Method for tissue culture of veratrum nigrum

Abstract

The present invention belongs to the technical field of tissue culture of medicinal plants. Provided in the present invention is a method for tissue culture of Veratrum nigrum. The method for tissue culture of Veratrum nigrum of the present invention comprises selection, disinfection, and inoculation and culture of an explant, wherein the explant is a Veratrum nigrum bulb, the disinfection comprises performing disinfection for 10-15 min using a Tween80 solution with a volume concentration of 0.3%-0.5%, performing disinfection for 2-5 min using an alcohol solution with a volume concentration of 95%, performing disinfection for 2-5 min using an NaClO solution with an available chlorine concentration of 2%-5%, and performing disinfection for 10-15 min using an NaClO solution with an available chlorine concentration of 0.2%-0.5%. In the present invention, the use of a Veratrum nigrum bulb as the explant for tissue culture is not seasonally restricted, and when combined with a proper method for explant disinfection, the short time requirement for obtaining aseptic Veratrum nigrum seedlings, and a high growth speed and a high seedling survival rate are achieved.

Description

Veratrum tissue culture method Technical Field

[0001] The invention belongs to the technical field of medicinal plant tissue culture, and in particular relates to a Veratrum tissue culture method. Background Art

[0002] Veratrum, a plant of the genus Veratrum in the Liliaceae family, is used medicinally, either as a root or as a whole plant with roots, for its expectorant, emetic, and insecticidal properties. In recent years, it has been further developed as a raw material for the production of biopesticides. Traditional Veratrum production typically involves seed propagation and seedling transplanting. Compared to seed and seedling cultivation, asexual propagation offers the advantages of faster growth, early flowering and fruiting, and the ability to maintain the beneficial characteristics of the parent plant.

[0003] But the current stage is less studied on the asexual propagation of Veratrum. CN 106105688 A (public date 2016.11.16) discloses a kind of cutting propagation method of Veratrum, and it is simple to operate, and propagation efficiency is fast. Except cuttage, setting up aseptic system is conducive to the collection and utilization of Veratrum germplasm resources, but the method that the sterile seedling of Veratrum is obtained is announced less, and conventional method uses seed or inflorescence as explant, and after aseptic decontamination, the sterile explant of Veratrum can be obtained, and then tissue culture is carried out. As CN 109452176 A (public date 2019.03.12) discloses a kind of medicinal plant Veratrum young embryo in vitro culture method, immature young embryo (seed) can be cultivated into seedling, promote Veratrum tissue culture seedling rooting rate. However, no matter using seed as explant, or using inflorescence as explant, its source all has obvious seasonality, hinders the efficiency that Veratrum sterile seedling is set up. In addition, the sterile seedlings obtained from seeds require a long time to grow and cannot provide sufficient explants for further genetic transformation treatment in the later stage; although the method of obtaining sterile seedlings through inflorescences can obtain a large number of sterile explants in a short time, it is not conducive to long-distance transportation, and there is a problem of contamination during transportation, which affects the efficiency of obtaining sterile seedlings.

[0004] Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a Veratrum tissue culture method, which uses Veratrum bulbs as explants for tissue culture, has no seasonal restrictions, is convenient to transport, has a low contamination rate, and takes a short seedling time.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A Veratrum tissue culture method comprises the following steps: selection, disinfection and inoculation of an explant, wherein the explant is a Veratrum bulb; the disinfection comprises disinfection with a Tween 80 solution having a volume concentration of 0.3% to 0.5% for 10 to 15 minutes, disinfection with a 95% alcohol solution having a volume concentration of 2 to 5 minutes, disinfection with a NaClO solution having an effective chlorine concentration of 2 to 5 minutes, and disinfection with a NaClO solution having an effective chlorine concentration of 0.2 to 0.5 minutes.

[0008] Preferably, the roots and rhizomes of Veratrum are removed, rinsed clean, and then excess leaves and leaf sheaths of the bulb are removed to obtain a clean bulb as an explant.

