Tissue culture method for inducing cluster buds from epimedium villosum leaves

Abstract

The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a tissue culture method for inducing cluster buds from epimedium hairy leaves, which comprises the following steps: disinfecting and segmenting the epimedium hairy leaves as explants, inoculating the explants into a callus induction culture medium, and carrying out dark culture to induce calluses; when a bud primordium is differentiated from the callus, subculturing the bud primordium into the callus induction culture medium again for dark culture; inoculating the callus with the bud primordium into a bud induction culture medium, carrying out dark culture for 7-9 days, then carrying out light culture for 30-50 days under the illumination intensity of 1000lx-2000lx, and inducing bud differentiation; and sequentially inoculating the induced buds into a bud proliferation culture medium and a rooting culture medium for culture to obtain tissue culture seedlings. According to the method disclosed by the invention, the epimedium hairy leaves are taken as explants, and the leaves can be directly induced to germinate under specific culture conditions, so that an efficient and stable tissue culture and rapid propagation system of the epimedium hairy is established.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant tissue culture and rapid propagation, and particularly relates to a leaf explant induction of clustered buds of Epimedium pubescens. BACKGROUND

[0002] As a traditional Chinese medicinal material recorded in Shennong's Herbal Classic, Epimedium pubescens is one of the four main sources of Epimedium, and has the effects of tonifying kidney and yang, strengthening muscles and bones, and dispelling wind and dampness. Modern studies have shown that flavonoids (such as icariin) contained in Epimedium pubescens have significant effects on anti-osteoporosis, improving cardiovascular function, and anti-tumor. However, due to long-term overexploitation, the natural population of Epimedium pubescens has been on the brink of extinction, which not only threatens the survival of the species, but also seriously restricts the development of modern medicine. In order to break this resource bottleneck, it is urgent to develop an efficient and stable artificial propagation technology of Epimedium pubescens.

[0003] Currently, the main tissue culture method of Epimedium pubescens relies on seeds, immature embryos or stem tips as explants, but there are the following problems: first, the acquisition of explants is seasonally limited, and seeds and embryos need to be collected according to seasons, and the number of stem tips is limited; second, the traditional tissue culture process is inefficient, it is difficult to break the seed dormancy, the efficiency of inducing clustered buds using seeds or stem tips is not high, the proliferation coefficient is not enough to meet the needs of large-scale production, and the operation steps are complicated and the cycle is long (the seed germination period is as long as more than 3 months), and long-term induction and subculture of callus is prone to genetic variation, which is not conducive to the preservation of excellent varieties, and limits the large-scale propagation and sustainable use of resources of Epimedium pubescens. SUMMARY

[0004] In order to solve the above problems, the present application provides a tissue culture method for inducing clustered buds of Epimedium pubescens leaf explants, which increases the differentiation rate of Epimedium pubescens clustered buds to more than 90% and the proliferation coefficient to more than 5 under the premise of maintaining cell genetic stability, and shortens the conventional culture period of more than 6 months to 126 days.

[0005] To achieve the above purpose, the specific technical solutions of the present application are as follows: The first aspect of the present application provides a tissue culture method for inducing clustered buds of Epimedium pubescens leaf explants, comprising the following steps: The leaf explants of Epimedium pubescens are divided into tissue blocks after being sterilized; The tissue blocks are inoculated into callus induction medium, and callus is induced by dark culture at 24-26 DEG C; When the callus differentiates into bud primordia, the callus with bud primordia is subcultured into callus induction medium, and dark culture is carried out at 23-27 DEG C for 12-15 days; The bud primordium of the callus is inoculated into bud induction medium, and after dark culture at 23-27°C for 7-9 days, light culture is conducted for 30-50 days to induce bud differentiation. The bud induction medium takes WPM medium as a basic medium, and further includes 1.0-2.5 mg 6-BA, 0.1-0.5 mg NAA, 30 g sucrose and 6 g agar per liter of the medium. The induced bud is inoculated into bud proliferation medium, and light culture is conducted at 23-27°C for 30-50 days to obtain multiple buds. The proliferated multiple buds are cut into single buds, which are inoculated into rooting medium to obtain tissue culture seedlings.

[0006] Further, the callus is inoculated into callus induction medium when the callus differentiates into bud primordium and after 12-15 days of culture.

