How to do a good job in plant tissue culture

Just as we can divide molecular biology experiments such as nucleic acid extraction into two steps: cell lysis and nucleic acid purification, plant tissue culture also has two core steps, namely, domestication of wild materials and morphogenesis.

Domestication of wild materials

Many times, it is often necessary to use plant tissue culture because of the need to genetically transform receptors, or to extract metabolites under different conditions for differential analysis. For this premise, experimenters often just copy the culture conditions given in the literature or make clear changes, and what they lack is only materials.

Therefore, sterile culture is the guarantee of this kind of tissue culture experiment, and the domestication process of wild materials is the concentrated expression of a series of anti-pollution struggles such as aseptic operation and explant disinfection.

Specific measures for domestication of wild materials are as follows:

1. Disinfection:

Usually we need to thoroughly disinfect the culture medium, Petri dish, tweezers, scalpel, deionized water and filter paper, which can completely kill the microorganisms in the equipment and culture medium;

Among them, culture medium, tweezers, scalpels, deionized water and other liquids and metal utensils are usually sterilized by 12 1℃ for 20-30 min, glassware by 180℃ for 2 h, and antibiotics, hormones and other reagents that are not resistant to high temperature are usually sterilized by filtration.

2. Disinfection:

Disinfection refers to the disinfection of explants and countertops, aiming at inhibiting and killing most microorganisms without harming plants. Among them, the disinfection methods of explants are mainly divided into surface disinfection and invasive disinfection. For the disinfection of operating table, it usually refers to the disinfection with 30-minute ultraviolet lamp in front of the clean working table and the air filtration disinfection during the operation. It is worth mentioning that it will be effective to wipe the countertop with 70% alcohol before using the ultra-clean countertop.

For the seeds, stems and leaves of most terrestrial plants, surface disinfection is enough to kill microorganisms that may cause pollution during tissue culture, such as 70% ethanol and 5% hydrogen peroxide, which can effectively kill microorganisms on the surface in a short time;

However, for some plants growing in humid and dark environment, there are usually a large number of tissue endophytes, which cannot be killed by surface disinfection. Some invasive disinfectants will be more effective, such as mercuric chloride and sodium hypochlorite.

3. Aseptic operation:

Sterile operation refers to the elimination of all pollution from the operator or other pollution caused by the operator's mistakes. Some common operation methods are as follows:

A) setting a tissue culture room and a buffer room separated from the external pollution area, changing clothes in the buffer room and entering the tissue culture room;

B) Before entering the tissue culture room, disinfect the whole body with alcohol spray in the buffer room, and put on special slippers for the tissue culture room at the same time;

C) During operation, do not wear gloves or limbs, and do not cross all open appliances;

D) Do not cover the air outlet of the ultra-clean table with appliances, and the opening of the ultra-clean table should not be too large;

E) Light the alcohol lamp in the ultra-clean table and try to operate around the flame of the alcohol lamp;

F) Incinerate and disinfect the used metal tools and glassware in time after each operation stage;

G) Tools that come into contact with the culture medium, such as tweezers and guns, are burned, disinfected or replaced immediately after they come into contact with other solid surfaces;

H) The ultra-clean platform used for other molecular experiments should be separated from the ultra-clean platform used for tissue culture and inoculation;