CN108849513B

CN108849513B - Primary culture method in water hyacinth tissue culture

Info

Publication number: CN108849513B
Application number: CN201810782133.7A
Authority: CN
Inventors: 杨志红; 张国贞
Original assignee: Huzhou University
Current assignee: Huzhou University
Priority date: 2018-07-12
Filing date: 2018-07-12
Publication date: 2021-06-04
Anticipated expiration: 2038-07-12
Also published as: CN108849513A

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a primary culture method in eichhornia crassipes tissue culture. The primary culture method in the water hyacinth tissue culture comprises the following steps: (1) and (3) disinfection: soaking stem of Eichhornia crassipes (Fr.) Crassifolia in 75% ethanol for 30S, sterilizing with 0.2% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times; (2) taking an explant: taking down axillary buds of the stems of the eichhornia crassipes under an aseptic condition, and peeling off tissues outside the axillary buds; taking the base of axillary bud as a base point, and taking a section of 0.4-0.7cm as an explant; (3) primary culture: transferring the explant into a culture medium for culture, wherein the culture medium takes MS as a basic culture medium, the concentration of sucrose is 30g/L, agar is 8g/L, the pH value is 5.8, and the hormone formula is as follows: 6-BA2mg/L + NAA0.2mg/L; the culture temperature is 25 ℃, the photoperiod is 16h of illumination and 8h of darkness, and the illumination intensity is 2000 Lx. The method for primary culture in the water hyacinth tissue culture can ensure that the browning rate and the death rate of the explant are both at a lower level.

Description

Primary culture method in water hyacinth tissue culture

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a primary culture method in eichhornia crassipes tissue culture.

Background

Eichhornia crassipes (Eichhornia crassipes), also called water hyacinth, is a perennial root floating herbaceous plant in the family of Podostachyaceae, and is originally produced in south America and introduced into China as a flower in 1901. The water hyacinth can be used as feed and fertilizer besides the ornamental effect, and the pollution treatment effect is gradually paid attention to under the condition that the current water pollution and the eutrophication degree are gradually serious. In addition to the above functions, the water hyacinth can be used for furniture and paper industry after being processed to a certain extent. The wide use of eichhornia crassipes benefits to a certain extent from its biological properties, and under the condition of proper external temperature, it can rapidly propagate to enable the biomass to rapidly increase in a short period of time. However, the growth of the plants is affected by seasonality, the water hyacinth can grow quickly at proper temperature, but when the temperature is reduced to below 10 ℃, the water hyacinth grows slowly and enters dormancy, and after the temperature is reduced continuously, most of the plants are rotten and die to cause water pollution, so that the application of the water hyacinth is limited. How to artificially cultivate a large number of plants indoors and put the plants in outdoor water to play the role when the outside temperature is proper is the problem which is attempted to be solved by the research. Plant tissue culture is an effective method and means for rapid propagation of plants. By utilizing the plant tissue culture technology, when the external temperature is not favorable for the growth of the eichhornia crassipes, the eichhornia crassipes is rapidly propagated and stored under the condition of artificial tissue culture. When the external temperature is proper, the artificially propagated eichhornia crassipes is released in natural water, so that the short-term rapid mass propagation of the eichhornia crassipes is realized, the natural geographical conditions are fully utilized, and the high-efficiency application in pollution control, feed and other aspects is realized.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a primary culture method in water hyacinth tissue culture, which ensures that the browning rate and the death rate of an explant are both at a lower level while establishing a primary culture mode of water hyacinth.

The scheme for solving the technical problems is as follows: the primary culture method in the tissue culture of the water hyacinth comprises the following steps:

(1) and (3) disinfection: soaking stem of Eichhornia crassipes (Fr.) Crassifolia in 75% ethanol for 30S, sterilizing with 0.2% mercuric chloride solution for 10min, and washing with sterile water for 3-5 times;

(2) taking an explant: taking down axillary buds of the stems of the eichhornia crassipes under an aseptic condition, and peeling off tissues outside the axillary buds; taking the base of axillary bud as a base point, and taking a section of 0.4-0.7cm as an explant;

(3) primary culture: transferring the explant into a culture medium for culture, wherein the culture medium takes MS as a basic culture medium, the concentration of sucrose is 30g/L, agar is 8g/L, the pH value is 5.8, and the hormone formula is as follows: 6-BA2mg/L + NAA0.2 mg/L; the culture temperature is 25 ℃, the photoperiod is 16h of illumination and 8h of darkness, and the illumination intensity is 2000 Lx.