[0009] Preferably, the disinfection includes disinfection with a 0.4% Tween 80 solution by volume for 12.5 minutes, disinfection with a 95% alcohol solution by volume for 3.5 minutes, disinfection with a 3.5% NaClO solution by volume for 3.5 minutes, and disinfection with a 0.35% NaClO solution by volume for 12.5 minutes.

[0010] Preferably, after the Tween 80 solution, alcohol solution, and NaClO solution are disinfected, the bulbs are rinsed 3-5 times with sterile water.

[0011] Preferably, after disinfection with a NaClO solution having an effective chlorine concentration of 2% to 5%, the bulb is peeled off its outer scales and cut into wedge-shaped bulb pieces in half without exposing the shoot apex meristem.

[0012] Preferably, after disinfection with the NaClO solution having an effective chlorine concentration of 0.2%-0.5%, the excess leaves on the bulb apex meristem are stripped off, leaving 3-5 scales for inoculation and culture.

[0013] Preferably, during the inoculation culture, the culture medium includes basal medium MS, sucrose 28-32 g / L, agar 7-9 g / L, 6-BA 0.8-1.2 mg / L, NAA 0.1-0.3 mg / L, and pH 5.8±0.2.

[0014] More preferably, during the inoculation culture, the culture medium includes basal medium MS, 30 g / L sucrose, 8 g / L agar, 1 mg / L 6-BA, 0.2 mg / L NAA, and pH 5.8.

[0015] More preferably, the culture medium is sterilized by high pressure sterilization at 121° C. for 30 min.

[0016] Preferably, during the inoculation culture, the illumination time is 16h / 8h, the illumination intensity is 1000-1500Lx, the temperature is 25±1°C, and the duration is 4-10 weeks.

[0017] More preferably, during the inoculation culture, the illumination time is 16h / 8h, the illumination intensity is 1250Lx, the temperature is 25°C, and the duration is 4-10 weeks.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a Veratrum tissue culture method. By using Veratrum bulbs as explants, the long-distance transportation of the explants can be achieved without seasonal restrictions. Furthermore, by combining appropriate explant disinfection methods, the contamination rate is low, and the time to obtain sterile seedlings can be effectively shortened. Furthermore, under sterile conditions, the explants do not experience growth dormancy, and the biomass growth rate is fast, thus providing sufficient material for later genetic transformation and other applications.

[0020] Therefore, the tissue culture method of the present invention can effectively shorten the time to obtain sterile Veratrum seedlings. At the same time, when Veratrum seed resources are limited, a variety of high-biomass sterile Veratrum seedlings can be obtained in a short time and without being affected by seasons, thereby reducing the cost of producing sterile Veratrum seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1: Growth of Veratrum bulb explants after inoculation and culture for 1 day, 10 days, and 50 days;

[0022] Figure 2: Growth of Veratrum seed explants after inoculation and culture for 1 day, 60 days, and 105 days. DETAILED DESCRIPTION

[0023] The invention provides a Veratrum tissue culture method, which comprises the selection, disinfection and inoculation culture of explants.

[0024] The explant of the present invention is a Veratrum bulb; preferably, the Veratrum root and rhizome are removed, rinsed clean, and then excess leaves and leaf sheaths of the bulb are removed to obtain a clean bulb as the explant.

[0025] The disinfection method comprises the following steps: disinfection with a Tween 80 solution having a volume concentration of 0.3% to 0.5% for 10 to 15 minutes, preferably disinfection with a Tween 80 solution having a volume concentration of 0.4% for 12.5 minutes; disinfection with an alcohol solution having a volume concentration of 95% for 2 to 5 minutes, preferably disinfection with a 95% alcohol solution having a volume concentration of 3.5 minutes; disinfection with a NaClO solution having an effective chlorine concentration of 2% to 5% for 2 to 5 minutes, preferably disinfection with a NaClO solution having an effective chlorine concentration of 3.5% for 3.5 minutes; disinfection with a NaClO solution having an effective chlorine concentration of 0.2% to 0.5% for 10 to 15 minutes, preferably disinfection with a NaClO solution having an effective chlorine concentration of 0.35% for 12.5 minutes.

