-green. Figure 5 ).

[0123] Table 7. Effects of different concentrations of KT on callus proliferation.

[0124]

[0125] In summary, among the single-factor assays for callus proliferation, 6-BA showed the best effect, producing a high callus proliferation coefficient and a strong granular texture. NAA was the next best, producing a firmer, whiter callus with a high proliferation coefficient. KT had no significant effect on callus proliferation, while 2,4-D resulted in a high callus proliferation coefficient but also a high degree of vitrification.

[0126] Single-factor experiments were conducted to determine that 6-BA and NAA had the greatest effect on callus proliferation, and experiments were carried out using different concentrations of the two hormones. Following the single-factor experiments, a two-factor experiment was conducted to investigate the effects of 6-BA and NAA on callus proliferation from *Haloxylon ammodendron* leaves. The concentrations of the two hormones were divided at 1.0 mg / L: 1.0–2.0 mg / L was designated as the high-concentration experimental group, and 0.5–1.0 mg / L as the low-concentration experimental group. Details are as follows.

[0127] Example 4: Effects of different concentrations of 6-BA and NAA combinations on the subculture proliferation of callus tissue from *Haloxylon ammodendron* leaves.

[0128] Callus induced from *Hypericum esculentum* explants was subcultured 1-2 times to obtain proliferated callus tissue, which was then used as experimental material to improve the callus proliferation experiment of *Hypericum esculentum* leaves.

[0129] K1 consists of WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0130] K2 consists of WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 1.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0131] K3 consists of WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 2.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0132] K4 consists of WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0133] K5 consists of WPM as the basic culture medium, supplemented with 1.5 mg / L 6-BA, 1.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0134] K6 consists of WPM as the basic medium, supplemented with 1.5 mg / L 6-BA, 2.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0135] K7 consists of WPM as the basic medium, supplemented with 2.0 mg / L 6-BA and 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0136] K8 consists of WPM as the basic culture medium, supplemented with 2.0 mg / L 6-BA, 1.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0137] K9 consists of WPM as the basic medium, supplemented with 2.0 mg / L 6-BA, 2.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0138] K10 consists of WPM as the basic culture medium, supplemented with 0.5 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0139] K11 consists of WPM as the basic medium, supplemented with 0.5 mg / L 6-BA, 0.8 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0140] K12 consists of WPM as the basic medium, supplemented with 0.5 mg / L 6-BA, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0141] K13 consists of WPM as the basic medium, supplemented with 0.8 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0142] K14 consists of WPM as the basic medium, supplemented with 0.8 mg / L 6-BA, 0.8 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0143] K15 consists of WPM as the basic medium, supplemented with 0.8 mg / L 6-BA, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0144] K16 consists of WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0145] K17 consists of WPM as the basic medium, supplemented with 1.0 mg / L 6-BA, 0.8 mg / L NAA, 30 g / L sucrose and 8 g / L agar;

[0146] K18 consists of WPM as the basic culture medium, supplemented with 1.0 mg / L 6-BA, 1.0 mg / L NAA, 30 g / L sucrose and 8 g / L agar.

[0147] Each treatment was inoculated into 20 culture dishes, with one callus mass inoculated into each dish, and repeated 3 times (a total of 60 culture dishes were inoculated). The callus proliferation coefficient and texture were calculated after 30 days. The results are shown in Tables 8 and 9.

[0148] Table 8. Effects of high concentrations of 6-BA and NAA on callus proliferation in *Hypericum sieboldii* leaves.

[0149]

[0150] Table 9. Effects of low concentrations of 6-BA and NAA on callus proliferation in *Hypericum sieboldii* leaves.

[0151]

[0152] It can be seen that in the high-concentration experimental group, the callus proliferation coefficient continuously increased with the increase of 6-BA and NAA concentrations, but the callus quality gradually decreased. When both were at low concentrations, the callus proliferation coefficient and texture were relatively good (Table 8). Figure 6 Table 8 shows that in the low-concentration experimental groups, when the 6-BA concentration was 0.5 mg / L, the callus proliferation coefficient gradually increased with increasing NAA concentration. When the 6-BA concentration was 0.8 mg / L, the callus proliferation coefficient showed a trend of first increasing and then decreasing with increasing NAA concentration. When the 6-BA concentration was 1.0 mg / L, the callus proliferation coefficient continuously decreased with increasing NAA concentration (Table 9). Adding 1.0 mg / L 6-BA + 0.5 mg / L NAA to the WPM basal medium resulted in the highest callus proliferation coefficient of *Haloxylon ammodendron* leaves, reaching 6.60, with good texture and a yellowish-white color (see Table 9). Figure 7 ).

[0153] In summary, plant growth regulators play a crucial role in regulating plant cell growth and differentiation during tissue culture. Common plant growth regulators include auxins such as NAA and IAA, and cytokinins such as 6-BA, KT, and TDZ. Through multiple single-factor and two-factor regulation experiments on callus tissue from *Haloxylon ammodendron* leaves, the single-factor results showed that 6-BA > NAA > 2, 4-D > KT in callus proliferation. Therefore, in the two-factor experiment, 6-BA and NAA were selected as the two plant growth regulators. Using 1.0 mg / L as the cutoff, the combined effects of high and low concentrations of the two regulators on callus proliferation were investigated. The experiment revealed that the callus condition and proliferation coefficient were better at low concentrations. Adding 1.0 mg / L 6-BA + 0.5 mg / L NAA to the basal culture medium resulted in the highest callus proliferation coefficient of 6.60 from *Haloxylon ammodendron* leaves, with a brittle texture, strong granular feel, and a yellowish-white color.

[0154] 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. A method for culturing leaf callus of Hypericum ascyron, characterized by, The method comprises the following steps: The leaf of the silverleaf rockfoil with incision is inoculated into an induction medium to induce callus, and then the callus is placed into a proliferation medium to perform proliferation culture, so as to obtain the callus; The induction medium takes WPM as a basic medium, and further adds 0.2-1.0 mg / L 6-BA, 0.5-1.5 mg / L 2,4-D, 0.1-1.0 mg / L NAA, 29-30 g / L sucrose and 7-8 g / L agar; and the proliferation medium takes WPM as a basic medium, and further adds 0.5-1.0 mg / L 6-BA, 0.5-1.0 mg / L NAA, 25-30 g / L sucrose and 7-8 g / L agar.

2. The culture method according to claim 1, characterized by, The initial pH value of the induction medium is 5.7-6.0, and the initial pH value of the proliferation medium is 5.7-6.

0.

3. The culturing method according to claim 1, wherein, Before inoculation, the leaf further comprises disinfection, and the disinfection comprises the following steps: first, soaking in 75% ethanol with a volume concentration for 25-35 s, washing with sterile water for 3-4 times, then soaking in a sodium hypochlorite solution with a mass concentration of 0.21%-0.30% for 6-10 min, and washing with sterile water for 5-6 times.

4. The culturing method according to claim 1, wherein The temperature of the induction culture and the proliferation culture is 23-27 ℃ respectively.

5. The culturing method according to claim 1, wherein The induction culture and the proliferation culture are light and dark alternating culture respectively.

6. The culture method according to claim 5, wherein The light time in the light and dark alternating culture is 14-16 h / d respectively; the light intensity is 2100-2600 lx respectively; and the dark time in the light and dark alternating culture is 8-10 h / d respectively.

7. The culture method according to claim 1 or 5, wherein The time of the induction culture is 28-35 d; and the time of the proliferation culture is 28-45 d.

8. The culture method according to claim 1 or 5, characterized by, Before the proliferation culture, the callus obtained by the induction culture is further subcultured.

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