Pangolin leopard tissue culture and rapid propagation method for inducing cluster buds by utilizing stem segments and application

Abstract

The invention relates to the technical field of rapid propagation of Chinese herbal medicines, in particular to a tissue culture and rapid propagation method for Pangolin by utilizing stem segments to induce cluster buds and application of the tissue culture and rapid propagation method. The stem segments with buds at the top ends of branches of Pangolin and terminal buds are selected as explants, and the explants are inoculated to a cluster bud induction culture medium to be cultured after being cleaned and disinfected; and inoculating the induced cluster buds on a cluster bud proliferation culture medium for culturing, inoculating the proliferated cluster buds on a rooting culture medium for culturing, and directly transplanting and domesticating. According to the method disclosed by the invention, a proper concentration combination of 6-BA, NAA, IAA and silver nitrate is adopted during primary induction, cluster buds can be directly induced, the production period is shortened, the production cost is reduced, the plant inheritable character is stable, the excellent quality and characteristics of stock plants are reserved, GA3 and TIBA are combined in the proliferation process, seedlings are more robust, and the survival rate of the seedlings is increased by combining with a proper concentration combination of 1 / 2 MS, IBA and SA. The method can promote the formation of base root primordia and the growth of root systems, increase the stress resistance of plants, and can form qualified rooting seedlings within 15-20 days.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid propagation of Chinese herbal medicines, and more specifically to a method for rapid propagation of tissue culture of Campanumoea javanica using stem segments to induce multiple shoots and its application. Background Art

[0002] Campanumoea javanica Bl., also known as Talinum paniculatum, abacus fruit, wild Codonopsis fruit, and Talinum root, is a herbaceous twining vine belonging to the family Campanulaceae and the genus Campanumoea. Its root is used as medicine and has the effects of clearing heat and calming the nerves. It is mainly used to treat neurasthenia and other symptoms, and can also be used as vegetables. It is a good medicinal resource with high development and utilization value. At present, the existing medicinal plant resources of Campanumoea javanica are extremely scarce, while the medical demand is increasing day by day. There is little research on the biotechnology of artificial propagation of Campanumoea javanica plants, but in recent years, there have been some research reports on tissue culture.

[0003] Publication No.: CN101869074A (Comparative Document 1), titled "A Tissue Culture and Rapid Propagation Method for Codonopsis lanceolata var. pilosula", which uses tender buds or bud-bearing stem segments as explants and conducts tissue culture and rapid propagation through induction culture, proliferation culture, rooting culture, etc. However, this method does not clearly state the proliferation rate and rooting rate, and the time for induction culture, proliferation culture, and rooting culture is relatively long. Liang Hanzhi et al. (Liang Hanzhi, Deng Sha, Wu Yongqing, et al. In vitro rapid propagation of Campanumoea javanica Blume stem segments through TDZ-induced multiple shoot formation [J]. Journal of Zhongkai University of Agriculture and Engineering, 2017, 30(3):6. DOI: 10.3969 / j.issn.1674-5663.2017.03.003.) used the bud-bearing stem segments of germinated mature seeds of Campanumoea javanica Blume as explants (Comparative Document 2), used MS as the basic medium, and added 6-benzyladenine (BA) and Thidiazuron (TDZ) at different mass concentrations to optimize the proliferation hormone formula; direct rooting and indirect rooting methods were used respectively to obtain complete plants. The combination of 0.5 mg / L BA and low concentrations of TDZ (0.01 - 0.05 mg / L) can obtain ideal multiple shoots, and has a high proliferation multiple and sustainable subculture times; the best plant growth regulator formula is 0.5 mg / L BA + 0.02 mg / L TDZ, the bud proliferation multiple at the 5th subculture reaches 10.9, and the subculture times can be 10 times; in the direct rooting method, the induced rooting rate and rooting coefficient are 28.5% - 30.5% and 2.5 - 3.3 respectively; in the indirect rooting method, the rooting rates and rooting coefficients induced by pretreatment with different mass concentrations of IBA are 30% - 100% and 1 - 5.3 respectively. Among them, when the stem segment ends are soaked in 300 mg / L IBA solution for 15 min, the rooting rate is 100% and the rooting coefficient is 5.3, and the survival rate after transplantation for 30 d is 90%. Wang Zhunian et al. (Wang Zhunian, Li Zhiying, Xu Li, et al. Tissue culture and rapid propagation of Millettia speciosa Champ. [J]. Acta Phytophysiologica Sinica, 2005(6).) also carried out proliferation culture and rooting culture after seed germination (Comparative Document 3). The seed germination medium is MS; the proliferation medium: MS + 6-BA 1.0 mg·L -1 (unit the same below)+NAA 0.1; the rooting medium is: MS + NAA0.5. The above media are all added with 0.6% agar and 3% sucrose, pH5.8; the culture temperature is (25±2)°C, the light time is 12h·d -1 , and the light intensity is 30 - 40 μmol·m -2 ·s -1, each bud of this method can further differentiate into 5 to 6 buds, proliferating once every half month on average, and the rooting rate is 100% after 15 days. The publication number is: CN106561453A (Comparative Document 4), and the name is: Method for rapid tissue propagation of Campanumoea javanica. This method mainly uses mature berries of Campanumoea javanica seeds as explants, takes their seeds for germination culture, and successively conducts primary culture, subculture, rooting culture and acclimatization transplantation on the obtained germinated seedlings to establish a tissue culture system. However, the genetic traits of seedling are unstable, and the offspring reproduced by seeds cannot fully inherit the traits of the mother plant, and often lose the original excellent qualities and characteristics. Moreover, its production steps are relatively cumbersome in actual production. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of the prior art and provide a rapid tissue culture and propagation method that directly induces cluster buds from stem segments and then obtains complete plants through rooting.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: a rapid tissue culture and propagation method of Campanumoea javanica that induces cluster buds from stem segments, and the steps include:

