Note: Descriptions are shown in the official language in which they were submitted.
Description 1 31 0928
Herbicide-resistant crop plants, process for their selec-
tion and their regeneration
It is possible to regenerate maize plants from callus
cultures derived from immature embryos [Green, C.E.,
Philips, R.L., Crop. Sci. 15, 417 (1975); EP 160,390;
EP 177,738]. Initially, however, this was only successful
in a few genotypes, for example in inbred lines A188, W64A
and Plack mexican sweet. Only improved nutrient media
permitted the establishment of morphogenic and embryogenic
callus cultures of a large number of different inbred
lines ~Duncan et al., Planta 165, 322 (1985)]. An impor-
tant requirement for this process is the addition of amino
acids to the nutrient medium.
Cell cultures which can be regenerated into intact plants
over a period of several months or years are suitable for
the selection of mutants and variant cell lines. If, for
example, a toxic dose of a herbicide ;s added tû the
nutrient medium, some callus sectors survive, and from
these it is possible to regenerate herbicide-resistant
plants. If the callus tissue is treated with mutagens,
the mutant yield is increased (US 4,443,971).
Herbicides which are inhibitors of enzymes which, in turn,
have a function in the biosynthesis of amino acids have
only a weakened effect on tissue cultures if the medium
contains amino acids, in particular in the case of amino
acids whose biosynthesis is inhibited by the particular
herbicide. In the presence of amino acids, that is to say
under conditions which to date have been considered as
particularly favorable for establishing and subculturing a
maize callus having the ability to regenerate, it is thus
very difficult to simultaneously find mutants which are
resistant to amino acid biosynthesis inhibitors.
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Surprisingly, ;t has been found that cell lines wh;ch are
derived fron crop plants and which are capable of regener-
ation can be cuLtivated in nutrient media which do not
contain amino acids. It is also possibLe, in such nutrient
media, to regnerate crop pLants from these cell lines.
This was particularly unexpected as amino acids are essen-
tiaL for grc~wth regeneration of the calLus cultures or the
plants. Such cuLtures can be employed in the selection of
herbicide-resistant celL Lines, caLlus cultures or pLants.
The invention thus reLates to a process for the reLiable
selection of crop plant ceLL Lines which are resistant to
amino acid biosynthesis inhibitors, by cultivation on
inhibitor-containing nutrient media, which process
comprises
a) seLecting callus cultures or cell suspension cultures
which grow on amino acid-free nutrient media while
maintaining their embryogenic and morphogenic compe-
tence,
b) cuLtivating these cuLtures on amino acid-free,
inhibitor-containing nutrient medium, and seLecting
inhibitor-resistant cultures from which intact plants
can be regenerated.
The invention is described in further detail below, in
particular in its preferred embodiments. Furthermore, the
invention is defined in the claims.
With the aid of the process according to the invention,
which is described below, it is possible to produce
herbicide-resistant regeneration-capable cell lines of
crop plants, preferably monocotyledon crop plants, in par-
ticular cereal plants. Maize cell lines are particularlypreferably used.
In order to be able to select herbicide-resistant cell
cultures, it is necessary, in a first step, to establish
cell lines which can be propagated reasonably well on
ammino acid-free media and which, in addition, can be
13tO928
3 -
induced in high frequency to regenerate the plant. For
this purpose, callus cultures are cultivated on amino
acid-free medium, for example modified Murashige Skoog
(MS) medium [Physiol. Plant 15, 473 (1962)] or modified
S N6 medium [Chu, C.C. et al., Sci. Sin. 16, 659 (1975)].
Nutrient media of this type essentially contain a carbon
source, such as, for example, sucrose, glucose, maltose
and raffinose, a nitrogen source, for example ammonium
salts or nitrates, and vitamins, hormones and mineral
salts known to those skilled in the art.
Preferably, one or more physiological organic acids or
their salts which have a positive influence on the growth
of the cultures are added to the relevant nutrient media,
in particular acids of the citric acid cycle, such as, for
example, citric acid, malic acid, oxalacetic acid, succin-
ic acid and pyruvic acid, or salts thereof. Sodium and
potassium salts are preferred, and ammonium salts are par-
ticularly preferred. These additives favor the growth of
callus or cell suspension cultures on the amino acid-free
media and facilitate subculturing over a long period, at
least 1 to 2 years, under standard culture conditions,
without losing the ability to regenerate into plants.