[0026] In the present invention, after disinfection with each disinfectant, the bulbs are preferably rinsed 3-5 times with sterile water, and more preferably, the bulbs are rinsed 4 times.

[0027] The present invention preferably uses a NaClO solution with an effective chlorine concentration of 2% to 5% to disinfect the bulb, then removes the external scales and cuts the bulb in half into wedge-shaped pieces without exposing the apical meristem to prevent subsequent disinfection from causing meristem death and affecting plant growth. Preferably, after disinfection with a NaClO solution with an effective chlorine concentration of 0.2% to 0.5%, remove excess leaves from the apical meristem of the bulb, leaving 3 to 5 scales for inoculation and culture.

[0028] The present invention preferably inoculates and cultures the culture medium comprising a basal culture medium MS, 28-32 g / L sucrose, 7-9 g / L agar, 0.8-1.2 mg / L 6-BA, 0.1-0.3 mg / L NAA, and a pH of 5.8 ± 0.2. Preferably, the basal culture medium MS, 30 g / L sucrose, 8 g / L agar, 1 mg / L 6-BA, and 0.2 mg / L NAA are used, with a pH of 5.8 ± 0.2. In an embodiment, the culture medium is prepared as follows: 4.74 g MS culture medium, 30 g sucrose, and 8 g / L agar are weighed, the volume is adjusted to 1 L with water, 1 mg 6-BA and 0.2 mg NAA are added, the pH is adjusted to 5.8, and the mixture is sterilized at 121°C for 30 min.

[0029] The present invention preferably inoculates and cultures the plant under illumination for 16 h / 8 h, with an illumination intensity of 1000-1500 Lx, preferably 1250 Lx, at a temperature of 25±1° C., preferably 25° C., and cultures are continued for 4-10 weeks to obtain sterile Veratrum seedlings.

[0030] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] A Veratrum tissue culture method comprises the following steps:

[0033] (1) Selection of explants:

[0034] The Veratrum plant is collected, the original bulb is removed from the root and rhizome, and then placed in running water for washing, and then the excess outer leaves and leaf sheaths are removed to obtain a clean bulb block;

[0035] (2) Disinfection:

[0036] (2.1) Immerse the bulbs in 0.4% Tween 80 solution with stirring for 12.5 minutes, and rinse four times with sterile water.

[0037] (2.2) Immerse the bulb in 95% alcohol solution by volume for 3.5 minutes, rinse four times with sterile water, and transfer the material to a sterile operating table;

[0038] (2.3) Soak the bulb in a 3.5% NaClO solution for 3.5 minutes, rinse with sterile water four times, remove the outer scales, and cut the bulb in half into wedge-shaped bulbs, making sure not to expose the shoot apical meristem.

[0039] (2.4) Place the cut wedge-shaped bulb pieces in a 0.35% NaClO solution for 12.5 minutes, then rinse four times with sterile water. Remove the excess leaves from the apical meristem, leaving only 3-5 scales to obtain the apical tissue.

[0040] (3) Inoculation and culture:

[0041] The shoot tip tissue from step (2.4) was inoculated into culture medium (weighing 4.74 g MS medium, 30 g sucrose, 8 g / L agar, adding water to 1 L, adding 1 mg 6-BA, 0.2 mg NAA, adjusting the pH to 5.8, and sterilizing at 121°C for 30 min), and cultured in a light incubator for 4-10 weeks under the culture conditions of 16 / 8h, 25±1°C, and 1250Lx.

[0042] Example 2

[0043] A Veratrum tissue culture method comprises the following steps:

[0044] (1) Selection of explants:

[0045] The Veratrum plant is collected, the original bulb is removed from the root and rhizome, and then placed in running water for washing, and then the excess outer leaves and leaf sheaths are removed to obtain a clean bulb block;

[0046] (2) Disinfection:

[0047] (2.1) Immerse the bulbs in 0.3% Tween 80 solution with stirring for 15 minutes, and rinse three times with sterile water;

[0048] (2.2) Immerse the bulb in 95% alcohol solution for 2 minutes, rinse three times with sterile water, and transfer the material to a sterile operating table;

[0049] (2.3) Soak the bulb in a 2% NaClO solution for 5 minutes, rinse three times with sterile water, remove the outer scales, and cut the bulb in half into wedge-shaped bulbs. Be careful not to expose the shoot apical meristem.