[0006] (1) Select the 40 - 50 cm bud-bearing stem segment at the top of the Campanumoea javanica branch and the apical bud as explants;

[0007] (2) Clean and disinfect, and then dry to remove moisture;

[0008] (3) Inoculate the disinfected explants on the cluster bud induction medium with the formula: MS + 6 - BA 1.0 - 5.0 mg / L + NAA 0.01 - 1.0 mg / L + IAA 0.05 - 0.15 mg / L + silver nitrate 0.5 - 1.5 mg / L, and culture in a light-dark alternating environment with a temperature of 26 ± 2 °C, a light intensity of 2000 - 3000 Lx, and a sunlight exposure of 10 h for 15 - 20 days;

[0009] (4) Inoculate the induced cluster buds on the cluster bud proliferation medium with the formula: MS + 6 - BA 1.0 - 3.0 mg / L + GA 3 0.5 - 1.5 mg / L + NAA 0.1 - 0.3 mg / L + TIBA 0.1 - 0.3 mg / L, and culture in a light-dark alternating environment with a temperature of 26 ± 2 °C, a light intensity of 2000 - 3000 Lx, and a sunlight exposure of 10 h for 15 - 20 days;

[0010] (5) Inoculate the induced cluster buds on the rooting medium with the formula: 1 / 2MS + IBA 1.0 - 3.0 mg / L + SA 0.5 - 2.0 mg / L + AC 0.5 g / L, and culture in a light-dark alternating environment with a temperature of 26 ± 2 °C, a light intensity of 2000 - 3000 Lx, and a sunlight exposure of 12 h for 10 - 18 days, and then directly transplant and acclimatize.

[0011] Further, in step (2), the cleaning is to scrub with 0.5% soapy water and then rinse with running water.

[0012] Further, in step (2), the disinfection steps are as follows: disinfect with 75% alcohol on the ultra-clean bench for 5 s to 15 s, rinse with sterile water for 3 to 5 times, 3 min for each time; disinfect with 0.05% mercuric chloride solution for 15 to 20 min, rinse with sterile water for 5 to 7 times, 2 min for each time.

