CN108668901B - Method for regenerating and propagating stem tip of lilac holly as Mongolian medicine - Google Patents
Method for regenerating and propagating stem tip of lilac holly as Mongolian medicine Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to the field of plant tissue culture, and relates to a research on a method for regenerating and propagating stem tips of a Mongolian drug Heilanshan clove, which is characterized by comprising the following steps: the method for regenerating and propagating the stem tip of the Syzygium aromaticum Hatch is as follows: cutting stem segments with buds from a robust stock plant, peeling stem tips after disinfection treatment, and placing the stem tips into a culture medium for primary culture; and (3) growing multiple shoots, cutting off single multiple shoots, carrying out subculture on the multiple shoots to obtain a large number of shoots of the Hailanshan clove, dividing the shoots into single plants, carrying out rooting culture, hardening seedlings and transplanting to obtain the transplanted seedlings of the Hailanshan clove. The beneficial effects are that: the method for regeneration and propagation of the stem tip of the Syzygium aromaticum is high in propagation speed, high in quality, stable in maternal genetic character, suitable for large-scale seedling propagation, simple, practicable, green and environment-friendly, and convenient to use and popularize, provides a large amount of high-quality seedling resources for production of rare Mongolian medicinal materials, provides product quality guarantee for industrialized unified management, reduces production cost of enterprises, and brings huge economic benefits.
Description
Technical Field
The invention belongs to the field of plant tissue culture, relates to research on a method for regenerating and propagating stem tips of a Mongolian drug Syzygium aromaticum, and particularly relates to research on a tissue culture technology of a plant inducing multiple buds by the stem tips of the Mongolian drug Syzygium aromaticum.
Background
The Syzygium aromaticum (Syringga pintifolia Hemsl. var. alashgnensis Maet s. Q. zhou) is a plant of Syzygium genus of Oleaceae family, is called Syzygium aromaticum, and is called alagha goldu bori, and is a variant of Syzygium pinnatifida (Syringga pintifolia Hemsley) which mainly grows on the lateral miscellaneous forest of the mountain ditch of the Hahland the filling altitude of 2000-3000 m, and roots and stems can be used as medicaments, so that the Syzygium aromaticum has the effects of relieving Heyi, clearing heat, relieving pain, relieving asthma and the like, has similar pharmacological action to the Chinese eaglewood, can also exceed the Chinese eaglewood in some aspects, can replace the Chinese eaglewood for use, and is a famous and precious medicinal material for Mongolian officinal medicine. In addition, the tender leaves can be used for making tea, the flowers can be used for extracting aromatic oil, and bonsais and cut flowers can be made to be used as landscaping ornamental plants. Therefore, the Heilanshan clove has higher medicinal value, ornamental value and market demand and is extremely urgent. However, in recent years, due to the deterioration of ecological environment and frequent and excessive intervention of human activities, the population of the Hailanshan clove is gradually reduced and even faces endangered risks, and the breeding work is very important. At present, the Hailashan clove is mainly bred by adopting the modes of sowing, cuttage and the like, but the seed breeding germination rate and the low seedling period are relatively long, the cuttage is difficult to root, a large amount of female parent materials are needed, the tree type is easy to damage, and the breeding rate of the Hailashan clove is far from meeting the market demand. However, the adoption of plant tissue culture can overcome the above disadvantages, but because different plant tissues have different difficulty degrees of culture, and the same material can also cause failure of tissue culture due to factors such as different organ sources, culture medium, temperature and the like, the finding of a rapid and effective propagation method for Heilanshan clove with stable hereditary character and capability of mass production is particularly important.
Disclosure of Invention
The invention aims to provide a rapid, effective and stable-genetic-trait propagation method for Heilanshan clove, namely a method for regenerating and propagating stem tips of Mongolian medicine Heilanshan clove.
The above purpose is realized by the following technical scheme, and the method for regenerating and propagating the stem tip of the lilac holly leaf, a Mongolian medicine, is provided, and is characterized in that: the method for regenerating and propagating the stem tip of the Syzygium aromaticum Hatch is as follows: selecting a Helianthus caryophyllus robust mother tree, cutting a stem section with buds from the robust mother tree, after disinfection treatment, stripping a stem tip as an explant material, and placing the stem tip into a stem tip meristem induction cluster bud primary culture medium for primary culture; after 30 days, cluster buds grow out, the single cluster buds are cut off and then subjected to subculture multiplication culture to obtain a large number of buds of the Hailanshan clove, the buds are divided into single plants, the single plants are inoculated to a rooting culture medium for rooting culture, and seedlings are hardened and transplanted to obtain transplanted seedlings of the Hailanshan clove.
