Establishment method of medicinal plant litsea cubeba tissue culture seedling system

Abstract

The invention discloses a method for establishing a medicinal plant litsea cubeba tissue culture seedling system. Aiming at the problems of difficulty in explant pollution and browning control, low proliferation and rooting efficiency, insufficient transplanting and domesticating survival rate and the like in establishment of a litsea cubeba tissue culture system in the prior art, the invention provides the establishment method of the medicinal plant litsea cubeba tissue culture seedling system, the germination rate of explants is 90% or above, and the transplanting survival rate in 30 days reaches 90.6%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for establishing a tissue culture seedling system of a medicinal plant Litsea cubeba. Background Art

[0002] Litsea cubeba is an important medicinal and spice plant. Its fruit is rich in citral (70-85%) and has antibacterial, antiviral, and antioxidant properties, making it widely used in medicine, food, and cosmetics. However, traditional propagation methods (seed sowing and cuttings) have significant limitations:

[0003] Seed propagation: The seeds have a long dormancy period (requiring stratification for more than 6 months), a low germination rate (<30%), and easy separation of genetic traits, making it difficult to meet the needs of large-scale planting;

[0004] Cutting propagation: Due to the age and season restrictions of the mother tree, the rooting rate fluctuates greatly (15%), the cuttings of mature trees are highly lignified, and the callus formation ability is poor.

[0005] Bottleneck problems of existing tissue culture technology:

[0006] In recent years, although some studies have attempted to establish a tissue culture system for Litsea cubeba, the following key issues still exist in practical applications:

[0007] (1) Problems in controlling explant contamination and browning:

[0008] Endophyte contamination: The contamination rate of endophytic fungi (such as Fusarium spp.) in the stems and leaves of Litsea cubeba is as high as 60-80%.

[0009] 2. Disinfection (0.1% for 8 minutes) can easily lead to explant death (mortality rate > 50%);

[0010] Oxidative browning of phenolic substances: The activity of polyphenol oxidase (PPO) surges after cutting, resulting in a browning rate of over 70% in the explants. Existing antioxidants (such as PVP and VC) can only inhibit the initial browning, and secondary browning still occurs during continued culture.

[0011] (2) Low proliferation and rooting efficiency:

[0012] Low axillary bud induction rate: The axillary bud induction rate of conventional MS+6-BA 2mg / L medium is only 30-40%, and vitrified seedlings are easily produced (incidence rate>15%);

[0013] Impaired adventitious root formation: During IBA-induced rooting, root primordium differentiation is delayed (>35 days), resulting in a short root system (<2 cm), and the root system is prone to inactivation after transplanting. Research suggests that impaired polar transport of endogenous auxin in Litsea cubeba is the primary cause (DOI: 10.1093 / aob / mcz145).

[0014] (3) Insufficient survival rate of transplanting and domestication:

[0015] The survival rate of tissue culture seedlings transplanted in the prior art is generally less than 50%, mainly due to:

[0016] Stomatal opening and closing disorder: Stomatal guard cell K in tissue culture seedlings + The channels are not fully functionalized, resulting in a water loss rate of >80% when the humidity drops sharply;

[0017] Lack of symbiotic microorganisms: Sterile seedlings lack growth-promoting rhizobacteria (PGPR), such as Pseudomonas, which affects the adaptability of the roots to the substrate.

[0018] Analysis on the difficulties in improving tissue culture technology of Litsea cubeba:

[0019] (1) Browning-contamination coordinated control: Although enhanced disinfection (e.g., HgCl2 concentration > .15%) can reduce the contamination rate, it will aggravate the oxidative stress of the explants, and a precise balance must be struck between sterilization intensity and cell activity protection;

[0020] (2) Root functionalization regulation: Conventional rooting culture medium is difficult to induce vessel molecule differentiation, and a new rooting culture medium ratio needs to be screened;

[0021] (3) Selection of transplanting medium and rooting agent. When the tissue culture seedlings of Litsea cubeba were transplanted into the traditional medium composed of nutrient soil, loess and vermiculite, the root system grew slowly and the survival rate was as low as 20%. Summary of the Invention

[0022] Aiming at the problems of establishing a tissue culture system of Litsea cubeba in the prior art, such as difficulty in controlling explant contamination and browning, low proliferation and rooting efficiency, and insufficient transplantation and acclimation survival rate, the present invention provides a method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba, with an explant germination rate of over 90% and a 30-day transplantation survival rate of 90.6%.

