Mesona chinensis tissue culture seedling forming method

Abstract

The invention belongs to the technical field of mesona chinensis tissue culture seedlings, and particularly relates to a mesona chinensis tissue culture seedling forming method. The invention discloses a mesona chinensis tissue culture seedling forming method. The method comprises the following steps: S1, treating an explant; s2, sterile culture: culturing the stem segments in a primary culture medium to obtain sterile seedlings; s3, cluster bud induction: cutting the aseptic seedling stems and culturing the aseptic seedling stems in a cluster bud induction culture medium to obtain cluster buds; s4, rooting induction: shearing cluster buds, inoculating the cluster buds into a rooting culture medium, and culturing to obtain tissue culture seedlings; s5, acclimatization and transplantation. According to the tissue culture seedling growing method, the growth coefficient can be increased, the vitrification rate can be reduced, the rooting number and the rooting length can be increased, the survival rate is high, meanwhile, the occurrence rate of field root rot can be reduced, compared with stem tip explants, induction materials can be enriched, and the tissue culture seedling growing work of mesona chinensis can be developed.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue culture seedling raising of Mesona chinensis, and particularly relates to a method for forming seedlings of tissue culture seedlings of Mesona chinensis. Background Art

[0002] Mesona chinensis is an annual herbaceous and perennial plant of the genus Mesona in the family Lamiaceae. It is an important medicinal and edible plant in China, with multiple effects such as clearing heat and detoxifying, detumescence and pain relief, and moistening the lungs and relieving cough. Mesona chinensis can be propagated by seeds, rhizome tillering, cuttings, etc. However, the germination rate of seeds is relatively low. Conventional propagations such as rhizome tillering propagation and cutting propagation are completed in the natural environment, and often problems such as the occurrence of pests and diseases will occur, and a suitable growth environment is required. Plant tissue culture is a technology that uses plant tissues, organs, cells and other materials as explants for culture to achieve their proliferation, growth and re-development, and finally form complete plants.

[0003] In the prior art, the tissue culture and propagation of Mesona chinensis have been disclosed. For example, the patent of CN112470926B discloses a rapid propagation method for virus-free seedlings of the shoot tips of Mesona chinensis. An in vitro culture system is established through shoot tip culture to induce the production of cluster buds, and the shoot tips of the cluster buds are taken to cultivate preliminary virus-free test-tube seedlings; then the shoot tips of the test-tube seedlings are used for the second shoot tip virus elimination to obtain virus-free seedling cluster buds; then the virus-free seedling cluster buds are used as mother seedlings to be cut into sections for rapid propagation to obtain virus-free propagated seedlings, and after growth, transplantation and field planting, Mesona chinensis plants are obtained. The method of this patent can rapidly proliferate and strengthen seedlings by alternately using a cluster bud induction and proliferation medium and a seedling strengthening and rooting medium, and avoid germplasm degradation caused by subculture; the virus-free seedlings cultivated have the advantages of high survival rate, good roots, strong growth potential, strong stress resistance, and few diseases.

[0004] However, in the actual process of tissue culture seedling raising, the shoot tip is the apical bud, and there is only one of it. The source of materials is limited, and the base number of induction materials is small, resulting in a low induction work efficiency, which will limit the development of tissue culture seedling raising of Mesona chinensis to a certain extent.

[0005] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for forming seedlings of tissue culture seedlings of Mesona chinensis to solve the problems existing in the prior art in the background art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A method for forming seedlings of tissue culture seedlings of Mesona chinensis, comprising the following steps:

[0009] S1. Process the explant

[0010] Select young and robust branches without diseases and pests, disinfect them, rinse them, and blot dry the surface moisture of the young branches;

[0011] S2. Axenic culture

[0012] Cut the stem segments of the young branches, vertically insert their biological lower ends into the primary medium and culture for 20 - 30 days to obtain axenic seedlings;

[0013] S3. Induction of cluster buds

[0014] Cut the stem segments of the axenic seedlings, vertically insert their biological lower ends into the cluster bud induction medium and culture for 30 - 45 days to obtain cluster buds;

[0015] S4. Rooting induction

[0016] Select cluster buds of 2 - 3 cm, cut and separate them from the base, inoculate them into the rooting medium and culture for 30 - 35 days to obtain tissue - cultured seedlings;

[0017] S5. Acclimatization and transplantation

[0018] Acclimatize the tissue - cultured seedlings at room temperature for 5 - 6 days, take out the seedlings, wash their roots, transplant them into a seedling - raising tray filled with a seedling - raising substrate, grow in a plastic greenhouse for 20 - 30 days, and then transplant them into the soil for growth.

