CN110946073B

CN110946073B - Method for rapidly propagating fraxinus mandshurica embryonic cells

Info

Publication number: CN110946073B
Application number: CN201811130201.8A
Authority: CN
Inventors: 齐凤慧; 詹亚光; 陈思齐; 刘林; 曾凡锁
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2018-09-27
Filing date: 2018-09-27
Publication date: 2021-04-16
Anticipated expiration: 2038-09-27
Also published as: CN110946073A

Abstract

The invention discloses a method for rapidly propagating fraxinus mandshurica embryonic cells, comprising the steps of selecting fraxinus mandshurica callus, wherein the source of the callus can be various explants such as roots, stems, leaves, seeds, embryos and the like, inoculating the callus in an F4 solid culture medium, and culturing for 1-2 subcultures to obtain the fraxinus mandshurica embryonic callus; inoculating the loose embryogenic callus to F4 liquid culture medium for 2-3 subcultures to obtain single cells and small cell masses of fraxinus mandshurica. The invention has the advantages that the F4 culture medium is selected, the culture medium selects 30g/L of sucrose and four hormone combinations of BA, NAA, IBA and TDZ besides the conventional WPM basic culture medium, the cell growth vigor on the culture medium is vigorous, and the phenomenon of browning and death cannot occur as long as subculture is carried out in the growth cycle. The establishment of the method lays a foundation for the scale production of the fraxinus mandshurica cells and the research of secondary metabolites.

Description

Method for rapidly propagating fraxinus mandshurica embryonic cells

Technical Field

The invention relates to a method for rapidly propagating fraxinus mandshurica embryonic cells, belonging to the technical field of cell engineering.

Background

Fraxinus mandshurica Rupr of Oleaceae, Fraxinus, tall deciduous trees. The basic characteristics are as follows: bark is thick, grey brown, big winter bud, conical, thick twig, quadrangular, leaf trace node-shaped and semicircular. Feathery compound leaves; the leaf stem is expanded near the base, the leaf implantation part is provided with joints and is made of paper, the leaf is long and round to be oval, the leaf edge is provided with fine sawteeth, the upper part is dark green, the lower part is yellow green, the conifer grows on the branches of the last year, the leaf is opened first, and the inflorescence stem and the branches are provided with narrow wing-shaped sharp edges; the male flowers and the amphoteric flowers are of different plants, and both have no corolla and no calyx; the male inflorescence is compact, the pedicel is thin and short, the anther is oval, the filament is very short, the ovary is flat and wide, the wing fruit is large and flat, the flower and the fruit can be obtained only 15 to 20 years under the natural growth environment, the flower can be obtained in 4 months, and the fruit can be obtained in 8 to 9 months.

The fraxinus mandshurica is one of the precious trees which are called 'three big hard and broad' in northeast China, has excellent wood material and beautiful texture, and is widely applied to various fields. According to investigation, the fraxinus mandshurica is a nursery stock basically propagated by seeds, and in recent years, some cutting propagation exists. At present, fraxinin, fraxinus chinensis extract and other substances are detected in the bark of ash tree, and the bark of ash tree is used for treating rheumatism, rheumatoid arthritis and osteodynia in folk, and has definite curative effect. However, the trees die after peeling, and the resource waste and damage can be caused by felling, which is not beneficial to sustainable development. Therefore, the rapid culture of the fraxinus mandshurica cells by using the cell engineering technology is one of the methods for solving the problem, and can provide conditions for industrial production and secondary metabolite research.

Disclosure of Invention

The invention aims to provide a method for rapidly propagating fraxinus mandshurica embryonic cells. The method can obviously promote differentiation of fraxinus mandshurica embryonic cells, formation of single cells and propagation of small cell masses, thereby providing important technical support for cultivation, industrial production and secondary metabolite extraction of fraxinus mandshurica embryonic cells.

In order to achieve the purpose, the callus of the fraxinus mandshurica is selected, and the induction source of the callus can be various explants such as roots, stems, leaves, seeds, embryos and the like. Inoculating the callus into F4 solid culture medium, culturing for 1-2 subcultures (each subculture for about 20 days) to obtain embryonic callus, and selecting loose embryonic callus for propagation to obtain large amount of embryonic cells. Loose embryonic cells are selected to be inoculated into an F4 liquid culture medium, shake culture is carried out for 2-3 subcultures (each subculture lasts for about 15 days), mixed cells of single cells and small cell clusters can be obtained, and the single cells and the small cell clusters can be separated by cell screening and filtering.