[0023] The purpose of the present invention is achieved through the following technical solutions:

[0024] A method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba, comprising the following steps:

[0025] S1. Selection and disinfection of explants: Select tender stem segments with liquid buds or terminal buds before germination, soak them in 75% alcohol for 2-5 seconds, then in 0.1% mercuric chloride for 10 minutes for sterilization, and then rinse with sterile water 3-4 times;

[0026] S2. Primary induction medium and culture method:

[0027] The primary induction culture medium is: WPM + CPPU 0.05mg / L + IBA 0.2mg / L, sucrose 30g / L, and agar 6g / L;

[0028] The primary induction culture condition is: temperature 26±2℃, dark culture for 7d, then light 1000-2000lx, light 14h / d;

[0029] S3, subculture proliferation medium and culture method: WPM + CPPU 0.02-0.05mg / L + IBA 0.2mg / L, sucrose 30g / L, and agar powder 6g / L, pH 5.5;

[0030] The subculture proliferation culture condition is: temperature 26±2℃, light 1000-2000lx, light 14h / d;

[0031] S4, rooting medium and culture method:

[0032] The rooting medium is: 1 / 2WPM + IBA 0.5mg / L + uniconazole 0.2mg / L, with sucrose 30g / L and agar powder 6g / L in the medium, pH 5.5;

[0033] The rooting culture condition is: temperature 26±2℃, light 2000-3000lx, light 14h / d;

[0034] S5, transplanting substrate and seedling training method:

[0035] The seedlings with good growth of 0.5-1.0cm root length obtained by rooting culture are opened and placed in a greenhouse for 5d of seedling training, then the medium on the plants is washed, and then soaked in 0.1% carbendazim solution for 1h before being transplanted into the substrate, and the rooting nutrient solution is selected as 500 times of silicious precision;

[0036] The volume ratio of the substrate is: V(coconut shell):V(wood fiber):V(peat):V(pine scale):V(vermiculite)=25:30:30:10:5.

[0037] Further, the WPM (wood plant medium containing calcium nitrate tetrahydrate) described in S2 has the basic formula shown in Table 1:

[0038] Table 1 WPM medium mother liquor formula

[0039]

[0040] Further, the primary induction culture medium described in S2 has a CPPU (plant growth regulator forchlorfenprop) concentration of 0.03mg / L.

[0041] Furthermore, the primary induction medium described in S2 was supplemented with 0.1 g / L activated carbon having a particle size of 20-40 μm.

[0042] Furthermore, using the primary induction medium and culture method of S2, the explant germination rate was ≥96.2%.

[0043] Furthermore, using the S3 subculture proliferation medium and culture method, the proliferation coefficient was ≥3.5 times after 30 days.

[0044] Furthermore, the rooting culture described in S4 adopts a light intensity gradient increasing mode:

[0045] First 10 days: 2000lx (light quality red: blue = 1:2)

[0046] Medium 10d: 2500lx (red:blue=1:1)

[0047] After 10d: 3000lx (red:blue=2:1).

[0048] Humidity control: maintain 95% RH for the first 15 days and reduce to 80% RH for the next 15 days.

[0049] Furthermore, using the rooting medium and culture method of S4, the rooting rate was ≥86.5% after 30 days.

[0050] Furthermore, a porosity grading system is established for the matrix described in S5:

[0051] Macropores (>50 μm): account for 15% of the total volume of the matrix and are composed of pine scales and vermiculite.

[0052] Mesopores (10-50 μm): account for 55% of the total volume of the matrix and are composed of coconut husks and wood fibers.

[0053] Micropores (<10 μm): account for 30% of the total volume of the matrix and are composed of peat.

[0054] The EC value is controlled at 0.8-1.2mS / cm, and the water holding capacity is 62%±3%.

[0055] Furthermore, using the transplanting medium and seedling training method of S5, the 30-day transplanting survival rate was ≥90.6%.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba described in the present invention systematically establishes a tissue culture technology system for Litsea cubeba, achieving the following effects:

[0058] 1. Explant contamination rate <1%.

[0059] 2. The explant germination rate is 96.2%, the 30-day proliferation coefficient is ≥3.5 times, and the 30-day rooting rate is ≥86.5%.

[0060] 3. The 30-day transplant survival rate is ≥90.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a bottom diagram of the rooting of Litsea cubeba tissue culture seedlings in the method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba described in an embodiment of the present invention;

[0062] Figure 2 This is a diagram of the rooting of Litsea cubeba tissue culture seedlings in the method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba described in an embodiment of the present invention;

[0063] Figure 3 This is a diagram of transplanting Litsea cubeba tissue culture seedlings in a method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba as described in an embodiment of the present invention;

[0064] Figure 4 This is a diagram showing the field growth over 180 days in a method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba as described in an embodiment of the present invention. DETAILED DESCRIPTION

[0065] The specific implementation of the present invention is further described below with reference to the examples.

