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Agronomy & Horticulture -- Faculty Publications Agronomy and Horticulture Department
9-1961
A Reappraisal of the Relationship Between Free and Bound A Reappraisal of the Relationship Between Free and Bound
Coumarin in Coumarin in
Melilotus Melilotus
Francis A. Haskins
University of Nebraska-Lincoln
, fhaskins@neb.rr.com
Herman J. Gorz
United States Department of Agriculture
Follow this and additional works at: https://digitalcommons.unl.edu/agronomyfacpub
Part of the Plant Sciences Commons
Haskins, Francis A. and Gorz, Herman J., "A Reappraisal of the Relationship Between Free and Bound
Coumarin in
Melilotus
" (1961).
Agronomy & Horticulture -- Faculty Publications
. 255.
https://digitalcommons.unl.edu/agronomyfacpub/255
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320
CROP
SCIENCE
A Reappraisal
of
the Relationship
Between
Free and Bound
Coumarin in
Melilotul
F. A.
Haskins
and
H.
J.
Gorz"
D
UN CAN
and
Dustman
(3,
4),
Clayton
and
Larmour
(1
),
and
Stevenson
and
Clayton
(13)
were
among
the
first
investigators
to
attempt
the
assay
of
coumarin
in
sweetclover
(j\felilotuJ
spp.).
All
of
these
workers
appar-
ently
believed
that
the
coumarin
measured
by
their
assay
methods
existed in
the
free
form
in
the
plant.
Roberts
and
Link
(8,
9)
recognized
the
presence
of
bound
coumarin
in
sweetclover
seeds
and
green
tissues
and
indicated
that
pro-
vision
must
be
made
for
the
hydrolysis
of
this
form
if reli-
able
values
for
total
coumarin
content
were
to be
obtained.
However,
they stated
that
in succulent,
green
tissues
the
free
form
usually
predominated
over
the
bound
form
(9).
Slatensek
and
Washburn
(12)
observed
that
Pioneer
sweet-
clover, a variety
described
by Stevenson
and
White
(14)
as
being
low in
coumarin
on
the
basis
of
colorimetric
anal-
ysis
of
alcoholic extracts,
appeared
to be
high
in
coumarin
when
assayed by a fluorometric
method
which
involved
heating
the
plant
tissue in alkali.
The
difference in values
obtained
by
the
two
methods
was
attributed
to
the
presence
of
bound
coumarin
which
was
hydrolyzed
in
the
Huoro-
metric
procedure
but
not
in
the
colorimetric
assay.
Although
the
fluorometric assay described by Slatensek
and
Washburn
(12)
did
not
permit
distinction
between
free
and
bound
coumarin,
it is
apparent
that
these
investigators
considered
coumarin
to be in
the
free
form
in all
coumarin-
containing
varieties
other
than
Pioneer.
In
recent years,
Goplen
et al.
(5)
have
reported
on
the
influence
of
two
pairs
of
alleles, Clfleu
and
Bib,
upon
the
level
and
form
of
coumarin
in sweetclover. A
qualitative
colorimetric
method
was
used
for
the
detection
of
free
coumarin
in alcoholic extracts;
and,
for
quantitative
meas-
urements
of
total
coumarin,
a fluorometric assay
similar
to
that
described by
Slatensek
and
Washburn
(12)
was used.
The
authors
concluded
that
the
Ce/c«
allelic
pair
governed
presence or absence
of
coumarin
and
that
the
Bib
alleles
determined
the
type
of
coumarin
(
free
or
bound)
present
in CII
individuals.
The
assay
methods
did
not
permit
deter-
1 Cooperative investigations of the Crops Research
Division,
ARS.
USDA,
and
the
Nebraska
Agricultural
Experiment
Station.
Research was
supported
in
part
by the
National
Science Founda-
tion
(Grant
No.
G131R2).
Published
with
the approval of
the
Director
as
Paper
No.
]091.
Journal
Series.
Nebraska
Agr. Exp.
Sta.
'Professor
of Agronomy. University of Nebraska. and Geneti-
cist.
Crops
Research Division. respectively.
The
technical assistance
of Larrv
\\'illiams
and
Walter
Akeson is gratefully acknowledged.
mination
of
free
coumarin
levels,
but
it
may
be inferred
from
their
discussion
that
the
authors
considered
the
cou-
marin
present
in CIICuBB
plant
tissues to be in
the
free
form.