[0013] Further, in step (3), the formula of the multiple shoot induction medium is: MS + 6-BA 2.5 mg / L + NAA 0.1 mg / L + IAA 0.10 mg / L + silver nitrate 1.0 mg / L.

[0014] Further, in step (4), the formula of the multiple shoot proliferation medium is: MS + 6-BA 1.0 - 3.0 mg / L + GA 3 0.5 - 1.5 mg / L + NAA 0.1 - 0.3 mg / L + TIBA (triiodobenzoic acid) 0.1 - 0.3 mg / L.

[0015] Further, in step (5), the formula of the rooting medium is: 1 / 2MS + IBA 2.0 mg / L + SA 1.5 mg / L + AC 0.5 g / L.

[0016] The beneficial technical effects of the present invention are as follows:

[0017] 1. In the disinfection of explants in the present invention, because the permeability of 75% alcohol is very strong, the young explants are easy to lose green and the plant cells are easy to be killed, so the disinfection time is shortened to 5 s, and the rinsing time is extended by 1 - 2 min compared with the conventional one each time; when using mercuric chloride for disinfection, the concentration is reduced by half compared with the conventional dosage (0.1%), and the time is extended from the conventional 10 - 15 min to 15 - 20 min. Because the penetration power of mercuric chloride is weak and the damage to the explants is small, extending the disinfection time hardly damages the explants, which not only saves the cost of disinfectants, but also effectively controls the disinfection pollution rate within 10%.

[0018] 2. In the primary induction of the present invention, an appropriate concentration combination of 6-BA, NAA, IAA, and silver nitrate is adopted, which can directly induce multiple shoots. The conventional callus induction and callus differentiation into buds pathways in the tissue culture process are not adopted, reducing the link of callus differentiation into buds, shortening the production cycle to a certain extent, simplifying the production process, reducing the production cost, effectively avoiding the variation risk caused by the influence of external factors on the callus, and stabilizing the genetic traits of the plants, retaining the excellent qualities and characteristics of the mother plants.

[0019] 3. During the proliferation process of the present invention, 6-BA, GA 3、 NAA, TIBA (triiodobenzoic acid) combination is used, which can stabilize the proliferation coefficient above 15 and make the seedlings stronger.

[0020] 4. When rooting, directly reduce the hormone ratio and use the combination of IBA and SA, which can effectively promote the rooting rate. During the proliferation process of the present invention, the combination of GA 3 and TIBA (triiodobenzoic acid) can make the seedlings stronger; at the same time, combined with the appropriate concentration combination of 1 / 2MS, IBA and SA, it promotes the formation of basal root primordia to a certain extent, promotes root growth, and increases the stress resistance of plants. Qualified rooted seedlings can be formed in 15 - 20 days.

[0021] 5. In the actual production process of the method of the present invention, after primary induction, rooting culture can be directly carried out without proliferation culture, further shortening the production time of tissue-cultured seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a photo of the primary cluster bud induction in Example 1 of the present invention.

[0024] Figure 2 It is a photo of the subculture and proliferation seedlings of cluster buds in Example 3 of the present invention.

[0025] Figure 3 It is a photo of the rooted seedlings in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0027] Example 1

[0028] A tissue culture and rapid propagation method for Codonopsis lanceolata using stem segments to induce cluster buds, the steps include:

[0029] (1) Select healthy Codonopsis lanceolata plants without pests and diseases, and take the 40 cm young stem segments at the top of the branches and the apical buds as explants.

[0030] (2) Remove the leaves from the excised explants and cut them into small segments with buds, each 3 cm long.

[0031] (3) Rinse them under running water for 80 min, soak them in 0.5% soap solution for 3 min, then gently brush them with a brush and thoroughly wash them with sterile water.

[0032] (4) Transfer the cleaned explants to a laminar flow bench, disinfect them with 75% alcohol for 5 s, rinse them 3 times with sterile water, 3 min for each rinse, disinfect them with 0.05% mercuric chloride solution for 20 min, and rinse them 7 times with sterile water, 2 min for each rinse.