The key point of the method for rapidly propagating the fraxinus mandshurica embryonic cells is that an F4 culture medium is selected, and the culture medium can be a solid culture medium or a liquid culture medium.

An F4 culture medium used for a technical method for rapidly propagating fraxinus mandshurica embryonic cells comprises the following components:

(1) f4 solid medium: comprises a basic culture medium, hormone, sucrose and agar.

The basal medium is selected from WPM medium and comprises NH₄NO₃ 400mg/L，KH₂PO₄ 170mg/L， K₂SO₄990mg/L，MgSO₄·7H₂O 370mg/L，Ca(NO₃)₂·4H₂O 556mg/L，CaCl₂·2H₂O 96mg/L， MnSO₄·4H₂O 22.5mg/L，ZnSO₄·7H₂O 8.6mg/L，CuSO₄·5H₂O 0.25mg/L，H₃BO₃ 6.2mg/L， Na₂MoO₄·2H₂O 0.25mg/L，FeSO₄·7H₂O 27.8mg/L，Na₂EDTA 37.3mg/L, nicotinic acid (VB)₃)0.5mg/L， VB₁ 1mg/L，VB₆0.5mg/L, glycine 2mg/L, inositol (general purpose) 100 mg/L.

The hormone is selected from BA, NAA, TDZ and IBA, and the content range of BA is 0.5-1.5mg/L, NAA 0.05-0.2mg/L, TDZ 0.1-0.5mg/L, IBA 0.1-0.5 mg/L.

The content of sucrose is 10-80g/L, and the content of agar is 3.0-6.8 mg/L.

(2) F4 liquid medium: comprises a basic culture medium, hormone and cane sugar.

The basal medium is selected from WPM medium and comprises NH₄NO₃ 400mg/L，KH₂PO₄ 170mg/L，K₂SO₄990mg/L，MgSO₄·7H₂O 370mg/L，Ca(NO₃)₂·4H₂O 556mg/L，CaCl₂·2H₂O 96mg/L，MnSO₄·4H₂O 22.5mg/L，ZnSO₄·7H₂O 8.6mg/L，CuSO₄·5H₂O 0.25mg/L，H₃BO₃ 6.2mg/L，Na₂MoO₄·2H₂O 0.25mg/L，FeSO₄·7H₂O 27.8mg/L，Na₂EDTA 37.3mg/L, nicotinic acid (VB)₃)0.5mg/L，VB₁ 1mg/L， VB₆0.5mg/L, glycine 2mg/L, inositol (general purpose) 100 mg/L.

The hormone is selected from BA, NAA, TDZ and IBA, and the content range of BA is 0.5-4.0mg/L, NAA 0.05-0.4mg/L, TDZ 0.1-0.6mg/L, IBA 0.1-2.0 mg/L.

The content of sucrose is 10-80 g/L.

Drawings

FIG. 1: fraxinus mandshurica embryonic cell under F4 solid culture condition

FIG. 2: fraxinus mandshurica unicell and small cell mass under F4 liquid culture condition

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

The invention adopts WPM as basic culture medium, 30g/L sucrose and 4 hormones with different concentrations are added:

solid medium: WPM basic culture medium + sucrose 30g/L + BA + NAA + TDZ + IBA + agar 4.5g/L, pH value 5.8-6.0;

liquid culture medium: WPM basic culture medium + sucrose 30g/L + BA + NAA + TDZ + IBA, pH value 5.8-6.0;

the hormone concentrations are shown in table 1 below:

TABLE 1 hormone concentration

The invention is carried out according to the following steps:

optimization of sucrose concentration:

adding sucrose with different concentrations of 10g/L, 20g/L, 30g/L, 60g/L and 80g/L, culturing for 20d, collecting cells, and weighing the growth amount. The growth rate of the cell dry weight was varied according to the concentration of sucrose to 30g/L >20g/L >10g/L > 60g/L >80g/L, and thus, 30g/L of sucrose was selected for cell culture.

