APPLICATIONS OF ROOT-ADMINISTERED CHEMICAL HYBRIDIZING AGENTS IN PLANT BREEDING

Abstract

A method of applying a Chemical Hybridization Agent (CHA) to a plant. The method includes providing a CHA source and bringing the CHA source in contact with a root of the plant. The method further includes having the root take up a part of the CHA. The disclosure further relates to methods of producing a haploid and/or doubled-haploid plant or hybrid seed. The disclosed method may be used in various aspects of plant breeding.

Claims

1. A method of applying a CHA to a plant comprising the steps of: (a) Providing a source comprising at least one CHA; (b) Bringing said source in contact with at least one root of a plant; and (c) Having the at least one root take up at least a part of said at least one CHA.

2. A method of inducing male sterility in a plant, comprising carrying out the steps according to claim 1, wherein said step (b) of bringing in contact is effected prior to flowering of said plant

3. A method of producing haploid embryos or plants comprising carrying out the steps according to claim 2, followed by: (d) Fertilizing said plant with an appropriate pollen of a remote plant, thereby generating haploid embryos; and (e) Optionally regenerating haploid plants from the haploid embryos generated in step (d).

4. A method of producing doubled-haploid plants comprising applying the steps according to the method of claim 3, followed by: (f) Treatment of the plants with an agent causing doubling of the chromosome set, such as colchicine or nitrous oxide.

5. A method according to claim 4, which is for producing hybrid seed or a line variety.

6. A method of producing a plant, such as a parent plant for hybrid seed production comprising: (g) Selecting from a pool of doubled-haploid plants produced by the method according to claim 4 a plant suitable as parent of a hybrid variety.

7. A doubled-haploid plant generated by the method according to claim 4.

8. A method of breeding comprising use of the plant obtained by the method of claim 3.

9. A method according to claim 1, wherein the plant is selected from cereals, fruits, vegetables, other crop plants and ornamentals.

10. A method according to claim 1, wherein said plant is a cereal plant.

11. A method according to claim 1, wherein said plant is selected from the group consisting of wheat spp. (Triticum spp.), rye, rice, barley, oats, millet and triticale.

12. A method according to claim 1, wherein said plant is wheat.

13. A method according to claim 1, wherein essentially no other plant part is in contact with said CHA.

14. A method according to claim 1, wherein the amount/concentration of CHA applied ranges between about 1.4 mg/plant and about 216 mg/plant.

15. A method according to claim 1, wherein said source is irrigation water.

16. A method according to claim 1, wherein said plant is cultivated in a substrate or as hydroponic culture.

17. A method according to claim 1, wherein said CHA is selected from clofencet, sintofen, azetidine-3-carboxylic acid, 2-chloroethylphosphonic acid (Ethrel), sodium 2,3-dichloroisobutyrate, triiodobenzoic acid, naphthalene acetic acid, maleic hydrazide, bromoxonil, glyphosate, giberrelic acid, iodosulfuron, flufenacet and nitroarylalkylsulfone derivatives and salts of any of the above.

18. A method according to claim 1, wherein CHA is selected from clofencet, sintofen, azetidine-3-carboxylic acid, sodium 2,3-dichloroisobutyrate, triiodobenzoic acid, naphthalene acetic acid, maleic hydrazide, bromoxonil, iodosulfuron, flufenacet and nitroarylalkylsulfone derivatives and salts of any of the above.

19. A method according to claim 1, wherein CHA is selected from clofencet, sintofen, azetidine-3-carboxylic acid, sodium 2,3-dichloroisobutyrate, triiodobenzoic acid, naphthalene acetic acid, maleic hydrazide, bromoxonil, iodosulfuron and nitroarylalkylsulfone derivatives and salts of any of the above.

20. A method according to claim 1, wherein CHA is selected from clofencet, sintofen or salts thereof.

21. A method according to claim 16, wherein for clofencet or azetidine-3-carboxylic acid when applied in cereals said contact with said CHA is between Zadoks stages 31 and 59.

Description

EXAMPLE SETUP

[0061] Chemical Hybridization Agents (CHA) are synthetic substances which, applied at a precise development stage of a plant, disturb the forming of grains of pollen and thus render the plant male-sterile. The sterilized plant may therefore only be fertilized by the pollen of another plant. Several CHAs have been developed in the course of time. Examples 1 and 2 demonstrate the application of two different CHAs in different doses and ways of treatment of the roots of single plants of two varieties and the effect regarding male sterility. Example 3 demonstrates that the proposed CHA treatment used in Example 2 and the resulting sterility of the plants is an effect of the caused male sterility and has little or no negative effect on female fertility in terms of seed set.