[0066] Example:

[0067] A method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba, comprising the following steps:

[0068] 1. Explant screening and sterilization system

[0069] 1.1 Explant Optimization

[0070] Select the tender stem segments with axillary buds (length 1-2 cm) of plants in the early spring budding stage or the terminal buds before the dormant buds are released as explants, which have a thin wax layer on the surface and high meristematic cell activity.

[0071] Microscopic dissection verified that the intactness rate of the selected explant bud primordium reached 98.3%, which was significantly better than that of old branch segments (34.7%) and lateral buds (72.5%).

[0072] 1.2 Sterilization process optimization

[0073] Establishment of gradient sterilization system: first use 75% ethanol to shake sterilization (250 rpm) for 2-5 s, then immediately transfer into the sterilization solution containing 0.1% HgCl2+0.05% Tween-80 for 10 min (frequency 120 times / min), rinse with sterile water for 4 times, each time for 3 min, verify by plate culture, the contamination rate is controlled below 3.8%, and the survival rate of explants is ≥95%;

[0074] 2. Primary induction culture system

[0075] 2.1 Primary induction medium formula

[0076] The basic medium is modified WPM, and the specific composition is as follows:

[0077] WPM basic salt (containing 2.2 g / L Ca(NO3)2·4H2O)

[0078] Cell division factor: CPPU 0.05 mg / L ± 0.002 mg / L

[0079] Auxin: IBA 0.2 mg / L ± 0.01 mg / L

[0080] Carbon source: sucrose 30 g / L (purity ≥99%)

[0081] Solidifying agent: agar powder 6 g / L (gel strength ≥120 g / cm 2 )

[0082] pH value 5.8±.1 (adjusted by.1M KOH / HCl)

[0083] 2.2 Culture parameters

[0084] Implement two-stage culture of dark induction-light differentiation:

[0085] Dark culture stage: temperature 26±.5℃, relative humidity 70%±5%, for 7 d

[0086] Light induction stage: transfer to light intensity 150±200 lx (red light: blue light = 3:1), light period 14 h / d, temperature maintained at 26±1℃

[0087] Culture container: PP material culture box with light transmittance of 92%, CO2 concentration automatically compensated to 400 ppm

[0088] By HPLC detection, the peak value of endogenous IAA under this condition reaches 3.2 nmol / g FW on the 5th day, and the bud differentiation rate is 96.2% (n=300);

[0089] 3. Subculture proliferation culture system

[0090] 3.1 Medium optimization

[0091] Adjust based on the primary culture medium:

[0092] CPPU concentration gradient: 0.02-0.05 mg / L

[0093] Add 0.1g / L activated carbon (particle size 20-40μm)

[0094] Osmotic pressure regulator: 0.05% polyvinylpyrrolidone

[0095] 3.2 Cultivate technological innovation

[0096] Adopt shaking-static alternating culture method:

[0097] The first 10 days were cultured with slow shaking at 50 rpm

[0098] After 20 days of static culture

[0099] Add 1 / 4 volume of fresh culture medium every 7 days

[0100] Flow cytometry showed that this regimen increased the cell division index to 35.7%, the proliferation coefficient to 3.5±.3 times at 30 days, and the coefficient of variation to <5%.

[0101] 4. Construction of efficient rooting system

[0102] 4.1 Culture medium composition

[0103] 1 / 2WPM basic salt (N content reduced to 15mM)

[0104] IBA 0.5mg / L and uniconazole 0.2mg / L act synergistically

[0105] Add 0.3g / L phloroglucinol (root inducer)

[0106] 0.5g / L humic acid (purity ≥85%)

[0107] 4.2 Environmental Control

[0108] Adopting light intensity gradient increasing mode:

[0109] First 10 days: 2000lx (light quality red: blue = 1:2)

[0110] Medium 10d: 2500lx (red:blue=1:1)

[0111] Rear 10d: 3000lx (red:blue=2:1)

[0112] Humidity control: maintain 95% RH for the first 15 days and reduce to 80% RH for the next 15 days

[0113] Figure 1 is a bottom view of rooting of the tissue culture seedlings of Chinese Litsea in the example, Figure 2 is a view of rooting of the tissue culture seedlings of Chinese Litsea in the example; anatomical observation shows that the scheme induces 3-5 lignified adventitious roots with a diameter >.5mm, the rooting rate is 86.5%(n=200), and the survival rate of transplanting is increased by 27.3%;

[0114] 5. Transplanting acclimatization technical system

[0115] 5.1 Substrate optimization

[0116] Establish a porosity classification system:

[0117] Large pores (>50μm): 15% of the total volume of the substrate, composed of pine needles and vermiculite

[0118] Medium pores (10-50μm): 55% of the total volume of the substrate, composed of coconut coir and wood fiber

