IL86358A - Biosynthesis-inhibitor resistant maize, process for its selection and its regeneration - Google Patents

Abstract

Callus cultures which are able to grow on nutrient medium containing no amino acids and are able to regenerate can be used for selection on inhibitor-containing nutrient media of cell lines which can be regenerated to crop plants and are resistant to the relevant inhibitor over generations. Cell suspension cultures likewise result in inhibitor-resistant cell lines under these conditions. [EP0290987A2]

Description

BIOSYNTHESIS-INHIBITOR RESISTANT MAIZE, PROCESS FOR ITS SELECTION AND ITS REGENERATION ,Desc r ipt i on - Herbicide-resistant crop plants, process for their selection 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 177,7383. Initially, however, this was only successful in a few genotypes, for example in inbred lines A188, W64A and Black 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)3. An important requirement for this process is the addition of amino acids to the nutrient medium.

Hibberd et al., teach a method of obtaining resistant cell lines by using a method wherein proline and glycine are included in the selection medium.

Gamborg and Shyluk teach that addition of TCA cycle acids permits growth of plant cells on ammonium as the sole nitrogen source.

Australian Patent 39507 is directed to the production of plants, plant tissues and plant seeds which are resistant to inhibition by an herbicide due to an altered acetohydroxyacid synthase. 86358/3 Cell cultures which can be regenerated into intact plants over a per od of several mont s or years are suitable for the selection of mutants and variant cell lines. If, for example, a toxic dose of a herbicide is added to 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 in ibitors 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, it has been found that cell lines which are derived from crop plants and which are capable of regeneration can be cult ated 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 essential for growth regeneration of the callus cultures or the plants. Such cultures can be employed in the selection of her b i c i de- r es i s t an t cell lines, callus cultures or plants.

SUMMARY OF THE INVENTION The invention thus relates to a process for the selection of maize cell lines which are resistant to amino acid biosynthesis inhibitors by cultivation on an inhibitor -containing nutrient medium, which rocess comprises a) select ng callus cultures or cell suspension cultures wh ch 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 containing acids or salts of the citric acid cycle, or peruvic acid, and selecting inhibitor-resistant cultures from which intact plants are regenerated, where the inhibitor is a herbicide of the general formula I Rx - J» - (CH2)n-RJ-COR* l R2 in which independently of one another R is hydroxyl or meth l, R is hydroxyl, methyl or ethyl, R 3 denotes aminomethylene, hydroxymethylene or a carbon yl group, CH, 0 + R denotes OH or j_ NH _ ^ _ C]xOH" ' where x is 1 to 5, and JJ n denotes 0 to A, Passages which are not in the ambit of the claims do not belong to the invention. The scope of protection is as defined in the claims, and as stipulated in the Patent Law ( 1968) .

In order to be able to select herb c i de- res i s t an t 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 - 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 _^5, 473 (1962)] or modified medium [Chu, C.C. et al., Sci. Sin. J_6, 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 ph siolog cal 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, fo 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 approx mately 0.1 to 10 mmol, in particular 0.5 to 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 35°C, preferably 20 to 30°C, with or without light. The transfer intervals chosen are usually approx. 10 to 30 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-res stant, callus cultures and cell suspension cultures. Using these cultures selection can in principle be carr ed out against all - 4 -inhibitors, but selection is preferably carried out against herbicides which inhibit amino acid biosynthesis. Within this group, in turn, selection is preferably carried out against herbicides of the general formula I in which independentl of one another 1 R is hydroxyl or methyl, ^ is hydroxyl, methyl or ethyl, and R^ denotes am i nome t hy I ene , hydroxymethy lene or a carbonyl group, CH, 0 .

R denotes OH or [- NH - C - C] OH" ' where x can be 1 H to 5, preferably 1 to 2, and n denotes 0 to 4, preferably 0 to 2. Particularly pre-ferred are glufosinate ( phosph not h r i c i n ) or its structural analogs, such as bialaphos, and dimethylphosph inyl-hydroxyacet i c acid. If the inhibitors are optically active, either the racemate of the compounds or the biologically active enantiomer can be used.