Haskins
and
Gorz
(6)
used
a
modification
of
the
Slaten-
sek
and
Washburn
(12)
procedure
for
the
determination
of
free
and
total
coumarin
in
aqueous
extracts
of
plant
tissues
representing
the
four
genotypes
homozygous
with
respect to
Cu/at
and
Bib.
Their
conclusions
regarding
the
influence
of
these
two
pairs
of
alleles
differed
somewhat
f
rom
the
conclusions
of
Goplen
et al.
(5).
Thus,
they
sug-
gested
that
the
Ca/c«
alleles
determined
whether
the
level
of
coumarin
would
be
high
or
low,
and
the
B/b
.illeles
influenced
the
presence
of
free
and
bound
coumarin
as
opposed
to
bound
coumarin
only.
It
was
reported
th.it in
young
leaves
of
the
CIIB
phenotype,
approximately
_)
7~(
of
the
coumarin
was in
the
free
form.
Similarly,
Clopton
(2)
has
indicated
that
approximately
one-third
of
the
cou-
marin
in
Hubam
sweetclover seeds occurs in
the
free
form.
Still
more
recently,
Rudorf
and
Schwarze
(10
l have
found
that
when
proper
precautions
are
taken
to
prevent
glycosidase activity
during
extraction,
extracts
of
"bitter"
Hubam
sweetclover
contain
little
if any
free
coumarin.
They
used
dilute
sulfuric
acid as an
extracting
solution,
and
reported
that
bound
coumarin,
which
was
presumed
to be
the
glycoside
of
ei.l-o-hydroxycinnamic acid. was
extracted
without
being
hydrolyzed.
Although
by no
means
a
complete
review
of
the
litera-
ture
on
coumarin
in sweetclover,
the
foregoing
is surticient
to indicate
that
the
point
of
view
regarding
the
form
in
which
coumarin
exists in
normal,
coumarin-containing
sweetrlover
plants
has
shifted.
Thus,
early
workers
appar-
ently
thought
that
the
free
form
predominated,
then
it was
believed
that
both
forms
were
present
in
substantial
quan-
tity,
and
most
recently it has
been
suggested
that
only the
bound
form
exists in
the
intact
plant.
The
present
study
substantiates
the
findings
of
Rudorf
and
Schwarze
(10),
but
employs
a
different
method
of
extraction
and
assay,
and
utilizes various
genotypes
of bien-
nial
sweetclover
rather
than
the
annual
variety,
Hub
rm.
MATERIALS
AND
METHODS
Plants
of biennial white-blossomed sweetclover (Meli/o!!, .rlb"
Desr.)
of the
four
genotypes. CuCuBB. CuCubb. cucuBB. and
cncub!».
were
used in this study.
The
plants were representative
Published in Crop Science (September-October 1961) v.1, no. 5
HASKINS
&
GORZ:
RELATIONSHIP
BETWEEN
FREE
AND
BOCND
COCMARli'\
321
Table
2-Mean
levels
of free
and
total
coumarin
in
young
leaves
from
9
varieties
of
sweerclover,
Procedure
"B"
was
used in
preparing
the
extracts;
5
plants
of each
variety
were
assayed.
Table
I-Mean
levels
of
free
and
total
coumarin
in
young
leaves
from
4
genotypes
of
sweetclovcr.
Results
are
based
on
extracts
made
by
two
procedures;
32
plants
of
each
genotype
were
assayed.
TI)t~tl
rm'au
~
SE
1).02
O.
III)]
11.02
(I.IIiI]
:i.
~2
U.1:::'
:1. 21
~-
0.12;-)
:L 10 i u, 221;
:L 02 ! O.
l;j~1
:{. 02 -;,
0.11;')
2.8·1:!:- O.
:-;:W
Total
mean
± SE
:1.:16 +
ll.206
4.
;(j
+ O. iOti
:l.
721
O.l'U
2.4:-
+
0.2:\0
4. :30:,- u, :\;'):l
Pro('t'dul'l'
"B"
Frvt
mean
::-:
SE
0.1)1+U.UU1
0.01
0.001
U.I)2
I
d.I)01
O.
()~
U.I)(I:!
Total
mean
.~
SE
O. U:l J
0.1)1)]
U.O:l U.OUI
:l.:l6
ll.164
:l. :Lj-'-
U.l1."