[0007] Further, the leaf is a leaf with 3-5 fully expanded leaves at the top of the Epimedium pubescens plant.

[0008] Further, the leaf is a leaf with initial leatherization but not fully hardened.

[0009] Further, the callus induction medium takes WPM medium as a basic medium, and further includes 1.0-3.0 mg 2,4-D, 1.0-3.0 mg 6-BA, 30 g sucrose and 6 g agar per liter of the medium.

[0010] Further, the bud proliferation medium takes WPM medium as a basic medium, and further includes 1.0-2.5 mg 6-BA, 0.1-0.5 mg NAA, 30 g sucrose and 6 g agar per liter of the medium.

[0011] Further, the rooting medium takes 1 / 2MS medium as a basic medium, and further includes 0.1-1 mg NAA and 0.05-0.1 g activated carbon per liter of the medium.

[0012] Further, the rooting medium is cultured at a temperature of 24-26°C, under an illumination intensity of 1000-2000 lx, and for a culture time of 20-50 days.

[0013] Further, the light culture is conducted under an illumination intensity of 1000-2000 lx.

[0014] Further, the tissue block is a tissue block with a size of 1-1.5 cm×1-1.5 cm.

[0015] Furthermore, the disinfection steps are as follows: soaking in 70% ethanol solution for 30-45 seconds, soaking in 2% sodium hypochlorite solution for 10-12 minutes, and rinsing with sterile water.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a tissue culture method for inducing shoot clusters from Epimedium pubescens leaves. The method includes the following steps: using Epimedium pubescens leaves as explants, the explants are disinfected, divided, and inoculated into callus induction medium, and cultured in the dark at 24℃~26℃ to induce callus tissue; when the callus tissue differentiates into shoot primordia, it is inoculated again into callus induction medium and cultured in the dark for 12d~15d; then the callus tissue with shoot primordia is inoculated into shoot induction medium and cultured in the dark for 7d~9d, followed by light culture for 30d~50d to induce shoot differentiation; the induced shoots are sequentially inoculated into shoot proliferation medium and rooting medium for culture to obtain tissue culture seedlings. The method provided by this invention is as follows: (1) It uses Epimedium pubescens leaves as explants, which are widely available, easy to obtain, and have high availability throughout the year; (2) It maintains genetic stability: it can directly induce buds from the leaves, avoiding the problem of gene mutation that easily occurs during long-term callus induction and passage; (3) It achieves efficient induction and rapid proliferation: it increases the differentiation rate of Epimedium pubescens clustered buds to over 90%, increases the proliferation coefficient to over 5, and shortens the conventional culture cycle of over 6 months to about 4 months (126 days); (4) It is easy to operate: it uses conventional culture containers and culture medium components, and the operation process is simple. This method is easy to promote and apply in Chinese medicinal material production bases. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Images show the experimental process of inducing shoot clusters from leaves of Epimedium brevicornu. Figure 1 In this context, A represents the induced callus tissue. Figure 1 In the image, B represents the initial appearance of red buds in the callus tissue. Figure 1 C in the text represents the further development of red buds in the callus tissue. Figure 1 In the diagram, D represents the gradual increase in red buds within the callus tissue. Figure 1 In the diagram, E represents a small cluster of young shoots induced from red buds. Figure 1 F in the diagram represents a clustered bud with more pronounced development. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0020] Epimedium pubescens is a traditional Chinese medicine, one of the four major sources of Epimedium, and is known for its kidney-tonifying and aphrodisiac effects. Its flavonoid components have significant applications in multiple fields. However, due to over-harvesting, its natural population is nearing depletion, threatening the species' survival and modern pharmaceutical development. Current tissue culture methods for Epimedium use explants such as seeds, embryos, and stem tips, but these methods suffer from seasonal limitations, low propagation efficiency, long cycles, and susceptibility to gene mutations, hindering large-scale propagation and resource utilization. Therefore, developing efficient artificial propagation techniques for Epimedium pubescens is urgently needed.

