Tissue culture and rapid propagation method for lilium brownii and application

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method for lilium marginatum and application of the tissue culture and rapid propagation method for lilium marginatum, a non-lignified tender stem section is selected as an explant, the cleaned explant is disinfected with 75% alcohol, 0.01% mercury bichloride and 0.05% mercury bichloride in sequence, the disinfected explant is inoculated into a primary culture medium for culture, and the primary culture medium is inoculated with a culture medium for culture; cluster buds obtained after primary culture are cut into single buds to be inoculated into a subculture medium to be cultured, subcultured cluster buds are cut into single buds to be inoculated into a rooting medium to be cultured, the primary induction rate reaches 83%, the subculture multiplication coefficient is 6 or above, and the rooting rate reaches 99% or above.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for rapid propagation of Phyllostachys edulis using tissue culture and its application. Background Technology

[0002] Golden-edged Dracaena reflexa 'Variegata' is a variety (strain) of Dracaena reflexa in the genus Dracaena of the family Asparagaceae. It is a perennial evergreen shrub. Its leaves grow in loose clusters, are linear or lanceolate, with an acuminate apex, a glossy bright green color, and golden-yellow longitudinal stripes along the leaf margins. The inflorescences are solitary or branched, with small white flowers. Native to Madagascar and Mauritius, it is widely cultivated in tropical regions. It prefers hot and humid climates, is drought-tolerant but also moisture-tolerant, and thrives in fertile, peat-rich, well-drained soil. Due to its vibrant foliage, it is often used as a landscaping plant or a large potted ornamental plant.

[0003] Currently, the most common propagation method for Phyllostachys edulis is cuttings. However, this method has drawbacks such as a low propagation coefficient and a long propagation cycle, which limits its large-scale promotion and propagation. Therefore, it is necessary to establish a rapid tissue culture propagation method for Phyllostachys edulis. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by providing a method for rapid propagation of Phyllostachys edulis via tissue culture and its application.

[0005] To achieve the above objectives, the technical solution adopted by this invention is: a method for rapid propagation of Phyllostachys edulis via tissue culture, comprising the following steps:

[0006] (1) Selection of explants: Select young, non-lignified stem segments as explants;

[0007] (2) Disinfection: After cleaning, the explants are disinfected with 75% alcohol for 30-40 seconds, rinsed with sterile water 2-3 times, disinfected with 0.01% mercuric chloride for 4 minutes and rinsed with sterile water 3-4 times, and disinfected with 0.05% mercuric chloride for 8 minutes and rinsed with sterile water 6-7 times.

[0008] (3) Primary culture: The sterilized explants were inoculated into the primary culture medium and cultured for 40 to 45 days under alternating light and dark conditions at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 2000 to 3000 lx.

[0009] The formulation of the primary culture medium is: modified MS + NAA 0.02-0.05 mg / L + CPPU 1.0-1.5 mg / L + TDZ 3.0-3.5 mg / L;

[0010] (4) Subculture: The clustered shoots of the primary culture were cut into single shoots, inoculated into the subculture medium, and cultured under alternating light and dark conditions at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 2000-3000 lx for 45-55 days.

[0011] The subculture medium was formulated as follows: modified MS + NAA 0.05–0.1 mg / L + BR 0.01–0.02 mg / L + TDZ 1.5–2.0 mg / L + peptone 3.0–3.2 g / L;

[0012] (5) Rooting culture: Cut the subcultured buds into single buds, inoculate them into rooting culture medium, and place them in a light-dark alternation environment at a temperature of 25±2℃, with light intensity of 2000~3000lx for 14 hours a day for 20~25 days.

[0013] The rooting medium formula is: 1 / 2 modified MS + NAA 0.2-0.4 mg / L + IBA 1.0-1.5 mg / L + peptone 1.0-1.5 g / L;

[0014] The modified MS was prepared with the following settings: EDTA-Na2 concentration adjusted to 18.65 mg / L, FeSO4·7H2O concentration adjusted to 13.9 mg / L, inositol concentration adjusted to 2.0 g / L, glycine concentration adjusted to 400.00 mg / L, and pH adjusted to 6.2–6.4.

[0015] Furthermore, the formulation of the primary culture medium is: modified MS + NAA 0.03 mg / L + CPPU 13 mg / L + TDZ 3.3 mg / L.