These compounds can thus be added to the nutrient medium
as a replacement for amino acids, preferably in concentra-
tions of approximately 0.1 to 10 mmol, in particular O.Sto 2 mmol. Standard culture conditions are taken to mean
conditions customary for those skilled in the art. Culti-
vation can be carried out at approx. 15 to 35C, prefer-
ably 20 to 30C, with or without light. The transfer
intervals chosen are usually approx. 10 to 3û days prefer-
ably 14 to 21 days, which of course must be dependent on
the growth of the cultures.
Cell lines which are able to grow on amino acid-free
nutrient media are then used for selecting inhibitor-
resistant, in particular herbicide-resistant~ callus cul-
tures and cell suspension cultures. Using these cultures,
selection can in principle be carried out against all
1 3 1 Oq28
-- 4
inhibitors, but selection is preferably carried out
against herbicides which inhibit amino acid biosynthesis.
~ithin this group, in turn, selection is preferably
carried out against herbicides Of the general formula I
R1 _ P - (CH2)n-R3-CoR4
in which independently of one another
R1 is hydroxyl or methyl,
R2 is hydroxyl, methyl or ethyl, and
R3 denotes aminomethylene, hydroxymethylene or a carbonyl
group,
CH3 0
R denotes OH or [_ NR - C - C] OR- ~ where x can be 1
to S, preferably 1 to 2, and
n denotes O to 4, preferably O to 2. Particularly pre-
ferred are glufosinate (phosphinothricin) or its struc-
tural analogs, such as bialaphos, and dimethylphosphinyl-
hydroxyacetic acid. If the inhibitors are optically
active, either the racemate of the compounds or the bio-
logically active enantiomer can be used.
In order to obtain higher yields during selection, the
seLected cell lines can be treated with mutagenic sub-
stances by methods known per se. This can be carried out
either using chemicals such as, for example, ethyl methane
sulfonate or N-methyl-N-nitrosoguanidine tMNG), or by
irradiation, such as, for example, with X-rays or UV-rays.
Mutagens of this type are used in concentrations such that
30 to 70% of the cells are killed.
The actual selection of the ;nhibitor-resistant cell sus-
pension cultures, preferably callus cultures, is carried
out on the abovementioned amino acid-free nutrient media,
to which the inhibitor has been added. Its concentration
in the medium can vary in a wide range and ;s essentially
chosen such that 70 to 99%, pre~erably 95 to 99%, of the
1 3 ~ 0928
calli die. The selection can be carried out repeatedly
using identical or increasing concentrations of herbicide.
The yield of the selection of resistant cells or calli
whlch are capable of regeneration can be increased by add-
ing to the nutrient medium the physiological organic acidsor their saLts listed above. Furthermore, the selectivity
can be improved by cultivating the callus caltures in the
light, approx. 8 to 16 h at 500 to 5,000 lux. Cultivation
is carried out under the customary conditions for one or
more passages, preferably 2 to 4 passages, suitable media
being both agar and liquid media. Transfer intervals vary
according to growth rate and are generally 1 to 5 weeks,
preferably 3 to 4 weeks.
In particular by repeated subculturing of the resistant,
morphogenic calli and cell suspensions, cell lines can be
established which tolerate herbicide concentrations of up
to 5 mM and which are still capable of regenerating plants.
With the aid of the process according to the invention it
is, of course, also possible to select cell suspension
2û cultures and regeneration-capable callus cultures which
are resistant to two herbicides with different sites of
action.
Regeneration of the herbicide-resistant plants is carried
out by methods known per se using nutrient media which can
also contain the herbicide against which selection has
previously been carried out. In certain circumstances,
however, regeneration on nutrient media without herbicide
may be advantageous. The invention hence also relates to
herbicide-resistant crop plants which can be obtained by
the previously listed cultivation methods, and to the use
of these plants for crossing with other genotypes, this
part of the invention also preferably relating to mono-
cotyledon crop plants, in particular cereal plants, and
particularly preferably to maize plants.
For examPle, it is possible, with the aid of crosses, to
1 3 1 092g
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produce hybrids which are resistant to one or two herbi-
cides. This is achieved by crossing a herbic;de-resistant
plant with a non-resistant inbred line, or by crossing two
herbicide-resistant plants which have been selected
against different herbicides having distinguishable sites
of action. Such crosses can also be carried out by known
methods. Herbicide-resistant plants are taken to mean
those which do not exhibit symptoms of damage at twice the
application rate necessary for combating weeds.
