CN113367063B

CN113367063B - Isolated culture method of akebia trifoliata

Info

Publication number: CN113367063B
Application number: CN202110455637.XA
Authority: CN
Inventors: 梁宏伟; 马鑫; 陈发菊; 王玉兵; 刘�文; 马江; 张德春; 杨敬元; 刘强; 汪迎春
Original assignee: Shennongjia National Park Research Institute; China Three Gorges University CTGU
Current assignee: Shennongjia National Park Research Institute; China Three Gorges University CTGU
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2023-03-10
Anticipated expiration: 2041-04-26
Also published as: CN113367063A

Abstract

The invention belongs to the technical field of cell culture, and relates to an in vitro culture method of akebia trifoliata. Taking zygotic embryos in newly-harvested mature seeds of akebia trifoliata as explants, and placing the explants on a callus induction culture medium to induce callus and proliferate so as to induce embryonic callus, differentiation of somatic embryos and plant regeneration. The method has the cultivation period of about 140 days, and the survival rate of the regeneration plants reaches more than 85 percent. The invention provides a feasible technical system for the high-efficiency in-vitro regeneration system and genetic improvement of akebia trifoliata.

Description

Isolated culture method of akebia trifoliata

Technical Field

The invention relates to the technical field of isolated culture of plants, in particular to an isolated culture method of akebia trifoliata.

Background

Caulis Akebiae (Trifolium Pratentis)Akebia trifoliate(Thunb.) Koidz is a wood vine fruit tree of Akebia of Lardizabalaceae, and the fruit can be used for processing food such as wine, fruit juice, preserved fruit, jelly, tea and the like except fresh food, and is a novel fruit with wide application. The whole plant can be used as medicine, and has effects of tonifying deficiency, relieving cough, clearing heat, promoting urination, dredging channels and activating collaterals, relieving pain, expelling pus, promoting lactation, etc. In addition, akebia trifoliata fruit and seedThe seeds and leaves contain a plurality of components such as protein flavonoids, grease, amino acid, polysaccharide, vitamins, steroids, lignin and the like, have an antioxidant function, can improve the immunity of a human body, delay aging, resist inflammation and cancer, have high health care value, and can be deeply developed as a medicine-food homologous plant. The akebia trifoliata has slender branches, strong climbing ability, high growth speed, suspended purple flowers and peculiar flower shapes, and has good ornamental value. In addition, the akebia trifoliata species has strong ecological adaptability. Therefore, the akebia trifoliata has high value in the aspects of eating, medicine, appreciation, ecology and the like and has huge development potential.

With the increase of market demand, wild fruit resources are excessively picked, so that the good seed source of akebia trifoliata is short in supply and short in demand. In addition, the akebia trifoliata has thick pericarp, the seed proportion in the fruits is large, the edible rate is low, the germination rate in a natural state is low, and the cultivation of new varieties with few seeds or no seeds is urgently needed. Therefore, an efficient and stable trilobate whole body cell embryogenesis technical system is established, and a feasible technical platform can be provided for rapid breeding of excellent seedlings and germplasm genetic improvement research. The establishment of the system can also effectively promote the development and utilization of the akebia trifoliata, protect natural resources and further promote the healthy and sustainable development of the akebia trifoliata industry.

Disclosure of Invention

In order to solve the technical problems, the invention provides an isolated culture method of akebia trifoliate, the culture period of the method is about 140 days, and the survival rate of regenerated plants reaches more than 85%.

The technical scheme adopted by the invention is as follows:

an isolated culture method of akebia trifoliate includes the following steps:

1) Induction of callus: taking zygote embryos in akebia trifoliata seeds as explants, and placing the explants on an induction culture medium to induce callus so as to obtain the induced primary callus;

2) Induction of embryogenic callus: placing the primary callus induced in the step 1) in an embryonic induction culture medium for culturing to obtain an embryonic callus;

3) Differentiation and proliferation induction of somatic embryos: placing the embryogenic callus obtained in the step 2) on a differentiation medium to induce somatic embryo differentiation and subculture proliferation;

4) Plant regeneration: placing the somatic embryos differentiated in the step 3) on a regeneration culture medium for inducing germination to obtain a whole plant of akebia trifoliata.

Preferably, in the step 1), the induction medium is the MS minimal medium added with 1.0-4.0 mg/L2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1-1.0 mg/L alpha-naphthylacetic acid (NAA).

Further preferably, in the step 1), the concentration of 2,4-D in the callus induction medium is 2.0 mg/L, and the concentration of NAA is 0.2 mg/L.