[0016] Furthermore, the subculture medium is formulated as follows: modified MS + NAA 0.08 mg / L + BR 0.01 mg / L + TDZ 1.8 mg / L + peptone 3.1 g / L.

[0017] Furthermore, the rooting medium is formulated as follows: 1 / 2 modified MS + NAA 0.3 mg / L + IBA 1.3 mg / L + peptone 1.3 g / L.

[0018] The beneficial technical effects of this invention are:

[0019] 1. In the initial induction of Phyllostachys edulis 'Aureomarginata', the induction rate of clustered shoots is low when using conventional combinations of auxin and 6-BA or KT, reaching a maximum of only 13%. However, when using CPPU and TDZ, the sensitivity is stronger. When using either CPPU or TDZ alone, the induction rate of clustered shoots can only reach a maximum of 32%. When the two are combined at appropriate concentrations, the induction rate of clustered shoots can reach up to 75%. When an appropriate concentration of NAA is added, the induction rate can reach as high as 83%. In the field of tissue culture, the initial induction of most varieties for industrial production is carried out in research laboratories. Improving the initial induction rate can effectively improve laboratory efficiency, thereby reducing research and development costs.

[0020] 2. When the combination of CPPU and TDZ is continued in subculture, the 50-day proliferation coefficient can be stabilized at 3-4. If the cytokinin concentration is further increased, vitrification will occur to a certain extent. As the concentration increases, the proportion of vitrified seedlings increases. However, in this invention, a certain concentration of BR is used in subculture, while the concentration of TDZ is reduced, which can increase the 50-day proliferation coefficient to 6 or above.

[0021] 3. The golden-edged bamboo has golden stripes on its leaf edges. Under natural conditions, it can be propagated by cuttings, and most of its leaf edges will show golden stripes. However, cutting propagation has disadvantages such as low propagation coefficient and long propagation cycle. When tissue culture is used for propagation, a large number of seedlings can be produced in a short period of time. However, in the research process of this invention, it was found that some seedlings propagated by tissue culture did not show golden stripes on their leaf edges. This invention uses a modified MS, by adjusting the concentration of iron salts, some organic matter and pH, and adding a certain concentration of peptone. On the one hand, it can make the seedlings stronger, and on the other hand, it can make 99% or more of the seedlings show golden stripes on their leaf edges. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is the initial culture result of Example 1 of the present invention.

[0024] Figure 2 This is the result of subculture in Example 1 of the present invention.

[0025] Figure 3 This is the result of subculture in Example 3 of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Part of the experimental process in this invention

[0028] (a) Initial culture experiment

[0029] Table 1 Initial Design of the First Generation Induction

[0030] serial number Basic culture medium NAA 6-BA KT 1-1 MS 0.05 mg / L 1.0 mg / L 0 1-2 MS 0.05 mg / L 2.0 mg / L 0 1-3 MS 0.05 mg / L 4.0 mg / L 0 1-4 MS 0.05 mg / L 0 2.0 mg / L 1-5 MS 0.05 mg / L 0 4.0 mg / L 1-6 MS 0.05 mg / L 0 8.0 mg / L

[0031] Table 2 Results of initial induction

[0032] serial number Induction rate serial number Induction rate 1-1 3.6% 1-4 2.9% 1-2 5.2% 1-5 10.8% 1-3 4.1% 1-6 8.7%

[0033] As shown in Table 2, when MS is used as the basic culture medium and 0.05 mg / L NAA is added, along with different concentrations of 6-BA or KT, the highest induction rate is only 10.8%, which is extremely low for most plant tissue cultures.

[0034] Table 3. Second Experimental Design for Primary Induction

[0035] serial number Basic culture medium NAA CPPU TDZ 2-1 MS 0.05 mg / L 0.5 mg / L 0 2-2 MS 0.05 mg / L 1.0 mg / L 0 2-3 MS 0.05 mg / L 2.0 mg / L 0 2-4 MS 0.05 mg / L 0 1.0 mg / L 2-5 MS 0.05 mg / L 0 2.0 mg / L 2-6 MS 0.05 mg / L 0 4.0 mg / L

[0036] Table 4 Results of the second induction of the first generation

[0037] serial number Induction rate serial number Induction rate 2-1 14.6% 2-4 8.9% 2-2 21.9% 2-5 21.0% 2-3 30.8% 2-6 32.7%

[0038] As shown in Table 4, when MS is used as the basic culture medium and 0.05 mg / L NAA is added, along with different concentrations of CPPU or TDZ, the induction rate is significantly increased, with the highest induction rate reaching 32.7%.