In order to obtain higher yields during selection, the selected cell lines can be treated with mutagenic substances by methods known per se. This can be carried out either using chemicals such as, for example, ethyl methane sulfonate or N-me t I -N-n i t rosoguan i d ne (MNG), 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 inhib tor-resistant cell suspension cultures, preferably callus cultures, is carried out on the abovement ioned amino acid-free nutrient media, to which the inhibitor has been added. Its concentrat on in the medium can vary in a wide range and is essentially chosen such that 70 to 99%, preferably 95 to 99%, of the calli die. The selection can be carried out repeatedly using identical or increasing c on c en t r a t i ons ■ of herbicide. The yield of the selection of resistant cells or calli which are capable of regeneration can be increased by adding to the nutrient medium the physiological organic acids or 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 subcultur ng 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 possibl e to select cell suspension cultures and r egene r a t i on-c apable callus cultures which are resistant to two herbic ides 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 monocotyledon crop plants, in particular cereal plants, and particularly preferably to maize plants.

For example, it is possible, with the aid of crosses, to - 6 -produce hybrids which are resistant to one or two herbicides. This is achieved by crossing a herbicide-resistant plant with a non-resistant inbred Line, or by crossing two h e r b c i de- r e s l s t an t plants which have been selected against different herbicides having distinguis able 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 protecting crop plants by selectively killing weeds using a herbicide on fields which are planted with plants obtainable in the process according to the invention and thus resistant to the herbicide. The herbicides preferably employed are substances which inhibit amino acid biosynthesis, in particular compounds having the abovemen t i oned 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 plants, until the weeds are sufficiently suppressed.

The invention is illustrated below with the aid of examples.

Examples 1. Establishing norphogenous maize cell cultures on amino acid-free nutrient medium a) Immature embryos were dissected from immature maize kernels of the inbred lines B73, W6 A 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 - 7 - sterile conditions and cultivated at 250C _+ 2°C in the dark. Suitable callus induction media are A: a modified Murashige and Skoog medium, or B: a modified N 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 ( itamin-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 K0H before autoclaving. The vitamins were filter-sterilized and added to the cooled medium. - 8 - Table 1: Nutrient medium modified MS medium (A) modified N^ medium (B) mg/ L mg/ L MgS0 .7H20 370 250 CaCl2- H20 440 166 NO3 1,900 2,830 (NH4>2S0 433 NH4NO3 1,650 KH2PO4 170 400 The compounds of the MS medium included in the bracket are added to the N0 medium.

Within 2 to 3 weeks, callus cultures which were capable of forming shoot primordia and somatic embryos, were formed.

These 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 nutrient media of this type for more than a year, while maintaining their regeneration 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), ct-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.

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 surviving morphogenic callus segments were subcultured on fresh medium. After a further 2 to 4 weeks, the calli could be used for selection experiments.

Selection of herbicide-resistant callus cultures The herbicide concentration in amino acid-free culture medium 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~ mol/l for both glufosinate and for d i methy Iphosph n I hydroxy ace t i c ac d. - 10 - 10,000 caLli in each case were transferred onto the herbicide-conta ning agar media (0.8%. of agar), and evaluation was carried out after 6 to 8 weeks. The Petri dishes were incubated at 25°C and at 1,000 to 2,000 Lux w th 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 d i me t hy I phos ph i ny I -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.

Regeneration of plants On the regeneration medium (medium A or B without 2,4-di chlorophenoxyacet i c acid or dicamba) and in the course of 3 to 5 weeks, mainly in the course of 3 to 4 weeks, the embryogenic h e rb i c i de- re s i s t a n t calli obtained d fferentiated complete plants. As soon as the leaves were 1 to 3 cm long, the plants were transplanted from the agar into a mineral culture substrate (vermiculite, perlite) and cultured at 90 to 100% relative 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 hyd ropon i c a 11 until a further 2 to 4 leaves are formed. The plants can then be transplanted into soil (sandy loam).

Application of herbicides When the plants have reached the 4- to 5-leaf stage, they are sprayed with herbicide solutions at application rates customary in practice (50 to 200 mg of active substance/m^, corresponding to 0.5 to 2 kg of ai/ha in the case of glufosinate and dimethylphos- - 11 - phinylhydroxyacetic acid). The herbicides are applied in the form of 0.1 to 1% strength aqueous solutions. 14 to 28 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 Table 2: Herbicidal action of d i me t h y I p h os ph i n y I hy d rox y a c e t i c and DL glufosinate on regnerant plants derived from he r b i c i de- r e s i s t an t callus. Scoring data 4 weeks after app I i c at i on .

