Tissue Culture without a Laboratory

If a laboratory is not available, hobby botanists often try to establish plant tissue culture with minimalistic equipments at home. It is possible at all, but several aspects have to be considered and the limitations can be very disillusioning.

Sterility

A main problem which appears in professional laboratories as well as in the “kitchen labs” is the necessity of sterilization. To establish a culture, in the first step plant parts (explants) are transferred to a vessel, which contains a media allowing the explant to survive. Explants, vessels and media have to be sterilized, and the transfer into the vessel must be performed in a sterile area.

Microorganisms like bacteria and fungi can grow very fast on plant tissue culture media. They change the composition of the medium, can secrete toxic substances or even grow into the tissue of the explant, which has predominantly lethal consequences. Especially during initiation stages (after explant sterilization), the success of sterilization always should be observed.

Obtaining a sterile working area

A sterile working area in a professional laboratory is given in a laminar flow cabinet. Informations about sucha device and how to build it can be found here:

Laminar flow cabinet

Another possibility is the usage of so called glove-boxes, which can consist of transparent containers, accessible through gloves connected with the casing. Building such boxes is very cheap, but handling can be difficult, especially for beginners. All surfaces in it and items used within the box have to be surface sterilized using disinfection sprays. Contaminations like fungi spores can enter within every time the box is opened, it often is necessary giving them some time to settle down on the area wetted with the disinfectant. Otherwise, if vessels are opened in the box, flying spores may can enter them and contaminate the culture.
Glove-boxes may allow a transplant of explants to a new medium. Mostly recommended if sterile cultures already are available. Establishment of clean cultures in such a box can be very challenging and the risk of contamination is very high.

Sterilization of media, glassware and tools

Typically done in an autoclave which sterilizes using water steam, reaching a temperature of up to 121 °C under pressure (approx. 15 PSI/1 Bar).

In home labs an autoclav is mostly overkill and too cost intensive, which makes hobby botanists using alternatives already available in the kitchen. Many users reported sufficient sterilization when using a pressure cooker, in which they put the vessels containing the media, or tools like forceps/scalpels wrapped in aluminium foil. The preparation of media is even possible in a microwave, but it needs permanent observation during the process to prevent media to overboil.

Sterilization of small volume stock solutions

Plant growth regulators (PGRs) are mostly not readily soluble in water. In this case a preparation of a stock solution is required. As some PGRs are temperature sensitive, a sterilization together with the media by autoclaving is not recommended. In this case sterile filtration allows a sterilization without reaching excessive temperatures. Syringe filters are used for such a procedure:

Syringe filter