[0062] Methods

[0063] The design of Example 1 and Example 2 is summarized in Table1

Example 1

[0064] Wheat plants of the wheat varieties JB Asano and Apache were grown in the greenhouse in standard planting pots, bottom perforated, edge length 12 cm, height 13 cm, with one plant per pot. Twelve pots were placed into a plastic tray, bottom non-perforated with fleece inlay (e.g. available at Hermann Meyer KG unter article no. 44 54 11). Standard greenhouse growing substrate soil (bedding substrate 1 of Klasmann-Deilmann GmbH) was filled into the pot ca. 2 cm below edge. Plants were grown under standard growing conditions with respect to fertilizer, watering, disease and light management. Each plant was reduced to 4 tillers. The necessary active amount of Clofencet per plant was calculated according to the standard foliar spray application protocol of 5.42 kg per hectare assuming 400 ears per square meter taking the higher tiller number of potted plants into account (average number of ears per plant in the field is typically 1.15 but it was 4 in our potted plants, so the dose rate for root application per plant, i.e. per pot, was adjusted upward). At Zadoks growth stage 31 the plants were watered with 0.0 mg (control), 10.84 mg, 16.26 mg and 21.68 mg, respectively, of Clofencet in 200 ml tap water per pot. Each treatment was repeated on 12 plants, whereas the highest concentration was repeated on 24 plants. Before flowering time (Zadoks stage 57 of control group) ears were bagged to avoid unwanted cross-pollination. Bags were removed at beginning of grain filling (Zadoks stage 75 and higher), seed set was determined at kernel ripeness of the control group. When ears carried mature seeds, the ears were threshed with a hand-threshing device (Baumann Dreschhexe #4.701.200) and the kernels per plant were counted. A wheat ear is called completely sterile (=100% sterility) when no kernel developed on a bagged ear. The average number of grains per plant of the control plants was set to 0% sterility.

TABLE-US-00001 TABLE 1 Experimental design for Examples 1 and 2. Example 1 Example 2 Factors Clofencet WL84811 2-(4-chlorophenyl)-3-ethyl-2,5-dihydro-5- azetidine-3-carboxylic acid oxopyridazine-4-carboxylic acid concentration 1 200 ml water (control) 100 ml water (control) concentration 2 200 ml water + 10.84 mg Clofencet 100 ml water + 1 mg WL84811 concentration 3 200 ml water + 16.26 mg Clofencet concentration 4 200 ml water + 21.68 mg Clofencet Varieties Apache, JB Asano KWS Target, JB Asano Number of 12 2 replications Number of treatments 1 11 (every week one application) per plant Treatment stage Zadok stage 31 Zadok stages 31 to 65 Growing Media Bedding substrate1 Sandy loam Klasmann-Deilmann GmbH

[0065] The results of Example 1 show that a single root application dose of 10.84 mg Clofencet (concentration 2) per plant with four tillers at growth stage 31 already gives a satisfying amount of average male sterility, and 16.26 mg (concentration 3) per plant are enough to ensure male sterility of almost 100%. Almost no plants watered once with an active dose of 16.26 mg of Clofencet or more in Zadoks stage 31 did set any seeds. At the lower concentration 2 a certain effect of the genotype (Apache vs. JB Asano) on average male sterility can be detected, which however is irrelevant at higher concentrations. Data not shown indicate that doses of active substance higher than 21.68 mg per plant do not have any further positive effect.

[0066] Results

TABLE-US-00002 TABLE 2 Results of Example 1 Clofencet Water per Number of Number of Average sterility per plant per plant per treatments per plants treated Treatment relative to Example 1 treatment treatment plant (replications) stage control Variety Apache JB Asano Concentration mg/plant ml Zadok stage [%] [%] 1 (control) 0 200 1 12 31 0 0 2 10.84 200 1 12 31 91.88 94.72 3 16.26 200 1 12 31 98.69 99.96 4 21.68 200 1 24 31 99.64 98.65

Example 2

[0067] Wheat plants of the wheat varieties JB Asano and KWS Target were grown in the greenhouse in standard planting pots, bottom perforated, edge length 12 cm, height 13 cm, with one plant per pot. Twelve pots were placed into a plastic tray, bottom non-perforated with fleece inlay. Sandy loam from the field was filled into the pots ca. 2 cm below edge. Plants were grown under standard growing conditions with respect to fertilizer, watering, disease and light management. Each plant per pot was reduced to 4 tillers. The treatment was watering each pot with 0.0 mg (control) or 1.0 mg WL84811 in 100 ml tap water per pot. Each treatment was repeated on 2 plants per variety, watering with the treatment was done once a week for 11 weeks starting at Zadoks growth stage 31 until growth stage 65 of the control group. Ears were bagged to avoid unwanted cross-pollination. Bags were removed at beginning of grain filling (Zadoks stage 75 and higher), seed set was determined at kernel maturity of the control group. When ears had set seed, the ears were threshed by hand and the kernels per plant were counted. A wheat ear is called completely sterile (=100% sterility) when no kernel was developed on a bagged ear. The average number of grains per plant of the control plants was set to 0% sterility.

