阅读说明：本技术 一种麻竹的组织培养方法 (Tissue culture method of dendrocalamus latiflorus ) 是由 姚强 于 2021-03-11 设计创作，主要内容包括：本发明涉及一种麻竹的组织培养方法,属于牡竹属麻竹组组培快繁技术领域。本发明提供的繁殖方法,包含以下步骤：1)形成愈伤组织；2)愈伤组织分化不定芽；3)不定芽生根培养；4)麻竹组培苗生根培养。本发明提供的繁殖方法选取麻竹幼枝作为外植体可在30d即可获得大量优质的麻竹组培苗,可快速、便捷、大量的为麻竹的研究及应用提供优良原材料。在保护麻竹自然资源、栽培对种苗的需求、以及基因库的建立等方面具有重要意义。(The invention relates to a tissue culture method of dendrocalamus latiflorus, belonging to the technical field of tissue culture and rapid propagation of dendrocalamus latiflorus groups. The propagation method provided by the invention comprises the following steps: 1) forming callus; 2) differentiating adventitious buds from the callus; 3) culturing adventitious buds for rooting; 4) and (5) rooting culture of the dendrocalamus latiflorus tissue culture seedling. According to the propagation method provided by the invention, the young dendrocalamus latiflorus branches are selected as explants, a large number of high-quality dendrocalamus latiflorus tissue culture seedlings can be obtained within 30 days, and good raw materials can be rapidly, conveniently and massively provided for research and application of dendrocalamus latiflorus. Has important significance in protecting natural resource of the dendrocalamus latiflorus, meeting the requirements of seedling cultivation, establishing a gene bank and the like.)

1. A tissue culture method of dendrocalamus latiflorus comprises the following steps:

1) forming callus on young dendrocalamus latiflorus branches: cutting young branches of 2-4 years old dendrocalamus latiflorus, sterilizing, cutting into small blocks of 4mm multiplied by 4mm, and inoculating the small blocks to a callus induction culture medium to obtain dendrocalamus latiflorus callus;

2) callus differentiation adventitious bud: cutting the callus obtained in the step 1) into small blocks of 4mm multiplied by 4mm, and transferring the small blocks to an induction culture medium for differentiating adventitious buds to obtain callus for generating adventitious buds;

3) adventitious bud rooting culture: transferring the adventitious bud callus growing to 2-4 cm obtained in the step 2) to a rooting induction culture medium to obtain rooted dendrocalamus latiflorus seedlings;

4) rooting culture: transferring the rooting dendrocalamus latiflorus seedlings with 1-3 cm of root hairs obtained in the step 3) to a rooting culture medium to obtain dendrocalamus latiflorus tissue culture seedlings;

the callus induction culture medium takes an MS solid culture medium as a reference, and comprises 31g/L of sucrose, 0.48-0.52 mg/L of indolebutyric acid and 8mg/L of 2, 4-dichlorophenoxyacetic acid;

the callus proliferation culture medium takes 3/4MS solid culture medium as a reference and comprises sucrose with the mass concentration of 31g/L, 2.8-3.1 g/L sorbitol, 250 mg/polyvinylpyrrolidone, 4.1g/L plant gel and 2 mg/L2, 4-dichlorophenoxyacetic acid;

the rooting induction culture medium takes 1/2MS solid culture medium as a reference, and comprises 1mg/L indole-3-acetic acid, 31g/L sucrose and 4.1g/L plant gel in mass concentration;

the rooting medium takes an MS solid medium as a reference and comprises 31g/L sucrose, 4.1g/L plant gel and 0.1mg/L TDZ by mass concentration, or takes the MS solid medium as a reference and comprises 0.1mg/L TDZ and 0.48-0.51 mg/L naphthylacetic acid;

the callus formation time of the dendrocalamus latiflorus is 10 days, the differentiation of adventitious buds from the callus is 14 days, and the rooting of the adventitious buds is 6 days.

2. The method according to claim 1, wherein the culture environment is an independent sterile environment, the pH value is 6.1, the culture temperature is 23 ± 2 ℃, the illumination intensity is 1800-2100 Lx, and the daily illumination is 16 h/d.

3. Use of the method according to claim 1 or 2 in tissue culture of a dendrocalamus latiflorus.

Technical Field

The invention relates to the field of plant tissue culture, in particular to a tissue culture method of dendrocalamus latiflorus.