[0039] First generation induction experiment design, third

[0040] Using MS as the basal medium, 0.05 mg / L NAA was added, and an orthogonal design was performed using CPPU and TDZ. The design is shown in Table 5.

[0041] Table 5 Orthogonal Experimental Design Table

[0042] serial number CPPU TDZ serial number CPPU TDZ 3-1 1.0 mg / L 3.0 mg / L 3-6 1.5 mg / L 6.0 mg / L 3-2 1.0 mg / L 4.0 mg / L 3-7 2.0 mg / L 3.0 mg / L 3-3 1.0 mg / L 6.0 mg / L 3-8 2.0 mg / L 4.0 mg / L 3-4 1.5 mg / L 3.0 mg / L 3-9 2.0 mg / L 6.0 mg / L 3-5 1.5 mg / L 4.0 mg / L

[0043] Table 6: Results of Orthogonal Experiments

[0044] serial number Induction rate serial number Induction rate 3-1 80.6% 3-6 37.2% 3-2 74.2% 3-7 66.7% 3-3 46.9% 3-8 40.8% 3-4 82.7% 3-9 20.3% 3-5 57.0%

[0045] As shown in Table 6, the induction rate is highest when CPPU 1.0 mg / L or CPPU 1.5 mg / L is combined with TDZ 3.0 mg / L, reaching 80.6-82.7%.

[0046] (ii) Subgeneration Proliferation Experiment

[0047] In general, during subculture in plant tissue culture, primary culture medium or appropriate adjustments to the primary culture medium are mostly used. However, in this invention, the original primary induction medium is used for subculture, and the highest proliferation coefficient is only 3.6. Moreover, the proliferation coefficient decreases with the number of subcultures, accompanied by severe vitrification. The same situation occurs when the concentrations of CPPU and TDZ are reduced. When MS is used as the basic medium and 0.05 mg / L NAA, 1.0 mg / L CPPU, and 2.0 mg / L TDZ are added, along with certain concentrations of 0.01 mg / L BR, 0.02 mg / L BR, or 0.04 mg / L BR, the proliferation coefficient can reach above 3.5, but all are accompanied by severe vitrification.

[0048] Subsequently, MS medium was selected as the basal medium, supplemented with 0.05 mg / L NAA, and different concentrations of CPPU, TDZ, and BR for orthogonal experimental design. The specific design is shown in Table 7.

[0049] Table 7. Successive orthogonal experimental design

[0050] serial number CPPU TDZ BR 4-1 0 0 0 4-2 0 2.0 mg / L 0.01 mg / L 4-3 0 3.0 mg / L 0.02 mg / L 4-4 1.0 mg / L 0 0.01 mg / L 4-5 1.0 mg / L 2.0 mg / L 0.02 mg / L 4-6 1.0 mg / L 3.0 mg / L 0 4-7 1.5 mg / L 0 0.02 mg / L 4-8 1.5 mg / L 2.0 mg / L 0 4-9 1.5 mg / L 3.0 mg / L 0.01 mg / L

[0051] Table 8 Results of successive orthogonal experiments

[0052] serial number Proliferation coefficient vitrification serial number Proliferation coefficient vitrification 4-1 1.8 none 4-6 4.1 +++ 4-2 6.3 + 4-7 3.9 +++ 4-3 6.4 ++ 4-8 3.9 ++++ 4-4 4.8 ++ 4-9 3.5 +++++ 4-5 4.3 ++

[0053] Note: "+" indicates that vitrification has occurred.

[0054] As shown in Table 8, the proliferation coefficient was the highest when using 2.0 mg / LTDZ + 0.01 mg / LBR and 3.0 mg / LTDZ + 0.02 mg / LBR, but the vitrification rate was also higher with the latter. The proliferation coefficient of 2.0 mg / LTDZ + 0.01 mg / LBR reached 6.3, and the vitrification rate was also lower.

[0055] Example 1

[0056] A method for rapid propagation of Phyllostachys edulis via tissue culture, comprising the following steps:

[0057] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0058] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 35 seconds, rinsed with sterile water 3 times, disinfected with 0.01% mercuric chloride for 4 minutes and rinsed with sterile water 4 times, and disinfected with 0.05% mercuric chloride for 8 minutes and rinsed with sterile water 7 times.

