阅读说明：本技术 一种复苏植物垫状卷柏的组培繁殖方法 (Tissue culture propagation method of revived plant selaginella pulvinata ) 是由 余蓉培 汪国鲜 浦艳飞 李帆 卢珍红 莫锡君 于 2019-12-16 设计创作，主要内容包括：一种复苏植物垫状卷柏的组培繁殖方法。该方法包括：垫状卷柏无菌外植体的获得、原生芽的诱导、不定芽的增殖以及组培苗壮苗和生根培养。本发明成功以复苏植物垫状卷柏幼嫩枝条尖端为外植体,构建了垫状卷柏组培繁殖体系,不定芽的增殖率达8倍以上,组培苗壮苗和生根培养过程中的单个不定芽的再次增殖率达11倍以上,1个垫状卷柏幼嫩枝条尖端在170～175天内可以扩繁得到88株以上的组培苗,获得了较高的繁殖效率,为垫状卷柏的规模化扩繁奠定了良好的基础,对复苏植物垫状卷柏的野生种群保护和资源开发利用具有重要意义。(A tissue culture propagation method of resuscitated plant Selaginella pulvinata (lour.) DC. The method comprises the following steps: obtaining a sterile tamariskoid spikemoss herb explant, inducing an original bud, proliferating an adventitious bud, and performing tissue culture seedling strengthening and rooting culture. The method successfully takes the tips of the young branches of the revived plant selaginella pulvinata as explants to construct a selaginella pulvinata tissue culture propagation system, the multiplication rate of the adventitious buds is more than 8 times, the re-multiplication rate of a single adventitious bud in the process of tissue culture seedling strengthening and rooting culture is more than 11 times, 1 young branch of the selaginella pulvinata can be propagated to obtain more than 88 tissue culture seedlings within 170-175 days, higher propagation efficiency is obtained, a good foundation is laid for the large-scale propagation of the selaginella pulvinata, and the method has important significance for the wild population protection and resource development and utilization of the revived plant selaginella pulvinata.)

1. A tissue culture propagation method of a revived plant selaginella pulvinata is characterized by comprising the following steps:

(1) obtaining of sterile explants

Selecting the tip of the young branch of the wild selaginella pulvinata as an explant, and sterilizing to obtain the sterile explant

(2) Induction of primary shoots

Inoculating the sterile explant obtained in the step (1) on a primary bud induction culture medium, and placing the sterile explant in a culture room at the temperature of 20-25 ℃ for dark culture, wherein the dark culture period is 30-45 days, and the primary bud induction culture medium is as follows: 1/2MS +6-BA 0.1-0.2 mg/L + NAA 0.1-0.2 mg/L + citric acid 400-500 mg/L + sucrose 30.0g/L + agar 7.0g/L, pH 5.80;

(3) proliferation of adventitious buds

Inoculating the primary bud obtained in the step (2) on an adventitious bud multiplication culture medium, and placing the primary bud in a culture chamber for multiplication culture, wherein the period of the multiplication culture is 50-60 days, and the adventitious bud multiplication culture medium is as follows: 1/2MS +6-BA 0.8-1.0 mg/L + NAA 0.05-0.1 mg/L + sucrose 30.0g/L + agar 7.0g/L, pH 5.80; the proliferation culture environmental conditions are as follows: 20 to 25 ℃, and the illumination intensity is 30 to 40 mu mol.m^-2·s^-1The illumination time is 16 hours per day;

(4) tissue culture seedling strengthening and rooting culture

When the height of the adventitious buds in the step (3) reaches 5-7 mm, cutting off single adventitious buds, inoculating the single adventitious buds on a tissue culture seedling strengthening and rooting culture medium, and culturing under the same culture environment condition as the step (3), wherein the culture period of the tissue culture seedling strengthening and rooting culture is 70-80 days, and the culture medium of the tissue culture seedling strengthening and rooting culture medium is as follows: 1/4-1/2 MS + 1-2 g/L of activated carbon + 30.0g/L of sucrose + 7.0g/L of agar, and the pH value is 5.80.