Background

The dendrocalamus latiflorus is a plant of the genus of the family Gramineae, is the widest bamboo species cultivated in the south of China, is 20-30 m high, 15-30 cm in diameter, 45-60 cm in internode length, high in rod-branch habit, and has multiple branches in each section. The dendrocalamus latiflorus is an excellent bamboo shoot and bamboo material dual-purpose bamboo, the bamboo shoot is sweet and delicious in taste and high in nutritional value, and a large amount of dried bamboo shoots and cans are sold in the market and abroad each year; the rod is thick and high, can be used as building material and paper-making material, and can be planted in a garden, so that it has high ornamental value.

Although the dendrocalamus latiflorus is a high-quality plant raw material, the natural propagation of the dendrocalamus latiflorus is difficult to meet the requirements of the current industry. In the breeding aspect of the dendrocalamus latiflorus, the research and development of the dendrocalamus latiflorus are limited due to the biological characteristics that the bamboos bloom irregularly and die quickly after blooming. Most of the current literature reports focus on the cuttage propagation and application value analysis of the dendrocalamus latiflorus, the literature reports on the tissue culture of the dendrocalamus latiflorus are very few, and the domestic reports about the tissue culture of the dendrocalamus latiflorus, such as the mushroom and industry (2014), the tissue culture research of the pluvialis camp on the dendrocalamus latiflorus (lehai camp, 2011) and the like are about the tissue culture of the dendrocalamus latiflorus. The tissue culture method of the dendrocalamus latiflorus can carry out mass rapid propagation, maintain the excellent quality of a female parent, provide a large amount of excellent raw materials for application of the dendrocalamus latiflorus, and lay a technical foundation for realizing cultivation of the dendrocalamus latiflorus, demands on dendrocalamus latiflorus seedlings and establishment of a gene library.

Disclosure of Invention

The method aims to solve the increasing commercial demand of the dendrocalamus latiflorus, protect dendrocalamus latiflorus resources, meet the requirements of scientific research, select excellent dendrocalamus latiflorus varieties, apply a tissue culture technology to carry out rapid mass propagation, maintain the excellent quality of female parents, provide a large amount of excellent raw materials for application of the dendrocalamus latiflorus, and lay a technical foundation for realizing the protection of wild resources of the dendrocalamus latiflorus, the requirements of cultivation on seedlings and the establishment of a gene library.

The tissue culture method of the dendrocalamus latiflorus provided by the invention comprises the following steps:

1) forming callus on young dendrocalamus latiflorus branches: and (3) cutting young branches of 2-4 years old dendrocalamus latiflorus, sterilizing, cutting into small blocks of 4mm multiplied by 4mm, and inoculating to a callus induction culture medium to obtain dendrocalamus latiflorus callus.

2) Callus differentiation adventitious bud: cutting the callus obtained in the step 1) into small blocks of 4mm multiplied by 4mm, and transferring the small blocks to an induction culture medium for differentiating adventitious buds to obtain the callus generating the adventitious buds.

3) Adventitious bud rooting culture: transferring the adventitious bud callus growing to 2-4 cm obtained in the step 2) to a rooting induction culture medium to obtain rooted dendrocalamus latiflorus seedlings.

4) Rooting culture: and (3) transferring the rooting dendrocalamus latiflorus seedlings with 1-3 cm of root hairs obtained in the step 3) to a rooting culture medium to obtain dendrocalamus latiflorus tissue culture seedlings.

The callus induction culture medium is based on an MS solid culture medium and comprises indolebutyric acid with the mass concentration of 0.48-0.52 mg/L and 2, 4-dichlorophenoxyacetic acid with the mass concentration of 8 mg/L.

The proliferation culture medium of the callus takes 3/4MS solid culture medium as a reference, and comprises sorbitol with the mass concentration of 2.8-3.1 g/L, 250 mg/L polyvinylpyrrolidone and 2 mg/L2, 4-dichlorophenoxyacetic acid.

Wherein the rooting induction culture medium takes 1/2MS solid culture medium as a reference and contains indole-3-acetic acid with the mass concentration of 1 mg/L.

The rooting medium takes an MS solid medium as a reference and contains 0.1mg/L TDZ, or takes the MS solid medium as a reference and contains 0.1mg/L TDZ + 0.48-0.51 mg/L naphthylacetic acid.

Wherein the concentration of sucrose in the MS culture medium is 31g/L, and the concentration of plant gel is 4.1 g/L.

The culture environment is an independent sterile environment, the pH value is 6.1, the culture temperature is 23 +/-2 ℃, the illumination intensity is 1800-2100 Lx, and the illumination is 16h/d every day.

Wherein the time for the young shoots to form a callus is 10 days.

Wherein the adventitious bud differentiation from the callus is 14 d.

Wherein the adventitious bud rooting is 6 d. .

