DE3825492A1 - METHOD FOR PRODUCING HYBRID SEEDS - Google Patents

Abstract

A method of hybrid seed production comprises cross pollinating male and female parent plants either a) the male plant being resistant to a phytotoxic chemical or b) each parent being resistant to a different phytotoxic chemical, the resistance being attributable solely to a homozygous dominant nuclear marker gene. In case (a) seed from the female plants only and in case (b) all seed is harvested. The harvested seed is dosed with the chemical(s) before planting or after seedling emergence therefrom. The chemical may be a herbicide or an antibiotic e.g. Kanamycin. The marker gene may be introduced by Agrobacterium-mediated transfer or direct DNA uptake or selected in a sematic cell culture transfer protoplast fusion or after inducing plant mutagenesis.

Description

This invention relates to an improved method of manufacture of hybrid seeds. In particular, it concerns the use one resistant to a phytotoxic chemical Genes in a male parent in a hybridization procedure, followed by the treatment of the resulting hybrid with a phytotoxic chemical, to eliminate plants from unwanted ones contaminating seeds.

For the commercial production of hybrid seeds, the male and female parents generally in planted an alternating pattern and the seed is just harvested by the female parent. A major problem with the  Manufacturing hybrid seeds is to ensure that the whole pollen that fertilizes the female parent from male parent. Seeds coming from each other Source of pollen will contaminate the hybrid seed population. Possible sources of contaminating pollen are Pollen from the female parent, causing self-pollination leads, or pollen from a neighboring field.

There are 6 ways to get fertile pollen from the female To exclude parent (Simmonds, Principle of Crop Improvement, 1979):

• 1. mechanical (e.g. tomato) - emasculation of the female Parent by hand, followed by pollination;
• 2. Separate sex (e.g. spinach) - Remove of the male plants of the genotype from which Field crop seeds with separated male and female parents are harvested;
• 3. Self-incompatibility (e.g. cabbage) - planting out of two self-incompatible but cross-compatible Parents and crops of all seeds, or Use of a self-incompatible genotype as female parent with a self-compatible Pollinating and harvesting the seed only from the female Parent.
• 4. Male core sterility (lots of fruits) - separate of the female parent for male sterility (ms ms) and fertility (ms ms); Remove of fertile male plants and pollination of sterile male plants with pollen from the male parent (Ms Ms),  
• 5. cytoplasmic male sterility (e.g. onions) - Use of sterile male lines as a female parent, and
• 6. Chemicals:
  • (a) male gametocids (e.g. wheat) - spraying the female parent with a chemical that causes male sterility;
  • (b) Sex reversal (e.g. pumpkin family) - spraying female parents with plant hormones to convert male flowers back into female flowers.

In connection with such procedures, however, there are several problems:

• (1) The physical separation of female and male Parents on alternating groups (Blocks) in the field make efficient pollen transfer difficult from male to female parents and can not produce to a maximum lead from hybrid seeds.
• 2. None of these procedures can guarantee the absolute exclusion of fertile pollen from the female parent (and thus the self-pollination of the female parent). There are two components:
  • (a) Human errors can occur during manual emasculation and removal of fertile male plants when the segregated sex and male core sterility are used. In addition, the work associated with these practices is very expensive.
  • (b) When chemical sprays are used to control gender expression, it is difficult to get a uniform application to completely eliminate female parent pollen production. In addition, there is often instability in the expression of the genes of self-incompatibility and male sterility. For example, it is known that elevated temperatures or high humidity reduce the self-incompatibility, which in particular leads to a high proportion of self-pollinated seeds in cabbage (Frankel and Galun, Pollination Mechanisms, Reproduction and Plant Breeding [1977]). This has led to the release of a number of "harsh hybrids" that contain many self-pollinated plants (Simmonds). Restoring fertility in certain male sterile lines of onions has made it uneconomical to produce hybrid seeds from otherwise excellent crosses (Grant, Onions, Plant Breeding in New Zealand [1983]).
• (3) When attempting contamination by someone else To avoid pollen from a neighboring field Hybrid seed groups raised in isolation plots. Recommended insulation distances vary from Field crop to field crop depending on the Way of pollination and can of 200 m for sorghum, corn and wheat up to 6.4 km for sunflowers  range (Wright, Commercial Hybrid Seed Production, Hybridization of Crop Plants [1980]). It Attempts are made to identify all sources of contaminants Pollen within these distances remove.
• A solution to the above problem (1) has been found in U.S. Patent Nos. 45 17 763, 46 58 084 and 46 58 085 (all by Beversdorf et al.).
• All involve making a combination of male sterility inherited from cytoplasm and either cytoplasmic herbicide resistance (45 17 763) or homozygosity for a dominant core-inherited herbicide-resistant gene (45 58 084) in the same female parent. The The concept allows the arbitrary mixing of two Parents to hybrid seed production, which the pollen transfer from the male to the female Parent is supported.
• If the female parent is male-sterile and herbicide-resistant and the male parent is male-fertile and herbicide-sensitive, then the seed made from such plants can be of two types:
  • (1) Real hybrid seeds from the female parent (herbicide resistant); and
  • (2) Non-hybrid seeds by self-pollination of the male parent.