The invention furthermore relates to a method for protec-
ting crop plants by selectively killing weeds using a
herbicide on fields which are planted with plants obtain-
able in the process according to the invention and thus
resistant to the herbicide. The herbicides preferably
employed are substances which inhibit amino acid biosyn-
thesis, in particular compounds having the abovementioned
general formula I. Glufosinate or its structural analogs,
such as, for example, di- and tripeptides, and dimethyl-
phosphinylhydroxyacetic acid, are particularly preferred.
If they are optically active inhibitors, both the racemate
and the optically active compound itself can be used. The
herbicide or mixtures of the herbicides are applied to the
field in a manner known per se, if possible at intervals,
preferably approximately 10 to 100 days after sowing of the
crop plan~s, until the weeds are sufficiently suppressed.
The invention is illustrated below with the aid of examples.
Examples
1~ EstabLishing morphogenous maize cel~ cultures on a~ino
acid-free nutrient mediun
a) Immature embryos were dissected from immature maize
kernels of the inbred lines B73, W64A and A 188 it
was also possible to employ hybrids thereof) 10 to
14 days after fertilization of the female flowers.
Preferably embryos of 1 to 1.5 mm in length were
transferred onto callus induction medium under
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sterile conditions and cultivated at 25C + 2C in
the dark. Su;table callus ;nduct;on med;a are
A: a modified Murashige and Skoog medium, or
~: a modified N6 medium,
whose composition is listed in Table 1, with the
addition of proline (1,500 mg/l), asparagine
(500 mg/l), glutamine (500 mg/l), casein hydrolysate
(vitamin-free, 500 mg/l) and sucrose (60 g/l).
0.7~ of agar was added to the nutrient media, and the
pH was adjusted to 5.8 using KOH before autocLaving.
The vitamins were filter-sterilized and added to the
cooled medium.
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Table 1: Nutrient medium
modified MS medium (A) modified N6 medium (B)
mg/l mg/l
M9504 7H20370 250
CaCl2.2H20440 166
KN03 1,900 2,830
(NH4)2S04 - 433
NH4N3 1,650
10 KH2P04 170 400
MnS04 22.3
KJ 0.86
COCl2 6H2o 0.025
ZnS04.7H20 8.6
15 CuS04.5H20 0.025
H3B03 6.2
Na2MoO4~2H2o 0.25
EDTA 37.3
FeS04.7H20 27.3 The compounds of
20 Thiamine.HCl û.5 the MS medium
Nicotinic acid 0.2 included in the
Cyanocobalamin 0.1 bracket are added
Pyridoxine HCl 0.2 to the N6 medium.
p-Aminobenzoic acid 0.05
Ca-Panthothenate 0.1
Biotin 0.1
Folic acid 0.05
Nicotinic acid 0.2
Choline HCl 0.1
30 Riboflavin 0.05
Inositol 100
Sucrose 30,000
2,4-Dichlorophenoxyacetic acid 1 mg
or 3,6-dichloroorthoanisic acid 2 m~
~ithin 2 to 3 weeks, callus cultures which were
capable of forming shoot primordia and somatic
embryos, were formed.
1 3 1 0~2~
.
Th~se callus cultures were then cultivated on the
amino acid-free nutrient media A and B. Starting
from 1,500 embryos, 5 cell lines were established
which were abLe to grow on nutr;ent med;a of th;s
type for more than a year, while maintaining their
regenerat;on capability. Subculturing was carried
out every 15 to 28 days.
b) The procedure is as in Example 1 a). However, the
following organic acids were added to the amino
acid-free nutrient media: citric acid (200 mg/l),
c~-ketoglutarate (150 mg/l), malic acid (130 mg/l),
oxalacetate (130 mg/l), succinic acid (120 mg/l) and
pyruvic acid (90 mg/l). The stock solution of the
organic acids was adjusted to a pH of 5.8 using NH3
solution before adding to the medium.
2. In vitro mutagenesis
Morphogenic pieces of maize callus which can grow on
amino acid-free nutrient medium were incubated for 10
to 120 minutes in liquid salt medium, comprising the
salts of nutrient solution B containing 0.1 to 1% of
ethyl methane sulfonate, washed 3 times with the salt
medium at intervals of 10 minutes, and then cultured on
culture medium A or B. After 2 to 4 weeks, the surviv-
ing morphogenic callus segments were subcultured on
fresh medium. After a further 2 to 4 weeks, the calli
could be used for selection experiments.
3. Selection of herbicide-resistant caLLus cultures
- The herbicide concentration in amino acid-free culturemedium A or B (according to Example 1b) at which 95 to
99% of the calli died was determined. This was the
case at a concentration of 2 x 10 4 mol/l for both
glufosinate and for dimethylphosphinylhydroxyacetic
acid.