Herbicide Application Damage in % app I i ed rate (kg a i / ha ) If 2 amounts Control Regenerant are indicated: plants derived split applicafrom resistant tion at an callus interval of 10 days D imethyl- 0 + 0 0% 0% phosphinyl- 0.5 + 1.0 95% 15% hydroxyacetic 0.75 + 0.75 90% 10% acid 1.0 + 0 75% 5% 0 0% 0% D,L-gluf os inate 0.75 90% 0% 1.0 98% 10% 6. Heritability of the herbicide resistance observed on regenerant plants Some of the gluf os nate-treated regenerant plants de- ve I oped into fertile plants. These were selfed and - 12 - used as the hybrid parent (pollen donator) in crossing experiments using herbicide-sensitive (wild type) genotypes, for example Karat (cultivar I) or Edo (cultivar II). The mature seeds were harvested 6 weeks after fertilization.

The F-| generation was grown in a growth cabinet at a day temperature of 25°C with a 14 h photo period at 30,000 lux, and a night temperature of 20°C, at 60% relative atmospheric humidity. The plants were grown in standard soil. 14 days after sowing, the plants were sprayed with g I uf os i na t e-ammon i urn (®BASTA, 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 herbicide treatment.

Table 3: Scoring data of F-| 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% Fi selfing progeny of 10 3 plants 85% 2 plants 95% of glufosinate- tested 7 plants 20% 6 plants 40% tolerant regenerant plants 2 plants 20% plants in each case C ross cultivar I x 6 plants 85% 7 plants 95% res istant 4 plants 20% 3 plants 30% regenerant plant

Claims (14)

- 13 - 86358/2 Patent Claims :

1. A process for the selection of maize cell lines which are resistant to amino acid b osynthesis inhibitors by cultivation on an nhibitor-containing nutrient medium, which process comprises a) selecting callus cultures or cell suspension cultures which grow on amino ac id-free nutrient medium while maintaining their embryogenic and morphogenic competence, b) cultivating these cultures on amino acid-free inhibitor-containing nutrient medium containing acids or salts of the citric acid cycle, or peruvic acid, and selectinq inhibitor-resistant cultures from which intact plants are regenerated, where the inhibitor, is a herbicide of the gen in which independentl of one another R1 is hydroxyl or methyl, R2 is hydroxyl, methyl or ethyl, R3 denotes aminomethylene, hydroxymethylene or carbonyl group, denotes . OH or [_ NH - - C]XOH" ' where x 1 to 5, and H ή denotes 0 to 4,

2. The process as claimed in claim 1, which comDrises addinq one or more physiological organic acids or salts thereof to the nutrient medium.

3. The process as claimed in claim 2, which comprises adding the acids and salts in concentrations of 0.1 to 10 mmol/1 - 14 -

4. The process as claimed in claim 3, which comprises adding the acids and salts in comcentrations of 0.5 to 2 mmol/l.

5. The process as claimed in claim 4, wherein the herbicide employed is glufos inate ( phos p h i no t h r i c i ne ) , bialaphos or d i me t hy pho s ph i ny I hyd r ox y a c e t i c acid.

6. The process as claimed in one or more of any of claims 1 to 5, which comprises adding the inhibitor in concentrations such that 70 to 99% of the cultures are killed.

7. The process as claimed in claim 6, which compris es adding the inhibitor in concentrat ons such that 95 to 99% of the cultures are killed.

8. An inhibitor-resistant cell line capable of regeneration, whenever obtained by cultivation by the process as claimed in one or more of claims 1 to 7.

9. Art inhibitor-resistant crop plant, whenever obtained by regeneration from a cell line as claimed in claim 8.

10. An inhibitor-resistant multiplication stock whenever obtained from the regenerant plant from a cell line as claimed in claim 8. - 15 -

11. The use of a crop plant as claimed in claim 9. for«the breeding of novel, i nh i b i t o r- r e s i s t an t crop plants » substantially as described in the specification.

12. A method for the protect ion of planted crop plants by treating the f ield with a herbicide, wherein the weed flora in a f ield which is planted with herbic ide- res istant crop plants as claimed in claim 9 are selectively destroyed by treatment wi th a herbicide which inhibits the biosynthes is of amino ac ids .

13. The method as claimed in claim 12, wherein the herbic ide is a compound of the formula I 0 1 I "I (CH2)n-RJ-COR- in which independently of one another 1 R is hydroxyl or methyl, R is hydroxyl, methyl or ethyl, R^ denotes aminomethylene, hydroxymethylene or a carbonyl group, CH, O . L I II R denotes OH or [- NH - C - C] ΟΥ ' where x s 1 to 5 and denotes 0 to 4.

14. The method as claimed in claim 12 or 13, wherein the herbic ide is glufosinate ( phosph no t h r c ne ) and/or bialaphos and/or dimethylphosph iny I hydroxyacet ic ac id, COHEN ZEDE & RAPAPORT By)