[0021] This invention provides a tissue culture method for inducing shoot clusters from Epimedium pubescens leaves, comprising the following steps: using Epimedium pubescens leaves as explants, the explants are disinfected, divided, and inoculated into callus induction medium, and cultured in the dark to induce callus tissue; when the callus tissue differentiates into shoot primordia, it is subcultured again into callus induction medium and cultured in the dark; the callus tissue with shoot primordia is inoculated into shoot induction medium and cultured in the dark for 7-9 days, and then cultured in light at a light intensity of 1000-2000 lx for 30-50 days to induce shoot differentiation; the induced shoots are sequentially inoculated into shoot proliferation medium and rooting medium for culture to obtain tissue culture seedlings. This invention uses Epimedium pubescens leaves as explants and, through specific culture conditions, can directly induce shoots from the leaves, establishing an efficient and stable tissue culture rapid propagation system for Epimedium pubescens.

[0022] Example 1: A tissue culture method for inducing clustered shoots from Epimedium brevicornu leaves, comprising the following steps: 1. Culture medium preparation (1) Callus induction medium (1L): WPM 2.68g + 2,4-D 1.0mg + 6-BA 1mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0023] (2) Bud induction medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.3mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0024] (3) Bud proliferation medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.5mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0025] (4) Rooting medium (1L): 1 / 2MS + 0.1mg NAA + 0.05g activated carbon.

[0026] 2. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0027] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0028] 3. Callus induction culture: Under aseptic conditions in a laminar flow hood, sterile leaves were divided into 1cm × 1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into callus induction medium using sterile forceps and cultured in the dark at 25℃ for 15 days. The callus differentiated into shoot primordia. The callus with shoot primordia was then subcultured into callus induction medium and cultured in the dark at 25℃ for 15 days. Figure 1 ).

[0029] 4. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 25℃ for 8 days. Then, it is cultured under weak light conditions with a light intensity of 1500 lx for another 30 days to induce bud differentiation.

[0030] 5. Bud subculture: The induced buds are transferred to bud proliferation culture and cultured for 30 days under weak light conditions of 25℃ and light intensity of 1500lx to obtain clustered buds.

[0031] 6. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm, inoculate them onto rooting medium, and culture them under weak light conditions of 25℃ and light intensity of 1500lx to obtain tissue culture seedlings.

[0032] Example 2: A tissue culture method for inducing clustered shoots from Epimedium brevicornu leaves, comprising the following steps: 1. Culture medium preparation (1) Callus induction medium (1L): WPM 2.68g + 2,4-D 1.0mg + 6-BA 1mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0033] (2) Bud induction medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.3mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0034] (3) Bud proliferation medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.5mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0035] (4) Rooting medium (1L): 1 / 2MS + 0.1mg NAA + 0.05g activated carbon.

[0036] 2. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0037] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 35 seconds, soak them in 2% v / v sodium hypochlorite solution for 12 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0038] 3. Callus induction culture: Under aseptic conditions in a clean bench, the sterilized leaves were divided into 0.5cm×0.5cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into callus induction medium using sterile forceps and cultured in the dark at 24℃ for 14 days. The callus differentiated into bud primordia. The callus with bud primordia was subcultured into callus induction medium and cultured in the dark at 27℃ for 12 days.

[0039] 4. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 23℃ for 9 days. Then, it is cultured under weak light conditions with a light intensity of 1000 lx for another 40 days to induce bud differentiation.

[0040] 5. Bud subculture: The induced buds are transferred to bud proliferation culture and cultured for 40 days under weak light conditions of 27℃ and light intensity of 1000lx to obtain clustered buds.

[0041] 6. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm, inoculate them onto rooting medium, and culture them under weak light conditions of 26℃ and light intensity of 1000lx to obtain tissue culture seedlings.

[0042] Example 3: A tissue culture method for inducing clustered shoots from Epimedium brevicornu leaves, comprising the following steps: 1. Culture medium preparation (1) Callus induction medium (1L): WPM 2.68g + 2,4-D 1.0mg + 6-BA 1mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0043] (2) Bud induction medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.3mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0044] (3) Bud proliferation medium (1L): WPM 2.68g + 6-BA 1mg + NAA 0.5mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0045] (4) Rooting medium (1L): 1 / 2MS + 0.1mg NAA + 0.05g activated carbon.

[0046] 2. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0047] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 45 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0048] 3. Callus induction culture: Under aseptic conditions in a clean bench, the sterilized leaves were divided into 1.5cm×1.5cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into callus induction culture medium using sterile forceps and cultured in the dark at 26℃ for 12 days to induce shoot primordia. The callus with shoot primordia was then subcultured into callus induction culture medium and cultured in the dark at 23℃ for 13 days.

