Method for inducing Tujia medicine blood tube callus

Abstract

The method comprises the following steps: respectively preparing a callus induction culture medium and a proliferation culture medium according to a formula, then carrying out high-temperature and high-pressure sterilization for 25-30 minutes, cooling to about 55-60 DEG C, and subpackaging; taking a blood cylinder as an explant, performing disinfection treatment, placing the blood cylinder on sterile filter paper after disinfection, and cutting the blood cylinder into stem segments of about 1-2cm for later use; and inoculating the disinfected blood cylinder tender stems which are cut into segments into a callus induction culture medium. According to the method, an optimized hormone proportion is adopted, the callus induction rate reaches 3.9%, a large number of loose-texture vigorous-growth calluses can be obtained after culture is conducted for 100 days, the propagation period is greatly shortened compared with natural growth, the biomass of the calluses can be increased by 5-12 times through a proliferation culture medium, the good activity is still kept after subculture, and by optimizing illumination, temperature and humidity, the survival rate of the calluses is improved. Browning is effectively avoided, long-term stable culture is ensured, only a conventional MS culture medium and common plant hormones are needed, special additives are not needed, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of plant callus induction and culture, and specifically provides a method for inducing the callus of the Tujia medicine Xue Tong Background Art

[0002] The Tujia medicine Xue Tong (Kadsura heteroclita (Roxb) Craib) belongs to the genus Kadsura of the Schisandraceae family and is an evergreen woody liana. Xue Tong is bitter in taste and warm in nature, with the effects of promoting qi and activating blood circulation, reducing swelling and relieving pain. It is commonly used in folk medicine with its vine stems and roots for treating rheumatoid arthritis, abdominal pain, qi pain, labor-induced low back pain, dysmenorrhea and other symptoms.

[0003] However, the growth rate of Xue Tong is slow and there is no artificial cultivation technology, resulting in its scarce resources and difficulty in meeting the development and utilization needs. The plant callus culture technology has the advantages of short reproduction cycle, low cost, and being not restricted by seasons, which is of great significance for the protection and utilization of wild plant resources. It can be seen that the callus of Xue Tong can be used as a new resource for the development and utilization of Xue Tong. Currently, there is no report on the induction and proliferation of the callus of Xue Tong. Therefore, a method for inducing the callus of the Tujia medicine Xue Tong is proposed to fill the technical gap in this field. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a method for inducing the callus of the Tujia medicine Xue Tong, which provides technical support for the rapid propagation of Xue Tong and lays an experimental foundation for the industrial production of the secondary metabolites of Xue Tong, and solves the problem that there is no report on the induction and proliferation of the callus of Xue Tong.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for inducing the callus of the Tujia medicine Xue Tong, comprising the following steps:

[0006] Step 1: Preparation of culture medium

[0007] Prepare the callus induction medium and proliferation medium according to the formula respectively, then sterilize them at high temperature and high pressure for 25 - 30 min, and subpackage after cooling to about 55 - 60 °C;

[0008] Step 2: Disinfection of explants

[0009] Using Xue Tong as the explant, conduct disinfection treatment, and after the disinfection is completed, place it on sterile filter paper and cut it into stem segments about 1 - 2 cm long for standby;

[0010] Step 3: Induction of callus

[0011] Inoculate the disinfected and segmented tender stems of Xue Tong into the callus induction medium and place them in the tissue culture room for cultivation;

[0012] Step 4: Callus Proliferation

[0013] Transfer the induced callus of the blood cylinder to a proliferation medium for proliferation culture.

[0014] Preferably, the proliferation medium is MS basal medium supplemented with 0.2 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar, with a pH of 5.8 - 6.0.

[0015] Preferably, the callus induction medium is MS basal medium supplemented with 4.0 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar, with a pH of 5.8 - 6.0.

[0016] Preferably, the MS is prepared by adding 30 g of sucrose and 6 g of agar to 1 L of MS medium.

