Method for establishing system for inducing rapid vegetative propagation of helleborus thibetanus

Abstract

The invention discloses a method for inducing establishment of a rapid asexual propagation system of helleborus thibetanus, and relates to the technical field of plant tissue culture, and the method comprises the following steps: obtaining axillary buds: taking current-year axillary buds as explants, and pretreating the explants; performing induction culture: inoculating the obtained pretreated explants to an induction culture medium for induction culture, and culturing for a period of time to obtain plantlets; multiplication culture: preparing a multiplication culture medium in advance, inoculating the obtained plantlets into the multiplication culture medium for multiplication culture, and culturing for a period of time to obtain rootless plantlets; and rooting culture: preparing a rooting culture medium in advance, inoculating the obtained rootless plantlets into the rooting culture medium for rooting culture, and culturing for a period of time to obtain a complete plant. According to the method for establishing the system for inducing the rapid vegetative propagation of the helleborus thibetanus, MS is used as a basic culture medium, and the bud induction rate of the helleborus thibetanus is increased to 30% or above by adjusting the concentrations of TDZ and 6-BA in the induction culture medium.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for inducing the establishment of a rapid asexual reproduction system of Helleborus fasciatus. Background Art

[0002] Helleborus thibetanus is a perennial evergreen herb with a short plant shape, peculiar flowers and leaves, and a flowering period from winter to the following spring with rich flower colors. In recent years, Helleborus thibetanus has been very popular and its price has remained high. The breeding of new varieties of Helleborus thibetanus in China is still rare, and there are few research reports on the large-scale breeding of Helleborus thibetanus as a garden flower plant. The development of rapid asexual propagation technology in vitro for Helleborus thibetanus is still in its infancy, and many key technologies in the tissue culture system need to be urgently resolved.

[0003] Therefore, in order to solve the above problems, a method for inducing the establishment of a rapid asexual reproduction system of Helleborus tatarinowii is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus, comprising the following steps: S1, axillary bud acquisition: take axillary buds of the current year as explants and perform pretreatment of the explants; S2, induction culture: prepare the induction culture medium in advance, inoculate the explants pretreated in S1 onto the induction culture medium for induction culture, and obtain seedlings after a period of culture; S3, value-added culture, preparing a value-added culture medium in advance, inoculating the seedlings obtained in step S2 into the value-added culture medium for value-added culture, and obtaining rootless seedlings after a period of culture; S4, rooting culture, pre-prepare rooting medium, inoculate the rootless seedlings obtained in S3 into the rooting medium for rooting culture, and obtain complete plants after a period of culture.

[0006] Preferably, in step S1, the pretreatment of the current year's axillary buds is specifically as follows: Take the axillary buds of the current year, wash them with a little detergent, and rinse them under sterile water for 20-40 minutes until the surface is smooth and free of obvious dirt; Then, place the washed axillary buds of the current year in alcohol and let them stand for 5s-14s, then wash them repeatedly with sterile water to remove the residual alcohol on the surface; Then soak it in mercuric chloride solution for 10-20 minutes and then wash it with sterile water several times to remove residual alcohol on the surface.

[0007] Preferably, the concentration of alcohol used for disinfection of the current year's axillary buds is 70%-80%, the concentration of mercuric chloride solution is 0.05%-0.15%, and the current year's axillary buds are washed 2-4 times with sterile water.

[0008] Preferably, in step S2, the induction medium is MS + 0.2 mg / L TDZ + 1 mg / L 6-BA + 30 g / L sucrose, and the pH of the induction medium is between 5.0 and 6.5.

[0009] Preferably, in step S3, the proliferation medium is MS++0.5 mg / L 6-BA+1 mg / L NAA+30 g / L sucrose, and the pH of the induction medium is between 5.0-6.5.

[0010] Preferably, in step S4, the rooting medium is 1 / 2MS+0.2mg / L 6-BA+2mg / L NAA+30g / L sucrose, and the pH of the rooting medium is between 5.0-6.5.

