Development of non-narcotic (opiumless and alkaloid-free) opium poppy, Papaver somniferum


Sharma, J.R.; Lal, R.K.; Gupta, A.P.; Misra, H.O.; Pant, V.; Singh, N.K.; Pandey, V.

Plant Breeding 118(5): 449-452

1999


A mutation breeding programme using gamma rays and ethyl methane sulphonate (EMS) was carried out for genetic conversion of narcotic `opium poppy' into non-narcotic 'seed poppy'. Two families contained 31 latexless (opiumless) and 23 partial latex-bearing plants which were identified in the M2 and confirmed in the M3 generations by the 'raypluck' method. Thin-layer chromatography (TLC)-densitometry of powdered capsule hulls (straw) from 22 representative plants also revealed that none of the five opium alkaloids (morphine, codeine, thebaine, papaverine and narcotine) was present in the straw. Six plants also possessed high seed yield (4.0-5.66 g/capsule vs. 3.39 g for control) and seven had a high seed oil content (50.7-53.5%). The best mutant genotype, LL-34 of the family CI-Comb-113-2, with 5.66 g/capsule seeds containing 52.6% oil was designated as cv. `Sujata'. This, perhaps the world's first opiumless and alkaloid-free seed poppy cultivar, offers a cheap and permanent (fundamental) solution to the global problem of opium-linked social abuse. Simultaneously, it serves as a safe and potential food crop with protein-rich seeds and healthy unsaturated seed oil.

Plant
Breeding
118,
449-452
(1999)
©
1999
Blackwell
Wissenschafts-Verlag,
Berlin
ISSN
0179-9541
Development
of
non
-narcotic
(opiumless
and
alkaloid
-free)
opium
poppy,
Papaver
somniferum
J.
R.
SHARMA,
R.
K.
LAL,
A.
P.
GUPTA,
H.
0.
MISRA,
V.
PANT,
N.
K.
SINGH
and
V.
PANDEY
Central
Institute
of
Medicinal
and
Aromatic
Plants
(CIMAP),
Lucknow-226015,
India
With
1
fi
gure
and
1
table
Received
July
9,
1999
/Accepted
July
19,
1999
Communicated
by
G.
Robbelen
Abstract
A
mutation
breeding
programme
using
gamma
rays
and
ethyl
methane
sulphonate
(EMS)
was
carried
out
for
genetic
conversion
of
narcotic
`opium
poppy'
into
non
-narcotic
'seed
poppy'.
Two
families
contained
31
latexless
(opiumless)
and
23
partial
latex
-bearing
plants
which
were
identified
in
the
M
2
and
confirmed
in
the
M
3
generations
by
the
'ray
-
pluck'
method.
Thin
-layer
chromatography
(TLC)-densitometry
of
powdered
capsule
hulls
(straw)
from
22
representative
plants
also
revealed
that
none
of
the
fi
ve
opium
alkaloids
(morphine,
codeine,
thebaine,
papaverine
and
narcotine)
was
present
in
the
straw.
Six
plants
also
possessed
high
seed
yield
(4.0-5.66
g/capsule
vs.
3.39
g
for
control)
and
seven
had
a
high
seed
oil
content
(50.7-53.5%).
The
best
mutant
genotype,
LL
-34
of
the
family
C
I
-Comb
-113-2,
with
5.66
g/capsule
seeds
containing
52.6%
oil
was
designated
as
cv.
`Sujata'.
This,
perhaps
the
world's
fi
rst
opiumless
and
alkaloid
-free
seed
poppy
cultivar,
offers
a
cheap
and
permanent
(fundamental)
solution
to
the
global
problem
of
opium
-linked
social
abuse.
Simultaneously,
it
serves
as
a
safe
and
potential
food
crop
with
protein
-rich
seeds
and
healthy
unsaturated
seed
oil.
Key
words:
Papaver
somniferum
combating
opium
abuse
mutation
breeding
oilseed
crop
opiumless
seed
poppy
Perhaps
no
medicinal
plant
possesses
such
a
high
food
value
together
with
an
excellent
pharmaceutical
property
as
does
the
opium
poppy,
Papaver
somniferum
L.
