CN108260526B

CN108260526B - Tissue culture method of early-falling marsdenia tenacissima

Info

Publication number: CN108260526B
Application number: CN201711267707.9A
Authority: CN
Inventors: 陈建荣; 刘芳; 唐映红; 唐伟卓
Original assignee: Changsha University
Current assignee: Changsha University
Priority date: 2017-12-05
Filing date: 2017-12-05
Publication date: 2021-01-12
Anticipated expiration: 2037-12-05
Also published as: CN108260526A

Abstract

The invention relates to the field of plant tissue culture, and discloses a tissue culture method of early-falling tetrapanax papyriferus, which comprises the following steps: 1) leaf callus formation: sterilizing leaf blades of wild long-stalked spring grass, cutting off veins and leaf margins, cutting into small blocks of about 5mm × 5mm, inoculating to callus induction culture medium, and conventionally culturing to form callus; 2) callus differentiation adventitious bud: cutting the callus formed in the step 1) into small blocks with the size of about 5mm multiplied by 5mm, inoculating the small blocks to an induction culture medium of a differentiated bud, and conventionally culturing until adventitious buds are formed on the callus; 3) adventitious bud rooting: cutting the adventitious bud with the size of about 3-5cm formed in the step 2), inoculating into a rooting induction culture medium, and conventionally culturing until a root is formed; 4) the tissue culture seedling blooms. The method can obtain a large amount of excellent early-falling spring grass tissue culture seedlings within 50 days, and has wide market application prospect.

Description

Tissue culture method of early-falling marsdenia tenacissima

Technical Field

The invention relates to the field of plant tissue culture, in particular to a tissue culture method of early-falling tetrapanax papyriferus.

Background

Early-falling Marsdenia tenacissima (Mazus caducifer Hance) belonging to Scrophulariaceae and belonging to genus Marsdenia, the height is 20-50cm, the Marsdenia tenacissima is strong, and the whole body is white and long and soft; the raceme grows at the top, the growth can reach 35cm, and the flowering phase is 4-5 months; the capsule is spherical, and the fruit period is 6-8 months; seeds are dark brown, much and small; growing on the wet road side, under the forest and on the grass slope below the elevation 1300 m. The plant of the genus Alternaria is a dwarf herb, most of which have medicinal value, have the functions of clearing heat and detoxicating, dispelling mad poison, relieving pain, invigorating stomach and the like, and are used for treating migraine, furuncle, running sore, scald, innominate swelling and pain and the like. The plant is rich in resources, is in a wild state in most regions, and has to select a variety with excellent properties from the wild state for practical application, such as medicinal application, garden application and the like. At present, no relevant documents and patents of the early-falling thermal groundsel herb tissue culture are seen, the tissue culture technology can be used for rapidly propagating a large number of selected excellent early-falling thermal groundsel herb varieties, the excellent quality of female parents is kept, and a large number of excellent raw materials are provided for the application of the early-falling thermal groundsel herb.

Disclosure of Invention

The invention provides a tissue culture method of early falling thermal weed, which aims to solve the problems that most of the early falling thermal weeds in wild state at present select excellent varieties and carry out mass rapid propagation on the early falling thermal weeds by applying a tissue culture technology, provide a large amount of excellent early falling thermal weed raw materials and lay a foundation for wide application of medicinal and garden application values of the early falling thermal weed raw materials.

The invention provides a tissue culture method of early-falling spring grass, which comprises the following steps:

1) leaf callus formation: sterilizing leaf blades of wild long-stalked spring grass, cutting off veins and leaf margins, cutting into small blocks of about 5mm × 5mm, inoculating to callus induction culture medium, and conventionally culturing to form callus;

2) callus differentiation adventitious bud: cutting the callus formed in the step 1) into small blocks with the size of about 5mm multiplied by 5mm, inoculating the small blocks to an induction culture medium of a differentiated bud, and conventionally culturing until adventitious buds are formed on the callus;

3) adventitious bud rooting: cutting the adventitious bud with the size of about 3-5cm formed in the step 2), inoculating into a rooting induction culture medium, and conventionally culturing until a root is formed;

4) and (3) blooming of the tissue culture seedlings: and (4) continuing to carry out conventional culture on the rooted tissue culture seedlings in a rooting culture medium to form buds until the buds bloom.

Wherein the induction culture medium for the leaf-forming callus is MS culture medium containing 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid.

Wherein the induction culture medium for differentiating the adventitious bud from the callus is an MS culture medium containing 2 mg/L6-benzylaminopurine and 0.1mg/L naphthylacetic acid.

Wherein the induction culture medium for the adventitious bud rooting is an MS culture medium containing 1mg/L of 6-benzylaminopurine and 0.3mg/L of naphthylacetic acid.

Wherein the induction culture medium for the tissue culture seedling to bloom is an MS culture medium containing 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid.

Wherein the concentration of sucrose in the MS culture medium is 30g/L, and the concentration of agar powder is 8 g/L.

Wherein the culture conditions of the conventional culture comprise that the pH value is 6.0, the illumination intensity is 1500lx, the illumination time is 10h/d, and the temperature of the constant-temperature incubator is 25 ℃.

Wherein the time for the leaf to form the callus is 20 days.

Wherein the time for differentiating adventitious buds from the callus is 10 days.

Wherein the time for the adventitious bud to root is 20 days.