[0011] Preferably, in S2, S3 and S4, the induction culture duration of the axillary buds is 20-40 days, the value-added culture duration of the seedlings is 20-40 days, and the rooting culture duration of the rootless seedlings is 20-40 days.

[0012] Preferably, the culture conditions in steps S1, S2, S3 and S4 are specifically: The temperature is 20℃-22℃; Light intensity is 1500-2000lx; Relative humidity is 65%-80%; The daily light duration is 12 hours.

[0013] Compared with the prior art, the technical effects of the present invention are: This scheme provides a method for inducing the establishment of a rapid asexual reproduction system for Helleborus. By formulating growth regulators in the culture medium, Helleborus can complete the entire process from bud induction, proliferation to rooting in only about 150 days, greatly shortening the asexual reproduction cycle of Helleborus and improving the reproduction efficiency.

[0014] This protocol provides a method for inducing the establishment of a rapid asexual propagation system for Helleborus chinensis. Using MS as the basic culture medium, the concentrations of TDZ and 6-BA in the induction culture medium are adjusted to increase the bud induction rate of Helleborus chinensis to more than 30%.

[0015] This scheme provides a method for inducing the establishment of a rapid asexual reproduction system of Helleborus thunbergii, using MS as the basic culture medium and adjusting the concentrations of 6-BA and NAA in the proliferation culture medium so that the proliferation coefficient of Helleborus thunbergii reaches 4 or more.

[0016] This protocol provides a method for inducing the establishment of a rapid asexual propagation system for Helleborus chinensis, using 1 / 2MS as the basic culture medium. By adjusting the concentrations of 6-BA and NAA in the rooting culture medium, the rooting rate reaches more than 70% and the number of roots reaches more than 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the axillary buds of the current year in an embodiment of the present invention.

[0018] Figure 2 This is a schematic diagram of the state of the axillary buds of Helleborus induced to proliferate 30 days in an embodiment of the present invention.

[0019] Figure 3 This is a schematic diagram of the state of the Helleborus officinalis seedlings 30 days after inducing rooting in an embodiment of the present invention. DETAILED DESCRIPTION

[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The present invention provides a method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus, comprising the following steps: S1. Explant acquisition and sterilization: Take the current year's axillary buds, add 0.1g of detergent to wash, rinse under running water for 30 minutes, disinfect with 75% alcohol for 30 seconds, rinse once with sterile water, disinfect with 0.1% Hgcl (mercuric chloride) for 20 minutes, rinse three times with sterile water, each time for 5 minutes, and finally obtain the sterilized axillary buds as shown in the figure. Figure 1 As shown; S2. Bud induction culture: Take the sterilized axillary buds, cut 1 cm in length from the top of the axillary buds, and inoculate them on the induction medium, which is MS + 0.2 mg / L TDZ + 1 mg / L 6-BA + 30 g / L sucrose, with a pH of 5.8. Seal the bottle and move the inoculated culture bottle into the culture room. Set the culture temperature to 21 ± 1 °C, the light intensity to 1500 lx, the relative humidity to 65%, and light for 12 hours per day. After about 90 days, new buds will be obtained. Test results: Table 1 Effects of different culture media on bud induction culture of Helleborus chinensis

[0022] S3. Proliferation culture: The induced buds were transferred to a proliferation medium, which contained: MS + 0.5 mg / L 6-BA + 1 mg / L NAA + 30 g / L sucrose, with a pH of 5.8, a culture temperature of 21 ± 1 °C, a light intensity of 1500 lx, a relative humidity of 65%, and 12 hours of light per day. After 30 days, a large number of proliferated seedlings were obtained. The state of the axillary buds after 30 days of proliferation culture is as follows: Figure 3 As shown; Test results: Table 2 Effects of different culture media on proliferation of Helleborus edulis

