Technical Protocol 2.0


Maintenance Of Virus-Elite Tissue Cultures

A clean culture line can, in theory, become infected or reinfected while in tissue culture. We have never experienced this directly, but have seen it on rare occasion, in our consultations. Here are some possible scenarios:

Infection via insects

Infestations of insects in test tube cultures does occur. The most common insects which are capable of thriving in tissue cultures are thrips and mites. Thrips are well known carriers of viruses. Thrip infestations are most hazardous if the original insect goes unnoticed and a population sterile offspring is developed. Sterile thrips can be easily subcultured along with the plantlets, as they are often very tiny and not noticeable. Still, thrips need a source of viruses in order to spread them. So the question of greenhouse or outside source of the viruses in question should always be addressed at the same time.

Infection via mechanical transfer

If virus-infected cultures co-exist in the laboratory with the virus indexed cultures, then it is possible to cross contaminate by operator error, typically from poor sterilization of tools between each line.

Reinitiation of lines from greenhouse plants or propagation cultures

If a germplasm line has been lost to some catastrophe, such as medium contamination, etc., then it is possible that the line was reinitiated into tissue culture from an infected source, such as a greenhouse plant, and insufficient testing was done to ascertain the virus status of the parent line and its tissue culture derivatives.

"Infection" via mislabeling or line identity errors

A line might appear to be re-infected when in fact it is a mislabeled line. The line in question should probably be grown out and verified as the actual plant it is supposed to be.

"Infection" via testing errors

Always question initial results. Testing labs are not infallible. Always make sure the tests have been repeated as blinds (coded labels) before accepting a result. Always examine the optical density readings for the positive and negative controls for indications of testing accuracy. ELISA based testing is rated on statistical significance in the difference between the positive and negative controls and the sample result. It is sometimes not a black and white answer. Some viruses are more problematic in testing than others.

Infection because the line was not perfectly virus-free originally

There are many different viruses and additional viruses may become relevant for testing which were not originally part of the elite line requirements. It is always important to check the prior test results for the viruses in question. Also, it is possible to have high titer negatives initially which rise over time to become positives. This is usually only the case over the first 6 months of a line in tissue culture.

Ideally, lines are tested using the broadest based testing system, such as electron microscopy or POTY virus tests, which can reduce the potential for non-obvious viruses present in the culture base.