Frt·(·
mean
j- SF:
0.04
-1:
0.00';
O.
O;j±
0.014
0.02
j-
0.002
0.02
±
0.00·1
0.02
+ O.O():!
0.02
±
U.OO;j
O. OS ±
U.020
U. 02 I U. (JOl
U. 02 ±
0.002
Frc(·
nu-an
' SE
1).02
:+-
0.001
U. OJ +
I).OO}
IJ.
«i
O.OUl
O.
i:
+
O.IFiU
cucubb
cucuHB
CuCubiJ
CuCullI3
Varfl·t~
Btonntal
vellow
Common
Y
l'!l()\~
Madrid
Goldtop
Erector
Bf
ennta
l
white
Common
Whit,
Spanish
Evc
rg
rcen
Arctic
Annual
whf tc
Israel
3 Schaeffer, G. W. Chemical genetics of coumarin metabolism in
Melilot
us.
Unpub.
Ph.D.
thesis, University of
Nebraska,
1960.
4
The
glucoside of tl'anJ-o-hrdroxycinnamic acid also occurs in
sweetclover tissues
(7),
and this isomer is included in the "total
coumarin"
figures obtained by the fluorometric procedure.
How-
ever.
Rudorf
and Schwarze
(10)
report
that
only small amounts
of the
/I'(WJ
isomer are normally
found
in the plant.
yielded results similar to those obtained when field-grown
material was used. In no case
did
procedure
"B"
produce
an extract containing an appreciable amount of free cou-
mann.
Results of assaying extracts obtained by using procedure
"B"
on leaves of nine sweetclover varieties are shown in
Table
2,
The
fact that very low free coumarin values were
observed for each of these varieties, all of which have the
CuB phenotype, indicates
that
the
virtual absence of free
coumarin is the rule rather than
the
exception in intact
sweetclover tissues. Furthermore, in preliminary tests
of
procedure
"B"
on two coumarin-containing grasses, sweet
vernal grass (Allthoxallth"lll odoratum
L.),
and
sweet grass
(Hierocbloe
odoraia
(L.)
Beauv.) essentially all of the
compound was found to be present in
the
bound form.
The
limited data available do not, of course,
permit
a general
statement as to whether a similar situation exists in all
species of plants which have been reported to contain
coumarin.
Evidence presented by
Rudorf
and Schwarze
(10)
and
Schaeffer" indicates
that
the bound coumarin present in
sweetclover is the glucoside
of
ciJ-o-hydroxycinnamic
acid"
Hydrolysis
of
this compound by ,S-glucosidase, an enzyme
known to occur in tissues
of
the C"C"BB genotype
(11).
yields ciJ-o-hydroxycinnamic acid which undergoes sponta-
neous lactonization to produce free coumarin. Accordingly.
the obvious conclusion to be drawn from results obtained
with extraction procedure
"A"
is
that
,S-glucosidase re-
leased a portion of the bound coumarin
during
the period
of time between addition of CuCuBB leaflets to water and
inactivation
of
,B-glucosidase in the autoclave. In an attempt
to determine the time
of
release of bound coumarin in pro-
of several F, lines
that
had been derived from a single, doublv
heterozygous
F" plant.
The
original cross from which this F"
plant
was derived involved a
cucuBB
plant
as the female
parent
and ,I
CuCubb
plant
as the male parent.
The
seed from which these two
parent plants
were
grown
was obtained from W. K. Smith.
Crops
Research Division, Agricultural Research Service, U. S.
Depart-
ment of Agriculture, and the University of Wisconsin.
Samples for coumarin assay
were
obtained from the first-year
growth of plants seeded in the field in rows
without
a companion
crop in the
spring
of 1960. Sixteen plants of each genotype
were
sampled on October 4, 1960, and 16 additional plants of each
genotype
were
sampled on
October
6. For each
plant
sampled. the
terminal
8- to lO-inch
portion
of one branch was removed and the
cut end was immediately immersed in a test
tube
of tap water.
Samples
were
then
carried to the laboratory,
where
a young, fullv
expanded leaf was selected on each of the branches.
This
leaf was
removed and the three leaflets were cut from the petiole
with
,1
sharp razor blade. Each leaflet was immediately weighed to the
nearest 0.1 mg. on a direct-reading balance.
