Tissue culture and rapid propagation method of scirpus mariqueter

Abstract

The invention discloses a scirpus mariqueter tissue culture and rapid propagation method which comprises the following steps: selecting a stem of scirpus mariqueter as an explant, performing soaking and sterilization treatment on the stem, and sequentially performing sterile seedling germination, multiplication culture, seedling strengthening and rooting culture. According to the method, the stems of the scirpus mariqueter serve as explants, robust and variation-free aseptic seedlings are obtained after strict disinfection and sterilization treatment, germination, proliferation, seedling strengthening and rooting culture, the propagation efficiency and growth efficiency of the scirpus mariqueter are effectively improved, diseased plants are reduced, large-scale propagation of the scirpus mariqueter can be achieved, and the method is suitable for large-scale popularization and application of the scirpus mariqueter. Therefore, the method meets the urgent needs of local species for vegetation restoration and providing plant baits and habitats for wetland waterfowls in ecological restoration engineering, and is high in practicability.

Description

Technical Field

[0001] The invention relates to the technical field of plant propagation, in particular to a tissue culture and rapid propagation method of Tripterygium wilfordii. Background Art

[0002] Scirpus mariqueter Tanget Wang is a perennial halophyte of the Cyperaceae family. It is a unique halophyte in my country. It is usually a pioneer species in the lower part of the intertidal zone and plays an important role in the wetland ecosystem. It is currently mainly distributed in the islands of the Yangtze River Estuary and the tidal wetlands on the south bank of Hangzhou Bay. It is a key species in the Yangtze River Estuary wetland ecosystem. Scirpus mariqueter Tanget Wang has a high sediment interception ability due to its special leaf structure. In addition, in addition to having a capture and adhesion effect on suspended sand, the Scirpus mariqueter community that is not submerged by the tide can significantly weaken the wind force. When the wind speed on the light beach exceeds 10m / s, the wind speed at the bottom of the Scirpus mariqueter is close to 0 (less than 1m / s). Since wind is the energy source of waves, the weakening of wind force by the Scirpus mariqueter community can inhibit the resuspension of sediments caused by wave action.

[0003] At present, the tissue culture research of Tripterygium seaum mainly uses its seeds as explants for cultivation. For example, Chinese patent CN106258074B discloses a method for germination of Tripterygium seaum seeds, which improves the germination rate by low-temperature stratification treatment and variable temperature germination; Chinese patent CN105409748B discloses a method for rapid propagation of Tripterygium seaum, which uses mature seeds of Tripterygium seaum with part of the seed coat removed as explants, and obtains Tripterygium seaum seedlings that can be used for standardized production through primary culture, proliferation culture, rooting culture and other processes.

[0004] However, in the process of implementing the technical solutions of the invention in the embodiments of the present application, the inventors of the present application discovered that the above technology has at least the following technical problems: the plants obtained by rapid tissue culture propagation using seeds as explants are prone to mutation, some excellent genetic characteristics of the mother plant of the sea sedge fade or disappear during the propagation process, the plants after propagation have a certain degree of disease, poor growth, and reduced ecological restoration function. Summary of the invention

[0005] The invention provides a tissue culture rapid propagation method for Tripterygium seaum, which can solve the problems of high seedling mutation rate and many virus strains in the tissue culture rapid propagation process of Tripterygium seaum.

[0006] In order to solve the above technical problems, the present invention provides a method for tissue culture and rapid propagation of Tripterygium wilfordii, comprising the following steps:

[0007] (1) Selection and treatment of explants: The stems of Tripterygium wilfordii were selected as explants, and were immersed and sterilized;

[0008] (2) Germination of sterile seedlings: The sterilized stems were inoculated into a primary culture medium containing MS + 0.5 mg / L 6-BA until germination. -1 +GA30.5mg·L -1 +3% sucrose + 0.6% agar;

[0009] (3) Proliferation culture: The explants germinated in the primary culture medium are inoculated into a proliferation culture medium to obtain a sterile sample. The proliferation culture medium is MS+6-BA 1.0-3.0 mg·L -1 +TDZ 0.002mg·L -1 +IBA 0.1~0.3mg·L -1 +3% sucrose + 0.6% agar;

[0010] (4) Seedling culture: The sterile sample after proliferation culture is inoculated into a seedling culture medium, wherein the seedling culture medium is 1 / 2MS + 6-BA 0.02-0.1 mg·L -1 +IBA 0.00~0.02mg·L -1 +3% sucrose +0.6% agar.

[0011] (5) Rooting culture: Select sterile samples with good growth from the seedling medium and inoculate them into the rooting medium. The rooting medium is 1 / 2MS + IBA 0.00-0.30 mg·L -1 +3% sucrose +0.6% agar.

