CA1310928C - Herbicide-resistant crop plants, process for their selection and theirregeneration - Google Patents
Herbicide-resistant crop plants, process for their selection and theirregenerationInfo
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- CA1310928C CA1310928C CA000566573A CA566573A CA1310928C CA 1310928 C CA1310928 C CA 1310928C CA 000566573 A CA000566573 A CA 000566573A CA 566573 A CA566573 A CA 566573A CA 1310928 C CA1310928 C CA 1310928C
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Abstract
Abstract of the disclosure With the aid of callus cultures which can grow on amino acid-free nutrient medium and which are capable of regen-eration, it is possible, on inhibitor-containing nutrient media, to select cell lines which can be regenerated to crop plants and which are resistant to the inhibitor con-cerned for generations. Under these conditions, cell sus-pension cultures also give inhibitor-resistant cell lines.
Description
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.
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.
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 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 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, 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 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 1 3 1 Oq28 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.
- 8 - I 3 1 0 q 2~
Table 1: Nutrient medium modified MS medium (A) modified N6 medium (B) mg/l mg/l CaCl2.2H20440 166 KN03 1,900 2,830 (NH4)2S04 - 433 NH4N3 1,650 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.
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.
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 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 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, 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 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 1 3 1 Oq28 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.
- 8 - I 3 1 0 q 2~
Table 1: Nutrient medium modified MS medium (A) modified N6 medium (B) mg/l mg/l CaCl2.2H20440 166 KN03 1,900 2,830 (NH4)2S04 - 433 NH4N3 1,650 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.
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-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%
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 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
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
Claims (17)
1. A process for the selection of crop plant cell lines which are resistant to amino acid biosynthesis inhibitors by cultivation on an inhibitor-containing nutrient medium, which process comprises a) selecting callus cultures or cell suspension cul-tures which grow on amino acid-free nutrient medium while maintaining their embryogenic and morphogenic competence, b) cultivating these cultures on amino acid-free, inhibitor-containing nutrient medium, and selecting inhibitor-resistant cultures from which intact plants are regenerated.
2. The process as claimed in claim 1, which comprises adding one or more physiological organic acids or salts thereof to the nutrient medium.
3. The process as claimed in claim 2, which comprises adding acids or salts of the citric acid cycle, or peruvic acid to the nutrient medium.
4. The process as claimed in claim 2 or 3, which comprises adding the acids and salts in concentrations of 0.1 to 10 mmol/l.
5. The process as claimed in claim 4, which comprises adding the acids and salts in concentrations of 0.5 to 2 mmol/l.
6. The process as claimed in claim 1, wherein the inhibitor added is a herbicide.
7. The process as claimed in claim 6, wherein the herbicide added is a compound of the general formula I
I
in which independently of one another R1 is hydroxyl or methyl, R2 is hydroxyl, methyl or ethyl, R3 denotes aminomethylene, hydroxymethylene or a carbonyl group, R4 denotes OH or , where x is 1 to 5, and n denotes 0 to 4,
I
in which independently of one another R1 is hydroxyl or methyl, R2 is hydroxyl, methyl or ethyl, R3 denotes aminomethylene, hydroxymethylene or a carbonyl group, R4 denotes OH or , where x is 1 to 5, and n denotes 0 to 4,
8. The process as claimed in claim 7, wherein the herbicide employed is glufosinate (phosphinothricine), bialaphos or dimethylphosphinylhydroxyacetic acid.
9. The process as claimed in claim 1, which comprises adding the inhibitor in concen-trations such that 70 to 99% of the cultures are killed.
10. The process as claimed in claim 9, which comprises adding the inhibitor in concentrations such that 95 to 99% of the cultures are killed.
11. A cell line resistant to an inhibitor of amino acid biosynthesis and capable of being cultured in an amino acid-free medium.
12. The use of a cell line claimed in claim 11 for the regeneration of a crop plant.
13. The use of a cell line as claimed in claim 12 for the breeding of novel, inhibitor-resistant crop plants.
14. The use of a crop plant obtainable by regeneration from a cell line as claimed in claim 11 for the breeding of novel, inhibitor-resistant crop plants.
15. A method for the protection of planted crop plants by treating the crop plants with a herbicide, wherein plants which are not herbicide resistant in a field which is planted with herbicide-resistant crop plants produced from the cell line of claim 11 are selectively destroyed by treatment with a herbicide which inhibits the biosynthesis of amino acids.
16. The method as claimed in claim 15, wherein the herbicide is a compound of the formula I
I
in which independently of one another R1 is hydroxyl or methyl, R2 is hydroxyl, methyl or ethyl, R3 denotes aminomethylene, hydroxymethylene or a carbonyl group, R4 denotes OH or , where x is 1 to 5 and n denotes 0 to 4.
I
in which independently of one another R1 is hydroxyl or methyl, R2 is hydroxyl, methyl or ethyl, R3 denotes aminomethylene, hydroxymethylene or a carbonyl group, R4 denotes OH or , where x is 1 to 5 and n denotes 0 to 4.
