A method for breeding of cattle ear seedlings and application thereof

Abstract

The application provides a breeding method and application of a young plant of a cow ear. The application adopts a leaf of the cow ear as an explant, optimizes a breeding technical system through induction of germination of double-face clustered buds of the leaf, and obtains a large amount of high-quality young plants. The method can overcome physiological time limitation of plant material taking, and will not affect species quantity and reproduction. Through induction of the double-face clustered buds of the leaf, a new way of asexual reproduction of the plant leaf is explored, utilization efficiency of the explant of the plant tissue culture is increased, and a simple, rapid and effective breeding system of the cow ear is established.

Description

TECHNICAL FIELD

[0001] The present application relates to biotechnology, in particular to a breeding method and application of Chirita eburnea seedling. BACKGROUND

[0002] Chirita eburnea, also known as Chirita eburnea, Shisanqi, Shihutie, Niueryan, Baicai, belongs to Gesneriaceae Chirita, is distributed in northern Guangdong, northern Guangxi, Guizhou, southeastern Hunan, southern and eastern Sichuan, and western Hubei, and is often grown on limestone hills in forests or under forest edges at an altitude of 100 to 1500 meters. It is a perennial herb with thick rhizome, leaves are all basal succulent, leaf blades are ovate or narrow ovate, 3.5-17 cm long and 2-9.5 cm wide, with entire margin, both surfaces covered with appressed short pubescence, sometimes sparsely pubescent on the upper surface. It is named after its leaf shape resembling a cow's ear. According to Guangxi Plant Checklist, Chirita eburnea whole grass or rhizome is a traditional Chinese medicine with the functions of clearing lung, stopping bleeding, detoxifying and tonifying deficiency and stopping bleeding, and is commonly used to treat pulmonary tuberculosis, cough, hemoptysis, and metrorrhagia. Fresh Chirita eburnea rhizome is cut into slices, mixed with honey, and eaten, which has good conditioning effect on qi deficiency and lung phlegm cough. Chirita eburnea rhizome or whole grass can also be used to treat cough, hemoptysis, and metrorrhagia. Chirita eburnea is not only a good medicine, but also a favorite of people due to its unique and bright flowers, long flowering period, evergreen, and beautiful shape, which has high ornamental and breeding value.

[0003] At present, due to the low natural propagation ability of Chirita eburnea itself, random collection and excavation, and continuous deterioration of the ecological environment in limestone areas, the number of wild Chirita eburnea individuals has been decreasing year by year, and protection work is urgently needed.

[0004] The literature (In vitro culture and rapid propagation of *Auricularia auricula-judae*, Plant Physiology Communications, 2006, 42(5):1) discloses a method for obtaining test-tube seedlings by using young leaves and flower stalks as explants for tissue culture. Among them, the callus induction rate of flower stalks reached 80%, and the callus induction rate of young leaves was 73%. Adventitious buds appeared on the dense, bright green callus tissue of both explants after about 30 days. Using flower stalks as explants can obtain more satisfactory results. The literature (In vitro culture and rapid propagation of *Auricularia auricula-judae*, Plant Physiology Communications, 2008, 44(2):2) discloses a method for obtaining test-tube seedlings by using flower stalks, bracts and young flower buds of *Auricularia auricula-judae* as explants for tissue culture. The induction pathway of regenerated plants of *Auricularia auricula-judae* in the literature is: callus tissue - adventitious bud induction - callus tissue - adventitious bud propagation. The whole process needs to go through the callus tissue stage, and the callus induction rate is 55%-76%. Using flower stalks as explants has significant drawbacks regarding the timing and location of collection. Firstly, flower stalks are reproductive organs and can only be collected during the plant's flowering period; collection at other times is impossible. Secondly, as the primary organ for plant reproduction, collecting flower stalks prevents pollination and fruit setting, impacting plant species and genetic diversity, and hindering biodiversity conservation. Using leaves as explants can effectively address these issues, but low propagation rates and long propagation cycles still exist. Summary of the Invention

[0005] This invention provides a method for propagating *C. cirrhosa* seedlings. Using *C. cirrhosa* leaves as explants, the propagation technology system is optimized by inducing the germination of double-sided clustered buds on the leaves, resulting in a large number of high-quality seedlings.