[0050] (2.4) Place the cut wedge-shaped bulb pieces in a 0.2% NaClO solution for 15 minutes, then rinse three times with sterile water. Remove the excess leaves from the apical meristem, leaving only 3-5 scales to obtain the apical tissue.

[0051] (3) Inoculation and culture:

[0052] The shoot tip tissue from step (2.4) was inoculated into culture medium (weighing 4.74 g of MS medium, 28 g of sucrose, 7 g / L of agar, diluting the volume to 1 L with water, adding 0.8 mg of 6-BA and 0.1 mg of NAA, adjusting the pH to 5.6, and sterilizing at 121°C for 30 min). Culture was carried out in a light incubator for 4-10 weeks under the conditions of 16 / 8 h, 25 ± 1°C, and 1000 Lx culture.

[0053] Example 3

[0054] A Veratrum tissue culture method comprises the following steps:

[0055] (1) Selection of explants:

[0056] The Veratrum plant is collected, the original bulb is removed from the root and rhizome, and then placed in running water for washing, and then the excess outer leaves and leaf sheaths are removed to obtain a clean bulb block;

[0057] (2) Disinfection:

[0058] (2.1) Immerse the bulbs in 0.5% Tween 80 solution with stirring for 10 minutes, and rinse with sterile water five times;

[0059] (2.2) Immerse the bulb in 95% alcohol solution for 5 minutes, rinse with sterile water five times, and transfer the material to a sterile operating table;

[0060] (2.3) Soak the bulb in a 5% NaClO solution for 2 minutes, rinse with sterile water five times, remove the outer scales, and cut the bulb in half into wedge-shaped bulbs, making sure not to expose the shoot apical meristem.

[0061] (2.4) Place the cut wedge-shaped bulb pieces in a 0.5% NaClO solution for 10 minutes, then rinse with sterile water five times. Remove the excess leaves from the apical meristem, leaving only 3-5 scales to obtain the apical tissue.

[0062] (3) Inoculation and culture:

[0063] The shoot tip tissue from step (2.4) was inoculated into culture medium (weighing 4.74 g MS medium, 32 g sucrose, 9 g / L agar, adding water to 1 L, adding 1.2 mg 6-BA, 0.3 mg / L NAA, adjusting the pH to 6.0, and sterilizing at 121°C for 30 min), and cultured in a light incubator for 4-10 weeks under the culture conditions of 16 / 8h, 25±1°C, and 1500 Lx culture.

[0064] Example 4

[0065] Differences in culture effects of different explants and disinfection methods of Veratrum

[0066] Method 1: Using the bulb as the explant, sterile seedlings were cultured according to the Veratrum tissue culture method of Example 1.

[0067] Method 2: Using Veratrum seeds as explants, sterile seedling culture is carried out, including the following steps:

[0068] Soak Veratrum seeds in sterile water twice, remove unfilled seeds floating on the water surface; disinfect with 75% alcohol for 30-60s, rinse with sterile water 3-5 times, vortex with 5% NaClO for 3-5min, rinse with sterile water 3-5 times, vortex with 5% H2O2 for 3-5min, rinse with sterile water 3-5 times, and inoculate into MS culture medium.

[0069] The culture results, shown in Figures 1 and 2, show that after sterilization, the collected Veratrum bulb explants achieved a sterile culture system after 10 days of culture. After 50 days, the Veratrum explants developed roots and significant leaf growth. Veratrum seeds, on the other hand, have a longer germination period, reaching the germination stage 60 days after sterilization and inoculation, and after three months of growth, the Veratrum shoots had not yet rooted. Compared to Veratrum seeds, using bulbs as explants is easier to obtain, requires a shorter sterile culture system, and produces significantly higher biomass than the control group.