[0019] More specifically, in step S1, soak and disinfect with 0.1% HgCl2 for 10 minutes, then disinfect with 75% alcohol for 50 seconds, then rinse with sterile water 3 - 4 times, 1 minute each time, and finally blot dry the surface moisture of the young branches with sterile filter paper.

[0020] More specifically, in step S2, the primary medium is: MS basal medium + 0.1 mg / L naphthaleneacetic acid + 0.2 mg / L 6 - benzylaminopurine + 25 g / L sucrose + 7 g / L agar, pH value 5.8.

[0021] More specifically, in step S3, the cluster bud induction medium is: MS basal medium + 0.05 mg / L naphthaleneacetic acid + 0.2 mg / L 6 - benzylaminopurine + 0.5 mg / L γ - aminobutyric acid + 25 g / L sucrose + 7 g / L agar, pH value 5.8.

[0022] More specifically, in step S4, the rooting induction medium is: 1 / 2 MS basal medium + 0.1 mg / L naphthaleneacetic acid + 0.5 mg / L γ - aminobutyric acid + 0.4 mg / L zeatin + 25 g / L sucrose + 7 g / L agar, pH value 5.8;

[0023] More specifically, in steps S2, S3 and S4, the culture conditions are as follows: temperature 26 ± 1°C, light duration 12 h / d, and light intensity 2000 lx.

[0024] More specifically, in steps S2 and S3, the stem segment length is 1.0 - 2.0 cm, and the top contains 1 pair of axillary buds.

[0025] More specifically, in step S5, during acclimatization, first do not open the bottle cap and acclimatize the seedlings for 2 - 3 d at room temperature, and then open the bottle cap and acclimatize the seedlings for 2 - 3 d at room temperature.

[0026] More specifically, the Mesona chinensis is Mesona chinensis Benth. var. paiwanensis H. W. Li.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention selects the stem segment of Mesona chinensis as the explant, and through the induction culture of the cluster bud induction medium of the present invention, it helps to improve the proliferation coefficient and reduce the vitrification rate, and further helps to induce the production of cluster buds from the stem segment of Mesona chinensis; subsequently, the rooting medium of the present invention is selected for induction culture, which helps to increase the number and length of roots, and further helps to induce the rooting of Mesona chinensis. The Mesona chinensis seedlings cultivated by the tissue culture and seedling formation method of the present invention have a high survival rate, and at the same time, the incidence of root rot in the field can be reduced. Moreover, compared with the shoot tip explant, the present invention can enrich the induction materials and help to carry out the work of tissue culture and seedling cultivation of Mesona chinensis. Detailed implementation manners

[0029] The technical solutions of the present invention patent will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. 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] 1. Materials and methods

[0031] 1.1 Tested Mesona chinensis

[0032] Mesona chinensis Benth. var. paiwanensis H. W. Li

[0033] 1.2 Processed explants

[0034] Select young and healthy Mesona chinensis branches without diseases and pests, soak and disinfect them with 0.1% HgCl2 for 10 min, then disinfect them with 75% alcohol for 50 s, then rinse them with sterile water 3 - 4 times, 1 min each time, and finally blot the surface moisture of the young branches with sterile filter paper.

[0035] 1.3 Sterile culture

[0036] Cut a stem segment with a pair of axillary buds at the top from young and tender branches. The stem segment is 2 cm long. Vertically insert its biological lower end into the primary medium and culture for 30 days to obtain aseptic seedlings;

[0037] Among them, the primary medium is: MS basal medium + 0.1 mg / L naphthaleneacetic acid + 0.2 mg / L 6-benzylaminopurine + 25 g / L sucrose + 7 g / L agar, pH value 5.8; The culture conditions are: temperature 26 ± 1 °C, light time 12 h / d, light intensity 2000 lx.

[0038] 1.4 Induction of clustered buds

[0039] Study the effects of different combinations of naphthaleneacetic acid, 6-benzylaminopurine and γ-aminobutyric acid on inducing clustered buds in the stem segments of Mesona chinensis.

[0040] Cut a stem segment with a pair of axillary buds at the top from the aseptic seedlings. The stem segment is 2 cm long. Vertically insert its biological lower end into the clustered bud induction medium (pH 5.8) in Table 1. Inoculate 4 segments in each bottle, and each medium treatment has 4 replicates, and each replicate inoculates 5 bottles. Culture for 30 days under the conditions of temperature 26 ± 1 °C, light time 12 h / d, and light intensity 2000 lx. Observe its growth situation and count the proliferation results. The results are shown in Table 2.