The non-hybrid seeds resulting from the self-pollination of the male parent can be sprayed with the herbicide, either after pollination but before harvesting the seeds (causing the male parent is destroyed) or after sowing the seed of the main harvest (resulting in the non-hybrid seedlings after germination be destroyed). This source of non-hybrid seeds arises not in the standard production of hybrid seeds because in the standard production of hybrid seeds, the female ones and male parents in alternating groups on the Field to be physically separated, and seeds only from the female Groups (blocks) is harvested. Hence the Production of a non-hybrid seed fraction due to the mixed Sowing the two parents on these patents inherent component.

U.S. Patent Nos. 46 58 084 and 46 58 085 also disclose the use of resistance to two different ones Herbicides are claimed to mix the arbitrary plants of the cytoplasmic male-sterile female parent, his line of maintenance and the male parent to allow. Plants from the maintenance line and the non-hybrid Seeds from the self-pollination of the male parent are sensitive to different Herbicides eliminated.

A possible solution to problem 2 (a) above was given by Wiebe provided (A proposal for Hybrid Barley, Agronomy Journal [1960]).

Wiebe interprets the possible finding of a close coupling between the male fertility gene and susceptibility towards a phytotoxic substance (DDT).  

This allows the early identification of fertile males Plants (msms) in populations of female Parents looking for male fertility (msms) and male sterility (msms) when using male Separate core sterility. Such plants can therefore the population of the female parent before flowering and the possible release of pollen, which leads to self-fertilization of the female parent will.

An identical proposal was made by the European patent application No. 86 104 213.3 (Advanced Genetic Sciences Inc). However, this approach involves the arbitrary Introduction of marker genes using transformation into the genome of fertile male plants (MsMs or Msms), and many independently transformed Plants are genetically analyzed to have an individual with them close bond between the marker gene and the location of the male To find fertility. Special weight is put on the use of suicide genes, resulting in sensitivity leads to certain chemicals. The most fertile Plants can then be replaced by a simple chemical Spraying at the seedling stage can be eliminated

However, the four patents described above still all have the problems associated with contamination by strangers Pollen and contamination through self-pollination of the female parent, caused by a breakdown in male sterility (above problems 2 (b) and 3).

It is an object of this invention, both of which are described above To overcome disadvantages of the prior art.  

These and other objects are achieved by the present invention solved by using a manufacturing process an essentially pure F₁ hybrid population of Provides plants, the method including:

• (a) planting alternate plots Of male and female parent plants, being the plants of the male Parent resistant to a phytotoxic Chemical and resistance are only one attributed to homozygous dominant core marker gene is what the plants of the female parent do not have:
• (b) fertilizing the female plants with pollen of male plants;
• (c) harvesting fertilized seed from the plots only (Places) of the female plants; and
• (d) treating those fertilized in step (c) Seeds with the phytotoxic chemical.

Step (d) can either be before planting or after growth of the seedlings are carried out. The phytotoxic Chemical consequently eliminates plants from self-pollination of the female parent or from external pollen sources originate and thereby leads to an essentially homogeneous F₁ hybrid population.

The above procedure can be used for plant species where both parents are self-compatible or where a parent is only self-incompatible.  

Preferably the phytotoxic chemical is a herbicide of any kind. The phytotoxic chemical can still be a Antibiotic, especially kanamycin (an antibiotic Complex produced by Streptomyces kanamyceticus).