[0017] Preferably, the explant is the tender stem of the blood cylinder. Rinse it with tap water to remove surface dust, drain the water, and transfer it to the laminar flow bench for operation.

[0018] Preferably, the culture conditions of the callus induction medium are: temperature 25 - 30 °C, light duration 12 - 14 h / d, relative air humidity 65 - 68%, light intensity 1500 - 1800 Lx. Callus of the blood cylinder can be obtained in 100 - 120 days.

[0019] Preferably, the culture conditions of the proliferation medium are: temperature 24 - 30 °C, light duration 10 - 14 h / d, relative air humidity 60 - 68%, light intensity 0 - 1800 Lx, and culture for 25 - 30 days.

[0020] Preferably, the explant is disinfected by soaking in 75 - 80% alcohol for 40 - 45 s.

[0021] Furthermore, during the disinfection process of the explant, it is also disinfected with 0.1% mercuric chloride (HgCl₂) solution for 7 - 9 min and rinsed with sterile water 4 - 5 times, 3 - 5 min each time.

[0022] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0023] 1. The present invention adopts an optimized hormone ratio, and the callus induction rate reaches 3.9%. A large amount of callus with loose texture and strong growth can be obtained after 100 days of culture, significantly shortening the propagation cycle compared with natural growth.

[0024] 2. The proliferation medium of the present invention can increase the biomass of callus by 5 - 12 times and still maintain good activity after subculture. By optimizing light, temperature and humidity, browning is effectively avoided to ensure long-term stable culture.

[0025] 3. The present invention only requires a conventional MS medium and common plant hormones, without the need for special additives, reducing production costs. The explant disinfection step is simple and efficient, and the contamination rate is less than 5%, which is suitable for industrial application.

[0026] 4. By optimizing the explant treatment, hormone ratio, and culture conditions, the present invention realizes the efficient induction and rapid proliferation of the callus of Xue Tong, providing a reliable technical solution for the large-scale culture and medicinal development of Xue Tong. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a diagram of the callus induction result with the tender stem of Xue Tong of the present invention as the explant;

[0028] Figure 2 It is a diagram of the proliferation culture result of the callus of Xue Tong of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Embodiment 1

[0031] A method for inducing the callus of the Tujia medicine Xue Tong, comprising the following steps:

[0032] Step 1. Medium preparation

[0033] Prepare the callus induction medium and the proliferation medium according to the formula respectively. Among them, the proliferation medium is the MS basic medium added with 0.2 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 6.5 g / L agar, with a pH of 6.0. The callus induction medium is the MS basic medium added with 4.0 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, and 6.5 g / L agar. The MS is prepared by adding 30 g of sucrose and 6 g of agar to 1 L of the MS medium, with a pH of 5.8, and then sterilized at high temperature and high pressure for 25 min, and cooled to about 55 °C for dispensing;

[0034] Step 2. Explant disinfection

[0035] Using the blood cylinder as the explant, the explant is the tender stem of the blood cylinder. Rinse it with tap water to remove the surface dust, drain the water, transfer it to the ultra-clean workbench for operation, and perform disinfection treatment. The disinfection method is to soak it in 75% alcohol for 40 s, disinfect it with 0.1% mercuric chloride (HgCl2) solution for 7 min, and rinse it with sterile water 4 times, 3 min each time. After disinfection, place it on sterile filter paper and cut it into stem segments about 1 cm long for standby;

[0036] Step 3: Callus induction

[0037] Inoculate the disinfected and segmented tender stems of the blood cylinder into the callus induction medium. The culture conditions are: temperature 25°C, light duration 12 h / d, relative air humidity 65%, light intensity 1500 Lx. After 100 days, the callus of the blood cylinder can be obtained and placed in the tissue culture room for culture;

[0038] Step 4: Callus proliferation

[0039] Transfer the induced callus of the blood cylinder to the proliferation medium for proliferation culture. The culture conditions are: temperature 24°C, light duration 10 h / d, relative air humidity 60%, light intensity 1560 Lx, and culture for 28 days.