The
ancient
Sumerians
(in
what
is
now
Iraq)
called
it
hul
gil
(Joy
plant')
and
used
its
seed
for
food
(Brownstein
1993).
The
'sleep
-inducing'
property
and
medicinal
value
of
the
latex
(raw
opium)
obtained
from
the
poppy
plant
have
also
been
known
and
used
throughout
human
history
(Veselovskaya
1976,
Husain
and
Sharma
1983).
The
narcotic
and
medicinal
attributes
of
opium
poppy
accrue
mainly
from
the
three
morphinane
alkaloids
(morphine,
codeine
and
thebaine),
one
benzylisoquinoline
alkaloid
(papaverine)
and
two
phthalideisoquinoline
alkaloids
(narcotine
and
narcine).
All
the
six
opium
alkaloids
are
present
in
the
latex
(raw
opium).
They
are
also
present
in
poppy
-straw
(capsule
hulls
or
some-
times
hulls
together
with
one-third
of
the
peduncle)
but
in
much
lower
concentrations
(Sharma
and
Gupta
1994).
However,
the
opium
per
se
and
the
morphinane
alkaloids
alone
are
addictively
narcotic
and
have
been
abused
for
centuries
(Husain
and
Sharma
1983,
Sharma
1996).
Now,
with
an
easy
chemical
con-
version
of
morphine
into
heroin,
the
opium
-linked
evils
have
even
assumed
a
menacing
global
proportion.
This
trend
is
a
dangerous
one
unless
effective
control
over
the
production
of
opium
and
morphine
is
exercised.
Under
the
auspices
of
the
International
Narcotic
Control
Board
(INCB)
which
regulates
opium
poppy
cultivation
world-
wide,
the
potential
poppy
growing
countries
in
Europe
and
Australia
have
shifted
their
emphasis
from
opium
or
gum
-
collection
to
chemical
extraction
of
the
alkaloids
from
the
straw
(concentrated
poppy
straw)
of CPS
varieties
of
opium
poppy
rich
in
opium
alkaloids
in
the
straw
(INCB
1981).
Alternatively,
Liersch
and
Krzymanski
(1993)
suggested
breeding
CPS
var-
ieties
with
negligible
or
no
narcotic
alkaloids
in
their
straw.
The
German
Federal
Health
Agency
has
fi
xed
the
maximum
morphine
content
to
0.01%
in
the
straw
of
cultivars
admitted
for
cultivation
in
their
country
(Nothnagel
et
al.
1996).
However,
CPS
varieties
may
only
partly
combat
the
opium
menace
since
they
are
not
free
from
opium,
and
hence
can
be
incised
(lanced)
by
unscrupulous
growers
to
collect
opium.
Therefore,
the
real
solution
is
genetic
inactivation
of
the
enzymes
responsible
for
the
biosynthesis
of
both
opium
and
opium
alkaloids,
thereby
evolving
an
opiumless
and
alkaloid
-
free
(non
-narcotic)
poppy
for
cultivation
(Sharma
and
Singh
1983,
Sharma
1996).
Such
a
non
-narcotic
poppy
could
also
serve
as
a
safe
and
potential
food
crop
with
protein
-rich
seeds
and
healthy
seed
oil
high
in
unsaturated
fatty
acids.
Opium
-deficient
plants
do
occur
in
natural
populations.
Long
ago,
such
plants,
locally
referred
to
as
`Bunjha',
were
reported
to
be
present
in
Indian
landraces
(Anonymous
1916).
Thirty-five
`Bunjha'
or
'blind'
plants
were
also
collected
in
our
germplasm
collection
of
opium
poppy
in
1982.
But
they
did
not
breed
true
and
all
proved
to
be
normal
latex
-flowing.
The
pre-
sent
report
deals
with
mutation
breeding
of
opium
poppy
using
gamma
rays
and
ethyl
methane
sulphonate
(EMS)
to
develop
a
non
-narcotic
(opiumless
and
alkaloid
-free)
seed
poppy
with
high
yields
of
seed
and
seed
oil.