The present invention further provides a method for Production of an essentially pure F 1 hybrid population of plants available in which both parents are self-incompatible, the process including:

• (a) planting alternate plots (Digits) or an arbitrary mix of first and second parent plants, the first parent plants resistant to one phytotoxic chemical A and the second Parent plants resistant to a phytotoxic Chemical B are and the resistance towards the phytotoxic chemicals A or B one homozygous dominant in each plant Core marker gene can be attributed to and being the gene resistant to chemical A in the second parent plants are missing and against the chemical B resistant gene at the first Parent plants missing;
• (b) fertilizing the first parent plants with Pollen from the second plants and fertilization the second parent plants with pollen from the first parent plants;
• (c) harvesting the fertilized seeds from the first Parent plants and the second parent plants; and  
• (d) treating the fertilized seeds contained in Step (c) were harvested with both phytotoxic Chemicals A and B.

Step (d) can either be before planting or after growth the seedlings are done to eliminate plants, the self-pollination of either the first or second parent plant or from foreign pollen sources come. This makes an essentially homogeneous F 1 hybrid population reached.

In particular, the phytotoxic chemicals A and B Herbicides of all kinds.

The phytotoxic chemicals can continue antibiotics be. A preferred antibiotic is kanamycin.

In particular, core marker genes are created using a plant transformation technique introduced.

A preferred plant transformation technique is the transfer with the help of agrobacterium.

Some aspects of transfer using Agrobacterium from Genes in plants are described in the European patent application No. 1 16 718 (P. Zambryski); as well as in European Patent application No. 84 116 036.9 (Plant Genetic Systems N.V., page 3, line 33); or in European Patent Application No. 86 104 213.3 (column 16, line 37) cited.

The plant transformation technique can continue Transformation by direct DNA uptake (into the protoplasm or cells, plant tissue or flowers).  

Another possible method is the core marker gene selected in a somatic cell culture. The patents by Beversdorf et al. concern such a procedure. At The Kernmaker gene is yet another possible method selected for a protoplast fusion transfer. To Another possible method is the core marker gene selected after inducing plant mutagenesis. The Nuclear marker gene can also be present by selecting one male or first or second parent plant that has the desired gene.

The present invention further provides a method for Testing the purity of F₁ hybrid populations of plants available where both parents are self-compatible or a parent is self-incompatible, using the procedure includes: performing steps (a) through (c) the first method described above; the planting a small amount of these seeds as a sample; treating the seedlings after outgrowth with the phytotoxic chemical; and determining the percentage of seedlings that are resistant to the phytotoxic chemical.

The present invention further provides a method for Testing the purity of F₁ hybrid populations of plants available in which both parents are self-incompatible which method includes: performing steps (a) to (c) of the second method described above; planting a small amount of seeds as a sample; treating the seedlings after growth with the phytotoxic chemicals A and B; and determining the Percentage of seedlings against the phytotoxic Chemicals A and B are resistant.

Further features of the invention result from FIGS. 1 and 2 and from the following description of preferred embodiments in conjunction with the subclaims.

The individual features can be used individually or individually to several in combination in one embodiment of the Invention to be realized.

The invention can be understood more fully under Reference to the accompanying drawings:

Fig. 1 is the representation of the inheritance of resistance to a phytotoxic chemical with respect to the production of hybrid seeds in the form of a diagram, according to a first preferred embodiment of the invention; and

Figure 2 is a graphical representation of the use of resistance to phytotoxic chemicals to produce hybrid seeds in accordance with a second preferred embodiment of the invention.

. With respect to Figure 1 (RR) is homozygous resistance; (Rr) means heterozygous resistance; (rr) means homozygous sensitivity.

The present invention comprises according to a first preferred one Embodiment the introduction of a dominant marker gene, what resistance to a phytotoxic Chemical transfers into the male parent of a hybrid Culture breed (RR). Both parents are self-compatible or only one parent is self-incompatible. Around That should maximize the efficiency of the process male parent homozygous for such a gene. In this embodiment, all the real hybrid seed, which is harvested by the female parent (rr), for the dominant marker gene heterozygous and against the corresponding one phytotoxic chemical. If the  Hybrid seed crop at the seedling stage with the appropriate Chemical is sprayed, only the seedlings that are made the real hybrid seeds (Rr) stem, survive, and all Seedlings that come from contaminating pollen be eliminated.

Real hybrid seed (Rr) production is illustrated by the route illustrated in (A) of FIG. 1. The seed produced by contaminating pollen (rr) is illustrated in (B) of FIG. 1. Usually the hybrid seed is predominantly of real F₁ origin, with fluctuating proportions of contaminating seeds. Contaminating seeds can be eliminated based on sensitivity to a phytotoxic chemical.