The method for subculture proliferation comprises the following steps: and (3) putting the single cluster bud into an axillary bud induction proliferation culture medium, carrying out subculture proliferation culture on the cluster bud, after 2 weeks, growing an axillary bud from the cluster bud, selecting an axillary bud with the length of 1.5-3cm, inoculating the axillary bud on the single cluster bud induction axillary bud proliferation culture medium again for subculture, and repeating the operation until the activity of the axillary bud is reduced to obtain a large number of Syzygium hybridum buds.
The rooting culture method comprises the following steps: taking the bud with the height of 2cm, dividing the bud into single plants, transferring the single plants into a rooting culture medium, and carrying out rooting culture at the culture temperature of 25 +/-2 ℃ under the conditions of dark culture or illumination time of 10-12h/d and illumination intensity of 2000 plus and 3000lux to obtain the test-tube plantlet of the Syzygium aromaticum Rehd.
The hardening seedling transplanting method comprises the following steps:
taking a test-tube seedling which is 2-5cm high and has 3-5 roots, 3-4 leaves and 1-2cm leaf length and is cultured by rooting, hardening the seedling under natural illumination for 5-7 days, then opening a sealing film on a bottle cover to harden the seedling for 2-3 days, spraying the leaves by a water spraying kettle in the period, then taking the test-tube seedling out of a culture bottle, cleaning a culture medium at the root, planting the test-tube seedling on a planting substrate, keeping the air humidity at more than 70%, and obtaining the transplanted seedling of the Hailashan clove after 25-35 days.
The formula of the planting matrix is as follows: humus soil, perlite and vermiculite are 3: 1.
The healthy Hailanshan clove mother tree is a healthy Hailanshan clove without diseases and insect pests introduced into a scientific and technological park of the national university of inner Mongolia.
The primary culture medium for inducing the shoot apical meristem into the cluster buds is as follows:
MS +0.1-1.0mg/L KT +0.05-1.0 mg/L6-BA +0-1.0mg/L NAA +25-30g/L sucrose +5-8g/L agar + 400-doped 600mg/L PVP; the pH value is adjusted to 5.8-6.0.
The culture medium for inducing the axillary bud proliferation by the single cluster bud is as follows:
MS +1.0-5.0 mg/L6-BA +0.1-0.9mg/L IBA +20-30g/L sucrose +5-8g/L agar +400mg/L PVP; the pH value is adjusted to 5.8-6.0.
The rooting culture medium comprises:
1/2MS, MS +0.1-1.0mg/L NAA +0.5-2.5 mg/L6-BA +25-30g/L sucrose +5-8g/L agar; the pH value is adjusted to 5.8-6.0.
The method for sterilizing the stem segments with buds comprises the following steps: putting the stem segments with buds into a beaker, adding a few drops of Tween-20, washing for 20-40min under tap water, and transferring into an ultra-clean workbench; sterilizing with 75% alcohol, shaking for 27-33s, pouring out alcohol, and washing with sterile water for 3-5 times; adding 1% sodium hypochlorite solution, treating for 10-15min, pouring out sodium hypochlorite, washing with sterile water for 6-8 times, and drying with sterile filter paper.
The culture conditions of the primary culture are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 10-12h/d, and the illumination intensity is 2000-.
The culture conditions of the subculture proliferation culture are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 10-12h/d, and the illumination intensity is 2000-.
Furthermore, the stem segment with the bud is treated by the following steps after being sterilized and before being stripped and put into a stem tip meristem induction cluster bud primary culture medium for primary culture: treating the stem with buds at 0-4 deg.C for 10-12 h.
The invention has the beneficial effects that: the provided tissue culture method for the stem tip regeneration and propagation of the Hailanshan clove can enable the Hailanshan clove to establish a sterile plant in a short time, carry out mass industrial production, can be produced annually, has high propagation speed, forms a propagation method which has high quality, stable maternal genetic character, is suitable for large-scale seedling propagation, and provides a large amount of high-quality seedling resources for the production of rare Mongolian medicinal materials; because the plants are consistent in size, the method provides product quality guarantee for industrialized unified management, reduces the production cost of enterprises, and brings huge economic benefits. And the method is simple and easy to implement, green and environment-friendly, and convenient to use and popularize.