Materials
and
Methods
Mutagen
treatment
and
screening:
Selfed
seeds
of
an
improved
strain,
Mass
-2B
(C
1
)
yielding
dark
pink
latex
(raw
opium)
and
another
high
-
morphine
commercial
cultivar
`Shweta'
(C
2
)
of
opium
poppy
were
irradiated
with
gamma
rays
(
60
Co)
at
100,
200,
400,
600
and
800
Gy
doses
or
treated
with
0.4%
aqueous
EMS.
In
addition,
one
combined
dose
of
200
Gy
+
0.4%
EMS
(Comb)
was
given
to
the
seeds
of
both
C
1
and
C2
parents.
Three
generations,
M
I
,
M2
and
M3,
were
screened
intensively
to
locate
opiumless
mutant
genotype(s).
The
M
1
generation
was
raised
in
3
m
long
paired
rows
and
both
the
M2
and
M3
generations
in
plant
-to
-progeny
rows
of
the
same
length.
Screening
of
opiumless
mutant
by
the
'ray
-pluck'
method:
A
'ray
-pluck'
method
was
devised
which
was
found
to
be
the
most
successful
for
screening
and
detection
of
opiumless
(latexless)
mutant
genotypes
in
the
M2
and
M3
generations.
This
method
entails
plucking
one
of
the
U.
S.
Copyright
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Center
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450
SHARMA
et
al.
I
stigmatic
rays
of
the
capsule
and
looking
for
the
flow
of
latex
(Fig.
1).
This
approach
was
very
rapid
in
locating
latexless
(LL)
or
partial
latex
(PL)
(watery
or
very
dilute
latex
-bearing)
plants
without
resorting
to
lancing
(incising)
their
capsules.
Analysis
of
opium
alkaloids
in
poppy
-straw:
The
fi
ve
major
opium
-
alkaloids,
namely
morphine,
codeine,
thebaine,
papaverine,
and
nar-
cotine
were
analysed
against
the
parental
control
by
a
thin
-layer
chro-
matography
(TLC)-densitometric
procedure
(Gupta
and
Verma
1996)
in
powdered
capsule
hulls
of
22
representative
plants
of
the
two
LL/PL
mutant
families,
C
I
-Comb
-101
and
C
I
-Comb
-113,
together
with
one
naturally
'blind'
(no
latex)
plant
that
had
coppery
capsules.
The
pro-
cedure
consisted
of
toluene
—acetone
—methanol
—ammonia
(40
:
40
:
2
:
2)
forming
the
mobile
phase
and
scanning
the
silica
gel
plates
60F
254
(Merck,
Darmstadt,
Germany)
after
staining
with
Dragendorff
reagent
no.
11C
(Wagner
et
al.
1984).
The
densitometric
scanning
profiles
of
the
fi
ve
opium
alkaloids
were
calibrated
against
the
corresponding
standards.
Determination
of
seed
oil:
The
oil
content
in
dried
seeds
of
seven
LL/PL
plants
marked
by
high
seed
yield
per
capsule
was
determined
by
the
rapid
and
non-destructive
pulsed
nuclear
magnetic
resonance
(NMR)
technique
(Tiwari
et
al.
1974).
Results
From
the
seeds
collected
in
the
M,
generation,
a
total
of
157
(115
C,
+
42
C
2
) M
2
families
were
screened
for
latexless
(LL)
mutant
genotypes.
Out
of
these,
only
fi
ve
families
(three
from
100
Gy
irradiation
of
`Shweta'
and
one
each
from
combined
treatment
of
Mass
-2B
and
`Shweta')
contained
one
to
three
LL
plants.
Six
others
(two
each
from
EMS
and
combined
treat-
ments
of
Mass
-2B
and
two
from
combined
treatment
of
`Shweta')
showed
partial
latex
(PL)
plants.
Seeds
of
all
the
LL/PL
plants
were
collected
from
the
above
11
M
2
families.
Since
such
opiumless
variants
may
or
may
not
be
true
mutants,
it
was
essential
to
confirm
their
inheritance/heritability.
Hence,
M
3
generations
of
all
the
LL/PL
plants
of
the
11
families
were
raised.