[0012] In a preferred embodiment of the present invention, the stem is a radial segment with nodes.

[0013] In a preferred embodiment of the present invention, in step (1), the method of soaking and disinfection and sterilization treatment is: soak the stems of the sea sedge in detergent water for 20 to 30 minutes, rinse them, soak them in 75% alcohol, rinse them, and then soak them in 10% sodium hypochlorite solution. After soaking, rinse them and set them aside.

[0014] In a preferred embodiment of the present invention, the immersion time in the 75% alcohol is 30 seconds, and the immersion time in the 10% sodium hypochlorite solution is 5 to 15 minutes.

[0015] In a preferred embodiment of the present invention, in steps (2) to (4), the culture conditions are: temperature 28-30°C, illumination time 12h, illumination intensity 2000Lx, and pH value 5.8-6.0.

[0016] In a preferred embodiment of the present invention, the proliferation medium is MS+6-BA 2.0 mg·L -1 +TDZ0.002mg·L -1 +IBA 0.2mg·L -1 +3% sucrose +0.6% agar.

[0017] In a preferred embodiment of the present invention, the seedling medium is 1 / 2MS + 6-BA 0.05mg·L -1 +IBA0.01mg·L -1 +3% sucrose +0.6% agar.

[0018] The beneficial effects of the present invention are as follows: the tissue culture rapid propagation method of the sea sclerotium of the present invention uses the stems of the sea sclerotium as explants, and obtains robust, non-variant sterile seedlings after strict disinfection and sterilization treatment, germination, proliferation, seedling strengthening and rooting culture, thereby effectively improving the reproduction efficiency and growth efficiency of the sea sclerotium, reducing diseased plants, and being able to achieve large-scale propagation of the sea sclerotium, thereby meeting the urgent needs of using the sea sclerotium as a native species for vegetation restoration in ecological restoration projects and providing planting bait and habitats for wetland water birds, and the method has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a photograph of sterile buds growing from the stems of the sea sedge of the present invention;

[0020] Figure 2 This is a photograph of the aseptic buds of the sea sedge of the present invention after proliferation;

[0021] Figure 3 This is a photograph of the sea sedge of the present invention after the seedlings have grown strong. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0023] The invention discloses a tissue culture and rapid propagation method of sea scutellaria, wherein the stem of sea scutellaria is used as an explant for tissue culture. Since the distribution of viruses in plants is uneven, the stem part is generally non-toxic or only carries viruses at a very low concentration. The stem is cut out under sterile conditions and then cultured on a suitable culture medium, which can regenerate virus-free plants on the one hand and maintain all genetic characteristics of the mother plant on the other hand.

[0024] The content of the present invention is described in detail below through specific embodiments.

[0025] Example 1

[0026] A tissue culture and rapid propagation method for Tripterygium wilfordii, the specific steps are as follows:

[0027] (1) Selection and processing of explants: Use tweezers and a scalpel to cut the stems of Tripterygium seaum as explants, specifically stem segments with nodes. Stem segments with nodes are immature plant tissues, are strong and have vigorous growth, can effectively avoid browning, and have a high germination rate.

[0028] After the cut stem segments are soaked in the foamed detergent water for 20 to 30 minutes, they are initially disinfected and sterilized, rinsed with running water for 20 minutes, placed in a clean beaker, and moved to a clean bench for use. In the clean bench, the soaked stem segments are first disinfected with 75% alcohol for 30 seconds, then rinsed with ddH2O for 3 times, then soaked with 10% sodium hypochlorite solution, and finally rinsed with ddH2O for 5 times.

[0029] When using sodium hypochlorite solution for disinfection and immersion, 4 groups were performed in parallel, as shown in Table 1, to obtain the optimal immersion time of sodium hypochlorite solution.

[0030] Table 1

[0031] Experimental Group 75% alcohol disinfection time NaClO disinfection time Contamination rate (%) Germination rate (%) Group 1 30s 5min 60 20 Group 2 30s 10min 60 20 Group 3 30s 15min 30 70 Group 4 30s 20min 10 0

[0032] The experimental results in Table 1 show that: based on the 75% alcohol disinfection for 30 seconds, the disinfection time of the sodium hypochlorite solution affects the contamination rate and germination rate of the stem segment. Among them, when the disinfection time of the sodium hypochlorite solution is 15 minutes, the disinfection and sterilization effect on the stem segment of the sea sedge is the best. In this embodiment, the stem segment after 75% alcohol disinfection for 30 seconds + 10% sodium hypochlorite solution disinfection for 15 minutes is selected as the explant for subsequent culture.