As an improvement, the water hyacinth growing in the field is fished from the nature and then placed in 0.1% potassium permanganate solution for 30min, and then washed by running water, and then the stem of the water hyacinth is cut.

The browning rate and death of explants during plant tissue culture are frequent phenomena and are problems to be concerned during tissue culture. The inventor finds that: when axillary buds of the stems of the eichhornia crassipes are used as explants, the browning rate and the death rate of the explants are high. The inventor further researches and discovers that: the length of the eichhornia crassipes explant has a remarkable influence on the browning rate and the death rate, and different hormone combinations have little influence on the browning rate and the death rate of the explant. The invention establishes a primary culture mode of the water hyacinth with higher culture success rate, and lays a certain foundation for biotechnological research of the water hyacinth and application of the water hyacinth.

Detailed Description

Example 1

The primary culture method in the tissue culture of the water hyacinth comprises the following steps:

(2) taking an explant: taking down axillary buds of the stems of the eichhornia crassipes under an aseptic condition, and peeling off tissues outside the axillary buds; taking the base of axillary bud as a base point, taking a section of 0.4-0.7cm as an explant, and throwing away the redundant part; that is, the length of each section of explant is kept between 0.4 cm and 0.7cm, and one end of the explant is originally the base of an axillary bud;

The water hyacinth growing in the field is fished from the nature and then placed in 0.1 percent potassium permanganate solution for 30min, and then washed by running water, and then the stem of the water hyacinth is intercepted

And (3) test results: (1) the browning rate of the explants was 12.2%, and the mortality rate was 7.5%. The influence of the browning rate and the death rate is analyzed in a correlation way to obtain the following results: the correlation coefficient of the browning rate and the death rate of the explant is 0.843, and is obviously correlated at the 0.01 level, which indicates that the browning of the explant directly influences the survival of the explant.

(2) The inductivity of the hormone combination to adventitious buds is 73.3 percent, and the inductivity to adventitious roots is 68.3 percent.

Comparative example 1

Compared to example 1, this comparative example adjusted the length of the explant to 0.8-1.1cm, with other conditions unchanged, resulting in an explant with a browning rate of 16.8% and a mortality rate of 10.8%.

Comparative example 2

Compared to example 1, this comparative example adjusted the length of the explant to 1.2-2.5cm, with other conditions unchanged, resulting in an explant with a browning rate of 28.5% and a mortality rate of 14.0%.

Comparative example 3

Compared to example 1, this comparative example adjusted the hormone formulation to: 2mg/L of 6-BA + 0.2mg/L of IBA, and the other conditions are unchanged, so that the induction rate of the adventitious bud is 49.3 percent, and the induction rate of the adventitious root is 42.0 percent.

Comparative example 4

Compared to example 1, this comparative example adjusted the hormone formulation to: 5mg/L of 6-BA + 0.5mg/L of NAA, and the other conditions are not changed, so that the induction rate of the adventitious bud is 46.7 percent, and the induction rate of the adventitious root is 36.0 percent.

Claims

1. The primary culture method in the water hyacinth tissue culture is characterized by comprising the following steps: the method comprises the following steps:

(2) taking an explant: taking down axillary buds of the stems of the eichhornia crassipes under aseptic conditions, and peeling off tissues outside the axillary buds; taking the base of axillary bud as a base point, and taking a section of 0.4-0.7cm as an explant;

2. The method for primary culture in eichhornia crassipes tissue culture according to claim 1, wherein the method comprises the following steps: the water hyacinth growing in the field is fished from the nature and then placed in 0.1 percent potassium permanganate solution for 30min and then washed by running water.