However,
only
three
plant
-progeny
rows
belonging
to
each
of
the
two
families,
namely
C,
-Comb
-101
and
C,
-Comb
-
113
of
Mass
-2B,
were
found
to
possess
31
LL
and
23
PL
plants.
None
of
the
LL
mutant
plants
oozed
latex,
while
normal
plants
profusely
secreted
dark
pink
latex
(characteristic
of
the
parent,
Mass
-2B)
as
shown
in
Fig.
1.
The
PL
plants
secreted
a
watery
latex
that
dwindled
when
their
capsules
reached
the
lancing
stage.
To
investigate
further
whether
these
LL/PL
plants
contained
opium
alkaloids
in
their
straw
(capsule
hull),
the
fi
ve
major
Fig.
1:
Capsules
of
the
opiumless
(no
fl
ow
of
latex)
mutant
(left)
and
the
normal
opium
containing
plants
(right)
of
opium
poppy
opium
alkaloids
were
estimated
by
densitometry
of
thin
-layer
chromatograms
from
the
powdered
straw
of
22
representative
LL/PL
plants
of
the
two
families,
along
with
the
two
other
plants.
The
relevant
data
are
presented
in
Table
1.
The
three
morphinane
(narcotic)
alkaloids,
i.e.
morphine,
codeine
and
thebaine,
were
virtually
absent
in
all
the
LL/PL
plants
except
that
the
two
plants
(PL
-48
and
LL
-55)
contained
0.01%
and
0.002%
thebaine,
respectively.
Similarly,
the
phthal-
ideisoquinoline
alkaloid,
narcotine
was
also
absent
in
all
plants
examined.
However,
the
benzylisoquinoline
alkaloid,
papaver-
ine,
was
absent
in
six
plants
(LL
-14,
16,
18,
31
and
34
and
PL
-
47),
occurred
in
trace
amounts
in
14
plants
(Table
1).
Two
plants
(LL
-12
and
SS)
had
0.03%
and
0.01%
papaverine,
respectively.
The
opiumless
coppery
capsule
(CC
-74)
was
also
free
from
all
alkaloids
except
thebaine
(0.15%)
and
papaverine
(in
trace
amounts).
In
contrast,
the
parental
control
(Mass
-2B)
contained
0.55%
morphine,
0.035%
codeine,
0.095%
papaver-
ine
and
both
thebaine
and
narcotine
in
trace
amounts.
Discussion
While
our
work
was
in
progress
two
reports
appeared
on
related
aspects:
First,
Nothnagel
et
al.
(1996),
in
Germany,
con-
fi
rmed
that
the
Swedish
var.
'Soma'
and
the
Polish
var.
Prz-
enko'
had
morphine
contents
as
low
as
0.01%
in
their
straw.
They
also
found
a
cross
-bred
line
RM9/95
possessing
less
than
0.01%
morphine,
which
conforms
to
the
requirement
of
the
German
Federal
Health
Agency.
The
second
report
from
India
pointed
out
that
an
accession
CIMAP-OP-1
of
opium
poppy
had
0.05%
morphine,
0.001%
codeine,
0.015%
thebaine,
0.130%
papaverine
and
0.087%
narcotine
(Bajpai
et
al.
1996).
However,
none
of
the
above
genotypes
was
free
from
latex
(opium)
and
hence
were
different
from
the
present
materials.
Our
fi
nding has
more
than
one
implication:
1.
The
non
-narcotic
'seed
poppy',
in
particular
the
family
C,
-
Comb
-113,
is
economically
highly
competitive
on
two
counts.
(1)
the
yield
of
its
(white)
seeds
per
capsule
was
considerably
higher
(the
highest
being
5.66
g/capsule
followed
by
4.40,
4.31
and
4.18
g)
than
the
parental
control
(3.39
g/capsule)
or
CIMAP-OP-1
(4.06
g/capsule),
and
`Sanchita'
(3.39
g/capsule)
(Bajpai
et
al.
1996)
or
the
line
RM
9/95
(2.09
g/capsule)
(Nothn-
agel
et
al.
1996).
Moreover,
the
seven
plants
evaluated
for
oil
content,
reached
>
50%
oil
in
their
seed
dry
matter
(50.7-
53.5%
compared
with
<50%
oil
in
the
usual
Indian
oil
seed
cultivars).