[0006] This invention also provides a method for breeding *Cephalotaxus fortunei* seedlings and its application in seedling propagation. Conducting research on the breeding and application of *Cephalotaxus fortunei* can not only stabilize the population size of this species and achieve resource protection, laying the foundation for its sustainable development and utilization, but also enhance the stability and resilience of the ecosystem. This has significant ecological and practical implications for biodiversity conservation and the protection of the Yangtze River.

[0007] The first aspect of this invention provides a method for propagating *Cephalotaxus fortunei* seedlings, comprising the following steps:

[0008] 1) Explant pretreatment: Select leaves of *Cephalotaxus fortunei* as explants, clean and disinfect them, and then blot dry with sterile filter paper;

[0009] 2) Induction culture: The pretreated explants were cut into blocks of (1.0-2.0)cm × (1.0-2.0)cm and inoculated into the first culture medium for induction culture to obtain induction culture with double-sided clustered buds;

[0010] The first culture medium includes at least three of the following: KT, TDZ, NAA, ε-polylysine, and GA3. The culture method includes: dark culture for days 1-7, and light culture after day 8, with a culture temperature of 23℃±2℃ and a culture time of 20-25 days. The light culture specifically includes: LED red and white composite light culture for days 8-15, and fluorescent lamp culture after day 15, with a light intensity of 2500-3000 lx and a light duration of 10-13 hours per day during the light culture period.

[0011] 3) Proliferation culture: Remove the induced culture from the first culture medium and cut it in a "+" shape along a direction perpendicular to the leaf surface to obtain 4 first cut parts; then cut the first cut parts horizontally along a direction parallel to the leaf surface to obtain 2 second cut parts containing single-sided clustered buds; inoculate the second cut parts containing single-sided clustered buds into the first culture medium for proliferation culture to obtain the proliferation culture;

[0012] 4) Seedling strengthening and rooting culture: The proliferation culture was taken out from the first culture medium and inoculated into the second culture medium for seedling strengthening and rooting culture. After the culture was completed, the cow ear seedlings were obtained.

[0013] This invention uses leaves of *Cephalotaxus fortunei* as explants, which can be obtained at any time of year without affecting the species’ quantity and reproduction. The invention optimizes the breeding technology system by inducing the growth of clustered buds on both sides of the leaves, and provides a method for breeding the medicinal plant *Cephalotaxus fortunei* by inducing the growth of clustered buds on both sides of the leaves, resulting in a large number of high-quality seedlings.

[0014] In one specific embodiment, the method for breeding cow ear seedlings provided by the present invention specifically includes the following steps:

[0015] Step 1, Explant pretreatment: Select leaves of *Cephalotaxus fortunei* as explants, clean and disinfect them, and then blot dry with sterile filter paper.

[0016] According to an embodiment of the present invention, selecting cow ear leaves specifically includes the following steps:

[0017] For wild or greenhouse-grown *Cephalotaxus fortunei* plants, following the principle of prioritizing young and tender leaves and pruning appropriately, the leaves of 1-3 month old plants are cut from the base and then cut along a direction basically perpendicular to the main vein of the leaf to obtain the lower part of the leaves near the base, the upper part of the leaves away from the base, and the middle part of the leaves located between the upper and lower parts of the leaves.

[0018] Because explants collected from the natural environment carry a large number of microorganisms on their surface, they need to be cleaned and disinfected to reduce the probability of culture failure due to culture medium contamination. However, during the cleaning and disinfection process, the upper and lower leaves are more susceptible to damage than the middle leaves after being soaked in the disinfectant. Therefore, this invention uses the middle leaves as explants to reduce the adverse effects of leaf damage on the experiment.

[0019] Furthermore, the middle leaves cut from the *Cephalotaxus fortunei* plant are cleaned and disinfected, specifically including the following steps:

[0020] Step 1-1: Wrap the explant in gauze and soak it in laundry detergent water for 3-6 minutes. Then remove the explant and rinse it under running water for 30-50 minutes. Wrapping the explant in gauze prevents it from floating in the liquid, which would reduce the cleaning and disinfection effect. Laundry detergent water is a surfactant that can improve the cleaning effect. Rinsing under running water can wash away contaminants and residual laundry detergent water.