Wherein the time for the tissue culture seedling to form buds is 10 days.

The invention has the beneficial effects that:

1) the method can obtain a large amount of excellent early-falling spring grass tissue culture seedlings within 50 days, and can quickly, massively and excellently provide a large amount of excellent raw materials for the application of the early-falling spring grass.

2) The method can obtain a large amount of excellent early-falling spring grass tissue culture seedlings within 60 days, and has wide market application prospect.

3) The method has important significance in protecting the natural resources of the wild early-falling spring grass, the good ecological environment and the like.

Drawings

FIG. 1: the callus is formed by the leaf of the early falling thermal weed after 20 days of cultivation in the embodiment of the invention (the early falling thermal weed is from field picking, the same below).

FIG. 2: the adventitious bud is differentiated after the callus of the early-falling tetrapanax papyriferus is cultured for 10 days in the embodiment of the invention.

FIG. 3: the rooting condition of the early-falling spring grass adventitious bud after 20 days of culture in the embodiment of the invention is shown.

FIG. 4: the flowering seedlings are obtained after the early-falling spring grass tissue culture seedlings are cultured for 10 days in the embodiment of the invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

A tissue culture method of early-falling Japanese tetrapanax comprises the following steps:

1) leaf callus formation: sterilizing leaf blades of wild long-stalked spring grass, cutting off veins and leaf margins, cutting into small blocks of about 5mm × 5mm, inoculating to callus induction culture medium, and conventionally culturing to form callus; the induction culture medium of the leaf forming callus is an MS culture medium containing 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid; the time for the leaf to form the callus is 20 days; the concentration of sucrose in the MS culture medium is 30g/L, and the concentration of agar powder is 8 g/L; the culture conditions of the conventional culture comprise that the pH value is 6.0, the illumination intensity is 1500lx, the illumination time is 10h/d, and the temperature of a constant-temperature incubator is 25 ℃.

2) Callus differentiation adventitious bud: cutting the callus formed in the step 1) into small blocks with the size of about 5mm multiplied by 5mm, inoculating the small blocks to an induction culture medium of a differentiated bud, and conventionally culturing until adventitious buds are formed on the callus; the induction culture medium for differentiating the adventitious buds from the callus is an MS culture medium containing 2 mg/L6-benzylaminopurine and 0.1mg/L naphthylacetic acid, and the time for differentiating the adventitious buds from the callus is 10 days; the culture conditions of the MS culture medium and the conventional culture are the same as the step 1).

3) Adventitious bud rooting: cutting the adventitious bud with the size of about 3-5cm formed in the step 2), inoculating into a rooting induction culture medium, and conventionally culturing until a root is formed; the induction culture medium for the adventitious bud rooting is an MS culture medium containing 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid; the time for differentiating adventitious buds from the callus is 20 days; the culture conditions of the MS culture medium and the conventional culture are the same as the step 1).

4) And (3) blooming of the tissue culture seedlings: and (4) continuing to carry out conventional culture on the rooted tissue culture seedlings in a rooting culture medium to form buds until the buds bloom. The induction culture medium for tissue culture seedling blooming is an MS culture medium containing 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid; the time for the tissue culture seedling to form buds is 10 days; the culture conditions of the MS culture medium and the conventional culture are the same as the step 1).

The result shows that the method can obtain a large amount of excellent early-falling spring grass tissue culture seedlings within 50 days, and can quickly, massively and excellently provide a large amount of excellent raw materials for the application of the early-falling spring grass; the method can obtain a large amount of excellent early-falling spring grass tissue culture seedlings within 60 days, and has wide market application prospect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A tissue culture method of early-falling Japanese tetrapanax comprises the following steps:

1) leaf callus formation: sterilizing leaf blades of wild long-stalked spring grass, cutting off veins and leaf margins, cutting into small blocks of 5mm × 5mm, inoculating to callus induction culture medium, and conventionally culturing to form callus;

2) callus differentiation adventitious bud: cutting the callus formed in the step 1) into small blocks of 5mm multiplied by 5mm, inoculating the small blocks to an induction culture medium of a differentiated bud, and conventionally culturing until adventitious buds are formed on the callus;

3) adventitious bud rooting: cutting the adventitious bud with the size of 3-5cm formed in the step 2), inoculating into a rooting induction culture medium, and conventionally culturing until a root is formed;

4) and (3) blooming of the tissue culture seedlings: the rooted tissue culture seedling is continued to be cultured in a rooting culture medium in a conventional way to form a bud until the bud blooms;

the callus induction culture medium for the leaf callus is an MS culture medium added with 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid;

the induction culture medium for differentiating adventitious buds from the callus is an MS culture medium added with 2 mg/L6-benzylaminopurine and 0.1mg/L naphthylacetic acid;

the rooting induction culture medium for the adventitious bud to root is an MS culture medium added with 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid;

the rooting culture medium for blooming of the tissue culture seedlings is an MS culture medium added with 1 mg/L6-benzylaminopurine and 0.3mg/L naphthylacetic acid;

the time for the leaf to form the callus is 20 days;

the time for differentiating adventitious buds from the callus is 10 days;

the rooting time of the adventitious bud is 20 days;

the time for the tissue culture seedling to form the buds is 10 days.

2. Use of the method of claim 1 in tissue culture of early-falling tetrapanax papyrifera.