阅读说明：本技术 一种以蒲公英叶片为外植体建立高效再生体系的方法 (Method for establishing efficient regeneration system by taking dandelion leaves as explants ) 是由 赵德刚 余思文 于 2020-07-21 设计创作，主要内容包括：本发明涉及了一种以蒲公英叶片为外植体建立高效再生体系的方法。本方法中以蒲公英叶片为外植体,具体操作步骤如下：(1)外植体的消毒；(2)丛生芽的诱导；(3)丛生芽的壮芽；(4)生根和移栽。本方法利用蒲公英叶片为外植体,建立了蒲公英高效的再生体系,使用本方法,可提高蒲公英的培养效率,为建立蒲公英稳定的遗传转化体系奠定了一定的基础。(The invention relates to a method for establishing a high-efficiency regeneration system by taking dandelion leaves as explants. In the method, dandelion leaves are taken as explants, and the specific operation steps are as follows: (1) sterilizing explants; (2) inducing cluster buds; (3) strong buds of cluster buds; (4) and (6) rooting and transplanting. The method utilizes dandelion leaves as explants to establish a dandelion efficient regeneration system, can improve the dandelion culture efficiency, and lays a certain foundation for establishing a dandelion stable genetic transformation system.)

1. A method for establishing a high-efficiency regeneration system by taking dandelion leaves as explants comprises the following steps:

(1) and (3) disinfection of explants: picking the second tender leaf of herba Taraxaci plant from the root, sterilizing in a super clean bench, and cutting into 1cm²Size;

(2) inducing and culturing cluster buds: inoculating the sterilized explant to an MS culture medium to induce cluster buds;

(3) bud strengthening of cluster buds: cutting off the cluster buds when the cluster buds grow to about 2cm, reserving 2-3 cluster buds for each plant, and inoculating to an MS culture medium for strong bud culture;

(4) rooting and transplanting: when the cluster buds grow to about 3cm, cutting off a single bud, inoculating the cut single bud to a rooting culture medium for rooting culture, and hardening and transplanting the bud after a root system grows out.

2. The method according to claim 1, wherein the sterilization treatment of the sterile material in step (1) comprises: washing the picked leaves with tap water, wiping, placing into a clean bench, sterilizing with 75% alcohol for 45s, washing with sterile water for 3 times, and absorbing surface water with sterile absorbent paper for inoculation.

3. The method of claim 1, wherein 1.5mg.L of the MS medium induced by the multiple shoots in step (2) is added^-16-BA and 0.2mg.L of^-1The NAA of (3), wherein the pH of the medium was 5.80, was cultured in induction medium for 35 d.

4. The method according to claim 1, wherein the rooting medium in step (4) is 1/2MS minimal medium containing 0.4mg.L^-1The NAA, the hardening-seedling time of the plant is 3-5d, and the matrix used for transplanting is vermiculite: the nutrient soil comprises the following components in percentage by mass 1: 2, uniformly mixing.

5. The method according to claim 1, wherein the culture conditions of steps (2) to (4) are all illumination intensity of 2000lx, daily illumination for 12h, and culture temperature (25 ± 2) ° c.

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for establishing a high-efficiency regeneration system by taking dandelion leaves as explants.

Background

Taraxacum mongolicum hand-Mazz is perennial herb of Compositae, is mainly produced in northern hemisphere temperate zone to subtropical zone, and is widely distributed in northeast, northwest, central and southwest provinces of China. The dandelion has high nutritive value, and the plant body contains various healthy nutritional ingredients such as taraxol, taraxacin, choline, organic acid, inulin and the like; the dandelion is a traditional Chinese medicinal material, and has the effects of clearing away heat and toxic materials, relieving swelling, resolving masses, inducing diuresis and treating stranguria. In addition, dandelion is increasingly popular among people as a high-quality nutritional health-care vegetable. Dandelion integrates edible and medicinal functions, and is called as a dietetic vegetable. The sexual propagation mode of the dandelion needs cross pollination, is easy to be polluted by hybridization, and is not beneficial to keeping the excellent characters and purity of seedlings.