1 3 I Q928
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10,000 calli in each case were transferred onto the
herbicide-containing agar media (0.8% of agar), and
evaluation was carried out after 6 to 8 weeks. The
Petri dishes were incubated at 25C and at 1,000 to
2,000 lux with a 12 hour photoperiod . 12 calli, which
grow while forming dark-green shoot primordia, were
formed on media containing glufosinate. 3 such calli
were obtained on media containing dimethylphosphinyl-
hydroxyacetic acid.
By repeated subculturing of the resistant morphogenic
calli, it was possible to establish cell lines which
tolerate herbicide concentrations of up to 5 mmol/l and
were still capable of regenerating plants.
4. Regeneration of plants
On the regeneration medium (medium A or B without
2,4-dichlorophenoxyacetic acid or dicamba) and in the
course of 3 to 5 weeks, mainly in the course of 3 to 4
weeks, the embryogenic herbicide-resistant calli ob-
tained differentiated complete plants. As soon as the
leaves were 1 to 3 cm long, the plants were transplan-
ted from the agar into a mineral culture substrate
(vermiculite, perlite) and cultured at 90 to 100% rela-
tive humidity during the first 4 to 7 days. Following
this, it was possible to cultivate the plants further
either in the growth cabinet or in the greenhouse. The
maize regenerates are grown hydroponically until a fur-
ther 2 to 4 leaves are formed. The plants can then be
transplanted into soil (sandy loam).
5. Application of herbicides
When the plants have reached the 4- to 5-leaf stage,
they are sprayed with herbicide solutions at applica-
tion rates customary in practice (50 to 200 mg of
active substance/m2, corresponding to 0.5 to 2 kg of
aiJha in the case of glufosinate and dimethylphos-
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phinylhydroxyacetic acid). The herbicides are applied
in the form of 0.1 to 1% strength aqueous solutions.
14 to Z8 days later, the treated plants are visually
scored. The test for herbicide resistance was carried
out under conditions which have led to severe damage
(degree of damage > 90%) in commercial maize hybrids
(control plants). The results of the application of
herbicides to regenerant plants are compiled in Table 2.
Tab~e 2:
Herbicidal action of dimethylphosphinyLhydroxyacetic acid
ancl DL glufosinate on regnerant plants derived from
herbicide-resistant callus. Scoring data 4 weeks after
application.
Herbicide Application Damage in %
15 applied rate
(kg ai/ha)
If 2 amounts Control Regenerant
are indicated: plants derived
split applica- from resistant
tion at an callus
interval of 10
days
Dimethyl- 0 + 0 0% 0%
phosphinyl- 0.5 + 1.0 95% 15%
hydroxyacetic 0.75 + 0.75 90% 10%
acid 1.0 + 0 75% 5%
O 0% 0%
D,L-glufosinate 0.75 90% 0%
1.0 98% 10%
6. Heritability of the herbicide resistance observed on
regenerant p~ants
Some of the glufosinate-treated regenerant plants de-
veloped into fertile plants. These were selfed and
1 3 1 0928
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used as the hybrid parent (pollen donator) ;n crossing
experiments using herbicide-sensitive (wild type) geno-
types, for example Karat (cultivar I) or Edo (cultivar
II). The mature seeds were harvested 6 weeks after
fertilization.
The F1 generation was grown in a growth cabinet at a
day temperature of 25C with a 14 h photo period at
30,000 lux, and a night temperature of 20C, at 60%
relative atmospheric humidity. The plants were grown
in standard soil. 14 days after sowing, the plants
were sprayed with gLufosinate-ammonium (~ASTA, Hoechst
AG, commercial formulation) in the 3- to 4-leaf stage.
The application rates tested correspond to 0.75 and
1.5 kg of ai/ha.
Table 3 shows the scoring data 4 weeks after the herbi-
cide treatment.
Table 3: Scoring data of F1 progeny of glufosinate-
resistant maize regenerant pLants
Application rate of herbicide:
0.75 kg of ai/ha 1.5 kg of ai/ha
Genotype Damage (%)
Cultivar I 80% 95%
Cultivar II 90% 100%
Wild type regenerant
plants 95% 100%
F1 selfing progeny of 10 3 plants 85% 2 plants 95%
of glufosinate- tested7 plants 20% 6 plants 40%
tolerant regenerant plants 2 plants 20%
plants in each
case
Cross cultivar I x 6 plants 85% 7 plants 95%
resistant 4 plants 20% 3 plants 30%
regenerant plant