After harvesting seeds from the female parent in one Hybrid seed block, can be a small seed sample (e.g. 1000 seeds) treated with the appropriate chemical, or the semen sample can be germinated and the seedlings with the appropriate Chemicals are sprayed to the percentage of To determine contamination. Then you can make recommendations for that required increase in sowing rates to be made Proportion of contaminating seedlings that is subsequently eliminated will be taken into account.

Referring to Fig. 2, in a second preferred embodiment of the invention, the self-incompatibility in both parents is used for hybrid seed production and the seed is harvested from both parents. Each parent must therefore be homozygous for different chemical resistance markers. The hybrid seedlings must then be sprayed with both appropriate chemicals to eliminate seedlings that come from contaminating pollen.

The first parent (AAbb) is opposite chemical A resistant, the second parent (aaBB) is against the chemical B resistant. Pure hybrid seeds (AaBb) are compared be resistant to both chemicals A and B. Non-hybrid Seeds that come from contaminating pollen (Fig or aaBb) only become resistant to chemical A or carry chemical B.

Since the methods of this invention include the step of Elimination of non-hybrid seeds during production of hybrid seeds, far less exists Need parcels (places) of parental plants isolate from contaminating pollen sources. These Methods allow the production of hybrid seeds to make it more efficient. Furthermore, many can be excellent female parents, their use for commercial Production of hybrid seeds because of irregular It has not been possible to regress to male fertility was to be used now.

Provided that germplasm sources are available conventional plant breeding methods are used, for resistance to phytotoxic chemicals in male Convict parents or hybrids. Resistance genes can take advantage of recent advances in plant cell genetics directly into specific plant genotypes be added. The applications of somatic cell selection, Protoplast fusion and transformation are genetic Manipulation of plants for resistance to certain Chemicals allowed (Conner and Meredith, Genetic Manipulation of Plant Cells, The Biochemistry of Plants. A comprehensive treatise Vol. 15, Molecular Biology [1988]).

In most examples, such techniques lead to individual Plants that are heterozygous for resistance and that need to be pollinated. Then the offspring have to  be tested to produce homozygotes. Although genes for resistance to all phytotoxic chemicals can be used is the most useful method Genes for resistance to herbicides. With Offers the help of Agrobacterium transformation a particularly advantageous method since several may be useful genes have been cloned that are resistant to certain herbicides are transmitted when in plants have been integrated.

The production of herbicide resistant plants transformation can induce resistance to glyphosate (Shah et al., Engineering Herbicide Tolerance in Transgenic Plants, Science Vol. 233 [1986]; Comai et al., Nature, Vol. 317 [1985]; U.S. Patent No. 4,535,060 [Comai]; Fillatti et al., Bio / Technology, Vol. 5 [1987]) or chlorosulfuron resistance (Haughn et al., Molecular and General Genetics, Vol. 211, [1988]); or phosphinothriun / bialaphos resistance (De Block, The EMBO Journal, Vol. 6 [1987]).

Example 1

The use of dominant marker genes with resistance to a phytotoxic chemical to bypass the Foreign pollen contamination during manufacture hybrid seeds are grown in Nicotiana plumbaginifolia plants, which are resistant to kanamycin.

A. Testing for kanamycin resistance

Kanamycin resistance can be convenient in N. plumbaginifolia by checking for seedlings with green cotyledons (kanamycin-resistant) compared to those with white cotyledons (kanamycin sensitive) can be studied in the presence of Kanamycin grow. The seeds were grown in 1 mM overnight  Gibberellic acid soaked, with 3% sodium hypochlorite Surface sterilized for 5 minutes and thoroughly sterile, distilled water. You were then on 1/2 MS salts (Murashige and Skoog, A Revised Medium for Rapid Growth and Bioassays With Tobacco Tissue Culture, Physiologia Plantarum, Vol. 15 [1962]) plus 0.8% sown agar, supplemented with 300 mg / L kanamycin.

The culture medium was sterilized in the autoclave at 121 kPa for 20 minutes and the filter-sterilized kanamycin was added after the autoclave process. The germinating seedlings were incubated at 26 ° C. under cold white fluorescent light (100 μmol × m ^-2 × sec ^-1 ; 16 h light; 8 h dark daily). Green versus white seedlings can be checked after 7 to 10 days.