The specific embodiment is as follows:
the first embodiment: selecting a Helianthus caryophyllus robust mother tree, cutting a stem section with buds from the robust mother tree, after disinfection treatment, stripping a stem tip as an explant material, and placing the stem tip into a stem tip meristem induction cluster bud primary culture medium for primary culture; after 30 days, cluster buds grow out, the single cluster buds are cut off and then subjected to subculture multiplication culture to obtain a large number of buds of the Hailanshan clove, the buds are divided into single plants, and the single plants are inoculated to a rooting culture medium for rooting culture and seedling hardening and transplanting to obtain a transplanted seedling of the Hailanshan clove.
The method for subculture proliferation comprises the following steps: and (3) putting the single cluster bud into an axillary bud induction proliferation culture medium, carrying out subculture proliferation culture on the cluster bud, after 2 weeks, growing an axillary bud from the cluster bud, selecting an axillary bud with the length of 1.5-3cm, inoculating the axillary bud on the single cluster bud induction axillary bud proliferation culture medium again for subculture, and repeating the operation until the activity of the axillary bud is reduced to obtain a large number of Syzygium hybridum buds.
The rooting culture method comprises the following steps: taking the bud with the height of 2cm, dividing the bud into single plants, transferring the single plants into a rooting culture medium, and carrying out rooting culture at the culture temperature of 25 +/-2 ℃ under the conditions of dark culture or illumination time of 10-12h/d and illumination intensity of 2000 plus and 3000lux to obtain the test-tube plantlet of the Syzygium aromaticum Rehd.
The hardening seedling transplanting method comprises the following steps:
taking a test-tube seedling which is 2-5cm high and has 3-5 roots, 3-4 leaves and 1-2cm leaf length and is cultured by the rooting culture, hardening the seedling under natural illumination for 5-7 days, then opening a sealing film on a bottle cover to harden the seedling for 2-3 days, spraying a water spray kettle on the leaves in the period, then taking out the test-tube seedling from a culture bottle, cleaning a culture medium at the root, planting the test-tube seedling in a planting matrix of humus soil, perlite and vermiculite, wherein the ratio of air humidity is 3: 1, keeping the air humidity above 70%, and obtaining the transplanted seedling of the Hailashan clove after 25-35 days.
The method specifically comprises the following steps:
1. the material taking method comprises the following steps:
selecting a strong Helianthus caryophyllus plant without diseases and insect pests introduced in the scientific and technological park of the national university of inner Mongolia, cutting a stem section with buds, and stripping a stem tip as an explant material.
2. Preparation of a culture medium:
(1) culture medium for inducing cluster buds at the primary generation of stem tips:
MS +1.0mg/L KT +1.0 mg/L6-BA +0-1.0mg/L NAA +30g/L sucrose +6g/L agar +400mg/L PVP; the pH was adjusted to 5.8.
(2) Single cluster bud induction axillary bud proliferation medium:
MS +5.0 mg/L6-BA +0.9mg/L IBA +20-30g/L sucrose +8g/L agar +400mg/L PVP; the pH was adjusted to 5.8.
(3) Rooting culture medium:
MS +1.0mg/L NAA +2.5 mg/L6-BA +30g/L sucrose +8g/L agar; the pH was adjusted to 5.8.
3. Material treatment:
(1) placing stem segments of the Hailanshan clove with lateral buds or terminal buds in a beaker, adding a few drops of Tween-20, washing for 20min under tap water, transferring to a super-clean workbench, disinfecting and shaking with 75% alcohol for 30s, pouring out the alcohol, washing with sterile water for 5 times, adding 1% sodium hypochlorite solution for treatment for 15min, pouring out the sodium hypochlorite, washing with the sterile water for 6 times, and sucking surface water with sterile filter paper; stripping the stem tip of about 3mm, and inoculating to primary culture medium for culture.
(2) And transferring the cluster buds cultured by the primary generation of the stem tip to an ultra-clean workbench, taking out the explant, placing the explant on sterile filter paper, cutting the cluster buds to form single buds, wherein the single buds can be used for subculture multiplication and rooting culture.