[0021] Steps 1-2: Transfer the cleaned explants to a clean bench and immerse them in 75% medical alcohol for 20-40 seconds. After immersion, rinse 1-2 times with sterile water. 75% medical alcohol is a surface disinfectant with strong penetrating power that denatures bacterial proteins and has a strong wetting effect, removing air from the material and facilitating the penetration of other disinfectants. Rinsing with sterile water reduces the damaging side effects of disinfectants on plant cells.

[0022] Steps 1-3: After rinsing with sterile water, immerse the explants in a cleaning solution containing mercuric chloride and Tween 80 for 5-10 minutes. After immersion, rinse 3-5 times with sterile water. Mercuric chloride is a disinfectant; its Hg content is... 2+ It can bind to negatively charged bacterial proteins, causing them to denature. Tween 80 is a surfactant; when added to disinfectants, it helps the disinfectant penetrate the explant surface more easily. The specific ratio of mercuric chloride to Tween 80 can be adjusted according to conventional techniques in the field.

[0023] After cleaning, use sterile filter paper to absorb excess moisture from the explants. Furthermore, cleaning and disinfection can damage the explants; therefore, after cleaning, damaged areas can be removed to obtain pre-treated explants.

[0024] Step 2, Induction Culture: Cut the pretreated explants into blocks of (1.0-2.0)cm × (1.0-2.0)cm and inoculate them into the first culture medium for induction culture to obtain an induced culture with double-sided clustered buds.

[0025] In the present invention, the pretreated explant is cut into blocks of (1.0-2.0)cm × (1.0-2.0)cm, that is, the length of the block is (1.0-2.0)cm and the width is (1.0-2.0)cm. For example, the size of the block can be at least one of 0.5cm × 0.5cm, 1cm × 1cm, 1.5cm × 1.5cm, and 2.0cm × 2.0cm, preferably 2.0cm × 2.0cm.

[0026] In the present invention, the first culture medium, in addition to the nutrients necessary for the growth of *C. brevicornu* (a type of plant), also includes inducing hormones, specifically at least three of the following: KT, TDZ, NAA, ε-polylysine, and GA3. KT (kinetin) is an endogenous cytokinin that promotes cell division, delays senescence of detached leaves, induces bud differentiation and development, and increases stomatal aperture. TDZ (thiabendazole) is a synthetically produced cytokinin that promotes bud differentiation in plants. NAA (naphthaleneacetic acid) is a plant growth regulator with auxin activity, used to promote bud proliferation and growth. ε-polylysine is a natural antibacterial preservative with a broad antibacterial spectrum and good stability. GA3 (gibberellin) is an endogenous plant growth regulator mainly used to promote plant growth and development.

[0027] Furthermore, based on each liter of the first culture medium, the mass of KT is 0.05-0.1 mg, the mass of TDZ is 0.1-0.5 mg, the mass of NAA is 0.5-1.0 mg, the mass of ε-polylysine is 0.01-0.05 g, and the mass of GA3 is 1.0-3.0 mg. It is understood that the above content refers to the content when the first culture medium includes the above components. That is, when the first culture medium includes KT, TDZ and NAA, the mass of KT in the first culture medium is 0.05-0.1 mg, the mass of TDZ is 0.1-0.5 mg, the mass of NAA is 0.5-1.0 mg, and the content of the other two substances is 0; when the first culture medium includes TDZ and NAA, the mass of TDZ in the first culture medium is 0.1-0.5 mg, the mass of NAA is 0.5-1.0 mg, and the content of the other three substances is 0; when the first culture medium includes KT and NAA, the mass of KT in the first culture medium is 0.05-0.1 mg, the mass of NAA is 0.5-1.0 mg, and the content of the other three substances is 0.

[0028] Furthermore, the first culture medium simultaneously includes KT, TDZ, NAA, ε-polylysine, and GA3, and based on each liter of the first culture medium, the mass of KT is 0.05-0.1 mg, the mass of TDZ is 0.1-0.5 mg, the mass of NAA is 0.5-1.0 mg, the mass of ε-polylysine is 0.01-0.05 g, and the mass of GA3 is 1.0-3.0 mg; through the synergistic effect of multiple hormones, it helps to improve the germination efficiency of clustered shoots.