[0033] (5) Cut off the wounds at both ends of the disinfected explants, retain the small segments with buds, 2 cm long, blot the surface moisture with sterile paper, and inoculate them according to the physiological polarity onto the cluster bud induction medium of MS + 6-BA 5.0 mg / L + NAA 0.01 mg / L + IAA 0.05 mg / L + silver nitrate 0.5 mg / L. Incubate them in an environment with an alternating light and dark cycle of 26 ± 2 °C, a light intensity of 2000 Lx, and a sunlight exposure of 10 h for 15 days. The culture results are as Figure 1 shown.

[0034] (6) Cut the induced cluster buds into single buds, and inoculate them according to the physiological polarity onto the cluster bud proliferation medium of MS + 6-BA 1.0 mg / L + GA 3 0.5 mg / L + NAA 0.1 mg / L + TIBA (triiodobenzoic acid) 0.1 mg / L. Incubate them in an environment with an alternating light and dark cycle of 26 ± 2 °C, a light intensity of 2000 Lx, and a sunlight exposure of 10 h for 15 days.

[0035] (7) Cut the subcultured and proliferated cluster buds into single buds, and inoculate them onto the rooting medium of 1 / 2MS + IBA 1.0 mg / L + SA 0.5 mg / L + AC 0.5 g / L. Incubate them in an environment with an alternating light and dark cycle of 26 ± 2 °C, a light intensity of 2000 Lx, and a sunlight exposure of 12 h for 10 days.

[0036] (8) After taking the rooted seedlings out of the bottle, wash the medium off, and plant them in a mixed substrate of peat soil, perlite, and vermiculite (1:1:1), and at the same time, carry out the conventional acclimation management of tissue culture seedlings.

[0037] Example 2

[0038] A tissue culture and rapid propagation method for Indocalamus tessellatus using stem segments to induce cluster buds, the steps include:

[0039] (1) Select healthy Indocalamus tessellatus plants without diseases and pests, and take the 45-cm young stem segments at the top of the branches and the apical buds as explants.

[0040] (2) Remove the leaves from the excised explants and cut them into small segments with buds, each 4 cm long.

[0041] (3) Rinse under running water for 70 min, soak in 0.5% soap solution for 4 min, then gently brush with a brush and thoroughly wash with sterile water.

[0042] (4) Transfer the cleaned explants to a laminar flow hood, disinfect with 75% ethanol for 10 s, rinse 3 times with sterile water, 3 min each time, disinfect with 0.05% mercuric chloride solution for 18 min, and rinse 7 times with sterile water, 2 min each time.

[0043] (5) Cut off the wounds at both ends of the disinfected explants, retain the small segments with buds, each 2.5 cm long, blot the surface moisture with sterile paper, and inoculate them onto the cluster bud induction medium of MS + 6-BA 4.0 mg / L + NAA 0.05 mg / L + IAA 0.1 mg / L + silver nitrate 1.0 mg / L according to the physiological polarity. Incubate in an environment with a temperature of 26 ± 2 °C, a light intensity of 2500 LX, and a light-dark cycle of 10 h of sunlight for 18 days.

[0044] (6) Cut the induced cluster buds into single buds and inoculate them onto the cluster bud proliferation medium of MS + 6-BA 2.0 mg / L + GA 3 1.0 mg / L + NAA 0.2 mg / L + TIBA (triiodobenzoic acid) 0.2 mg / L according to the physiological polarity. Incubate in an environment with a temperature of 26 ± 2 °C, a light intensity of 2500 Lx, and a light-dark cycle of 10 h of sunlight for 18 days.

[0045] (7) Cut the subcultured and proliferated cluster buds into single buds and inoculate them onto the rooting medium of 1 / 2MS + IBA 2.0 mg / L + SA 1.5 mg / L + AC 0.5 g. Incubate in an environment with a temperature of 26 ± 2 °C, a light intensity of 2500 Lx, and a light-dark cycle of 12 h of sunlight for 15 days.

[0046] (8) After taking the rooted seedlings out of the bottle, wash the medium clean and plant them in a mixed substrate of peat soil, perlite, and vermiculite (1:1:1), and at the same time carry out the conventional acclimatization management of tissue culture seedlings.