2. The tissue culture propagation method of the revived plant Selaginella pulvinata as claimed in claim 1, wherein: specifically, the surface of the explant is cleaned by 1% v/v of liquid detergent, then the explant is placed in a beaker, gauze is covered on the beaker, and the beaker is washed for 2-2.5 hours by flowing water; and sterilizing the explant by using a disinfectant for 12-15 minutes under an aseptic condition, and then washing the explant by using aseptic water for 5-6 times, wherein the washing time is 2-3 minutes each time, so that the aseptic explant is obtained.

3. The tissue culture propagation method of the revived plant Selaginella pulvinata as claimed in claim 1, wherein: the disinfectant is HgCl with the concentration of 0.1% w/v₂And (3) solution.

Technical Field

The invention belongs to the field of plant propagation, and particularly relates to a tissue culture propagation method of a revived plant selaginella pulvinata.

Background

Selaginella pulvinata (Hook. & Grev) Maxim.) belongs to Selaginella of Selaginellaceae, and is in cushion shape, and main stem is not in creeping shape, and mainly grows on the surface of exposed limestone or stone cracks, and the growing environment is dry. Selaginella pulvinata is commonly called as 'Jiu died hempleaf groundsel herb', is a typical resuscitation plant, has extremely strong dehydration resistance, can keep long-term survival through lower metabolism under the condition of extreme dehydration, and can quickly recover normal growth when the water condition is proper. Selaginella pulvinata is a special ornamental plant resource due to the miraculous resuscitation characteristic. Meanwhile, the selaginella pulvinata maxim contains medicinal components such as flavone, biflavone, alkynol and the like, has the effects of resisting cancer, viruses, oxidation, sugar reduction and the like, and is an important medicinal plant resource.

Selaginella pulvinata is an allosporidium fern, has megaspores and microspores, in a humid environment, the megaspores develop into female gametophytes with a cervicales, the microspores develop into male gametophytes with spermatids, in a water environment, the sperms in the spermatids enter the cervicales and are combined with ova to form fertilized ova, and finally, Selaginella pulvinata seedlings are developed. However, Selaginella pulvinata is arid in the habitat, has a short humid period only in rainy season, is not beneficial to spore propagation of Selaginella pulvinata, and is difficult to form spore propagation offspring. Continuous field investigation finds that the natural reproduction rate of the wild population of the selaginella pulvinata maxim is low, and the number of individuals in the population is not obviously increased. In addition, the selaginella pulvinata maxim has ornamental and medicinal values, wild resources are greatly mined, so that the field population quantity of the selaginella pulvinata maxim is reduced sharply, and the wild resources cannot meet the current requirements.

In Selaginella, part of the main stem is of creeping type, such as: the small emerald green clouds have adventitious roots at the stem nodes and can be used for cuttage propagation, but the main stem of the selaginella pulvinata maxim is not creeping and is extremely difficult to perform cuttage propagation. In addition, in the previous research, the tissue culture process of the selaginella pulvinata maxim is easy to brown, and the tissue culture propagation difficulty is higher. At present, no report about the related propagation technology of the revived plant selaginella pulvinata or a tissue culture propagation method of the revived plant selaginella pulvinata is available.

Disclosure of Invention

The invention provides a tissue culture propagation method of a revived plant selaginella pulvinata, aiming at solving the problems of rapid reduction of wild population of the revived plant selaginella pulvinata, low natural fertility and the like.