[0049] 4. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 27℃ for 7 days. Then, it is cultured under weak light conditions with a light intensity of 2000 lx for another 50 days to induce bud differentiation.

[0050] 5. Bud subculture: The induced buds are transferred to bud proliferation culture and cultured for 50 days under weak light conditions of 23℃ and light intensity of 2000lx to obtain clustered buds.

[0051] 6. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm, inoculate them onto rooting medium, and culture them under weak light conditions of 24℃ and 2000lx light intensity to obtain tissue culture seedlings.

[0052] Example 4: A tissue culture method for inducing clustered shoots from Epimedium brevicornu leaves, comprising the following steps: 1. Culture medium preparation (1) Callus induction medium (1L): WPM 2.68g + 2,4-D 2.0mg + 6-BA 3mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0053] (2) Bud induction medium (1L): WPM 2.68g + 6-BA 2.5mg + NAA 0.5mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0054] (3) Bud proliferation medium (1L): WPM 2.68g + 6-BA 2.5mg + NAA 0.3mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0055] (4) Rooting medium (1L): 1 / 2MS + 1mg NAA + 0.05g activated carbon.

[0056] 2. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0057] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0058] 3. Callus induction culture: Under aseptic conditions in a clean bench, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into callus induction culture medium using sterile forceps and cultured in the dark at 25℃ for 15 days. The callus differentiated into bud primordia. The callus with bud primordia was subcultured into callus induction culture medium and cultured in the dark at 25℃ for 15 days.

[0059] 4. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 25℃ for 8 days. Then, it is cultured under weak light conditions with a light intensity of 1500 lx for another 30 days to induce bud differentiation.

[0060] 5. Bud subculture: The induced buds are transferred to bud proliferation culture and cultured for 30 days under weak light conditions of 25℃ and light intensity of 1500lx to obtain clustered buds.

[0061] 6. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm, inoculate them onto rooting medium, and culture them under weak light conditions of 25℃ and light intensity of 1500lx to obtain tissue culture seedlings.

[0062] Example 5: A tissue culture method for inducing clustered shoots from Epimedium brevicornu leaves, comprising the following steps: 1. Culture medium preparation (1) Callus induction medium (1L): WPM 2.68g + 2,4-D 3.0mg + 6-BA 2mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0063] (2) Bud induction medium (1L): WPM 2.68g + 6-BA 1.5mg + NAA 0.1mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0064] (3) Bud proliferation medium (1L): WPM 2.68g + 6-BA 1.5mg + NAA 0.1mg + sucrose 30g + agar 6g, pH adjusted to 5.8.

[0065] (4) Rooting medium (1L): 1 / 2MS + 0.5mg NAA + 0.1g activated carbon.

[0066] 2. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0067] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0068] 3. Callus induction culture: Under aseptic conditions in a clean bench, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into callus induction culture medium using sterile forceps and cultured in the dark at 25℃ for 15 days. The callus differentiated into bud primordia. The callus with bud primordia was subcultured into callus induction culture medium and cultured in the dark at 25℃ for 15 days.

[0069] 4. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 25℃ for 8 days. Then, it is cultured under weak light conditions with a light intensity of 1500 lx for another 30 days to induce bud differentiation.

[0070] 5. Bud subculture: The induced buds are transferred to bud proliferation culture and cultured for 30 days under weak light conditions of 25℃ and light intensity of 1500lx to obtain clustered buds.

[0071] 6. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm, inoculate them onto rooting medium, and culture them under weak light conditions of 25℃ and light intensity of 1500lx to obtain tissue culture seedlings.

[0072] Comparative Example 1: A tissue culture method for inducing clustered shoots from leaves of Epimedium brevicornu, comprising the following steps: 1. Selection and disinfection of explants Leaf collection: Select tender leaves of Epimedium brevicornu that are reddish and soft to the touch, and rinse them under running tap water to remove surface dust before use.

[0073] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0074] 2. Induction and culture of callus: Under aseptic conditions, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into the callus induction medium prepared in Example 1 using sterile forceps and cultured in the dark at 25°C for 15 days. Then, the induced callus was transferred to the callus induction medium and cultured in the dark at 25°C for 15 days.

[0075] 3. Bud induction culture: The callus tissue was transferred to the bud proliferation medium prepared in Example 1 and cultured under weak light conditions of 25°C and light intensity of 1500 lx.