[0029] Considering the normal growth of cow ears, the pH value of the first culture medium is 5.5-6.0.

[0030] Furthermore, the basal medium for the first culture medium is MS medium, which is the most commonly used plant tissue culture medium. It is characterized by high concentrations of inorganic salts and ions, high nitrate content, and suitable quantity and ratio of nutrients, which can meet the nutritional and physiological needs of plant cells. MS medium contains sucrose and agar, and those skilled in the art can adjust the content of sucrose and agar according to the actual situation.

[0031] Based on the cleaned and disinfected explants and the prepared first culture medium, the explants were laid flat in the first culture medium to induce shoot clustering. Research has shown that placing the leaves with the underside facing upwards helps improve the germination efficiency of both lateral buds; the underside of the leaf refers to the lower surface of the leaf facing downwards in its natural growth state.

[0032] The inoculated explants are placed in an incubator for induction culture, which specifically includes the following steps:

[0033] Dark culture was used for days 1-7, and light culture was used after day 8. The culture temperature was 23℃±2℃, and the culture time was 20-25 days. The light culture specifically included: LED red and white composite light culture was used for days 8-15, and fluorescent lamp culture was used after day 15. The light intensity during the light culture period was 2500-3000 lx, and the light time was 10-13 hours per day.

[0034] Dark culture can effectively reduce explant browning, while light culture can ensure normal dedifferentiation and redifferentiation of explants. By combining dark and light culture, it is possible to achieve variable light culture of explants and induce the germination of bifacial clustered shoots.

[0035] Step 3, Proliferation Culture: Remove the induced culture from the first culture medium and cut it in a "+" shape along the direction perpendicular to the leaf surface to obtain 4 first cut parts; then cut the first cut parts horizontally along the direction parallel to the leaf surface to obtain 2 second cut parts containing single-sided clustered buds; inoculate the second cut parts containing single-sided clustered buds into the first culture medium for proliferation culture to obtain the proliferation culture.

[0036] After 20-25 days of culture, clustered buds, i.e. double-sided clustered buds, grow on both the upper and lower surfaces of the leaves. The induction culture is removed from the first culture medium and cut twice to obtain 8 leaves with single-sided clustered buds. The 8 leaves with single-sided clustered buds are then further cultured for proliferation, thereby effectively improving the propagation efficiency of the explants.

[0037] The proliferation culture process can continue using the first culture medium, and the specific culture method includes the following steps:

[0038] The cells were cultured under fluorescent lamps with a light intensity of 2500-3000 lx, a light duration of 10-13 hours per day, a culture temperature of 23℃±2℃, and a culture time of 10-20 days.

[0039] After 10-20 days of propagation culture, the culture can be further divided to improve explant propagation efficiency. The specific steps include:

[0040] The proliferating culture was cut to obtain a third cut portion with clustered buds, and the third cut portion was inoculated into the first culture medium for proliferating culture.

[0041] Step 4, Seedling Strengthening and Rooting Culture: The proliferation culture is taken out from the first culture medium and inoculated into the second culture medium for seedling strengthening and rooting culture. After the culture is completed, the cow ear seedlings are obtained.

[0042] After the proliferation culture is completed, the proliferation culture can be removed from the first culture medium for seedling strengthening and rooting culture to obtain *C. oxear* seedlings suitable for soil growth. To further simplify the culture process, this invention provides a seedling strengthening and rooting culture medium that can simultaneously achieve seedling strengthening and rooting culture, helping to shorten the seedling cycle and further improve the propagation efficiency of *C. oxear* seedlings.

[0043] Specifically, the seedling and rooting medium is the second culture medium, which, in addition to the nutrients necessary for the growth of *C. oxear* (a type of plant), also includes one or two of IBA and Ac. IBA (indolebutyric acid) is a auxin-like plant hormone that promotes rooting in cuttings and increases germination and survival rates. The addition of Ac (activated carbon) utilizes adsorption to reduce the impact of some harmful substances, such as preventing tissue browning and death caused by phenolic substances.