[0040] By adopting the above technical scheme, the proliferation medium can increase the biomass of the callus by 3 - 5 times, and still maintain good activity after subculture. By optimizing the light, temperature and humidity, browning is effectively avoided, ensuring long-term stable culture.

[0041] Example 2

[0042] A method for inducing the callus of the Tujia medicine blood cylinder, comprising the following steps:

[0043] Step 1: Medium preparation

[0044] Prepare the callus induction medium and the proliferation medium according to the formula respectively. Among them, the proliferation medium is the MS basic medium added with 0.2 mg / L 6 - BA, 0.5 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar, pH 6.0. The callus induction medium is the MS basic medium added with 4.0 mg / L 6 - BA, 0.2 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar. The MS is prepared by adding 30 g sucrose and 6 g agar to 1 L of MS medium, pH 6.0, and then sterilized at high temperature and high pressure for 30 min, and cooled to about 55 - 60°C for subpackaging;

[0045] Step 2: Explant disinfection

[0046] Using the blood cylinder as the explant, the explant is the tender stem of the blood cylinder. Rinse it with tap water to remove the surface dust, drain the water, transfer it to the laminar flow bench for operation, and perform disinfection treatment. The disinfection method is to soak it in 80% alcohol for 45 s, disinfect it with 0.1% mercuric chloride (HgCl2) solution for 9 min, and rinse it with sterile water 5 times, 5 min each time. After disinfection, place it on sterile filter paper and cut it into stem segments about 2 cm long for standby;

[0047] Step 3: Callus induction

[0048] Inoculate the disinfected and segmented tender stems of the blood cylinder into the callus induction medium. The culture conditions are: temperature 30 °C, light time 14 h / d, relative air humidity 68%, light intensity 1800 Lx. The blood cylinder callus can be obtained after 120 days and placed in the tissue culture room for culture;

[0049] Step 4: Callus proliferation

[0050] Transfer the induced blood cylinder callus to the proliferation medium for proliferation culture. The culture conditions are: temperature 30 °C, light time 14 h / d, relative air humidity 68%, light intensity 0 Lx, and culture for 28 days.

[0051] Specifically, the proliferation medium can increase the biomass of the callus by 10 - 12 times, and still maintain good activity after subculture. By optimizing the light, temperature and humidity, browning can be effectively avoided to ensure long-term stable culture.

[0052] Example 3

[0053] Please refer to Figure 1 , the induction of the blood cylinder callus in this example is as follows:

[0054] 1) Material preparation

[0055] Explant: Select tender stems of the blood cylinder with strong growth, about 0.3 - 0.5 cm in diameter.

[0056] Medium:

[0057] Induction medium: MS + 4.0 mg / L 6 - BA + 0.2 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar (pH 5.8 - 6.0).

[0058] Proliferation medium: MS + 0.2 mg / L 6 - BA + 0.5 mg / L NAA + 30 g / L sucrose + 6.5 g / L agar (pH 5.8 - 6.0).

[0059] 2) Explant disinfection

[0060] Rinse the tender stems of the blood cylinder with tap water for 30 minutes to remove the surface impurities. In the laminar flow bench, perform the following treatments in sequence:

[0061] S1. Soak in 75% alcohol for 45 seconds;

[0062] S2. Disinfect with 0.1% HgCl2 solution for 7 minutes;

[0063] S3. Rinse with sterile water 4 - 5 times, 3 minutes each time. Blot dry with sterile filter paper and cut into stem segments 1 cm long for standby.

[0064] 3) Inoculation and culture

[0065] Inoculate the disinfected stem segments into the induction medium, 3 - 4 segments per bottle.

[0066] Culture conditions:

[0067] Temperature: 25 ± 1°C;

[0068] Light: 12 h / d, light intensity 1800 Lx (white LED light source);

[0069] Humidity: 65%.