[0041]

[0042] Table 1 Effects of media with different hormone combinations on inducing clustered buds in the stem segments of Mesona chinensis

[0043]

[0044] Table 2 Results of the effects of media with different hormone combinations on inducing clustered buds in the stem segments of Mesona chinensis

[0045] Number Inoculation number (section) Proliferation coefficient Vitrification rate (%) Cluster bud induction medium 1 80 1.6 33.4 Cluster bud induction medium 2 80 2.7 41.9 Cluster bud induction medium 3 80 1.3 56.2 Cluster bud induction medium 4 80 5.5 23.4 Cluster bud induction medium 5 80 4.9 29.6 Cluster bud induction medium 6 80 6.8 38.2 Cluster bud induction medium 7 80 9.5 11.3

[0046] As can be seen from Table 2, different hormone tissue culture media have different effects on inducing clustered buds in the stem segments of Mesona chinensis. When using naphthaleneacetic acid, 6-benzylaminopurine or γ-aminobutyric acid alone, the proliferation coefficient is low and the vitrification rate is high, which is not conducive to inducing clustered buds in the stem segments of Mesona chinensis. When using them in pairwise combinations, the proliferation coefficient can be increased and the vitrification rate of the clustered buds can be reduced. When using all three at the same time, the proliferation coefficient can be significantly increased, and at the same time, the vitrification rate can be significantly reduced. It shows that the combination of naphthaleneacetic acid, 6-benzylaminopurine and γ-aminobutyric acid selected in the present invention helps to increase the proliferation coefficient and reduce the vitrification rate, and thus helps to induce clustered buds in the stem segments of Mesona chinensis, providing support for subsequent experiments.

[0047] 1.5 Rooting induction

[0048] The effects of different combinations of naphthylacetic acid, γ-aminobutyric acid and zeatin on the rooting induction of Agar-agar Grass were studied.

[0049] 2 cm clustered buds were selected and cut from the base and separated, and inoculated into the rooting medium (pH 5.8) in Table 3. Four segments were inoculated into each bottle, and each medium was treated 4 times, and each repetition was inoculated into 5 bottles. The seedlings were cultured for 30 days under the conditions of temperature 26±1°C, light duration 12h / d, and light intensity 2000lx to obtain tissue culture seedlings. The growth was observed and the rooting effect was statistically analyzed. The results are shown in Table 4.

[0050] Table 3 Effects of different hormone combination culture media on rooting induction of Agar-agar grass

[0051]

[0052] Table 4 Effects of different hormone combination culture media on rooting induction of Agar-agar grass

[0053] Number Inoculation number (piece) Average number of roots per plant (piece / plant) Average root length (cm) Root induction medium 1 80 6.3 0.36 Root induction medium 2 80 4.1 0.19 Root induction medium 3 80 2.7 0.28 Root induction medium 4 80 8.2 0.57 Root induction medium 5 80 5.3 0.84 Root induction medium 6 80 4.6 0.53 Root induction medium 7 80 12.2 1.42

[0054] As shown in Table 4, different hormone tissue culture medium has different effects on inducing the rooting of Agar-agar grass. When naphthylacetic acid, γ-aminobutyric acid or zeatin is used alone, the number of roots is small and the roots are short, which is not conducive to inducing the rooting of Agar-agar grass. When the three are used in combination in pairs, the number of roots and the length of roots can be increased, and when the three are used simultaneously, the number of roots and the length of roots can be significantly increased. It is shown that the present invention selects naphthylacetic acid, γ-aminobutyric acid and zeatin combination, which helps to increase the number of roots and the length of roots, and then helps to induce the rooting of Agar-agar grass, providing support for subsequent experiments.

[0055] 1.6 Hardening and transplanting

[0056] The tissue culture seedlings of the present invention were hardened at room temperature for 3 days without opening the bottle cap, and then hardened at room temperature for 3 days with the bottle cap opened; the seedlings were taken out, the roots were cleaned, and transplanted to a seedling tray containing a seedling medium (mixed by peat, fine sand and yellow mud in a mass ratio of 1:1:2). When the plant height was about 20 cm in a plastic greenhouse, it was transplanted to the field soil of the test base of Guangxi South Asian Tropical Agricultural Science Research Institute in Longzhou County, Chongzuo City, Guangxi, and 2,500 plants were transplanted per mu. At the same time, the Paitan agar grass cuttings that grew to about 20 cm were transplanted in the same test base as a control. After 2 months, a 5-point sampling method was adopted, 5 points were randomly selected per mu, and 100 plants were selected at each point to investigate the survival rate of the agar grass seedlings and the situation affected by root rot. The results are shown in Table 5.