[0044] Furthermore, based on each liter of the second culture medium, the volume fraction of IBA is 0.3-0.7 ml / L, and the mass fraction of Ac is 0.05-0.2%. It can be understood that the above contents refer to the contents when the second culture medium includes the above components; that is, when the second culture medium includes IBA, the volume fraction of IBA in the second culture medium is 0.3-0.7 ml / L, and the content of Ac is 0; when the second culture medium includes Ac, the mass fraction of Ac in the second culture medium is 0.05-0.2%, and the content of IBA is 0.

[0045] Furthermore, the second culture medium includes both IBA and Ac, which helps to further improve the rooting rate and growth of *Cephalotaxus fortunei* seedlings.

[0046] Considering that the seedlings of *Cephalotaxus fortunei* can grow normally, the pH value of the second culture medium is 5.5-6.0.

[0047] In addition, the basal medium for the second culture medium is MS medium, which contains sucrose and agar. Those skilled in the art can adjust the content of sucrose and agar according to the actual situation.

[0048] In the present invention, the cultivation method for robust seedlings and rooting culture specifically includes the following steps:

[0049] Cultivation was carried out using fluorescent lamps with a light intensity of 2000-3000 lx and a light duration of 12 hours per day. The cultivation temperature was 25℃±2℃. Roots began to develop after 3-5 days of cultivation. After 20-30 days of cultivation, the seedlings reached a height of 2-3 cm and had 5-10 roots, thus obtaining the "cow ear" seedlings.

[0050] Finally, according to a specific embodiment of the present invention, in steps 2-3, the induction rate is not less than 70% and the proliferation rate is not less than 80%.

[0051] The induction rate is expressed as the proportion of the number of induction cultures with double-sided clustered buds to the total number of experimental explants, and the proliferation rate is expressed as the proportion of the number of induction cultures that can produce proliferation cultures to the total number of induction cultures participating in proliferation culture.

[0052] The second aspect of this invention provides the application of the method for propagating *C. oxeye* seedlings provided in the first aspect in the propagation of *C. oxeye* seedlings. Conducting research on the propagation and application of *C. oxeye* can not only stabilize the population size of *C. oxeye* and achieve resource protection for this species, laying the foundation for its sustainable development and utilization, but also enhance the stability and resilience of the ecosystem, which has significant ecological and practical significance in protecting biodiversity and the Yangtze River protection.

[0053] In summary, this invention provides a method for breeding cow ear seedlings and its application, which has the following advantages:

[0054] 1) Using leaves from the vegetative organs of the cow's ear as explants instead of flower stalks which can only be harvested during the reproductive stage, this overcomes the physiological time limitation of plant harvesting and allows for harvesting at any time throughout the year.

[0055] 2) Using cow's ear leaves as explants has little impact on the plant and will not affect the species' population or reproduction.

[0056] 3) By inducing clustered shoots on both sides of the leaves, a new approach to asexual propagation of plant leaves was explored, while the utilization efficiency of plant tissue culture explants was increased several times. Attached Figure Description

[0057] Figure 1 The parent plant from which the explants were obtained in Example 1;

[0058] Figure 2 The induction culture with bifacial clustered buds obtained from the explant induction culture in Example 2;

[0059] Figure 3A This is a longitudinal section of the induction culture obtained by cutting a cross shape along a direction perpendicular to the leaf surface in Example 3;

[0060] Figure 3B This is a cross-section of a culture containing single-sided clustered buds, obtained by transversely cutting the longitudinal section of the induced culture along a direction parallel to the leaf surface in Example 3.

[0061] Figure 3C The culture obtained after proliferation culture in Example 3;

[0062] Figure 4A In Example 4, roots began to develop in the propagation culture 3-5 days after the seedlings were vigorous and rooted.

[0063] Figure 4B The seedlings of *Cephalotaxus fortunei* obtained in Example 4 were cultured for 20-30 days after the seedlings were vigorous and rooted. Detailed Implementation

[0064] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. 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.