This
gives
the
promise
of
a
high
yield
of
poppy
seed
oil,
which
is
largely
unsaturated
(Singh
et
al.
1990,
Nergiz
and
Development
of
non
-narcotic
poppy
451
Table
1:
Opium
alkaloids,
seed
yield
and
oil
content
of
latexless
(LL)
and
partial
latex
(PL)
plants
of
opium
poppy'
Plant/genotype
No.
Family
MO
Opium
alkaloids'
(%)
CO
TH
PA
NA
seeds/capsule
(g)
Oil
(%)
LL
-1
LL
-11
LL
-12
LL
-13
LL
-14
LL
-15
LL
-16
LL
-17
LL
-18
LL
-19
PL
-23
LL
-31
LL
-32
LL
-33
LL
-34
LL
-36
PL
-45
PL
-47
PL
-48
LL
-55
LL
-56
LL
-57
CC
-74
Control
C
I
-Comb
-101C
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-1
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-2
C
I
-Comb
-113-3
C
I
-Comb
-113-3
C
I
-Comb
-113-3
Coppery
capsule
Mass
-2B
(parent)
0.55
0.035
0.01
0.002
0.15
t
t
t
0.03
t
t
t
t
t
t
t
t
t
t
0.01
t
t
t
0.095
1.10
3.93
4.40
2.72
4.06
2.77
4.40
2.95
3.35
3.81
3.90
3.08
3.31
2.48
5.66
3.11
4.31
2.89
4.18
1.69
1.17
2.38
2.39
3.39
52.5
51.5
53.5
50.7
52.6
51.2
52.7
47.5
'Abbreviations:
MO
=
morphine,
CO
=
codeine,
TH
=
thebaine,
PA
=
papaverine
and
NA
=
narcotine
alkaloids
in
percentage
of
hull
dry
matter;
-
=
nil;
t
=
traces,
based
on
spot
detection
on
TLC
plate;
oil
in
per
cent
of
seed
dry
matter.
Otles
1994),
i.e.
beneficial
for
dietary
control
of
coronary
heart
disease.
(2)
Being
free
from
opium
and
narcotic
alkaloids,
these
mutant
genotypes
might
serve
as
a
cheap
and
permanent
(fun-
damental)
control
of
the
global
problem
of
opium
-linked
social
abuses.
2.
As
a
further
consequence,
the
non
-narcotic
opium
poppy
would
obviously
deprive
the
use
of
opium
alkaloids
which
are
very
important
for
pharmaceutical
purposes.
This
is
now
the
core
problem
we
are
dealing
with
and
certain
clues
are
at
hand
(Fairbairn
and
Djote
1970,
Fairbairn
and
Steele
1981,
Roberts
et
al.
1983,
Facchini
and
de
Luca
1995)
which
might
allow
us
to
develop
a
CPS
chemotype/cultivar
that
is
opiumless
but
enriched
with
the
desired
alkaloid/s
in
the
straw
to
meet
the
pharmaceutical
requirements.
3.
Our
opiumless
and
alkaloid
-free
genotypes
open
new
per-
spectives
for
genetic
research
on
the
biosynthesis
of
opium
(latex)
and
its
alkaloids
in
opium
poppy
(Sharma
et
al.
1988,
Nessler
et
al.
1990,
Lal
and
Sharma
1991).
The
best
non
-nar-
cotic
mutant,
LL
-34
named
cv.
`Sujata',
with
a
high
potential
for
both
seed
and
seed
oil
yields,
is
now
in
the
pipeline
for
commercial
exploitation.
The
var.
`Sujata'
has
been
patented
in
the
USA
under
the
US
Patent
application
No.
09/276,720
dated
26
March
1999.
Acknowledgements
The
authors
are
grateful
to
the
Director,
CIMAP,
Lucknow
for
pro-
viding
the
necessary
facilities
and
to
Mr
Ram
Chandra
and
Mohd.
Rashid
for
helping
in
the
fi
eld
operations.
Thanks
are
also
due
to
Dr
M. M.
Gupta
for
valuable
suggestions
relating
to
the
chemical
analysis.
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