[0070] After culturing for 30 days, observe the formation of callus (see Figure 1 ).

[0071] 4) Results

[0072] 30 days after inoculating the explants, the incision starts to swell and form light yellow callus. After 100 days, the callus is light yellow or light green, with loose texture and vigorous growth, and the induction rate reaches over 3.9%.

[0073] Example 4

[0074] Please refer to Figure 2 , the proliferation culture of the blood cylinder callus in this example

[0075] 1) Materials

[0076] Take the callus induced in Example 1 and cut it into small pieces about 0.5 cm × 0.5 cm.

[0077] 2) Inoculation and culture

[0078] Inoculate the callus pieces into the proliferation medium, 3 - 4 pieces per bottle.

[0079] The culture conditions are the same as those in Example 1, temperature 25°C, light 12 h / d, light intensity 0 Lx, humidity 65%, and subculture every 28 days.

[0080] 3) Results

[0081] After 15 days of culture, the volume of the callus increased significantly, showing a light yellow or light green color, with uniform texture. After 3 subcultures, the biomass of the callus increased by about 5 - 12 times, and the proliferation coefficient reached 11.05.

[0082] Experimental Example 1: Effects of Different Hormone Ratios on Callus Induction

[0083] 1) Experimental Design

[0084] Based on the MS medium, different combinations of 6 - BA and NAA concentrations were set, with 20 bottles in each group and repeated 3 times.

[0085] Group A: MS + 2.0 mg / L 6 - BA + 0.1 mg / L NAA.

[0086] Group B: MS + 4.0 mg / L 6 - BA + 0.2 mg / L NAA

[0087] Group C: MS + 6.0 mg / L 6 - BA + 0.5 mg / L NAA

[0088] Explant Treatment and Culture:

[0089] The tender stems of the blood cylinder were rinsed with tap water for 30 minutes to remove surface impurities. In the ultra - clean workbench, the following treatments were carried out in sequence:

[0090] S1. Soak in 75% alcohol for 45 seconds;

[0091] S2. Disinfect with 0.1% HgCl2 solution for 7 minutes;

[0092] S3. Rinse with sterile water 4 - 5 times, 3 minutes each time. Blot dry with sterile filter paper and cut into 1 - cm - long stem segments for standby

[0093] 2) Experimental Results

[0094] Group 6-BA (mg / L) NAA (mg / L) Callus induction rate (%) Callus state Group A 2.0 0.1 1.8％ Grows slowly, texture is hard Group B 4.0 0.2 3.9％ Grows vigorously, texture is loose Group C 6.0 0.5 2％ Partially browned, growth is uneven

[0095] After the experiment, it was proved that Group B (4.0 mg / L 6 - BA + 0.2 mg / L NAA) had the highest induction rate (3.9%), and the growth state of the callus was the best. Too high a 6 - BA concentration (Group C) might cause browning of the explants and reduce the induction efficiency.

[0096] Experimental Example 2: Effects of Different Light Intensities on Callus Proliferation

[0097] 1) Experimental Design

[0098] The proliferation medium (MS + 0.2 mg / L 6 - BA + 0.5 mg / L NAA) was used, and different light intensities were set;

[0099] Group D: 1800 Lx;

[0100] Group E: 0 Lx;

[0101] After culturing for 28 days, the fresh weight increment of the callus was measured, and the fresh weight growth rate was calculated. Five bottles were used each time, and the experiment was repeated 3 times.

[0102] 2) Experimental results

[0103]

[0104] After the experiment, it was proved that the light intensity of 0 Lx (Group E) was the most suitable for the proliferation of the callus of Xue Tong, and the fresh weight growth rate reached 1105%.