[0065] Example 1: Explant Pretreatment

[0066] (1) As Figure 1As shown, *Caulis Auricularia auricula-judae* (YZB1168) planted in the Cultivation Resource Nursery of the Yangtze Rare Plant Research Institute of China Three Gorges Corporation, located in Yichang City, Hubei Province, was used as the explant source. Tender leaves from 1-3 month old *Caulis Auricularia auricula-judae* were selected and cut from the base, yielding lower leaves near the base, upper leaves further from the base, and middle leaves located between the upper and lower leaves; the middle leaves were used as explants. The explants were wrapped in gauze and soaked in a beaker containing detergent water for 5 minutes, then rinsed under running water for 45 minutes. The cleaned explants were transferred to a laminar flow hood and soaked in 75% medical alcohol for 30 seconds, followed immediately by rinsing with sterile water. The rinsed explants were then soaked in a cleaning solution containing 0.1% mercuric chloride and 5 drops of Tween 80 for 8 minutes, followed by rinsing three times with sterile water. Finally, the leaves were blotted dry with sterile filter paper.

[0067] (2) Remove the damaged parts of the explant caused by cleaning and disinfection to obtain the pretreated explant.

[0068] Example 2: Induction Culture

[0069] (1) Investigating the effect of explant size on explant budding:

[0070] Pretreated explants were cut into pieces of 0.5cm×0.5cm, 1cm×1cm, 1.5cm×1.5cm, or 2.0cm×2.0cm and inoculated into sterile MS medium containing 28.0mg / L ε-polylysine, 1.0mg / L GA3, 0.06mg / L KT, 0.11mg / L TDZ, and 1.0mg / L NAA. The MS medium contained 30g / L sucrose and 7g / L agar, and the pH of the sterile medium was 5.8.

[0071] Inoculation was started on sterile culture medium. Dark culture was used for days 1-7, followed by LED red-white combined light culture for days 8-15, and then fluorescent light culture from day 15 onwards. During the light culture period, the light intensity was 2500-3000 lx, and the light duration was 12 hours per day. The induction culture temperature was 23℃±2℃. Induced cultures with double-sided clustered buds were obtained on days 20-25. Figure 2 As shown.

[0072] This invention uses the ratio of the number of inducible cultures with bifacial shoot clusters (induced number) to the number of experimental explants (explant quantity), i.e., the induction rate, to evaluate the effect of explant size on explant germination. Table 1 shows that the highest explant germination rate, reaching 88.9%, was achieved when the leaf size was 2.0 cm × 2.0 cm and the explants were inoculated with the dorsal side facing upwards. Therefore, subsequent experiments all used cultures with a leaf size of 2.0 cm × 2.0 cm.

[0073] Table 1

[0074]

[0075] (2) Investigating the effect of sterile culture medium formulation on explant germination:

[0076] Pretreated explants were cut into 2.0cm × 2.0cm pieces and inoculated into sterile culture media: ①MS + 0.05mg / L KT + 0.5mg / L TDZ + 0.8mg / L NAA, ②MS + 0.4mg / L TDZ + 1.0mg / L NAA, ③MS + 0.1mg / L KT + 0.6mg / L NAA, ④MS + 28.0mg / L ε-polylysine + 1.0mg / L GA3 + 0.06mg / L KT + 0.11mg / L TDZ + 1.0mg / L NAA. All MS media contained 30g / L sucrose and 7g / L agar, and the pH of all sterile culture media was 5.8.

[0077] Inoculation was started on sterile culture medium. Dark culture was used for days 1-7, LED red and white combined light culture was used for days 8-15, and fluorescent light culture was used after day 15. The light intensity during the light culture period was 2500-3000 lx, the light time was 12 hours per day, and the induction culture temperature was 23℃±2℃. Induction culture with double-sided clustered buds was obtained on days 20-25.

[0078] This invention uses the ratio of the number of inducible cultures with bifacial shoot clusters (induced number) to the total number of experimental explants (explant quantity), i.e., the induction rate, to evaluate the effect of sterile culture medium formulation on explant germination. Table 2 shows that the highest explant germination rate, reaching 98.9%, was achieved at a sterile culture medium formulation of MS + 28.0 mg / L ε-polylysine + 1.0 mg / L GA3 + 0.06 mg / L KT + 0.11 mg / L TDZ + 1.0 mg / L NAA, with a pH of 5.8.