For genetic transformation of dandelion, establishment of an in vitro high-efficiency regeneration system is an indispensable prerequisite. The efficient regeneration system established by using the dandelion leaves as the explant has great significance for more in-depth research on the effective components of the dandelion and maintaining excellent characters, improves the culture efficiency of the dandelion, and provides a certain basis for establishing a dandelion genetic transformation system.

Disclosure of Invention

The invention provides a method for establishing a high-efficiency regeneration system by taking dandelion leaves as explants, which can induce callus and regenerate plants by taking the dandelion leaves as materials, thereby realizing a high-efficiency tissue culture system of the dandelion and providing technical support for establishing a genetic transformation system of the dandelion.

A method for establishing a high-efficiency regeneration system by taking dandelion leaves as explants comprises the following steps:

(1) and (3) disinfection of explants: picking the second tender leaf of herba Taraxaci plant from the root, sterilizing in a super clean bench, and cutting into 1cm²Size;

(2) inducing and culturing cluster buds: inoculating the sterilized explant to an MS culture medium to induce cluster buds;

(3) bud strengthening of cluster buds: cutting off the cluster buds when the cluster buds grow to about 2cm, reserving 2-3 cluster buds for each plant, and inoculating to an MS culture medium for strong bud culture;

(4) rooting and transplanting: when the cluster buds grow to about 3cm, cutting off a single bud, inoculating the cut single bud to a rooting culture medium for rooting culture, and hardening and transplanting the bud after a root system grows out.

The sterilization treatment method of the sterile material in the step (1) comprises the following steps: washing the picked leaves with tap water, wiping to dry, placing in a superclean workbench, sterilizing with 75% alcohol for 45s, washing with sterile water for 3 times, and absorbing surface water with sterile absorbent paper for inoculation;

adding 1.5mg.L of MS culture medium for inducing cluster buds in the step (2)^-16-BA and 0.2mg.L of^-1The NAA of (4), wherein the pH of the culture medium is 5.80, and culturing is performed in an induction medium for 35 d;

the steps are(4) The medium rooting culture medium is 1/2MS minimal medium added with 0.4mg.L^-1The NAA, the hardening-seedling time of the plant is 3-5d, and the matrix used for transplanting is vermiculite: the nutrient soil comprises the following components in percentage by mass 1: 2, uniformly mixing.

In each stage of the plant tissue culture, the illumination intensity is 2000lx, the illumination is 12h every day, and the culture temperature is (25 +/-2) DEG C.

The invention adopts dandelion leaves as material, and 1.5mg.L of the dandelion leaves are added after the explant is disinfected^-16-BA and 0.2mg.L of^-1Inducing cluster buds under the action of the MS culture medium of NAA, adding 0.4mg.L^-1Inducing rooting under the action of an MS culture medium of NAA; the invention successfully establishes a dandelion efficient regeneration system, and can be used in the steps of tissue culture and genetic transformation.

Drawings

FIG. 1 morphology of explants at various time periods,

wherein, A, the induction condition of the cluster buds of the dandelion is realized when the dandelion is cultured for 10 days;

B. inducing cluster buds of dandelion when the dandelion is cultured for 25 days;

C. inducing cluster buds of dandelion when cultured for 35 days;

D. bud strengthening of cluster bud of herba Taraxaci;

E. inducing the cluster buds of the dandelion to root;

F. transplanting seedlings of the dandelion.

Detailed Description

The present invention will be described in further detail with reference to examples.

Culture medium and culture conditions: the culture medium for inducing the cluster buds comprises: MS +1.5mg.L^-16-BA+0.2mg.L^-1NAA; the rooting culture medium of the cluster buds comprises: MS +0.4mg.L^-1NAA. The illumination intensity is 2000lx at each stage of the culture, the illumination is 12h every day, and the temperature is 25 +/-2 ℃.