Using this approach, a single one green, kanamycin-resistant seedling of N. plumbaginifolia among thousands of white, kanamycin sensitive seedlings observed. This seedling was made into a kanamycin-free Medium transferred. After 8 weeks it was in earth transferred. Controlled pollinations were used during flowering carried out and the resulting progeny for kanamycin resistance checked.

The originally isolated seedling (NpKR) was on one Site heterozygous for spontaneous mutation to kanamycin resistance. This is from the self-pollinated and back-crossed Progeny clearly visible, which are in proportion of 3: 1 and 1: 1 in kanamycin-resistant and kanamycin-sensitive Separate seedlings (Table 1). From the self-pollinated offspring became a single plant (NpKR B) identified that produced genuine kanamycin resistance (Table 1). This plant was undoubtedly on a single site homozygous for kanamycin resistance.  

Table 1

Inheritance of kanamycin resistance in a wild kanamycin-resistant mutant of Nicotiana plumbaginifolia (Np KR). Seedling progeny was checked for resistance to 300 mg / L kanamycin

B. Production of hybrid seeds

The production of hybrid seeds was carried out using a N. plumbaginifolia plant of wild type than the female Parent and NpKR B (homozygous for kanamycin resistance) simulated as the male parent. Three levels of Control over contamination by foreign pollen was made by pollinating the emasculated female parent manufactured with:

• 1. Pollen only from NpKR B (strict control),
• 2. Pollen primarily from NpKR B, with a small one Percentage from a wild-type plant (less strict control), and
• 3. Considerable pollen fractions from both NpKR B and a wild-type plant (loose control).

When NpKR B is used to produce flowers from wild-type plants these different levels of pollination control pollinate the green, kanamycin-resistant seedlings, the real F₁ hybrids are, without further ado, the white ones Kanamycin-sensitive seedlings, which come from the "foreign" wild-type pollen are differentiated (Table 2). As expected the percentage of contaminating seeds increases at when control of the foreign pollen is relaxed becomes. This experiment shows the principle of elimination of non-hybrid seeds when there is a risk of pollen contamination during the production of hybrid seeds.

Example 2

The use of dominant marker genes with resistance to a phytotoxic chemical to bypass the Self-pollination in the female parent during manufacture hybrid seeds is produced by Nicotiana plumbaginifolia plants illustrates which resistance to Kanamycin are genetically engineered.

Table 2 Using kanamycin resistance to eliminate seeds, which come from contaminating pollen during the Production of hybrid seeds in Nicotiana plumbaginifolia. Female parent = kanamyin sensitive plant from Wild type with different levels of self-pollination. Male parent = NpKR B (homozygous for kanamycin resistance). Seedling offspring for resistance to 300 mg / L Kanamycin checked

A. Transformation

A binary vector system, the Agrobacterium tumefaciens (Race LBA 4404, Hoekema et al., A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Tiplasmid, Nature, 303, pages 179-180 [1983]) and harbors the plasmid pKIWI-6, was used for transformation. This plasmid contains between the right and left edges of the T-DNA, two chimeric genes, which are suitable expressed in plant cells to become. One transmits resistance to the antibiotic Kanamycin and consists of the code region of the neomycin phosphotransferase II gene (from the bacterial transposon Tn5) under the control of the octopine synthase promoter and Poly-A signal (OCS-NPTII-OCS). The other gene transmits Chloramphenicol acetyltransferase (CAT) activity and exists from the code region of CAT (from the bacterial transposon Tn9) under the control of the mannopine synthase promoter and the octopine synthase poly A signal.  

Leaf parts from in vitro plants of N. plumbaginifolia were immersed in a suspension of A. tumefaciens (one Overnight culture in MG / L broth -Garfinkel and Nests, Agrobacterium tumefaciens Affected in Crown Gall Tumorigenesis and Optopine Metabolism, Journal of Bacteriology, Vol. 144 [1980]), patted dry and on RMOP medium cultivated (Sidorov et al, Isoleucine-requiring Nicotiana Plant Deficient in Threonine Deaminase, Nature, Vol. 294, [1981]). After 2 days, the leaf segments were the same Medium transferred, which with 500 mg / L cefotaxime (around the To prevent overgrowth of Agrobacterium) and 300 mg / L Kanamycin (to select for transformed plant cells) was added. Regenerated kanamycin-resistant germs on MS salts (Murashige & Skoog, 1962) plus 3% Sucrose and 0.8% agar take root and were then placed in soil transferred. Controlled pollinations were used during flowering carried out and the resulting offspring was on Kanamycin resistance checked as described in Example 1 is.