Preferably, the embryogenic induction medium of step 2) is supplemented with 0.01-0.5 mg/L NAA in MS minimal medium.

Further preferably, the NAA concentration in the embryogenic callus induction medium in the step 2) is 0.2 mg/L.

Preferably, the differentiation medium of step 3) is MS minimal medium supplemented with 6-benzylamino adenine (6-BA) (0.1-0.6 mg/L) and NAA (0.05-0.2 mg/L).

Further preferably, the concentration of NAA in the somatic embryo differentiation culture medium in the step 3) is 0.1 mg/L, and the concentration of 6-BA is 0.2 mg/L; the NAA concentration of the secondary embryo multiplication culture medium is 0.05 mg/L, and the 6-BA concentration is 0.1 mg/L.

Preferably, the regeneration medium for germination of the somatic embryos into whole plants in step 4) is an MS minimal medium without exogenous hormone addition.

The invention has the beneficial effects that:

1. the invention uses zygotic embryo in mature seed of akebia trifoliata as explant to obtain regenerated plant via somatic embryogenesis. The zygotic embryo is taken as the explant to carry out in vitro regeneration, and has the advantages of convenient material taking, no time limitation and sufficient available materials. The somatic embryogenesis can greatly improve the propagation coefficient of akebia trifoliata, provides a good technical platform for the rapid propagation and germplasm innovation research of the species, and has wide application prospect.

2. The cultivation period of the method is about 140 days, and the survival rate of the regeneration plants reaches more than 85 percent.

Drawings

FIG. 1 induction of primary callus by Akebia trifoliata zygotic embryo; a. mature fruits of akebia trifoliata, seeds and young embryos are placed in a callus induction culture medium for 10 days, callus induction is carried out for 40 days, and callus is subcultured for 1 time in the steps of e and f.

The scale in FIG. 1a is 1cm, and the scale in FIGS. 1b to 1f is 0.5 cm.

FIG. 2 embryogenic callus induction and somatic embryo differentiation; a. yellowish, slightly soft surface water-blotted non-callus, b. brown, compact embryogenic callus, c. early somatic embryo, d. cotyledon embryo and secondary embryo.

In FIGS. 2a and 2b, the scale is 0.5 cm, and in FIGS. 2c and 2d, the scale is 0.5 mm;

FIG. 3 regeneration of Akebia trifoliate plants; a. germinating somatic embryos, completely regenerating plants, and transplanting regenerated seedlings into a matrix.

The scale in the figure is 1cm.

Detailed Description

The present invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.

Example 1

MS minimal medium: reference is made to the literature (Murashige T, skoog F. A recycled medium for rapid group and bioassays with superbactco tissue cultures. Physiol. Plant, 1962, 15: 473-497).

The specific implementation mode is as follows:

1. sterilization of explants

Peeling seeds of mature fruits of Akebia trifoliata freshly harvested in 8 months, cleaning, sterilizing in an ultra-clean workbench, treating with 75% ethanol for 30 s, cleaning with sterile water for 2 times, and then using 0.1% HgCl ₂ The solution (mercuric chloride) was sterilized for 18 minutes and washed with sterile water 6 times for use. Longitudinally cutting the seeds along the back suture line with a scalpel, picking the zygotic embryo out with the scalpel for inoculation (FIGS. 1a, 1b,1c）。

2. Induction of Primary callus

15 zygotic embryos are inoculated into each culture dish, 20 bottles of each combination are inoculated, the process is repeated for 3 times, and the callus is induced under the conditions that the temperature is 25 +/-2 ℃, the illumination time is 12-16 h/day and the illumination intensity is 2000-2500 lux. The embryo is initiated to be callus after 5 days of inoculation, cotyledons are opened at 10 days, the color is light yellow, then the embryo gradually expands to be callus, the volume is obviously increased when the embryo is cultured to 40 days, and the embryo completely forms callus with yellow brown color and more brown color (figure 1d, 1e and 1 f).

Statistical analysis shows that the added 2,4-D concentration in the MS minimal medium is 2.0 mg/L, and the callus induction rate is the highest when the NAA concentration is 0.2 mg/L and can reach 95.67%.

3. Induction of embryogenic callus

Transferring the obtained primary callus into an embryonic callus induction culture medium, and performing subculture for 1-2 times to obtain brown and dark brown embryonic callus with compact texture; the non-embryogenic callus was milky white, yellowish green, loose in texture, water-stained or thin and soft on the surface (FIGS. 2a and 2 b). Statistical analysis shows that when 0.2 mg/L NAA is added into the MS minimal medium, the embryogenic callus has the highest induction rate.