[0059] (3) Primary culture: After sterilization, the explants were inoculated into primary culture medium (formulation: modified MS + NAA 0.03 mg / L + CPPU 1.3 mg / L + TDZ 3.3 mg / L), and cultured at 25±2℃ under alternating light and dark conditions for 43 days with 12 hours of light per day and a light intensity of 2500 lx. Figure 1 As shown.

[0060] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into subculture medium (formulation: modified MS + NAA 0.08 mg / L + BR 0.01 mg / L + TDZ 1.8 mg / L + peptone 3.1 g / L), and cultured at 25 ± 2℃ under alternating light and dark conditions for 50 days with 12 hours of light per day and a light intensity of 2500 lx. Figure 2 As shown.

[0061] (5) Rooting culture: The subcultured shoots were cut into single shoots and inoculated into rooting medium (formulation: 1 / 2 modified MS + NAA 0.3 mg / L + IBA 1.3 mg / L + peptone 1.3 g / L). The medium was placed at a temperature of 25±2℃, with light for 14 hours a day and light intensity of 2500 lx under alternating light and dark conditions for 23 days.

[0062] The modified MS was as follows: the concentration of EDTA-Na2 was adjusted to 18.65 mg / L, the concentration of FeSO4·7H2O was adjusted to 13.9 mg / L, the concentration of inositol was adjusted to 2.0 g / L, the concentration of glycine was adjusted to 400.00 mg / L, and the pH was adjusted to 6.3.

[0063] Example 2

[0064] A method for rapid propagation of Phyllostachys edulis via tissue culture, comprising the following steps:

[0065] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0066] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 30 seconds, rinsed twice with sterile water, disinfected with 0.01% mercuric chloride for 4 minutes and rinsed three times with sterile water, and disinfected with 0.05% mercuric chloride for 8 minutes and rinsed six times with sterile water.

[0067] (3) Primary culture: The sterilized explants were inoculated into the primary culture medium (formulation: modified MS + NAA 0.02 mg / L + CPPU 1.0 mg / L + TDZ 3.0 mg / L) and cultured for 45 days under light and dark alternation at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 2000 lx.

[0068] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into subculture medium (formulation: modified MS + NAA 0.05 mg / L + BR 0.01 mg / L + TDZ 1.5 mg / L + peptone 3.0 g / L), and cultured at 25 ± 2℃ under alternating light and dark conditions for 55 days with 12 hours of light per day and a light intensity of 2000 lx. Figure 3 As shown.

[0069] (5) Rooting culture: The subcultured shoots were cut into single shoots and inoculated into rooting medium (formulation: 1 / 2 modified MS + NAA 0.2 mg / L + IBA 1.0 mg / L + peptone 1.0 g / L). The medium was placed at a temperature of 25±2℃, with light for 14 hours a day and light intensity of 2000~3000 lx under alternating light and dark conditions for 25 days.

[0070] The modified MS was as follows: the concentration of EDTA-Na2 was adjusted to 18.65 mg / L, the concentration of FeSO4·7H2O was adjusted to 13.9 mg / L, the concentration of inositol was adjusted to 2.0 g / L, the concentration of glycine was adjusted to 400.00 mg / L, and the pH was adjusted to 6.2.

[0071] Example 3

[0072] A method for rapid propagation of Phyllostachys edulis via tissue culture, comprising the following steps:

[0073] (1) Selection of explants: Select young, non-lignified stem segments as explants.

[0074] (2) Disinfection: After cleaning, the explants were disinfected with 75% alcohol for 40 seconds, rinsed with sterile water 3 times, disinfected with 0.01% mercuric chloride for 4 minutes and rinsed with sterile water 4 times, and disinfected with 0.05% mercuric chloride for 8 minutes and rinsed with sterile water 7 times.

[0075] (3) Primary culture: The sterilized explants were inoculated into the primary culture medium (formulation: modified MS + NAA 0.05 mg / L + CPPU 1.5 mg / L + TDZ 3.5 mg / L) and cultured for 40 days under light and dark alternation at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 3000 lx.