The method has the beneficial effects that:

1) the method can obtain a large amount of high-quality dendrocalamus latiflorus tissue culture seedlings within 30 days, and can quickly, conveniently and massively provide excellent raw materials for application and research of dendrocalamus latiflorus.

2) The method has important significance in protecting natural resource of dendrocalamus latiflorus, meeting the requirements of seedling cultivation, establishing a gene bank and the like.

Drawings

FIG. 1: in the present example, the young dendrocalamus latiflorus branches after 10 days of cultivation formed callus (the dendrocalamus latiflorus comes from field picking, the same applies below).

FIG. 2: in the embodiment of the invention, adventitious buds are differentiated from the dendrocalamus latiflorus callus after the dendrocalamus latiflorus young branches form the callus for 14 days.

FIG. 3: in the embodiment of the invention, the adventitious buds of the dendrocalamus latiflorus are cultured to root after the adventitious buds of the dendrocalamus latiflorus are differentiated from the callus of the dendrocalamus latiflorus for 6 days.

FIG. 4: in the embodiment of the invention, the seedling condition of the dendrocalamus latiflorus is shown after adventitious buds of the dendrocalamus latiflorus are cultured and rooted for 7 days.

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.

The plant growth regulators used in the following examples, indolebutyric acid, 2, 4-dichlorophenoxyacetic acid, sorbitol, polyvinylpyrrolidone, indole-3-acetic acid, naphthylacetic acid, were different from those reported for use in the tissue culture of dendrocalamus latiflorus.

Example 1

A tissue culture method of dendrocalamus latiflorus comprises the following steps:

1) callus formed by young branches: sterilizing wild hemp bamboo leaves, cutting into small pieces of about 4mm × 4mm, inoculating to callus induction culture medium, and conventionally culturing to form callus; the callus forming time of the young branches is 10 days; the induction culture medium of the dendrocalamus latiflorus callus is an MS solid culture medium containing indolebutyric acid with the mass concentration of 0.48-0.52 mg/L and 2, 4-dichlorophenoxyacetic acid with the mass concentration of 8 mg/L; the concentration of sucrose in the MS culture medium is 31g/L, and the concentration of plant gel is 4.1 g/L; the culture environment is an independent sterile environment, the pH value is 6.1, the culture temperature is 23 +/-2 ℃, the illumination intensity is 1800-2100 Lx, and the illumination is 16h/d every day.

2) Callus differentiation adventitious bud: cutting the callus obtained in the step 1) into small blocks with the size of 4mm multiplied by 4mm, inoculating the small blocks to an induction culture medium of differentiated adventitious buds, and conventionally culturing the small blocks until the callus forms adventitious buds; the adventitious bud differentiation of the callus is 14 d; the differentiation induction adventitious bud culture medium is 3/4MS solid culture medium containing 2.8-3.1 g/L sorbitol, 250 mg/L polyvinylpyrrolidone and 2 mg/L2, 4-dichlorophenoxyacetic acid; the concentration of sucrose in the 3/4MS culture medium is 31g/L, and the concentration of plant gel is 4.1 g/L; the culture conditions of the conventional culture are the same as those of the step 1).

3) Adventitious bud rooting culture: transferring the adventitious bud callus growing to 2-4 cm obtained in the step 2) to a rooting induction culture medium, and conventionally culturing until root hairs are primarily formed to obtain rooting dendrocalamus latiflorus seedlings; the rooting induction culture medium is 1/2MS culture medium containing 1mg/L indole-3-acetic acid; the adventitious bud rooting culture is 6 d; the concentration of sucrose in the 1/2MS culture medium is 31g/L, and the concentration of plant gel is 4.1 g/L; the culture conditions of the conventional culture are the same as those of the step 1).

4) Rooting culture: transplanting the rooting dendrocalamus latiflorus seedlings with 1-3 cm of root hairs obtained in the step 3) to a rooting culture medium, and conventionally culturing until the root hairs are formed to obtain dendrocalamus latiflorus tissue culture seedlings; the dendrocalamus latiflorus rooting culture medium is an MS culture medium containing 0.1mg/L of TDZ, or an MS culture medium containing 0.1mg/L of TDZ and 0.48-0.51 mg/L of naphthylacetic acid; the MS medium and the conventional culture conditions are the same as those in step 1)

The result shows that the method can obtain a large amount of excellent dendrocalamus latiflorus tissue culture seedlings within 30 days, provide a large amount of excellent raw materials for application of dendrocalamus latiflorus, lay a technical foundation for realizing the protection of natural resource of dendrocalamus latiflorus, the demand of cultivation on seedlings and the establishment of a gene bank, and have wide market application prospect.

Although the invention has been described in detail above with reference to specific embodiments and illustrative embodiments, it is apparent that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.