[0076] Results: The dehydration and curling rate of explants was >60%, the tissue gradually turned brown, and the surviving explants only formed transparent water-soaked callus tissue without bud differentiation.

[0077] Comparative Example 2: A tissue culture method for inducing clustered shoots from leaves of Epimedium brevicornu, comprising the following steps: 1. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0078] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0079] 2. Induction and culture of callus: Under aseptic conditions, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were then inoculated into the callus induction culture medium prepared in Example 1 using sterile forceps and cultured in the dark at 25°C for 15 days to induce callus.

[0080] 3. Bud induction culture: The callus tissue was transferred to the bud induction culture medium prepared in Example 1 and cultured in the dark for 8 days. Then, it was moved to a low light condition of 25°C and light intensity of 1500 lx and cultured for another 30 days to induce bud differentiation.

[0081] 4. Bud subculture: The induced buds were transferred to the bud proliferation culture prepared in Example 1 and cultured for 30 days under weak light conditions of 25℃ and light intensity of 1500lx. The experimental results were then observed.

[0082] Results: Callus tissue appeared at the edge of the explant, but the callus tissue grew slowly and turned brown (browning rate 85%), and did not form a complete bud.

[0083] Comparative Example 3: A tissue culture method for inducing clustered shoots from leaves of Epimedium brevicornu, comprising the following steps: 1. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0084] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0085] 2. Induction and culture of callus: Under aseptic conditions, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into the callus induction medium prepared in Example 1 using sterile forceps. The callus was cultured in the dark at 25°C for 15 days to differentiate into bud primordia. The callus with bud primordia was then subcultured into the callus induction medium and cultured in the dark at 25°C for 15 days.

[0086] 3. Bud induction culture: When the red bud primordia protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium prepared in Example 1 and cultured in the dark for 30 days. Then, it is transferred to a low light condition of 25°C and light intensity of 1500 lx for culture.

[0087] Results: The tissue around the formed red buds gradually turned brown, and the buds could not grow after being transferred to the proliferation medium, with a browning rate of over 70%.

[0088] Comparative Example 4: A tissue culture method for inducing clustered shoots from leaves of Epimedium brevicornu, comprising the following steps: Shoot proliferation medium (1L): WPM 2.68g + 6-BA 1.0mg + NAA 1.0mg, pH adjusted to 5.8.

[0089] 1. Selection and disinfection of explants Leaf collection: Select the 3rd to 5th fully unfolded, dark green leaves at the top of the Epimedium plant that are beginning to show leatheriness but not yet fully hardened, and rinse off the surface dust under tap water for later use.

[0090] Leaf disinfection: Disinfect the collected leaves in 70% v / v ethanol solution for 30 seconds, soak them in 2% v / v sodium hypochlorite solution for 10 minutes, rinse them 5 times with sterile water, and place them on sterile filter paper to absorb the moisture.

[0091] 2. Induction and culture of callus: Under aseptic conditions, the sterilized leaves were divided into 1cm×1cm tissue blocks using a sterile scalpel. The tissue blocks were inoculated into the callus induction medium prepared in Example 1 using sterile forceps. The callus was cultured in the dark at 25°C for 15 days to differentiate into bud primordia. The callus with bud primordia was then subcultured into the callus induction medium and cultured in the dark at 25°C for 15 days.

[0092] 3. Bud induction culture: When red buds protrude from the callus tissue, the tissue block is transferred to the bud induction culture medium and cultured in the dark at 25℃ for 8 days. Then, it is cultured under weak light conditions with a light intensity of 1500 lx for another 30 days to induce bud differentiation.

[0093] 4. Bud subculture: The cultured buds are transferred to bud proliferation medium and cultured for 30 days under weak light conditions of 25℃ and light intensity of 1500lx to obtain clustered buds.

[0094] 5. Rooting culture: Under aseptic conditions, cut off individual buds that have grown to 3cm~5cm and inoculate them onto the rooting culture medium prepared in Example 1. Culture them under weak light conditions of 25℃ and light intensity of 1500lx to obtain tissue culture seedlings.

[0095] Results: A large amount of callus was produced at the base of the bud (callus index 3.2), which turned brown and lignified. The bud proliferation coefficient was only 1.8±0.3, and the height of the new buds was stagnant at 1cm~2cm.