[0033] (2) Germination of sterile seedlings: The sterilized stems were inoculated into a primary culture medium containing MS + 0.5 mg / L 6-BA. -1 +GA30.5 mg·L -1 +3% sucrose +0.6% agar, pH 5.8; after inoculation, transfer the experimental bottle to a climate incubator, irradiate for 12 hours at a temperature of 28°C and a light intensity of 2000Lx, and culture until the stems sprout. Figure 1 shown.

[0034] (3) Proliferation culture: The explants sprouted in the primary culture medium were inoculated in groups into a proliferation culture medium containing different concentrations of plant growth regulators. After inoculation, the experimental bottles were transferred to a climate incubator and irradiated for 12 h at a temperature of 28° C. and a light intensity of 2000 Lx. The conditions after proliferation culture were as shown in the attached figure. Figure 2 shown.

[0035] The concentration of the plant growth regulator in the proliferation medium is shown in Table 2 below.

[0036] Table 2 Effects of plant growth regulators on proliferation

[0037] Experimental Group <![CDATA[Regulator / mg·L -1 > Number of vaccinations Proliferation number Proliferation coefficient / % Group 5 6-BA1.0+IBA0.1 10 10 1.0 Group 6 6-BA1.0+IBA0.2 10 14 1.4 Group 7 6-BA1.0+IBA0.3 10 12 1.2 Group 8 6-BA2.0+IBA0.1 10 19 1.9 Group 9 6-BA2.0+IBA0.2 10 27 2.7 Group 10 6-BA2.0+IBA0.3 10 23 2.3 Group 11 6-BA3.0+IBA0.1 10 19 1.9 Group 12 6-BA3.0+IBA0.2 10 20 2.0 Group 13 6-BA3.0+IBA0.3 10 17 1.7

[0038] Note: In addition to the regulators listed in the table above, the proliferation medium of Tripterygium seaweed was supplemented with 1x MS medium, 0.002 mg·L -1 TDZ, 3% sucrose and 0.6% agar.

[0039] From the data in Table 2, it can be seen that within a certain dosage range, different ratios of growth regulators 6-BA and IBA affect the proliferation effect of the explants of Tripterygium wilfordii. -1 +IBA 0.2mg·L -1 When used in combination in the following amounts, the proliferation effect on the explants of Tripterygium wilfordii is the best.

[0040] The proliferation medium used in this example is MS+6-BA 2.0 mg·L -1 +TDZ 0.002mg·L -1 +IBA0.2mg·L -1 +3% sucrose + 0.6% agar, pH 5.8, sterile samples were obtained after incubation.

[0041] (4) Seedling cultivation: The sterile samples after proliferation culture were inoculated into seedling cultivation medium containing different concentrations of plant growth regulators. After inoculation, the experimental bottles were transferred to a climate incubator and irradiated for 12 hours at a temperature of 28°C and a light intensity of 2000 Lx. The conditions after seedling cultivation were as shown in the attached figure. Figure 3 shown.

[0042] The concentration of the plant growth regulator in the seedling-strengthening culture medium is shown in Table 3 below.

[0043] Table 3 Effects of plant growth regulators on seedling growth

[0044] Experimental Group <![CDATA[Regulator / mg·L -1 > Plant height (cm) Growth status Group 14 6-BA0.02+IBA0.00 1～2 The seedlings are relatively short, and some have yellow leaves. Group 15 6-BA0.02+IBA0.01 2～3 Growth is average, some leaves have yellow Group 16 6-BA0.02+IBA0.02 1～2 The seedlings are relatively short, and some have yellow leaves. Group 17 6-BA0.05+IBA0.00 2～3 Growth is average, some leaves have yellow Group 18 6-BA0.05+IBA0.01 4～5 Good growth, dark green leaves Group 19 6-BA0.05+IBA0.02 3～4 Good growth, with small buds Group 20 6-BA0.10+IBA0.00 2～3 Average growth, with small buds Group 21 6-BA0.10+IBA0.01 2～3 Average growth, with small buds Group 22 6-BA0.10+IBA0.02 1～2 The seedlings are shorter and have more small buds.

[0045] Note: In addition to the regulators listed in the above table, the medium for growing seedlings of Tripterygium wilfordii contains 1 / 2 times of MS medium, 3% sucrose and 0.6% agar.

[0046] From the data in Table 3, it can be seen that the plant growth regulator 6-BA has a significant effect on the seedling growth of the explants of Tripterygium wilfordii, and 0.05 mg·L -1 6-BA and 0.01mg·L -1 When IBA is used in combination, the seedling strengthening effect is best.

[0047] The seedling medium used in this example is 1 / 2MS + 6-BA 0.05mg·L -1 +IBA 0.01mg·L -1 +3% sucrose +0.6% agar.

