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
Infection via insects
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
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
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
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
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
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
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