The invention relates to a tissue culture propagation method of a revived plant selaginella pulvinata maxim, which comprises the following steps:

(1) obtaining of sterile explants

Selecting the tips of young branches of wild selaginella pulvinata plants as explants, and sterilizing to obtain sterile explants;

(2) induction of primary shoots

Inoculating the sterile explant obtained in the step (1) on a primary bud induction culture medium, and placing the sterile explant in a culture room at the temperature of 20-25 ℃ for dark culture, wherein the dark culture period is 30-45 days, and the primary bud induction culture medium is as follows: 1/2MS +6-BA 0.1-0.2 mg/L + NAA 0.1-0.2 mg/L + citric acid 400-500 mg/L + sucrose 30.0g/L + agar 7.0g/L, pH 5.80;

(3) proliferation of adventitious buds

Inoculating the primary bud obtained in the step (2) on an adventitious bud multiplication culture medium, and placing the primary bud in a culture chamber for multiplication culture, wherein the period of the multiplication culture is 50-60 days, and the adventitious bud multiplication culture medium is as follows: 1/2MS +6-BA 0.8-1.0 mg/L + NAA 0.05-0.1 mg/L + sucrose 30.0g/L + agar 7.0g/L, pH 5.80; the proliferation culture environmental conditions are as follows: 20 to 25 ℃, and the illumination intensity is 30 to 40 mu mol.m^-2·s^-1The illumination time is 16 hours per day;

(4) tissue culture seedling strengthening and rooting culture

When the height of the adventitious buds in the step (3) reaches 5-7 mm, cutting off single adventitious buds, inoculating the single adventitious buds on a tissue culture seedling strengthening and rooting culture medium, and culturing under the same culture environment condition as the step (3), wherein the culture period of the tissue culture seedling strengthening and rooting culture is 70-80 days, and the culture medium of the tissue culture seedling strengthening and rooting culture medium is as follows: 1/4-1/2 MS + 1-2 g/L of activated carbon + 30.0g/L of sucrose + 7.0g/L of agar, and the pH value is 5.80.

Further, the disinfection and sterilization mode is that 1% v/v of liquid detergent is used for cleaning the surface of the explant, then the explant is placed in a beaker, gauze is covered on the beaker, and the beaker is washed for 2-2.5 hours by flowing water; and sterilizing the explant by using a disinfectant for 12-15 minutes under an aseptic condition, and then washing the explant by using aseptic water for 5-6 times, wherein the washing time is 2-3 minutes each time, so that the aseptic explant is obtained.

Further, the disinfectant is HgCl with the concentration of 0.1% w/v₂And (3) solution.

The invention has the main innovation points and beneficial effects that:

1. the invention successfully constructs the tissue culture propagation technology of the selaginella pulvinata maxim.

According to the method, a selaginella pulvinata protogenic bud induction culture medium, an adventitious bud propagation culture medium, a tissue culture seedling strengthening and rooting culture medium are screened out, the browning problem in the selaginella pulvinata tissue culture is solved, a selaginella pulvinata tissue culture propagation technology is constructed, and the blank of the selaginella pulvinata tissue culture propagation technology is filled.

2. The tissue culture propagation efficiency of the selaginella pulvinata maxim is higher, and a foundation is laid for large-scale propagation of the selaginella pulvinata maxim.

The multiplication rate of the adventitious buds reaches more than 8 times, the re-multiplication rate of single adventitious buds in the processes of tissue culture seedling strengthening and rooting culture in the step (4) reaches more than 11 times, the tissue culture propagation technology constructed by the invention can realize the propagation of more than 88 tissue culture seedlings from the tips of 1 tender twigs of selaginella pulvinata maxim within 170-175 days, higher propagation efficiency is obtained, and a good foundation is laid for the large-scale propagation of selaginella pulvinata maxim.

Drawings

FIG. 1 shows the tip of tender shoot of Selaginella pulvinata (Willd.) Ohwi. In FIG. 1, the line segment is a scale and the length is 5 mm.

FIG. 2 shows the induction culture of Selaginella pulvinata prototropha. In FIG. 2, the line segment is a scale bar and the length is 1 cm.

Detailed description of the invention

The following examples facilitate a better understanding of the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available. In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The preparation of the culture medium in the following examples is conventional, i.e.: mixing the components and the content thereof according to the formula of the culture medium, adjusting the pH value to the pH value required by the culture medium by using a pH meter, and performing conventional disinfection and sterilization operation on the culture medium to obtain the culture medium.