[0068] Results

TABLE-US-00003 TABLE 3 Results of Example 2 Total WL84811 per Water per Number of amount Number of Average sterility plant per plant per treatments of plants treated Treatment relative to control Example 2 treatment treatment per plant WL84811 (replications) stage after CHA Variety KWS JB Target Asano Concentration mg/plant ml mg/plant Zadok stage [%] [%] 1 (control) 0.0 100 11 0.0 2 31 to 65 0 0 2 1.0 100 11 11.0 2 31 to 65 100 100

[0069] The result of Example 2 shows, that also a root treatment by regular weekly watering with small doses of 1.0 mg of WL84811 is very effective to generate male sterility. No plant treated with WL84811 and bagged before flowering set any seeds.

Example 3

[0070] Male sterile plants generated by Clofencet treatment as described in Example 1 were used. Before single bagging the 4 ears of each plant at Zadoks stages 55-59, flowers were clipped to facilitate pollination. At flowering time (spikelets wide open) three male-sterile ears (no pollen in the transparent bags) were pollinated with pollen from an untreated control plant of the same wheat variety. One sterile ear was not pollinated to cross check the efficacy of the Clofencet treatment. Bags were removed at beginning of grain filling (Zadoks stage 75 and higher), seed set was determined at kernel maturity. If ears had set seed, the ears were threshed with a hand-threshing device (Baumann Dreschhexe #4.701.200) and the kernels per plant were counted. The average number of grains per plant of the control plants was set to 100% seed set.

[0071] Example 3 shows that the root treatment with Clofencet does not harm the female fertility of the treated plants. Plants treated once with Clofencet and pollinated with pollen of untreated control plants produced seed. Compared to the control with 100% seed set, Clofencet treatment of plants of the variety Apache even seems to enhance seed set.

[0072] Results

TABLE-US-00004 TABLE 4 Results of Example 3 Average seed set of Clofencet treated plants after Clofencet Water per Number of Number of pollination with per plant per plant per treatments plants treated Treatment wheat pollen Example 3 treatment treatment per plant (replications) stage (rel. to control) Variety Apache JB Asano Concentration mg/plant ml Zadok stage [%] [%] 1 (control) 0 200 1 12 31 100 100 2 10.84 200 1 12 31 101 98 3 16.26 200 1 12 31 115 94 4 21.68 200 1 24 31 107 75

DISCUSSION

[0073] Examples 1 and 2 show that CHA applied to plants via root by watering is effective. This method is especially valuable in greenhouse applications, where waste water and soil recycling can be controlled. It prevents the greenhouse staff from aerosol pollution and limits the risk of unwanted exposure.

[0074] Example 1 shows, that even a single root application of CHA can be effective compared to the previously known foliar application. However, this single dose has to be a higher dose per plant compared to foliar application. One reason for this could be that the xylem-mediated transfer of the CHA from root to inflorescence is less effective than the leaf-to-spike phloem transport. The idea to soak the plants with a relatively high amount of solution (200 ml of water for a pot of 1728 ccm) and to retain the water with the diluted CHA within the growing media (soil or substrate), has the effect that the CHA may be successively incorporated into the plant over a period of time, thus making sure to hit the optimal physiological time point for inducing the male sterility. The varieties used did not show clear genotype-specific reactions to the treatments and a tenfold dose of CHA in a single application, i.e. a 10 concentration of CHA in potted plants with root application compared to field plants with spray application, proved to be very effective.

[0075] Example 2 shows that lower concentrations of CHA can be also effective, but they need to be applied over a longer time period.

[0076] Example 3 shows that CHA root treatment of wheat plants with adequate doses does not substantially harm the fertility of the female organs. After CHA-mediated emasculation and subsequent pollination with wheat pollen, the tested wheat varieties set morphologically normal seeds. Kernel numbers per spike were somewhat different between varieties, which confirms previous observations made with foliar CHA application.