[0105] In summary, the present invention adopts an optimized hormone ratio, and the callus induction rate reaches 3.9%. A large amount of callus with loose texture and strong growth can be obtained after culturing for 100 days, significantly shortening the propagation cycle compared with natural growth. The proliferation medium can increase the biomass of the callus by 5 - 12 times, and still maintain good activity after subculture. By optimizing the light, temperature and humidity, browning is effectively avoided, ensuring long-term stable culture. Only conventional MS medium and common plant hormones are required, without special additives, reducing production costs. The explant disinfection step is simple and efficient, and the contamination rate is less than 5%, which is suitable for industrial application. By optimizing the explant treatment, hormone ratio and culture conditions, the efficient induction and rapid proliferation of the callus of Xue Tong are realized, providing a reliable technical solution for the large-scale culture and medicinal development of Xue Tong, providing technical support for the rapid breeding of Xue Tong, and at the same time laying an experimental foundation for the industrial production of the secondary metabolites of Xue Tong, solving the problem that there is no report on the research of the induction and proliferation of the callus of Xue Tong.

[0106] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article or device including the said element.

[0107] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing callus of Tubo ethnic medicine Xue Tong, characterized in that, It includes the following steps: Step 1: Medium preparation Prepare the callus induction medium and the proliferation medium according to the formula respectively, then sterilize them at high temperature and high pressure for 25 - 30 min, and cool to about 55 - 60 °C for sub-packaging; Step 2: Explant disinfection Use the blood cylinder as the explant, carry out disinfection treatment, and after disinfection, place it on a sterile filter paper and cut it into stem segments about 1 - 2 cm long for standby; Step 3: Callus induction Inoculate the disinfected and segmented young stems of the blood cylinder into the callus induction medium, and place it in the tissue culture room for cultivation; Step 4: Callus proliferation Transfer the induced callus of the blood cylinder to the proliferation medium for proliferation culture.

2. The method for inducing callus of the Tujia medicine Xue Tong according to claim 1, characterized in that, The proliferation medium is MS basal medium supplemented with 0.2 mg / L 6 - BA, 0.5 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar, with a pH of 5.8 - 6.

0.

3. A method for inducing callus of Tubo ethnic medicine Xue Tong according to claim 1, characterized in that, The callus induction medium is MS basal medium supplemented with 4.0 mg / L 6 - BA, 0.2 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar, with a pH of 5.8 - 6.

0.

4. A method for inducing callus of the Tujia medicine Xue Tong according to claim 3, characterized in that, The MS is prepared by adding 30 g of sucrose and 6 g of agar to 1 L of MS medium.

5. A method for inducing callus of Tubo ethnic medicine Xue Tong according to claim 1, characterized in that, The explant is the young stem of the blood cylinder. Wash it with tap water to remove the surface dust, drain the water, and transfer it to the ultra-clean workbench for operation.

6. A method for inducing callus of Tubo ethnic medicine Xue Tong according to claim 1, characterized in that, The culture conditions of the callus induction medium are: temperature 25 - 30 °C, light time 12 - 14 h / d, relative air humidity 65 - 68%, light intensity 1500 - 1800 Lx, and callus tissue of the blood cylinder can be obtained in 100 - 120 days.

7. A method for inducing callus of the Tujia medicine Xue Tong, according to claim 1, characterized in that The culture conditions of the proliferation medium are: temperature 24 - 30 °C, light time 10 - 14 h / d, relative air humidity 60 - 68%, light intensity 0 - 1800 Lx, and culture for 25 - 30 days.

8. A method for inducing callus of Tubo ethnic medicine Xue Tong according to claim 1, characterized in that, The explant disinfection is carried out by soaking it in 75 - 80% alcohol for 40 - 45 s.

9. A method for inducing callus of Tubo ethnic group's medicine Xue Tong as claimed in claim 8, characterized in that, During the explant disinfection process, it is also disinfected with 0.1% mercuric chloride (HgCl2) solution for 7 - 9 min, and rinsed with sterile water 4 - 5 times, 3 - 5 min each time.

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