The
mid-leaflet was
oven-dried for the determination of percentage dry matter.
One
side leaflet was
dropped
into 2 ml. of distilled
water
at room
temperature. contained in a test tube.
Two
drops of
95%
ethyl
alcohol
were
applied to the leaflet to facilitate submersion, and
the tube and contents
were
then autoclaved for 15 min. at approxi-
marely 15 psi.
This
method of extraction. designated procedure
"A".
has been described in an
earlier
publication
(6).
The
other
side leaflet was subjected to extraction procedure
"B."
which
dif-
fered from procedure
"A" in
that
the leaflet was
dropped
into
2 ml. of water
that
had been heated in a boiling water bath for
10 to 15 min.
Addition
of alcohol was not required for submer-
sion of the leaflet
under
these conditions.
The
tube was
left
in
the boiling
water
for approximately 10 sec. after the leaflet had
been added and was then allowed to stand at room temperature
until it was placed in the autoclave. Leaflets to be extracted
by
procedure
"A"
and
"B"
were
autoclaved simultaneously. Auto-
c1aved extracts
were
cooled. leaflets
were
removed and discarded.
and the extracts
were
stored in a freezer for
later
assay.
The
assay
method used was essentially the fluorornetric procedure for free
and total
coumarin
which
was described by Haskins and
Gorz
(6).
In both extraction procedures. care was taken to avoid
bruising
the leaflets.
Operations
were
scheduled in such a way
that
the
maximum time elapsing between removal of a leaf from
the
branch
and addition of the leaflets to
water
was approximately 40 min ..
and the maximum time elapsing between
addition
of the leaflets
to water and the start of autoclaving was
8 min. In an earlier
experiment it was found
that
leaflets could stand in dry tubes at
room
temperature
for at least 6 hours
without
appreciable altera-
tion in levels of free and total coumarin.
Extraction
procedure
"B"
also was used in obtaining extracts
for assaying 5 plants of each of 9 sweetclover varieties.
Four
yellow.blossomed
varieties-Common
Yellow, Madrid,
Goldtop,
and Erector
(M.
o/ficillttlir
(L.)
Desr.)
and 5 white-blossomed
varieties-Common
White,
Spanish, Evergreen, Arctic, and Israel
(M.
albJ)-were
used in this
portion
of the study.
With
the
exception of the annual variety. Israel. these varieties are biennial
in
,erowth habit. Samples were collected on October 10. 1960, from
field-grown plants
seeded in rows
without
a companion crop in
the
~Ning
of
1960.
RESUL
TS
AND
DISCUSSION
Mean values for free and total coumarin in
"A"
and
"B" extracts
of
young leaves from 32 field-grown plants
of each homozygous genotype are shown in Table 1.
For
mcuhh.
mc"BB.
and CuCuhh leaves, differences in results
obtained by the two extraction procedures were insignifi-
cant.
A.s
expected, leaves of the
mm
genotypes contained
very small amounts of either free or bound coumarin, and
assays of
CrcCubb
extracts indicated substantial amounts of
coumarin, essentially all of which was in the bound form.
In the case of CttCuBB leaves, on the other hand, extracts
obtained by procedure
"A"
contained substantial amounts
of free coumarin whereas
"B"
extracts contained little if
any ()f the compound in this form. Total coumarin levels
observed in the two types of extract were not appreciably
different.
Ar plicarion of the 2 extraction procedures to greenhouse-
grown plants of the 4 doubly-homozygous genotypes
Published in Crop Science (September-October 1961) v.1, no. 5
:'>22
CROP
SCIENCE
cedure
"A,"
CuCuBB
leaflets were
added
to water, treated
with
small amounts
of
alcohol to cause submersion, and
allowed to
stand
at
room
temperature
for
various times up
to
one
hour
before
removal
from
the water
and
subjection
to extraction procedure
"B."
Little
if
any free coumarin
was
found,
either
in the leaflet extracts or in
the
water
in
which the leaflets were allowed to stand
prior
to extraction.
Apparently, then,
the
release of free coumarin
noted
in
procedure
"A"
occurred in
the
autoclave in advance
of
heat
inactivation of ,B-glucosidase in
the
leaflet tissues.
The
pos-
sible involvement of alcohol in hydrolyzing
bound
cou-
marin
during
autoclaving in
procedure
"A"
is precluded
by
the
fact
that
omission
of
the alcohol did
not
prevent
the
release of free coumarin.