[0023] S4. Seedling rooting culture: The obtained seedlings were transferred to the rooting medium, which was: 1 / 2MS + 0.2mg / L 6-BA + 2mg / L NAA + 30g / L sucrose, pH 5.8, culture temperature was set at 21±1℃, light intensity was set at 1500lx, relative humidity was set at 65%, light was on for 12h per day, and the seedlings had strong roots after 30d. The status of the seedlings after 30d of rooting culture was as follows Figure 3 As shown; Test results: Table 3 Effects of different culture media on rooting culture of Helleborus chinensis

[0024] Combined with the above experimental data, it can be known that the method for inducing the establishment of a rapid asexual reproduction system of Helleborus provided in this embodiment can realize the complete process from bud induction, proliferation to rooting of Helleborus by adjusting the growth regulator in the culture medium, which only takes about 150 days; using MS as the basic culture medium, by adjusting the concentrations of TDZ and 6-BA in the induction culture medium, the concentrations of 6-BA and NAA in the proliferation culture medium, and the concentrations of 6-BA and NAA in the rooting culture medium, the bud induction rate of Helleborus is increased to more than 30%, the proliferation coefficient reaches more than 4, the rooting rate reaches more than 70%, and the number of roots reaches more than 3, thereby greatly shortening the asexual reproduction cycle of Helleborus and improving the reproduction efficiency; Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatula, characterized in that: The following steps are involved: S1, axillary bud acquisition: take axillary buds of the current year as explants and perform pretreatment of the explants; S2, induction culture: prepare the induction culture medium in advance, inoculate the explants pretreated in S1 onto the induction culture medium for induction culture, and obtain seedlings after a period of culture; S3, value-added culture, preparing a value-added culture medium in advance, inoculating the seedlings obtained in step S2 into the value-added culture medium for value-added culture, and obtaining rootless seedlings after a period of culture; S4, rooting culture, pre-prepare rooting medium, inoculate the rootless seedlings obtained in S3 into the rooting medium for rooting culture, and obtain complete plants after a period of culture.

2. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 1, characterized in that: In step S1, the pretreatment of the axillary buds of the current year is specifically as follows: Take the axillary buds of the current year, wash them with a little detergent, and rinse them under sterile water for 20-40 minutes until the surface is smooth and free of obvious dirt; Then, place the washed axillary buds of the current year in alcohol and let them stand for 5s-14s, then wash them repeatedly with sterile water to remove the residual alcohol on the surface; Then soak it in mercuric chloride solution for 10-20 minutes and then wash it with sterile water several times to remove residual alcohol on the surface.

3. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 2, characterized in that: The alcohol concentration used for disinfection of the current year's axillary buds is 70%-80%, the concentration of the mercuric chloride solution is 0.05%-0.15%, and the current year's axillary buds are washed 2-4 times with sterile water.

4. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 3, characterized in that: In step S2, the induction medium is MS + 0.2 mg / L TDZ + 1 mg / L 6-BA + 30 g / L sucrose, and the pH of the induction medium is between 5.0 and 6.

5.

5. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 4, characterized in that: In step S3, the proliferation medium is MS++0.5 mg / L 6-BA+1 mg / L NAA+30 g / L sucrose, and the pH of the induction medium is between 5.0-6.

5.

6. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 5, characterized in that: In step S4, the rooting medium is 1 / 2MS+0.2mg / L 6-BA+2mg / L NAA+30g / L sucrose, and the pH of the rooting medium is between 5.0-6.

5.

7. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 6, characterized in that: In S2, S3 and S4, the induction culture time of the axillary buds is 20-40 days, the value-added culture time of the seedlings is 20-40 days, and the rooting culture time of the rootless seedlings is 20-40 days.

8. The method for inducing the establishment of a rapid asexual reproduction system of Helleborus truncatus according to claim 7, characterized in that: The culture conditions in steps S1, S2, S3 and S4 are specifically: The temperature is 20℃-22℃; Light intensity is 1500-2000lx; Relative humidity is 65%-80%; The daily light duration is 12 hours.

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