[0047] Example 3

[0048] A method for rapid tissue culture and propagation of Sinopodophyllum hexandrum using stem segments to induce cluster buds, the steps include:

[0049] (1) Select healthy Sinopodophyllum hexandrum plants without pests and diseases, and take the top 50 cm young stem segments and apical buds of the branches as explants.

[0050] (2) Remove the leaves from the excised explants and cut them into small segments with buds, each 5 cm long.

[0051] (3) Rinse under running water for 60 min, soak in 0.5% soap solution for 5 min, then gently scrub with a brush and thoroughly wash with sterile water.

[0052] (4) Transfer the cleaned explants to a laminar flow hood, disinfect with 75% alcohol for 15 s, rinse 3 times with sterile water for 3 min each time, disinfect with 0.05% mercuric chloride solution for 15 min, and rinse 7 times with sterile water for 2 min each time.

[0053] (5) Cut off the wounds at both ends of the disinfected explants, retain the 3-cm bud-containing small segments, blot the surface moisture with sterile paper, and inoculate them onto the cluster bud induction medium of MS + 6-BA 3.0 mg / L + NAA 1.0 mg / L + IAA 0.15 mg / L + silver nitrate 1.5 mg / L according to the physiological polarity. Incubate in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a sunlight exposure of 10 h for 20 days.

[0054] (6) Cut the induced cluster buds into single buds and inoculate them onto the cluster bud proliferation medium of MS + 6-BA 3.0 mg / L + GA 3 1.5 mg / L + NAA 0.3 mg / L + TIBA (triiodobenzoic acid) 0.3 mg / L according to the physiological polarity. Incubate in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a sunlight exposure of 10 h for 20 days. The culture results are as Figure 2 shown. No callus appears in the cluster buds, no vitrified seedlings, and the proliferation coefficient is high.

[0055] (7) Cut the subcultured and proliferated cluster buds into single buds and inoculate them onto the rooting medium of 1 / 2MS + IBA 3.0 mg / L + SA 2.0 mg / L + AC 0.5 g / L. Incubate in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a sunlight exposure of 12 h for 18 days. The culture results are as Figure 3 shown. The seedlings and roots are robust, and the rooting rate is 100%.

[0056] (8) After taking the rooted seedlings out of the bottle, wash the medium clean and plant them in a mixed substrate of peat soil, perlite, and vermiculite (1:1:1), and at the same time carry out the conventional acclimation management of tissue culture seedlings.

[0057] Comparative Example 1

[0058] Refer to Example 1 in the patent "A Tissue Culture and Rapid Propagation Method for Codonopsis lanceolata Wall. ex DC." (CN101869074A), using tender buds or bud-bearing stem segments as explants, and carrying out tissue culture and rapid propagation through induction culture, proliferation culture, rooting culture, etc.

[0059] Comparative Example 2

[0060] Using the tender buds or bud-bearing stem segments of Example 1 as explants, the operation was carried out according to Liang Hanzhi, Deng Sha, Wu Yongqing, etc. In vitro rapid propagation of Campanumoea javanica stem segments through TDZ-induced multiple shoot formation [J]. Journal of Zhongkai University of Agriculture and Engineering, 2017, 30(3):6. DOI: 10.3969 / j.issn.1674-5663.2017.03.003.

[0061] Comparative Example 3

[0062] Using the tender buds or bud-bearing stem segments of Example 1 as explants, the operation was carried out according to Wang Zhunian, Xu Li, Huang Bilan, etc. In vitro rapid propagation of Platycodon grandiflorus var. glaucus [J]. Acta Phytophysiologica Sinica, 2007, 043(004):745.

[0063] Comparative Example 4

[0064] Using the tender buds or bud-bearing stem segments of Example 1 as explants, as described in Example 2 of the patent "Rapid Propagation Method of Campanumoea javanica" (CN106561453A), multiple shoots could not be induced.