[0096] Examples 1-5 have similar effects. For ease of subsequent discussion and reference, the experimental results of Example 1 are used as an example. This invention statistically analyzes the tissue culture results of Example 1 and Comparative Examples 1-4. The statistical indicators are the differentiation rate of clustered shoots, the proliferation coefficient, the browning / vitrification rate, the proportion of effective shoots, and the culture cycle.

[0097] Cluster shoot differentiation rate (%) = number of shoot-forming explants / number of surviving explants × 100%; Proliferation coefficient = Number of new shoots / Number of inoculated shoots; Effective buds are those that are ≥3cm in height, have dark green leaves, and are free from deformities; Data are the mean ± SD of three replicate experiments (n = 30 bottles / group).

[0098] Table 1. Effects of different tissue culture methods on the induction of clustered shoots from leaves of Epimedium brevicornu. Note: - indicates that the corresponding indicator is not included in the statistics.

[0099] The results are shown in Table 1. Example 1 showed the best performance in terms of shoot differentiation rate, proliferation coefficient, effective shoot ratio, and culture period, and had the lowest browning / vitrification rate. This indicates that the tissue culture method provided by the present invention can successfully increase the differentiation rate of shoots of Epimedium pubescens to over 90%, increase the proliferation coefficient to over 5, and shorten the conventional culture period of more than 6 months to about 4 months (126 days), avoiding the problem of gene mutation that is prone to occur during long-term callus induction and passage.

[0100] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0101] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A tissue culture method for inducing clustered shoots from leaves of Epimedium brevicornu, characterized in that, Includes the following steps: Using leaves of Epimedium pubescens as explants, the explants were disinfected and divided into tissue blocks; Tissue blocks were inoculated into callus induction medium and cultured in the dark at 24℃~26℃ to induce callus tissue. When the callus differentiates into bud primordia, the callus with bud primordia is subcultured into the callus induction medium and cultured in the dark at 23℃~27℃ for 12d~15d. Callus tissue with bud primordia was inoculated into bud induction medium and cultured in the dark at 23℃~27℃ for 7d~9d, followed by light culture for 30d~50d to induce bud differentiation. Each liter of the bud induction medium uses WPM medium as the basic medium and also includes 1.0 mg to 2.5 mg of 6-BA, 0.1 mg to 0.5 mg of NAA, 30 g of sucrose and 6 g of agar; The induced buds were inoculated into bud proliferation medium and cultured under light at 23℃~27℃ for 30d~50d to obtain clustered buds; The clustered buds were cut into single buds and inoculated onto rooting medium to obtain tissue culture seedlings.

2. The tissue culture method according to claim 1, characterized in that, The leaves in question are the 3rd to 5th fully unfolded leaves at the top of the Epimedium brevicornu plant.

3. The tissue culture method according to claim 2, characterized in that, The blades are those that have begun to show signs of leatheriness but have not yet fully hardened.

4. The tissue culture method according to claim 1, characterized in that, Each liter of the callus induction medium uses WPM medium as the basal medium and also includes 1.0 mg to 3.0 mg of 2,4-D, 1.0 mg to 3.0 mg of 6-BA, 30 g of sucrose and 6 g of agar.

5. The tissue culture method according to claim 1, characterized in that, Each liter of the bud proliferation medium is based on WPM medium and also includes 1.0 mg to 2.5 mg of 6-BA, 0.1 mg to 0.5 mg of NAA, 30 g of sucrose and 6 g of agar.

6. The tissue culture method according to claim 1, characterized in that, Each liter of the rooting medium contains 1 / 2 MS medium as the basic medium, and also includes 0.1 mg to 1 mg of NAA and 0.05 g to 0.1 g of activated carbon.

7. The tissue culture method according to claim 1, characterized in that, The rooting medium is cultured at a temperature of 24℃~26℃ and a light intensity of 1000lx~2000lx.

8. The tissue culture method according to claim 1, characterized in that, The light intensity for photoculture is 1000 lx to 2000 lx.

9. The tissue culture method according to claim 1, characterized in that, The tissue block is a tissue block measuring 1cm~1.5cm × 1cm~1.5cm.

10. The tissue culture method according to claim 1, characterized in that, The disinfection steps are as follows: soak in 70% ethanol solution for 30-45 seconds, soak in 2% sodium hypochlorite solution for 10-12 minutes, and rinse with sterile water.

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