[0119] Micro-pores (<10μm): 30% of the total volume of the substrate, composed of peat

[0120] EC value is controlled at 0.8-1.2mS / cm, water holding capacity is 62%±3%

[0121] 5.2 Acclimatization process

[0122] Seedling training stage: CO2 concentration gradient decreases (200→400ppm), light intensity increases (300→15000lx)

[0123] Transplanting treatment: root system pretreatment with 500 times diatomite solution (containing 0.05% trehalose)

[0124] Environmental transition: air flow rate is maintained at 0.2m / s for the first 3d, and then gradually increased to 0.8m / s

[0125] Figure 3 is a view of transplanting of the tissue culture seedlings of Chinese Litsea in the example; Figure 4 is a view of 180d field growth in the example; through infrared thermal imaging monitoring, the scheme makes stomatal opening complete adaptive adjustment within 7d, the survival rate is 90.6% at 30d, and the new shoot growth amount is 4.2cm.

[0126] Example verification:

[0127] 500 samples were cultured for the whole cycle, the RSD value of the key indicators at each stage was <6.5%, the culture period was shortened by 22 days compared with the conventional method, the regenerated plants with uniform phenotype were obtained, and the genetic stability was 99.3% detected by SSR molecular markers.

[0128] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, it is possible to make several improvements and changes without departing from the creative concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba, characterized in that: The steps include: S1. Selection and disinfection of explants: Select tender stem segments with liquid buds or terminal buds before germination, soak them in 75% alcohol for 2-5 seconds, then in 0.1% mercuric chloride for 10 minutes for sterilization, and then rinse with sterile water 3-4 times; S2. Primary induction medium and culture method: The primary induction medium was: WPM+CPPU 0.05 mg / L+IBA 0.2 mg / L, sucrose 30 g / L, agar 6 g / L; The primary induction culture conditions were as follows: temperature 26 ± 2 °C, dark culture for 7 days, then switched to a light intensity of 1000-2000 lx, with a light intensity of 14 h / d; S3, Subculture proliferation medium and culture method: WPM + CPPU 0.02-0.05 mg / L + IBA 0.2 mg / L, sucrose 30 g / L and agar powder 6 g / L, pH 5.5; The conditions for subculture proliferation were as follows: temperature 26 ± 2°C, light intensity 1000–2000 lx, and light intensity 14 h / d; S4. Rooting medium and culture method: The rooting medium was 1 / 2WPM + IBA 0.5 mg / L + uniconazole 0.2 mg / L, with sucrose 30 g / L and agar powder 6 g / L added to the medium, and the pH value was 5.

5. The rooting culture conditions were as follows: temperature 26 ± 2 °C, light intensity 2000–3000 lx, and photoperiod 14 h / d; S5. Transplanting medium and seedling training method: Select the well-growing seedlings with 0.5-1.0 cm long roots obtained by root culture, open the culture bottles and place them in the greenhouse for 5 days to harden the seedlings, wash the culture medium on the plants, and then soak them in 0.1% carbendazim solution for 1 hour before transplanting them into the substrate. The rooting nutrient solution is 500 times dilution of diatomaceous earth; The volume ratio of the substrate is: V coconut husk: V wood fiber: V peat: V pine scale: V vermiculite = 25:30:30:10:

5.

2. The method for establishing a tissue culture seedling system of a medicinal plant Litsea cubeba according to claim 1, characterized in that: The primary induction medium described in S2, wherein the CPPU concentration is 0.03 mg / L.

3. The method for establishing a tissue culture seedling system of a medicinal plant Litsea cubeba according to claim 1, characterized in that: The primary induction medium described in S2 was supplemented with 0.1 g / L activated carbon with a particle size of 20-40 μm.

4. The method for establishing a tissue culture seedling system of a medicinal plant Litsea cubeba according to claim 1, characterized in that: The rooting culture described in S4 adopts a light intensity gradient increasing mode: First 10 days: 2000lx light quality red:blue=1:2; Medium 10d: 2500lx red: blue = 1:1; After 10 days: 3000lx red: blue = 2:1; Humidity control: maintain 95% RH for the first 15 days and reduce to 80% RH for the next 15 days.

5. The method for establishing a tissue culture seedling system of the medicinal plant Litsea cubeba according to claim 1, characterized in that: The matrix described in S5 was used to establish a porosity classification system: Macropores > 50 μm: account for 15% of the total volume of the matrix and are composed of pine scales and vermiculite; Mesopores 10-50 μm: account for 55% of the total volume of the matrix and are composed of coconut pith and wood fibers; Micropores <10 μm: account for 30% of the total volume of the matrix and are composed of peat; The EC value is controlled at 0.8-1.2mS / cm, and the water holding capacity is 62%±3%.

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