[0065] Primary induction time / seed germination time (days) Primary induction multiple shoot rate / seed germination rate Proliferation culture time (days) Subculture multiple shoot proliferation coefficient Rooting culture time (days) Rooting rate at 20 days Example 1 15 99% 15 15.7 10 100% Example 2 18 98% 18 14.2 15 100% Example 3 20 95.4% 20 13.5 18 100% Comparative Example 1 30-45 - 35-45 10.4 20-30 98.6% Comparative Example 2 30 - 30 10.9 30 30.5% Comparative Example 3 21 95% 15 6 15 100% Comparative Example 4 - - - - - -

[0066] As can be seen from the above table, the seed germination time of the comparative examples was relatively long, the primary induction multiple shoot rate was relatively low, Comparative Example 4 could not directly induce multiple shoots, and the proliferation coefficients were all relatively low. In contrast, the primary induction time of Examples 1, 2, and 3 was short, the multiple shoot rates were all very high, approaching 100%. At the same time, in terms of rooting, the present invention can achieve 100% formation of rooted seedlings that can be taken out of the bottle in 20 days, significantly shortening the rooting and seedling formation time.

[0067] In the actual production of tissue culture seedlings, the proliferation coefficient of multiple shoots, the culture time, and the rooting time almost determine the length of the production cycle and the level of production cost. Finally, it should be noted that the above examples are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above examples, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced. Any modification or partial replacement without departing from the spirit and scope of the present invention should be covered by the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Leopardus leopardus by inducing clustered buds through stem segments, characterized in that the steps include: (1) Select the 40-50 cm stem segments with buds and terminal buds of Leopardus chinensis branches as explants; (2) After cleaning, disinfect and dry the water; (3) Inoculate the sterilized explants on a clustered bud induction medium with the following formula: MS + 6-BA 1.0-5.0 mg / L + NAA 0.01-1.0 mg / L + IAA 0.05-0.15 mg / L + silver nitrate 0.5-1.5 mg / L, and culture them in a light-dark alternating environment with a temperature of 26±2°C, a light intensity of 2000-3000 Lx, and a daylight of 10 h for 15-20 days; (4) Inoculate the induced clustered buds on a clustered bud proliferation medium with the formula: MS + 6-BA 1.0-3.0 mg / L + GA3 0.5-1.5 mg / L + NAA 0.1-0.3 mg / L + TIBA 0.1-0.3 mg / L, and culture them in a light-dark alternating environment with a temperature of 26±2°C, a light intensity of 2000-3000 Lx, and a daylight of 10 h for 15-20 days; (5) Inoculate the proliferated clustered buds onto a rooting medium with the formula: 1 / 2MS + IBA 1.0-3.0mg / L + SA 0.5-2.0mg / L + AC 0.5g / L, place it in a light-dark alternating environment with a temperature of 26±2℃, a light intensity of 2000-3000Lx, and 12h of sunlight for 10-18 days, and then directly transplant and acclimate.

2. The method according to claim 1, characterized in that: In step (2), the cleaning is performed by scrubbing with 0.5% soap solution and then rinsing with running water.

3. The method according to claim 1, characterized in that: In step (2), the disinfection step is: disinfect with 75% alcohol on a clean bench for 5s to 15s, rinse with sterile water 3 to 5 times, each time for 3 minutes; disinfect with 0.05% mercuric chloride solution for 15 to 20min, rinse with sterile water 5 to 7 times, each time for 2 minutes.

4. The method according to claim 1, characterized in that: In step (3), the formula of the cluster bud induction medium is: MS+6-BA2.5mg / L+NAA0.1mg / L+IAA0.10mg / L+silver nitrate 1.0mg / L.

5. The method according to claim 1, characterized in that: In step (4), the formula of the cluster bud proliferation culture medium is: MS+6-BA2.0mg / L+GA31.0mg / L+NAA0.2mg / L+TIBA0.2mg / L.

6. The method according to claim 1, characterized in that: In step (5), the formula of the rooting medium is: 1 / 2MS+IBA 2.0mg / L+SA1.5mg / L+AC 0.5g / L.

7. Application of the method according to any one of claims 1 to 6 in tissue culture rapid propagation of Leopardus leopardus utilizing stem segments to induce clustered buds.

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