[0057] Table 5 Growth of tissue culture seedlings and cutting seedlings

[0058] Seedling Number of plants (plant) Survival rate (%) Incidence of root rot (%) Tissue culture seedling 500 97.6 6.2 Cutting seedling 500 98.4 10.8

[0059] As can be seen from Table 5, the survival rates of the tissue-cultured seedlings and cuttage seedlings of the present invention after transplantation to the field are 97.6% and 98.4% respectively. The survival rates of both are relatively good and the difference is not significant. However, in terms of being affected by root rot disease, the incidence rate of root rot disease of the tissue-cultured seedlings of the present invention is only 6.2%, which is significantly lower than 10.8% of the cuttage seedlings, indicating that the tissue-cultured seedlings of the present invention can reduce the incidence rate of root rot disease in the field.

[0060] It should be noted that in the acclimatization and transplantation, the tissue-cultured seedlings of the present invention use the cluster bud induction medium 7 for cluster bud induction and the rooting induction medium 7 for rooting induction.

[0061] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the present invention to the precise forms disclosed, and obviously, many changes and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the present invention, as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for growing seedlings of agar-agar grass tissue culture seedlings, characterized in that: The following steps are involved: S1. Handling of explants Select young and tender branches that are healthy and free of diseases and insect pests, disinfect, rinse, and dry the surface moisture of the young and tender branches; S2. Sterile culture Cut the stem segments of young branches and vertically insert their biological lower ends into the primary culture medium and culture for 20-30 days to obtain sterile seedlings; S3. Induction of budding Cut the sterile seedling stem segment, insert the biological lower end of the segment vertically into the cluster bud induction medium and culture it for 30-45 days to obtain cluster buds; S4. Rooting Induction Select 2-3 cm clustered buds and cut them from the base, inoculate them into rooting medium and culture them for 30-35 days to obtain tissue culture seedlings; S5. Hardening and transplanting Harden the tissue culture seedlings at room temperature for 5-6 days, take out the seedlings, clean the roots, and transplant them into the seedling tray filled with seedling medium. After growing in the plastic greenhouse for 20-30 days, transplant them into the soil for growth.

2. The method for growing seedlings of grass agarwood tissue culture seedlings according to claim 1, characterized in that: In step S1, the branches are immersed in 0.1% HgCl2 for 10 minutes and then disinfected with 75% alcohol for 50 seconds, and then rinsed with sterile water for 3-4 times, each time for 1 minute, and finally the surface moisture of the young branches is absorbed with sterile filter paper.

3. The method for growing seedlings of grass agarwood tissue culture seedlings according to claim 1, characterized in that: In step S2, the primary culture medium is: MS basal medium + 0.1 mg / L naphthaleneacetic acid + 0.2 mg / L 6-benzylaminopurine + 25 g / L sucrose + 7 g / L agar, pH 5.

8.

4. The method for growing seedlings of grass agarwood tissue culture seedlings according to claim 1, characterized in that: In step S3, the clustered shoot induction medium is: MS basal medium + 0.05 mg / L naphthaleneacetic acid + 0.2 mg / L 6-benzylaminopurine + 0.5 mg / L γ-aminobutyric acid + 25 g / L sucrose + 7 g / L agar, pH 5.

8.

5. The method for growing seedlings of grass agarwood tissue culture seedlings according to claim 1, characterized in that: In step S4, the root induction medium is: 1 / 2MS basal medium + 0.1 mg / L naphthaleneacetic acid + 0.5 mg / L γ-aminobutyric acid + 0.4 mg / L zeatin + 25 g / L sucrose + 7 g / L agar, pH 5.

8.

6. The method for growing seedlings of agar-agar grass tissue culture seedlings according to claim 1, characterized in that: In steps S2, S3 and S4, the culture conditions are: temperature 26±1°C, light exposure time 12h / d, and light intensity 2000lx.

7. The method for growing seedlings of agar-agar grass tissue culture seedlings according to claim 1, characterized in that: In step S2, the stem segment is 1.0-2.0 cm in length and contains a pair of axillary buds at the top.

8. The method for growing seedlings of agar-agar grass tissue culture seedlings according to claim 1, characterized in that: In step S5, during the seedling hardening, the seedlings are first hardened at room temperature for 2-3 days without opening the bottle cap, and then hardened at room temperature for 2-3 days with the bottle cap opened.

9. The method for growing seedlings of agar-agar grass tissue culture seedlings according to claim 1, characterized in that: The agar-agar grass is Paitan agar-agar grass.

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