Solid culture:

(1) selecting fraxinus mandshurica callus;

(2) under the aseptic condition, the callus is inoculated into an F-type solid culture medium and placed in a tissue culture room, and the day and night illumination period culture condition is as follows: the illumination intensity in the daytime is 2000-3000Lux, the illumination time is 12-14h/d, and the temperature is 25 +/-1 ℃; the temperature at night is 18 +/-1 ℃; carrying out subculture once for about 20 days, and observing an experimental result after 1-2 subcultures;

(3) according to the statistics of results, in F1-9, the cell vigor on the F4 culture medium is fastest, more embryogenic callus is basically more than 90%, the cell vigor on the F5-6 culture medium is also proper, and the embryogenic callus can also reach 70-80%, but the cell vigor is lower than that of the F4 culture medium, so that the F4 is selected as the propagation culture medium of the embryonic callus of the fraxinus mandshurica, the culture medium is applied by 7-8 subcultures, the cell vigor is still vigorous, and the phenomenon of browning death cannot occur.

Liquid culture:

(1) selecting the vigorous callus with loose expansion in the F4 solid medium.

(2) Inoculating the loose callus into F4 liquid culture medium under aseptic condition,

(3) filtering the single cells and the small cell clusters by using a cell sieve under the aseptic condition, and respectively putting the single cells and the small cell clusters into F4 liquid nutrient medium for culturing to obtain the fraxinus mandshurica single cells and the embryonic cell clusters.

(4) The filtered cells in (3) can also be inoculated in F4 solid culture medium, and fresh cells can be seen to grow out after about 10 days.

The invention screens out a culture system for promoting the rapid propagation of fraxinus mandshurica callus and cells by optimizing the culture system of fraxinus mandshurica embryonic callus, single cell and small cell mass, and provides technical support for large-scale production and production of secondary metabolites.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.

Compared with the prior art, the method selects the calli of the fraxinus mandshurica from different sources, carries out technical optimization of different solid and liquid culture modes, selects the culture medium which can quickly grow in any mode, lays a foundation for later culture and industrial production by using a bioreactor, and provides conditions for later research of secondary metabolites of the fraxinus mandshurica.

Claims

1. A method for rapidly propagating fraxinus mandshurica embryonic cells is characterized in that fraxinus mandshurica callus is inoculated in an F4 culture medium for culture, and the method comprises the following steps:

(1) selecting a culture medium: f4 solid culture medium, the components are WPM + sucrose 10-80g/L + BA1.5mg/L + NAA0.1mg/L + TDZ0.3mg/L + IBA0.5mg/L + agar 4.5g/L, pH value is 5.8-6.0;

f4 liquid culture medium, wherein the components are WPM + sucrose 10-80g/L + BA1.5mg/L + NAA0.1mg/L + TDZ0.3mg/L + IBA0.5mg/L, and the pH value is 5.8-6.0;

(2) inoculating the fraxinus mandshurica callus into the F4 solid culture medium in the step (1), and placing the fraxinus mandshurica callus in a culture room for culture, wherein the culture conditions of day-night lighting period are as follows: the illumination intensity in the daytime is 2000-3000Lux, the illumination time is 12-14h/d, and the temperature is 25+1 deg.C; night temperature 18 deg.C+1 deg.C; subculturing for about 20 days, and performing 1-2 subcultures to obtain embryonic callus;

(3) selecting the loose embryogenic callus in the step (2), inoculating the loose embryogenic callus in the F4 liquid culture medium in the step (1), and placing the embryogenic callus on a shaking table for shake culture at the rotating speed of 120 rpm; the day and night illumination period culture conditions are as follows: the illumination intensity in the daytime is 2000-3000Lux, the illumination time is 12-14h/d, and the temperature is 25+1 deg.C; night temperature 18 deg.C+1 deg.C; performing subculture for 15-20 days for 2-3 times to obtain a mixture of single cells and small cell clusters;

(4) filtering the single cell and the small cell mass by using a cell sieve under the aseptic condition, and respectively putting the single cell and the small cell mass into an F4 liquid culture medium or an F4 solid culture medium to obtain the fraxinus mandshurica single cell and the embryogenic cell mass.