17. The method as claimed in claim 15 or 16, wherein the herbicide is glufosinate (phosphinothricine) and bialaphos and dimethylphosphinylhydroxyacetic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19873715958 DE3715958A1 (en) | 1987-05-13 | 1987-05-13 | HERBICIDE-RESISTANT CULTURAL PLANTS, METHOD FOR THEIR SELECTION AND REGENERATION |
| DEP3715958.5 | 1987-05-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1310928C true CA1310928C (en) | 1992-12-01 |
Family
ID=6327445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000566573A Expired - Fee Related CA1310928C (en) | 1987-05-13 | 1988-05-12 | Herbicide-resistant crop plants, process for their selection and theirregeneration |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0290987B1 (en) |
| JP (1) | JPS63304927A (en) |
| CN (1) | CN1026205C (en) |
| AT (1) | ATE114331T1 (en) |
| AU (1) | AU616405B2 (en) |
| CA (1) | CA1310928C (en) |
| DE (2) | DE3715958A1 (en) |
| DK (1) | DK259688A (en) |
| ES (1) | ES2066769T3 (en) |
| FI (1) | FI882203A (en) |
| HU (1) | HU202709B (en) |
| IL (1) | IL86358A (en) |
| NZ (1) | NZ224576A (en) |
| ZA (1) | ZA883345B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA906806B (en) * | 1989-08-31 | 1991-06-26 | Ici Australia Operations | Plants |
| DE4013099A1 (en) * | 1990-04-25 | 1991-10-31 | Hoechst Ag | Transforming immature somatic plant, esp. maize, embryos - by treating, in dry state, with nucleic acid soln., esp. for introducing resistance to phosphinothricin |
| US5767367A (en) * | 1990-06-23 | 1998-06-16 | Hoechst Aktiengesellschaft | Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation |
| DK0469273T3 (en) * | 1990-06-23 | 2004-04-13 | Bayer Cropscience Gmbh | Fertile transgenic maize plants with species promoted gene as well as methods for their preparation |
| US5760021A (en) * | 1992-05-29 | 1998-06-02 | The Procter & Gamble Company | Phosphonocarboxylate compounds pharmaceutical compositions, and methods for treating abnormal calcium and phosphate metabolism |
| CN114891717A (en) * | 2022-06-13 | 2022-08-12 | 上海龙殷生物科技有限公司 | Suspension cell culture medium, fresh and sweet tobacco cells and application |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4443971A (en) * | 1979-10-16 | 1984-04-24 | Cornell Research Foundation, Inc. | Herbicide-tolerant plants |
| ATE100141T1 (en) * | 1984-03-06 | 1994-01-15 | Mgi Pharma Inc | HERBICIDE RESISTANCE IN PLANTS. |
| WO1986002097A1 (en) * | 1984-10-01 | 1986-04-10 | The General Hospital Corporation | Plant cells resistant to herbicidal glutamine synthetase inhibitors |
| EP0242236B2 (en) * | 1986-03-11 | 1996-08-21 | Plant Genetic Systems N.V. | Plant cells resistant to glutamine synthetase inhibitors, made by genetic engineering |
| US4806483A (en) * | 1986-08-18 | 1989-02-21 | Sungene Technologies Corporation | Process for regenerating corn |
| US4843005A (en) * | 1986-08-18 | 1989-06-27 | Sungene Technologies Corporation | Process for regenerating corn |
| US4857465A (en) * | 1986-12-02 | 1989-08-15 | Lubrizol Genetics, Inc. | Whole plant regeneration via organogenesis and somaclonal variation in glycine species |
-
1987
- 1987-05-13 DE DE19873715958 patent/DE3715958A1/en not_active Withdrawn
-
1988
- 1988-05-07 AT AT88107373T patent/ATE114331T1/en not_active IP Right Cessation
- 1988-05-07 DE DE3852146T patent/DE3852146D1/en not_active Expired - Fee Related
- 1988-05-07 ES ES88107373T patent/ES2066769T3/en not_active Expired - Lifetime
- 1988-05-07 EP EP88107373A patent/EP0290987B1/en not_active Expired - Lifetime
- 1988-05-11 ZA ZA883345A patent/ZA883345B/en unknown
- 1988-05-11 HU HU882369A patent/HU202709B/en not_active IP Right Cessation
- 1988-05-11 NZ NZ224576A patent/NZ224576A/en unknown
- 1988-05-11 FI FI882203A patent/FI882203A/en not_active Application Discontinuation
- 1988-05-11 DK DK259688A patent/DK259688A/en not_active Application Discontinuation
- 1988-05-12 CA CA000566573A patent/CA1310928C/en not_active Expired - Fee Related
- 1988-05-12 JP JP63113724A patent/JPS63304927A/en active Pending
- 1988-05-12 IL IL8635888A patent/IL86358A/en unknown
- 1988-05-12 CN CN88102755A patent/CN1026205C/en not_active Expired - Fee Related
- 1988-05-12 AU AU16095/88A patent/AU616405B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP0290987A2 (en) | 1988-11-17 |
| DK259688A (en) | 1988-11-14 |
| NZ224576A (en) | 1990-10-26 |
| CN1026205C (en) | 1994-10-19 |
| CN88102755A (en) | 1988-11-30 |
| ES2066769T3 (en) | 1995-03-16 |
| EP0290987B1 (en) | 1994-11-23 |
| IL86358A (en) | 1994-12-29 |
| FI882203A0 (en) | 1988-05-11 |
| DK259688D0 (en) | 1988-05-11 |
| JPS63304927A (en) | 1988-12-13 |
| FI882203A (en) | 1988-11-14 |
| AU1609588A (en) | 1988-11-17 |
| IL86358A0 (en) | 1988-11-15 |
| HU202709B (en) | 1991-04-29 |
| DE3715958A1 (en) | 1988-11-24 |
| ATE114331T1 (en) | 1994-12-15 |
| EP0290987A3 (en) | 1991-09-04 |
| ZA883345B (en) | 1988-11-09 |
| DE3852146D1 (en) | 1995-01-05 |
| AU616405B2 (en) | 1991-10-31 |
| HUT49977A (en) | 1989-12-28 |
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