[0079] Table 2

[0080]

[0081] Example 3: Proliferation Culture

[0082] Take out the induction culture with double-sided clustered buds, and cut a cross shape along a direction perpendicular to the leaf surface of the induction culture with a leaf surface area of ​​2.0cm × 2.0cm, obtaining four 1.0cm × 1.0cm first cut parts, as shown. Figure 3A As shown; then, make a transverse cut along a direction parallel to the leaf surface, dividing the first 1.0cm × 1.0cm section into two second sections containing single-sided clustered buds, as shown. Figure 3B As shown.

[0083] The portions containing single-sided clustered buds were inoculated into sterile culture media: ① MS + 0.05 mg / L KT + 0.5 mg / L TDZ + 0.8 mg / L NAA, ② MS + 0.4 mg / L TDZ + 1.0 mg / L NAA, ③ MS + 0.1 mg / L KT + 0.6 mg / L NAA, and ④ MS + 28.0 mg / L ε-polylysine + 1.0 mg / L GA3 + 0.06 mg / L KT + 0.11 mg / L TDZ + 1.0 mg / L NAA. All MS media contained 30 g / L sucrose and 7 g / L agar, and the pH of all sterile media was 5.8. Fluorescent lamp cultivation was used, with a light intensity of 2500-3000 lx, a light duration of 12 hours per day, and a cultivation temperature of 23℃±2℃. After 10-20 days of proliferation culture, proliferated cultures were obtained. Figure 3C As shown.

[0084] This invention evaluates the effect of sterile culture medium formulation on the proliferation effect of proliferating cultures by using the proportion of induced cultures that yield proliferating cultures to the total number of induced cultures participating in the proliferation culture (i.e., the proliferation rate) and the proliferation coefficient obtained by dividing the number of proliferating cultures by the number of induced cultures participating in the proliferation culture. Table 3 shows that the highest proliferation rate and proliferation coefficient, reaching 99.3% and 6.8, respectively, were achieved when the sterile culture medium formulation was MS + 28.0 mg / L ε-polylysine + 1.0 mg / L GA3 + 0.06 mg / L KT + 0.11 mg / L TDZ + 1.0 mg / L NAA, with a pH of 5.8.

[0085] Table 3

[0086]

[0087] Example 4: Seedling Strengthening and Rooting Culture

[0088] The proliferation cultures obtained in Example 3 were inoculated into seedling rooting media: A, MS + 0.5 ml / L IBA + 0.1% Ac; B, MS + 0.1% Ac; C, MS + 0.5 ml / L IBA. All MS media contained 30 g / L sucrose and 7 g / L agar, and the pH of all seedling rooting media was 5.8. Culture was performed under fluorescent lamps at a light intensity of 2000-3000 lx, a light duration of 12 hours per day, and a culture temperature of 25℃ ± 2℃. Roots began to develop after 3-5 days of culture. Figure 4A As shown; after 20-30 days of cultivation, the seedlings reach a height of 2-3cm and have 5-10 roots, thus obtaining the "cow ear" seedlings, as shown. Figure 4B As shown.

[0089] This invention uses the ratio of rooted seedlings to the total number of seedlings, i.e., the rooting rate, to evaluate the effect of the rooting medium formulation on seedling rooting. Table 4 shows that different rooting medium formulations did not significantly affect rooting induction. However, the 30-day seedling growth data indicated that the seedlings grew most vigorously when the rooting medium formulation was MS + 0.5 ml / L IBA + 0.1% Ac at pH 5.8.

[0090] Table 4

[0091]