It
follows
that
in procedure
"B"
the
boiling
water
inactivated ,B-glucosidase
with
suffi-
cient rapidity to
prevent
appreciable hydrolysis
of
bound
coumann.
Hydrolysis
of
bound
coumarin can be induced by several
means
other
than
extraction procedure
"A".
Thus,
the fol-
lowing treatments of
CuCtiBB leaflets resulted in
the
for-
mation of substantial amounts of free coumarin: mechan-
ical maceration; freezing in liquid
nitrogen
followed by
thawing
in water, acetone, methyl ethyl ketone, methyl
alcohol,
95~/;;
ethyl alcolhol, n-propyl alcohol, n-butyl
alcohol, ethyl ether, ethyl acetate, benzene, or
chloroform
at room temperature; and
brief
submersion in
the
foregoing
organic solvents followed by addition of water
and
auto-
claving. In some instances the
extent
of hydrolysis exceeded
90(/c. Replacement
of
water
in
procedure
"A"
by
10-"
M
solutions
of
the
glucosidase inhibitors,
AgNO"
,
HgCl
z'
and CuCl
z'
failed to effect complete inhibition of hydroly-
sis. In these
and
other
extraction trials,
the
treatments most
effective in
preventing
hydrolysis of
bound
coumarin
were
those involving submersion of the tissue in a
hot
solvent;
and, of
the
solvents tested,
none
was
more
effective
than
hot
water
as used in procedure
"B."
However, inhibition
of
hydrolysis can be effected by
the
use
of
low tempera-
tures, as shown in an
experiment
in
which
leaves
of
the
CtlCtiBB genotype were
ground
in liquid
nitrogen
in a
mortar
after which
the
frozen
ground
material was ex-
tracted first
with
100'/; methyl alcohol and then with
50%
methyl alcohol in a deep freeze.
The
extracts
thus
obtained
accounted
for
approximately
99%
of
the
total coumarin
present in the leaves, and
had
free
coumarin levels only
slightly
higher
than those observed in extracts
made
by
procedure
"B."
The
simultaneous existence of
bound
coumarin and
,B-glucosidase in intact tissues
of
the
CtlCtiBB genotype
apparently
depends
upon
a
pattern
of
cellular or tissue
organization
that
limits hydrolysis
of
the
bound
coumarin
by the enzyme.
Anything
that
interferes
with
the postu-
lated
pattern
of organization
without
immediate inactiva-
tion of
the
enzyme, then, may lead to rapid formation
of
free coumarin.
The
virtual absence of free coumarin in intact sweet-
clover tissue
of
the CuCtiBB genotype may result
from
either
(a)
failure
of
,B-glucosidase to hydrolyze
bound
cou-
marin in such tissue, or
(b)
failure
of
free coumarin to
accumulate because of rapid conversion to
other
com-
pounds.
Available evidence does
not
permit
a conclusive
choice between these alternatives. If
the
first is correct, the
postulation
that
the
B/ b allelic pair controls
the
conver-
sion of
bound
coumarin to
the
free form in
the
intact
plant
(5,
6)
obviously is in error, as is
the
suggestion
of
Schaef-
fer et al.
(11)
that ,B-glucosidase is responsible
for
this
conversion in
the
intact
plant.
If
the
second alternative is
correct, on the
other
hand,
no
change
in
the
suggested roles
of
the
B/
b alleles
and
,B-glucosidase is required.
The
report
of
Kosuge
and
Conn
(7)
that
exogenously-supplied cou-
marin is metabolized at a
rapid
rate,
with
melilotic acid
being one
of
the
principal products, gives some credence
to this alternative. Regardless
of
which
alternative
IS COf-
rect, or
whether
both
are partially correct,
there
IS no
apparent
reason to
doubt
the
influence of
the
B/
b alleles
on level
of
,B-glucosidase activity in
preparations
of
sweet-
clover tissues, as demonstrated by Schaeffer et al.
rt
l
):
SUMMARY
In early
work
on coumarin in sweetclover,
the
assump-
tion was
made
that
the
free
form
of
the
compound
pre-
dominated
in
the
intact plant. Subsequent investigations
demonstrated the presence
of
bound
coumarin
in addition
to free, and it was
then
thought
that
both
forms
were nor-
mally present. Recent
work
indicates
that
when
suitable
extraction procedures are used, virtually all
the
coumarin
is obtained in
the
bound
form. Such extraction procedures
must provide for
the
rapid inactivation
of
,B-glucosidase,
thus
preventing
the hydrolysis
of
bound
coumarin (appar-
ently
the
glucoside
of
ciJ-o-hydroxycinnamic
acid)
Juring
the extraction process.