[0070] Method 3: Using Veratrum inflorescence as explant, sterile seedling culture is carried out, including the following steps:

[0071] The collected inflorescences were first removed from the main stem and then placed in running water for 1-3 hours to remove excess external stem segments to obtain clean inflorescences. Subsequently, subsequent tissue culture was performed according to the disinfection method and culture method of Example 1 to obtain sterile seedlings.

[0072] Seven days after inflorescence disinfection and inoculation, statistical results showed that 39 of the 45 inflorescence explants tested were contaminated, a contamination rate of 87%. Ten days later, the contamination rate of Veratrum inflorescences reached 100%. Using bulbs as explants is easier to disinfect than using Veratrum inflorescences, and the sterile system requires less time.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for tissue culture of Veratrum nigrum, including the selection, disinfection and inoculation culture of explants, characterized in that, The explant is the bulb of Veratrum nigrum; the disinfection includes disinfecting with a Tween 80 solution with a volume concentration of 0.3%-0.5% for 10-15 minutes, disinfecting with an alcohol solution with a volume concentration of 95% for 2-5 minutes, disinfecting with an NaClO solution with an available chlorine concentration of 2%-5% for 2-5 minutes, and disinfecting with an NaClO solution with an available chlorine concentration of 0.2%-0.5% for 10-15 minutes.

2. The method for tissue culture of Veratrum nigrum L. according to claim 1, characterized in that, Remove the roots and rhizomes of Veratrum nigrum, rinse them thoroughly, and then remove the redundant leaves and leaf sheaths of the bulb to obtain a clean bulb as the explant.

3. The veratrum tissue culture method according to claim 1, characterized in that, The disinfection includes disinfecting with a Tween 80 solution with a volume concentration of 0.4% for 12.5 minutes, disinfecting with an alcohol solution with a volume concentration of 95% for 3.5 minutes, disinfecting with an NaClO solution with an available chlorine concentration of 3.5% for 3.5 minutes, and disinfecting with an NaClO solution with an available chlorine concentration of 0.35% for 12.5 minutes.

4. The method for tissue culture of Veratrum nigrum according to claim 1 or 3, characterized in that After disinfecting with the Tween 80 solution, alcohol solution, and NaClO solution, rinse the bulb with sterile water 3-5 times.

5. The method for tissue culture of Veratrum nigrum L. according to claim 1, wherein, After disinfecting with the NaClO solution with an available chlorine concentration of 2%-5%, peel off the outer scales of the bulb and cut it in half into wedge-shaped bulb pieces without exposing the shoot apical meristem.

6. The veratrum tissue culture method according to claim 1, characterized in that After disinfecting with the NaClO solution with an available chlorine concentration of 0.2%-0.5%, peel off the redundant leaves on the shoot apical meristem of the bulb and leave 3-5 scales for inoculation and culture.

7. The method for tissue culture of Veratrum nigrum L. according to claim 1, wherein During the inoculation and culture, the culture medium includes the basic medium MS, sucrose 28-32 g / L, agar 7-9 g / L, 6-BA 0.8-1.2 mg / L, NAA 0.1-0.3 mg / L, and pH 5.8±0.

2.

8. The method for tissue culture of Veratrum nigrum L. according to claim 7, characterized in that, During the inoculation and culture, the culture medium includes the basic medium MS, sucrose 30 g / L, agar 8 g / L, 6-BA 1 mg / L, NAA 0.2 mg / L, and pH 5.

8.

9. The method for tissue culture of Veratrum nigrum L. according to claim 7 or 8, characterized in that, The culture medium is sterilized by high pressure at 121°C for 30 minutes.

10. The method for tissue culture of Veratrum nigrum according to claim 1 or 7, characterized in that, During the inoculation and culture, the light time is 16 h / 8 h, the light intensity is 1000-1500 Lx, the temperature is 25±1°C, and it lasts for 4-10 weeks.

11. The method for tissue culture of Veratrum nigrum according to claim 10, characterized in that, During the inoculation and culture, the light time is 16 h / 8 h, the light intensity is 1250 Lx, the temperature is 25°C, and it lasts for 4-10 weeks.

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