Potential
limitations
with
using
rhodamine
B
for
the
quantification
of
epicuticular
acylsugars
Willmann,
M
.
R
.
and
Mutschler,
M
.
A
.
Department
of
Plant
Breeding
and
Biometry,
Cornell
University,
Ithaca,
New
York
Control
of
insect
pests
is
an
important
factor
in
tomato
production
.
The
acylsugars
produced
by
the
wild
tomato
L
.
pennellii
mediate
that
species
resistance
to
a
number
of
important
pests
of
tomato,
including
potato
aphid,
green
peach
aphid,
leaf
miner,
fruit
worm,
army
worm,
and
silverleaf
whitefly
.
The
goal
of
this
project
is
the
transfer
of
the
acylsugar
-
mediated
pest
resistance
to
horticulturally
acceptable
varieties
of
cultivated
tomato
.
The
breeding
program
has
progressed
through
5
backcross
generations,
using
for
selection
an
acylsugar
assay
(Goffreda
et
al
.
1990)
that
is
based
on
Nelson’s
copper
reagent
.
This
assay
measures
acylsugars
by
measuring
the
total
sugar
content
of
the
acylsugars
produced
.
Lin
and
Wagner
(1994)
suggested
that
the
affinity
of
rhodamine
B
for
acylsugars
could
be
used
to
detect
and
quantify
acylsucroses
and
acylglucoses
.
We
developed
a
miniprep
protocol
based
on
their
paper
to
rapidly
screen
large
segregating
populations
for
individuals
accumulating
acylsugars
.
The
assay
is
essentially
that
of
Lin
and
Wagner
(1994),
downscaled
to
permit
the
test
to
be
run
in
Elisa
plates
.
The
Rhodamine
-
based
assay
does
not
give
separate
estimations
of
acylsucroses
&
acylglucoses,
but
it
has
several
advantages
over
the
Nelson
-
based
assay
.
The
Rhodamine
assay
has
a
much
greater
sensitivity
to
lower
levels
of
acylsugars
than
the
Nelson
-
based
acylsugar
assay
.
As
a
result,
the
Rhodamine
-
based
assay
can
be
used
at
a
younger
stage
of
plant
development
than
possible
using
previous
methods
.
In
addition,
the
rhodamine
assay
only
requires
approximately
1/3
the
labor
and
1/3
the
supplies
cost
of
the
Nelson’s
-
based
acylsugar
assay
.
Also,
rhodamine
B
does
not
detect
free
sugars,
greatly
reducing
the
background
.
It
was
soon
apparent,
however,
that
the
measurement
of
acylsugars
using
the
Rhodamine
B
assay
is
biased
in
some
way
.
The
acylsugars
of
four
segregating
populations
originating
from
interspecific
crosses
between
L
.
pennellii
and
L
.
esculentum
were
quantified
using
both
the
Nelson’s
copper
reagent
and
rhodamine
assays
.
In
all
cases,
a
characteristic
bifurcation
in
the
data
was
seen,
such
that
two
intersecting
lines
were
present
when