[0092] In summary, the preferred explants are the middle part of tender leaves from 1-3 month old *C. brevicornu* leaves; the preferred leaf size is 2.0cm × 2.0cm; the preferred aseptic culture medium formula is MS + 28.0mg / L ε-polylysine + 1.0mg / L GA3 + 0.06mg / L KT + 0.11mg / L TDZ + 1.0mg / L NAA, wherein the MS medium contains 30g / L sucrose and 7g / L agar, and the pH of the aseptic medium is 5.8; the induction culture method is to use dark culture for days 1-7, LED red-white combined light culture for days 8-15, and fluorescent lamp culture after day 15; the proliferation culture method is to cut the induced culture vertically and parallel to the leaf surface, and then inoculate it into the aseptic culture medium; the preferred seedling and rooting culture medium is MS + 0.5ml / L IBA + 0.1% Ac, wherein the MS medium contains 30g / L sucrose and 7g / L agar, and the pH of the seedling and rooting culture medium is 5.8. The above-mentioned preferred propagation method can overcome the physiological time limitations of plant material acquisition in the existing technology, and will not affect the species quantity and reproduction. By inducing clustered buds on both sides of the leaves, a new way of asexual propagation of plant leaves has been explored, and the utilization efficiency of plant tissue culture explants has been increased. At the same time, subculture and rooting culture are combined into a one-step method to directly produce seedlings, simplifying the experimental technical process. Finally, a simple, fast and effective propagation system for cow's ear is established.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for propagating *Cephalotaxus fortunei* seedlings, characterized in that, Includes the following steps: Step 1, Explant pretreatment: Select leaves of *Cephalotaxus fortunei* as explants, clean and disinfect them, and then blot dry with sterile filter paper; Step 2, Induction Culture: The pretreated explants were cut into 2.0cm×2.0cm pieces and inoculated into the first culture medium for induction culture to obtain an induced culture with double-sided clustered buds; The block-shaped material is laid flat in the first culture medium, with the leaves placed facing upwards; The cultivation method includes: dark cultivation for days 1-7, followed by light cultivation from day 8 onwards, with a cultivation temperature of 23 ℃ ± 2 ℃ and a cultivation time of 20-25 days; the light cultivation specifically includes: LED red-white composite light cultivation from day 8-15, followed by fluorescent lamp cultivation from day 15 onwards, with a light intensity of 2500-3000 lx and a light duration of 10-13 hours per day during the light cultivation period; the first culture medium is a sterile culture medium, which consists of: MS + 28.0 mg / L, ε-polylysine + 1.0 mg / L, GA3 + 0.06 mg / L, KT + 0.11 mg / L, TDZ + 1.0 mg / L NAA; the pH value of the sterile culture medium is 5.8; Step 3, Proliferation Culture: Remove the induced culture from the first culture medium and cut it in a "+" shape along a direction perpendicular to the leaf surface to obtain 4 first cut parts; then cut the first cut parts horizontally along a direction parallel to the leaf surface to obtain 2 second cut parts containing single-sided clustered buds; inoculate the second cut parts containing single-sided clustered buds into the first culture medium for proliferation culture to obtain a proliferation culture; Step 4, Seedling Strengthening and Rooting Culture: The proliferation culture is removed from the first culture medium and inoculated into the second culture medium for seedling strengthening and rooting culture. After the culture is completed, ox ear seedlings are obtained. The second culture medium is a seedling strengthening and rooting culture medium, which is: A, MS + 0.5 ml / L IBA + 0.1% Ac, B, MS + 0.1% Ac, C, MS + 0.5 ml / L IBA; the pH value of the seedling strengthening and rooting culture medium is 5.

8.

2. The method according to claim 1, characterized in that, The method further includes: cutting the proliferation culture to obtain a third cut portion with clustered buds, and inoculating the third cut portion into a first culture medium for proliferation culture.

3. The method according to claim 1, characterized in that, Selecting leaves from cow ears involves the following steps: cutting leaves from the base of 1-3 month old cow ears, and then cutting the cut leaves to obtain the lower part of the leaves near the base, the upper part of the leaves away from the base, and the middle part of the leaves located between the upper and lower parts of the leaves. The middle leaf was used as the explant.

4. The method according to any one of claims 1-3, characterized in that, The cleaning and disinfection process includes the following steps: Step 1-1: Wrap the explant in gauze and soak it in laundry detergent water for 3-6 minutes. Then remove the explant and rinse it under running water for 30-50 minutes. Steps 1-2: Transfer the cleaned explants to a clean bench and immerse them in 75% medical alcohol for 20-40 seconds. After immersion, rinse them 1-2 times with sterile water. Steps 1-3: After rinsing with sterile water, place the explants in a cleaning solution containing mercuric chloride and Tween 80 for 5-10 minutes. After soaking, rinse with sterile water 3-5 times.

5. The application of the method according to any one of claims 1-4 in the breeding of bovine ear seedlings.

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