4. And (3) inducing and culturing cluster buds:
and (3) inoculating the stripped stem tip with the diameter of 3mm into a primary culture medium for culture, wherein the culture temperature is 25 +/-2 ℃, the illumination time is 12h/d, and the illumination intensity is 2000-3000 lux.
5. And (3) proliferation culture:
cutting the cluster buds cultured by the primary generation of the stem tip to form single buds, inoculating the single buds into a subculture multiplication medium, and culturing at the temperature of 25 +/-2 ℃, with the illumination time of 12h/d and the illumination intensity of 2000-3000 lux.
6. Inducing and rooting:
when the cluster buds grow to 3cm high, the buds are divided into single plants and transferred to a rooting culture medium; the culture temperature is 25 +/-2 ℃, the illumination time is 12h/d, and the illumination intensity is 2000-.
7. Transplanting the plantlets:
when the test-tube plantlet grows to 3cm high and has 5 roots and 3 leaves, and the length of the leaves is 1cm, hardening the plantlet, putting the test-tube plantlet under natural light to harden the plantlet for 7d, opening a sealing film on a bottle cap to harden the plantlet for 3d, and spraying a sprinkling can to the leaves to ensure that the leaves do not lose water so as to enhance the adaptability of the test-tube plantlet to the outdoor environment; then taking out the plantlet from the culture bottle, cleaning the root culture medium, and then planting in a matrix for field planting: humus, perlite and vermiculite are 3: 1, and the transplanted seedlings are obtained after 30 days. Keeping the air humidity above 70%, and placing in a cool and ventilated place with the light from weak to strong in the early stage. The survival rate of the transplanted seedlings can reach 80 percent.
A large number of experiments prove that the tissue propagation method for the Hailanshan clove can enable the Hailanshan clove to establish sterile plants in a short time, carry out large-scale industrial production, can be produced annually and propagated quickly, forms a propagation method which has high quality, stable maternal genetic character and is suitable for large-scale seedling propagation, provides a large amount of high-quality seedling resources for production and garden beautification of rare Mongolian medicine Hailanshan clove, solves the problems that the seed propagation germination rate (the germination rate of the Hailanshan seeds is only 14 percent reported by relevant documents) and the period of young seedlings is relatively long, and cuttage is difficult to root so as to cause the Hailanshan clove to be endangered, improves the drought resistance, cold resistance and stress resistance of the Hailanshan clove, provides excellent plants and fills in the blank of the propagation field of the Hailanshan clove.
The second embodiment: in addition to the first embodiment, further, the stem segments with buds are treated as follows after being sterilized and before being stripped and put into a primary medium for primary culture of shoot apical meristem-inducing multiple shoots: treating the stem with buds at 0-4 deg.C for 10-12 h. The method comprises the following specific steps: selecting a Hailan caryophyllus robust mother tree, cutting a stem section with buds from a robust mother plant, carrying out low-temperature treatment for 10-12 hours, preferably 11 hours, at the temperature of 0-4 ℃ after disinfection treatment, stripping a stem tip as an explant material, and placing the explant material into a stem tip meristem induction cluster bud primary culture medium for primary culture; after 30 days, cluster buds grow out, the single cluster buds are cut off and then subjected to subculture multiplication culture to obtain a large number of buds of the Hailanshan clove, the buds are divided into single plants, the single plants are inoculated to a rooting culture medium for rooting culture, and seedlings are hardened and transplanted to obtain transplanted seedlings of the Hailanshan clove.
Through a large number of repeated and comparative experiments, compared with the first embodiment, under the same conditions, the budding rate of the multiple shoots of the shoot apical meristem induced multiple shoots is 1.1-1.3 times that of the first embodiment, and the comparison of the survival rate data of the transplanted seedlings shows that the survival rate of the transplanted seedlings in the embodiment is averagely improved by 3 percent.