In the
present
study, inactivation
of
,B-glucosidase was
achieved by submersion
of
the
plant
tissue in
boiling
water.
When
this step was incorporated into
the
extraction pro-
cedure,
leaf
samples
of
4 homozygous genotypes (cuetibb,
etictiBB.
CuCtlbb, and
CuCuBB)
and 9 coumarin-contain-
ing varieties
of
sweetclover,
and
of sweet vernal grass and
sweet grass were
found
to be essentially devoid
of
free
coumarin. In
ge?eral, any
treatment
that
caused disruption
of
cellular or tissue organization
prior
to inactivation of
,B-glucosidase resulted in conversion
of
bound
coumarin to
the
free form.
Therefore,
care in
handline
the
plant
tissue
is essential.
b
Implications of the
apparent
absence of free coumarin in
the intact sweetclover
plant
are discussed
with
respect to
the
possible roles
of
the
B/b
alleles
and
,B-glucosidase in
coumarin biosynthesis.
LITERATURE CITED
1. CLAYTON.
].
S..
and
LARMOUR. R. K. A
comparative
color
test
for
coumarin
and
melilotic
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in Me/ilotus
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Can.
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Res., Sec. C
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1935.
2. 'CLOPTON,
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R.
Determination
of
coumarin
in
the
presence
of
sterols.
].
Agr.
and
Food
Chern.
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1958.-
3. DUNCAN, 1.
].,
and
DUSTMAN, R. B.
Quantitative
determina-
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of
coumarin
in
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material.
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and
Eng.
Chem.,
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1934.
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and
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Determination
of
coumarin
in
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the
steam-distillation
and
alcoholic-extraction
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Anal.
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1937.
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P.,
GREJ'NSHIHDS,
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E. R.,
and
BAEKZIGER, H.
The
inheritance
of
coumarin
in
sweet
clover.
'Can. J.
Botany
35:583-593.
1957.
(). HASKINS, F. A.,
and
GORZ,
H.
].
F1uorometric
assav
eJt
free
and
bound
coumarin
in
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Agron.
].
49:
~9
.\-497.
1957.
7. KOSUGE,
T.,
and
CONN, E. E.
The
metabolism
01'
.ir.nnati::
compounds
in
higher
plants.
1.
Coumarin
and
»-roumanc
acid.
].
BioI.
Chern.
234:2/33-2137.
1959.
8.
ROBERTS,
W.
1.,
and
LINK, K. P. A
precise
method
f"j[
the
determination
of
coumarin,
melilotic
acid,
and
coumaric
acid
in
plant
tissue.
].
BioI.
Chem.
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1937.
9. ,
and
.
Determination
of
coumarin
and
melilotic
acid. A
rapid
micromethod
for
determin.ir.on
in
Published in Crop Science (September-October 1961) v.1, no. 5
Melilotus
seed and
green
tissue. Ind. and
Eng.
Chem.,
Anal.
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9:438-441.
19.37.
10.
RUDORF,
W.,
and
SCHWARZE.
P.
Beitrage
zur
Ziichtung
cines
cumarin-Freien Steinklees und
Untersuchungen
iiber
Cumarin
und
verwandte
Verbindungen.
Z. f. Pflanzenzucht.
39:245-
274. 1958.
II.
SCHAEFFER,
G.
W.,
HASKINS,
F. A., and GORZ, H.
].
Genetic
control of
coumarin
biosynthesis
and
(:i.glucosidase
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3:268-271.
19!iO.
323
12.
SLATENSEK,
]. M.,
and
WASHBURN, E. R. A
rapid,
fluoro-
metric
method
for
the
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of
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and
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compounds
in
sweet
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].
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Agron.
31i:704-708.
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13.
STEVENSON,
T. M., and
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].
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tt.
..
~--~~-~,
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W.
].
Investigations
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Published in Crop Science (September-October 1961) v.1, no. 5