4. Differentiation induction and proliferation of somatic embryos

The embryonic callus obtained by 1-2 times of subculture is placed on a differentiation medium to induce the differentiation and subculture proliferation of the somatic embryo. After 30-40 days of culture, the embryogenic callus was differentiated to produce early somatic embryos (FIG. 2 c). Statistical analysis shows that when 0.1 mg/L NAA and 0.2 mg/L6-BA are added into the MS minimal medium, the somatic embryo differentiation incidence rate is the highest, and the somatic embryo induction rate is 55%.

During subsequent subculture propagation, secondary embryos occurred on the portion of the mature cotyledon embryos next to the medium (FIG. 2 d). The cells were placed in a proliferation medium, and the results showed that the proliferation rate was the highest on MS medium supplemented with 0.05 mg/L NAA and 0.1 mg/L6-BA, and the proliferation rate reached 19.84.

5. Plant regeneration

The differentiated mature somatic embryos are placed on a plant regeneration medium for germination (figure 3 a), and then the whole plant of akebia trifoliata can be obtained (figure 3 b). The results show that effective rooting is not seen in the rooting culture medium added with different exogenous hormones, and the germinated seedlings die due to different degrees of withering; while the MS minimal medium without exogenous hormone can grow strongly, and the height of the somatic embryo seedlings and the growth of leaves are obvious.

6. Hardening and transplanting seedlings

When the regenerated seedlings grow to 3 to 5 cm, transferring the tissue culture bottle seedlings from the culture room to the buffer room, gradually opening the seal cover of the tissue culture bottle mouth to enable the regenerated seedlings to be in contact with air, and hardening the seedlings for 7 days at the temperature of 20-25 ℃. Taking out the regenerated seedlings, thoroughly cleaning the culture medium at the root under running water, and finally transferring the seedlings to charcoal soil: vermiculite: the perlite is 2:1:1 (fig. 3 c), the culture medium is watered thoroughly and placed in a greenhouse for culture, and the survival rate is more than 85 percent.

The above-described embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and features in the embodiments and examples in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.

Claims

1. An isolated culture method of akebia trifoliate is characterized by comprising the following steps:

1) Induction of callus: taking zygotic embryos in mature akebia trifoliata seeds as explants, placing the explants on an induction culture medium to induce callus, wherein the induction culture medium is obtained by adding 1.0-4.0 mg/L of 2, 4-dichlorophenoxyacetic acid and 0.1-1.0 mg/L of alpha-naphthylacetic acid into an MS basic culture medium to obtain the induced primary callus;

2) Induction of embryogenic callus: placing the primary callus induced in the step 1) into an embryogenic induction culture medium for culturing, wherein the embryogenic induction culture medium is obtained by adding 0.01-0.5 mg/L alpha-naphthylacetic acid into an MS minimal medium;

3) Differentiation and proliferation induction of somatic embryos: placing the embryogenic callus obtained in the step 2) on a differentiation culture medium to induce somatic embryo differentiation and subculture proliferation, wherein the differentiation and proliferation culture medium is obtained by adding 0.1-0.6 mg/L6-benzylamino adenine and 0.05-0.2 mg/L alpha-naphthylacetic acid into an MS minimal medium;

2. The isolated culture method of akebia trifoliate as claimed in claim 1, wherein: in the step 1), the concentration of 2, 4-dichlorophenoxyacetic acid in the callus induction culture medium is 2.0 mg/L, and the concentration of alpha-naphthylacetic acid is 0.2 mg/L.

3. The isolated culture method of akebia trifoliate as claimed in claim 1, wherein: the concentration of alpha-naphthylacetic acid in the embryogenic callus induction culture medium in the step 2) is 0.2 mg/L.

4. The isolated culture method of akebia trifoliate as claimed in claim 1, wherein: the concentration of alpha-naphthylacetic acid in the somatic embryo differentiation culture medium in the step 3) is 0.1 mg/L, and the concentration of 6-benzylaminopurine is 0.2 mg/L; the concentration of the alpha-naphthylacetic acid in the secondary embryo multiplication culture medium is 0.05 mg/L, and the concentration of 6-benzylamino adenine is 0.1 mg/L.

5. The isolated culture method of akebia trifoliata according to claim 1, wherein the culture method comprises: and 4) the regeneration culture medium for germinating the somatic embryo into a complete plant is an MS basic culture medium without exogenous hormone addition.