Claims (3)
1. A method for regenerating and propagating the stem tip of a Heilanshan clove serving as a Mongolian medicine is characterized by comprising the following steps: the method for regenerating and propagating the stem tip of the Syzygium aromaticum Hatch is as follows: selecting a Helianthus caryophyllus robust mother tree, cutting a stem section with buds from the robust mother tree, after disinfection treatment, stripping a stem tip as an explant material, and placing the stem tip into a stem tip meristem induction cluster bud primary culture medium for primary culture; after 30 days, cluster buds grow out, the cluster buds are cut off and then subjected to subculture multiplication culture to obtain a large number of buds of the Hailanshan clove, the buds are divided into single plants, the single plants are inoculated to a rooting culture medium for rooting culture, hardening seedlings and transplanting are carried out to obtain transplanted seedlings of the Hailanshan clove,
the method for subculture comprises the following steps: placing the single cluster bud into an axillary bud proliferation induction culture medium, carrying out subculture on the cluster bud proliferation induction culture medium, after 2 weeks, allowing the cluster bud to grow axillary buds, selecting 1.5-3cm axillary buds, inoculating the axillary buds on the single cluster bud proliferation induction culture medium for subculture, repeating the above operation until the axillary bud activity is reduced to obtain a large amount of Syzygium aromaticum buds,
the rooting culture method comprises the following steps: taking a bud with the height of 2cm, dividing the bud into individual plants, transferring the individual plants into a rooting culture medium, carrying out rooting culture at the culture temperature of 25 +/-2 ℃, under the conditions of dark culture or illumination time of 10-12h/d and illumination intensity of 2000-3000lux to obtain a test-tube plantlet of the Syzygium aromaticum,
the hardening seedling transplanting method comprises the following steps: taking a test-tube seedling which is 2-5cm high and has 3-5 roots, 3-4 leaves and 1-2cm leaf length and is cultured by rooting, hardening the seedling under natural illumination for 5-7 days, then opening a sealing film on a bottle cover to harden the seedling for 2-3 days, spraying a water spray kettle on the leaves, taking out the test-tube seedling from a culture bottle, cleaning a culture medium at the root, planting the test-tube seedling on a planting substrate, keeping the air humidity at more than 70 percent, and obtaining a transplanted seedling of the Hailan caryophyllata after 25-35 days,
the formula of the planting matrix is as follows: humus soil: perlite: vermiculite is 3: 1,
the primary culture medium for inducing the shoot apical meristem into the cluster buds is as follows: MS +0.1-1.0mg/L KT +0.05-1.0 mg/L6-BA +0-1.0mg/L NAA +25-30g/L sucrose +5-8g/L agar + 400-doped 600mg/L PVP; the pH value is adjusted to be between 5.8 and 6.0,
the culture medium for inducing the axillary bud proliferation by the single cluster bud is as follows: MS +1.0-5.0 mg/L6-BA +0.1-0.9mg/L IBA +20-30g/L sucrose +5-8g/L agar +400mg/L PVP; the pH value is adjusted to be between 5.8 and 6.0,
the rooting culture medium comprises: 1/2MS, MS +0.1-1.0mg/L NAA +0.5-2.5 mg/L6-BA +25-30g/L sucrose +5-8g/L agar; the pH value is adjusted to be between 5.8 and 6.0,
the method for sterilizing the stem segments with buds comprises the following steps: putting the stem segments with buds into a beaker, adding a few drops of Tween-20, washing for 20-40min under tap water, and transferring into an ultra-clean workbench; sterilizing with 75% alcohol, shaking for 27-33s, pouring out alcohol, and washing with sterile water for 3-5 times; adding 1% sodium hypochlorite solution, treating for 10-15min, pouring out sodium hypochlorite, washing with sterile water for 6-8 times, drying with sterile filter paper,
the culture conditions of the primary culture are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 10-12h/d, the illumination intensity is 2000-,
the culture conditions of the subculture proliferation culture are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 10-12h/d, and the illumination intensity is 2000-.
2. The method for regenerating and propagating the stem tip of the Syzygium Helianthi as the Mongolian medicine according to claim 1, which is characterized in that: the healthy Hailanshan clove mother tree is a healthy Hailanshan clove without diseases and insect pests introduced into a scientific and technological park of the national university of inner Mongolia.
3. The method for regenerating and propagating the stem tip of the Syzygium Helianthi as the Mongolian medicine according to claim 1, which is characterized in that: after the stem segment with the bud is subjected to disinfection treatment, the stem tip is stripped and placed into a stem tip meristem induction cluster bud primary culture medium for primary culture, and the following treatment is carried out: treating the stem with buds at 0-4 deg.C for 10-12 h.
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| CN116034871A (en) * | 2022-11-18 | 2023-05-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Tissue culture rapid propagation method of glabrous tarragon |
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