[0048] (5) Rooting culture: After seedling culture, sterile samples with good growth were selected and inoculated into rooting medium containing different concentrations of plant growth hormone. After inoculation, the experimental bottles were transferred to a climate incubator and irradiated for 12 hours at a temperature of 28°C and a light intensity of 2000Lx. The concentration of the plant growth regulator in the seedling culture medium is shown in Table 4 below.

[0049] Table 4 Effects of plant growth regulators on rooting

[0050]

[0051]

[0052] Note: In addition to the regulators listed in the above table, the medium for growing seedlings of Tripterygium wilfordii contains 1 / 2 times of MS medium, 3% sucrose and 0.6% agar.

[0053] From the data in Table 4, we can see that the plant growth hormone IBA has a significant effect on the rooting effect of the explants of Tripterygium wilfordii. When the concentration is 0.10 mg·L -1 Rooting effect is best when

[0054] The rooting medium used in this example is 1 / 2MS + IBA 0.10mg·L -1 +3% sucrose +0.6% agar. When the sterile sample is inoculated into the rooting medium and the plant root system grows, the sterile seedlings of Tripterygium wilfordii can be obtained.

[0055] The tissue culture and rapid propagation method of the sea sclerotium of the present invention uses the stems of the sea sclerotium as explants for tissue culture, which can effectively reduce the toxin content of the explant itself, and then through strict disinfection and sterilization treatment, the risk of seedling infection can be further effectively reduced to obtain a sterile sample, thereby improving the reproduction efficiency and growth efficiency of the sea sclerotium, effectively reducing diseased plants, and realizing large-scale propagation of the sea sclerotium to meet the urgent needs of using it as a native species for vegetation restoration in ecological restoration projects and providing planting bait and habitats for wetland water birds.

[0056] In addition, the present invention uses the stems of the sea sedge as explants for tissue culture, which can maintain all the genetic characteristics of the mother plant and has a low mutation rate, thereby ensuring that the excellent performance of the sea sedge is continued during the propagation process.

[0057] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for rapid propagation of Tripterygium wilfordii by tissue culture, characterized in that: The steps include: (1) Selection and treatment of explants: The stems of Tripterygium wilfordii were selected as explants, and were immersed and sterilized; (2) Germination of sterile seedlings: The sterilized stems were inoculated into a primary culture medium containing MS + 0.5 mg / L 6-BA until germination. -1 +GA30.5mg·L -1 +3% sucrose + 0.6% agar; (3) Proliferation culture: The explants germinated in the primary culture medium are inoculated into a proliferation culture medium to obtain a sterile sample. The proliferation culture medium is MS+6-BA 1.0-3.0 mg·L -1 +TDZ 0.002mg·L -1 +IBA 0.1~0.3mg·L -1 +3% sucrose + 0.6% agar; (4) Seedling culture: The sterile sample after proliferation culture is inoculated into a seedling culture medium, wherein the seedling culture medium is 1 / 2MS + 6-BA 0.02-0.1 mg·L -1 +IBA 0.00~0.02mg·L -1 +3% sucrose +0.6% agar. (5) Rooting culture: Select sterile samples with good growth in the seedling medium and inoculate them into the rooting medium, and culture them until roots grow.

2. The method for rapid propagation of Tripterygium wilfordii according to claim 1, characterized in that: The stem is a stem segment with nodes.

3. The method for rapid propagation of Tripterygium wilfordii according to claim 2, characterized in that: In step (1), the soaking and disinfection and sterilization treatment method is: soak the stems of the sea sedge in detergent water for 20 to 30 minutes, rinse them, soak them in 75% alcohol, rinse them, soak them in 10% sodium hypochlorite solution, rinse them after soaking and set them aside.

4. The method for rapid propagation of Tripterygium wilfordii according to claim 3, characterized in that: The immersion time of the 75% alcohol is 30 seconds, and the immersion time of the 10% sodium hypochlorite solution is 5 to 15 minutes.

5. The method for rapid propagation of Tripterygium wilfordii according to claim 2, characterized in that: In steps (2) to (4), the culture conditions are: temperature 28-30°C, illumination time 12h, illumination intensity 2000Lx, and pH value 5.8-6.

0.

6. The method for rapid propagation of Tripterygium wilfordii according to claim 2, characterized in that: The proliferation medium is MS+6-BA2.0 mg·L -1 +TDZ 0.002mg·L -1 +IBA 0.2mg·L -1 +3% sucrose +0.6% agar.

7. The method for rapid propagation of Tripterygium wilfordii according to claim 2, characterized in that: The seedling medium is 1 / 2MS+6-BA 0.05mg·L -1 +IBA 0.01mg·L -1 +3% sucrose +0.6% agar.

8. The method for rapid propagation of Tripterygium wilfordii according to claim 2, characterized in that: The rooting medium is 1 / 2MS+IBA 0.10mg·L -1 +3% sucrose +0.6% agar.

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