Reeves, A. F. Tomato leaf extracts
and thiamine-requiring mutants
Three years ago a study was
initiated to explore the
feasibility of working out
the pathway of thiamine synthesis in tomato with the use of the thiamine
requiring mutants tl and spa. It was felt that by treating one mutant with the
extract of another, and vice versa, the sequence of blocks could be
established. Further, it might be possible to refine the extract to determine
the active agent, and therefore, an intermediate in the pathway.
Our first step was to determine if an extract of normal tomato plants
could be used to produce a response in thiamine-requiring mutants. Leaves of
mature VF-36 plants (at least four months old) were ground in water with
mortar and pestle and the extract concentrated by freeze drying. 18 grams of
fresh tissue yielded 1 ml of extract. This extract was added to the cotyledons
of tl plants at the cotyledon stage of growth, one drop twice a week for two
weeks. Although the extract "burned" the cotyledons at the point of
application, the plants did respond somewhat; that is, they became greener and
grew somewhat faster than untreated controls. However, there was still marked
chlorosis at a time when control tl plants treated with 100 ppm thiamine were
fully green. At least there was a response that we could detect visually, and
the method seemed feasible.
In our first experimental run, however, we had to use young seedlings for
the extract of the mutants (they would die without thiamine treatment).
Therefore, we also used VF-36 seedlings (one month old) for our control
extract. Here we obtained the strange and unexpected results shown in the
accompanying table. In less than two weeks all of the extracts had killed our
tl test plants! At a time when untreated control tl seedlings were still
limping along, many of the treated plants had turned brown and withered almost
to the point of total decay.
However, overzealous spraying with insecticides had interfered with the
previous test, and we were not sure how many plants had been killed by
extract, and how many by insecticide. A small re-test was set up in the
following summer. An extract of three-week-old VF-36 plants (70.9 g -3.7 ml)
and one from six-month-old VF-36 (21.1 g - 0.8 ml) were compared directly.
In 12 days the three tl test plants treated with the first extract died,
while the four untreated tl plants were still alive. After 15 days the
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