[0076] (4) Subculture: The clustered shoots from the primary culture were cut into single shoots and inoculated into subculture medium (formulation: modified MS + NAA 0.1mg / L + BR 0.02mg / L + TDZ 2.0mg / L + peptone 3.2g / L). The medium was placed at a temperature of 25±2℃, with light for 12 hours per day and light intensity of 3000lx, and cultured under alternating light and dark conditions for 45 days.

[0077] (5) Rooting culture: The subcultured shoots were cut into single shoots and inoculated into rooting medium (formulation: 1 / 2 modified MS + NAA 0.4 mg / L + IBA 1.5 mg / L + peptone 1.5 g / L). The medium was placed at a temperature of 25±2℃, with light for 14 hours a day and light intensity of 3000 lx under alternating light and dark conditions for 20 days.

[0078] The modified MS was as follows: the concentration of EDTA-Na2 was adjusted to 18.65 mg / L, the concentration of FeSO4·7H2O was adjusted to 13.9 mg / L, the concentration of inositol was adjusted to 2.0 g / L, the concentration of glycine was adjusted to 400.00 mg / L, and the pH was adjusted to 6.4.

[0079] Examples 1-3 were statistically compared in terms of disinfection contamination rate, disinfection mortality rate, primary induction rate, proliferation coefficient, and rooting rate. The results are shown in Table 9 below.

[0080] Table 9 Statistical Results

[0081] Disinfection contamination rate Disinfection mortality rate Primary induction rate Proliferation coefficient Rooting rate Example 1 24.6% 10.7% 83.0% 7.2 99.8% Example 2 29.8% 9.7% 81.8% 6.6 99.2% Example 3 27.4% 8.9% 82.4% 6.9 99.5%

[0082] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Phyllostachys edulis via tissue culture, characterized by the following steps: include: (1) Selection of explants: Select young, non-lignified stem segments as explants; (2) Disinfection: After cleaning, the explants are disinfected with 75% alcohol for 30-40 seconds, rinsed with sterile water 2-3 times, disinfected with 0.01% mercuric chloride for 4 minutes and rinsed with sterile water 3-4 times, and disinfected with 0.05% mercuric chloride for 8 minutes and rinsed with sterile water 6-7 times. (3) Primary culture: The sterilized explants were inoculated into the primary culture medium and cultured for 40 to 45 days under alternating light and dark conditions at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 2000 to 3000 lx. The formulation of the primary culture medium is: modified MS + NAA 0.02-0.05 mg / L + CPPU 1.0-1.5 mg / L + TDZ 3.0-3.5 mg / L; (4) Subculture: The clustered shoots of the primary culture were cut into single shoots, inoculated into the subculture medium, and cultured under alternating light and dark conditions at a temperature of 25±2℃, with 12 hours of light per day and a light intensity of 2000-3000 lx for 45-55 days. The subculture medium was formulated as follows: modified MS medium + NAA 0.05–0.1 mg / L + BR 0.01–0.02 mg / L + TDZ 1.5–2.0 mg / L + peptone 3.0–3.2 g / L; (5) Rooting culture: Cut the subcultured buds into single buds, inoculate them into rooting culture medium, and place them in a light-dark alternation environment at a temperature of 25±2℃, with light intensity of 2000~3000lx for 14 hours a day for 20~25 days. The rooting medium formula is: 1 / 2 modified MS + NAA 0.2-0.4 mg / L + IBA 1.0-1.5 mg / L + peptone 1.0-1.5 g / L; The modified MS was prepared with the following settings: EDTA-Na2 concentration adjusted to 18.65 mg / L, FeSO4·7H2O concentration adjusted to 13.9 mg / L, inositol concentration adjusted to 2.0 g / L, glycine concentration adjusted to 400.00 mg / L, and pH adjusted to 6.2–6.

4.

2. The method according to claim 1, characterized in that, The formulation of the primary culture medium was: modified MS + NAA 0.03 mg / L + CPPU 13 mg / L + TDZ 3.3 mg / L.

3. The method according to claim 1, characterized in that, The subculture medium was formulated as follows: modified MS + NAA 0.08 mg / L + BR 0.01 mg / L + TDZ 1.8 mg / L + peptone 3.1 g / L.

4. The method according to claim 1, characterized in that, The rooting medium formula is: 1 / 2 modified MS + NAA 0.3 mg / L + IBA 1.3 mg / L + peptone 1.3 g / L.

5. The application of the method according to any one of claims 1-4 in the rapid propagation of Phyllostachys edulis tissue culture.

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