Tissue culture rapid propagation method and application of Campanumoea javanica Blume using stem segments to induce multiple buds

Abstract

The present invention relates to the technical field of rapid propagation of Chinese herbal medicines. Specifically, it is a tissue culture and rapid propagation method and application of Campanumoea javanica Blume using stem segments to induce cluster buds. The stem segments with buds at the top and terminal buds of Campanumoea javanica Blume branches are selected as explants. After cleaning and disinfection, the explants are inoculated on the cluster bud induction medium for culture. The induced cluster buds are inoculated on the cluster bud proliferation medium for culture, and the proliferated cluster buds are inoculated on the rooting medium for culture and then directly transplanted and domesticated. In the initial induction of the present invention, an appropriate concentration combination of 6-BA, NAA, IAA, and silver nitrate is used, which can directly induce cluster buds, shorten the production cycle, reduce the production cost, the genetic traits of the plants are stable, and the excellent qualities and characteristics of the mother plants are retained. In the proliferation process, the combination of GA3 and TIBA makes the seedlings stronger. Combining the appropriate concentration combination of 1 / 2MS with IBA and SA promotes the formation of basal root primordia and root growth, increases the stress resistance of plants, and qualified rooting seedlings can be formed in 15-20 days.

Description

Technical Field

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

[0002] Campanumoea javanica Bl., also known as Talinum paniculatum, abacus fruit, wild codonopsis fruit, and soil codonopsis, is a herbaceous twining vine of the family Campanulaceae and the genus Campanumoea. Its roots are used as medicine and have the effects of clearing heat and calming the nerves. It is mainly used to treat neurasthenia and other diseases, and can also be used as vegetables. It is a good medicinal resource with high development and utilization value. 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 there have been some research reports on tissue culture in recent years.

[0003] Publication No.: CN101869074A (Comparative Document 1) Title: A method for rapid tissue culture propagation of Codonopsis lanceolata var. pilosula. It uses tender buds or stem segments with buds as explants and conducts tissue culture 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 Platycodon grandiflorus through TDZ-induced adventitious bud formation from stem segments [J]. Journal of Zhongkai University of Agriculture and Engineering, 2017, 30(3):6. DOI: 10.3969 / j.issn.1674-5663.2017.03.003.) used stem segments with buds after germination of mature seeds of Platycodon grandiflorus as explants (Comparative Document 2), used MS as the basic medium, and added 6-benzyladenine (BA) and thidiazuron (TDZ) with 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) could obtain ideal cluster buds, and had a high proliferation multiple and sustainable subculture times; the best plant growth regulator formula was 0.5 mg / L BA + 0.02 mg / L TDZ. The bud proliferation multiple at the 5th subculture reached 10.9, and the subculture times could be 10 times; in the direct rooting method, the induced rooting rate and rooting coefficient were 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 were 30% - 100% and 1 - 5.3 respectively. Among them, when the stem segment ends were soaked in 300 mg / L IBA solution for 15 min, the rooting rate was 100%, the rooting coefficient was 5.3, and the survival rate after transplantation for 30 d was 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 was MS; the proliferation medium: MS + 6-BA 1.0 mg·L -1 (unit the same below)+NAA 0.1; the rooting medium was: MS + NAA0.5. The above media were all added with 0.6% agar and 3% sucrose, pH5.8; the culture temperature was (25±2)℃, the light time was 12h·d -1 , and the light intensity was 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 the mature berries of Campanumoea javanica seeds as explants, takes its 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 of seed propagation cannot fully inherit the traits of the mother plant, often losing the original excellent qualities and characteristics, and its production steps are relatively cumbersome in actual production. Summary of the Invention

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

[0005] To achieve the above object, the technical solution adopted by the present invention is: a tissue culture and rapid propagation method of Campanumoea javanica using stem segments to induce cluster buds, 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 the 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 + GA3 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 successively, rinse with sterile water for 3 to 5 times, 3 min 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 each time.

[0013] Further, in step (3), the formula of the cluster bud 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 cluster bud proliferation medium is: MS + 6-BA 1.0 - 3.0 mg / L + GA3 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, since 75% alcohol has strong permeability, young explants are prone to chlorosis and plant cells are easily killed, so the disinfection time is shortened to 5 s, and the rinsing time is extended by 1 - 2 min compared with the conventional 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. Since the penetration power of mercuric chloride is weak and the damage to 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 cluster buds, without adopting the conventional callus induction and callus differentiation into buds pathways in the tissue culture process, 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 brought by the influence of external factors on callus, and stabilizing the genetic traits of plants, retaining the excellent qualities and characteristics of the mother plants.

[0019] 3. In the proliferation process of the present invention, 6-BA, GA 3、The combination of NAA and TIBA (triiodobenzoic acid) can stabilize the proliferation coefficient above 15, and the seedlings are 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. In the proliferation process of the present invention, the combination of GA3 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, to a certain extent, it promotes the formation of basal root primordia, promotes root growth, and at the same time 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 culture 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 the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[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 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 some embodiments of the present invention, rather than all 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 protection scope 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 and pest-free Codonopsis lanceolata plants, and take the 40 cm young stem segments and apical buds at the top of the branches as explants.

[0030] (2)The excised explants are defoliated and cut into small segments with buds, each 3 cm long.

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

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

[0033] (5)Cut off the wounds at both ends of the disinfected explants, retain small segments with buds, each 2 cm long, blot the surface moisture with sterile paper, and inoculate them 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 according to the physiological polarity. Incubate in an environment with a temperature of 26 ± 2 °C, a light intensity of 2000 Lx, and a light-dark cycle of 10 h of sunlight for 15 days. The culture results are as Figure 1 shown.

[0034] (6)Cut the induced cluster buds into single buds, and inoculate them onto the cluster bud proliferation medium of MS + 6-BA 1.0 mg / L + GA3 0.5 mg / L + NAA 0.1 mg / L + TIBA (triiodobenzoic acid) 0.1 mg / L according to the physiological polarity. Incubate in an environment with a temperature of 26 ± 2 °C, a light intensity of 2000 Lx, and a light-dark cycle of 10 h of sunlight 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 in an environment with a temperature of 26 ± 2 °C, a light intensity of 2000 Lx, and a light-dark cycle of 12 h of sunlight for 10 days.

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

[0037] Example 2

[0038] A method for rapid propagation of tissue culture of Sinopodophyllum hexandrum using stem segments to induce cluster buds, the steps include:

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

[0040] (2)The excised explants are defoliated and cut 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 scrub with a brush and thoroughly wash with sterile water.

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

[0043] (5) Cut off the wounds at both ends of the disinfected explants, retain the 2.5-cm bud-containing segments, 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, and culture them 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, inoculate them onto the cluster bud proliferation medium of MS + 6-BA 2.0 mg / L + GA3 1.0 mg / L + NAA 0.2 mg / L + TIBA (triiodobenzoic acid) 0.2 mg / L according to the physiological polarity, and culture them 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, inoculate them onto the rooting medium of 1 / 2MS + IBA 2.0 mg / L + SA 1.5 mg / L + AC 0.5 g, and culture them 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, 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.

[0047] Example 3

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

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

[0050] (2) Remove the leaves from the cut explants and cut them into 5-cm bud-containing segments.

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

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

[0053] (5) Cut off the wound ends of the disinfected explants, retain 3-cm bud-containing small segments, absorb 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, and culture them in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a daylight of 10 h for 20 days.

[0054] (6) Cut the induced cluster buds into single buds, inoculate them onto the cluster bud proliferation medium of MS + 6-BA 3.0 mg / L + GA3 1.5 mg / L + NAA 0.3 mg / L + TIBA (triiodobenzoic acid) 0.3 mg / L according to the physiological polarity, and culture them in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a daylight of 10 h for 20 days. The culture results are as Figure 2 shown. There is no callus formation 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, inoculate them onto the rooting medium of 1 / 2MS + IBA 3.0 mg / L + SA 2.0 mg / L + AC 0.5 g / L, and culture them in a light-dark alternating environment at a temperature of 26 ± 2 °C, a light intensity of 3000 Lx, and a daylight of 12 h for 18 days. The culture results are as Figure 3 shown. The seedlings and roots are strong, and the rooting rate is 100%.

[0056] (8) After taking the rooted seedlings out of the bottle, wash the medium clean, 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] According to Example 1 in the patent "A Tissue Culture and Rapid Propagation Method of Codonopsis lanceolata Wall. ex DC.", using tender buds or bud-bearing stem segments as explants, tissue culture and rapid propagation are carried out 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 operations were carried out according to Liang Hanzhi, Deng Sha, Wu Yongqing, etc. In vitro rapid propagation of Campanumoea javanica Blume stem segments through TDZ-induced multiple shoots [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 operations were carried out according to Wang Zhunian, Xu Li, Huang Bilan, etc. In vitro rapid propagation of Platycodon grandiflorum [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 in Example 2 of the patent "Rapid Propagation Method of Campanumoea javanica Blume" (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, close to 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, greatly shortening the rooting and seedling formation time.

[0067] In the actual production process of tissue culture seedlings, the number of multiple shoot proliferation coefficients, the culture time, and the rooting time almost determine the length of the production cycle and the level of production costs. 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 tissue culture and rapid propagation method of Campanumoea javanica Blume using stem segments to induce multiple shoots, characterized in that the steps Including: (1) Selecting the 40-50 cm stem segments with buds and apical buds at the top of the leopard branches as explants; (2) Cleaning and disinfecting, and then sucking dry the moisture; (3) Inoculating the disinfected explants onto 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 culturing 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; (4) Inoculating the induced cluster buds onto the cluster 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 culturing 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; (5) Inoculating the proliferated cluster buds onto 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 culturing 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 transplanting and domesticating.

2. The method according to claim 1, wherein In step (2), the cleaning is to brush with 0.5% soap solution and then rinse with running water.

3. The method according to claim 1, wherein In step (2), the disinfection steps are: disinfecting with 75% alcohol for 5 s-15 s in a super clean bench, rinsing with sterile water for 3-5 times, 3 min for each time; disinfecting with 0.05% mercuric chloride solution for 15-20 min, and rinsing with sterile water for 5-7 times, 2 min for each time.

4. The method according to claim 1, wherein In step (3), the formula of the cluster bud 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.

5. The method according to claim 1, characterized in that In step (4), the formula of the cluster bud proliferation medium is: MS + 6-BA 2.0 mg / L + GA3 1.0 mg / L + NAA 0.2 mg / L + TIBA 0.2 mg / L.

6. The method according to claim 1, wherein 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.

7. Application of the method according to any one of claims 1-6 in the tissue culture rapid propagation of leopard using stem segments to induce cluster buds.

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