B. Testing for kanamycin resistance

The inheritance of kanamycin resistance can be found in N. plumbaginifolia 7-10 days after sowing on a medium containing 300 mg / L Kanamycin contains can be studied comfortably by checking on Seedlings with green cotyledons (kanamycin resistant) against those with white cotyledons (kanamycin sensitive). The Inheritance has been studied in ten transformed plants; here is only about results from one of these plants (NpT 17) reported.

The originally transformed plant of NpT 17 was heterozygous for insertion into a single site of kanamycin-resistant Genes. This is from the self-pollinated and backcrossed offspring clearly visible  in a ratio of 3: 1 or 1: 1 for kanamycin-resistant and divides sensitive seedlings (Table 1). From the self-pollinated Progeny became a single plant (NpT 17D) identified which genuine kanamycin resistance spawned (Table 3). Obviously, this plant was homozygous in a single location for kanamycin resistance.

NpT 17D was crossed back to a large in the wild type To produce population of seedlings (over 5000) which heterozygous for inserted kanamycin-resistant genes was. Any genetic instability of kanamycin resistance would be caused by the appearance of rare white, kanamycin sensitive Seedlings recognized within such a population will. All seedlings were resistant to kanamycin (Table 3), which shows high genetic stability. That's why are genes with resistance to phytotoxic chemicals, which in plants by means of transformation via Agrobacterium were introduced to be sufficiently stable to be considered genetic Markers to be used that indicate the purity of the seeds of crop crops.

Table 3

Inheritance of kanamycin resistance in a transformed Nicotiana plumbaginifolia plant (NpT 17). The seedling progeny was checked for resistance to 300 mg / L kanamycin

C. Hybrid Seed Production

The production of hybrid seeds was carried out using a N. plumbaginifolia plant of wild type than the female Parent and NpT 17D (homozygous for kanamycin resistance) simulated as the male parent. Different levels control of female pollen contamination were created by emasculating the female parent in different ways Stages of development of flowering established. N. plumbaginifolia is self-pollinating and pollinating the flowers yourself just before opening the flower crown and the development of the pigmentation of the corolla. Three levels control of selfing (self-fertilization) of the female parent were executed.

• 1. strict control was exercised by Emasculation of 2-3 cm long flower buds, sufficient long before the pollen is dropped,  
• 2. less stringent control was exercised by Emasculation of a 4-5 cm long flower, straight to the time when the maximum length of the flower crown is reached and when the pollen begins to be thrown off to become, and
• 3. loose control was exercised through "emasculation" after the development of the pigment of the flower crown, exactly when the flowers open.

At this stage it has considerable self-pollination took place.

When NpT 17D is used to make flowers of wild-type plants with these different levels of pollination control can pollinate green, kanamycin-resistant seedlings, which are real F₁ hybrids, light from the white ones, Kanamycin-sensitive seedlings resulting from self-pollination of the female parent (Table 4). As expected, the percentage of contaminants increases Seeds when in control of the female Self-fertilization is loosened. This experiment shows the principle of eliminating non-hybrid seeds, if the risk of pollen contamination through self-pollination of the female parent during the manufacture of hybrid seeds exist.

Seeds of NpKR B (Example 1) and NpT 17D (Example 2) were deposited and are available at the Crop Germplasm Resource Center, Crop Research Division, DSIR, Private Bag, Christchurch, New Zealand. They are on request from the curator available in the collection.  

Table 4 Using kanamycin resistance to eliminate seeds, from the self-pollination of the female parent in Nicotiana plumbaginifolia Female parent = kanamycin sensitive plant from Wild type with different levels of self-pollination. Male parent = NpT 17D (homozygous for kanamycin resistance). Seedling offspring for resistance to 300 mg / L Kanamycin checked

Claims (26)

1. A process for producing a substantially pure F₁ hybrid population of plants, which process includes:

• (a) planting alternate plots of male and female parent plants;
• (b) fertilizing the female plants with pollen from the male plants;
• (c) harvesting fertilized seeds only from the plots of the female plants,

characterized in that

• - the male parent plants (RR) are resistant to a phytotoxic chemical, the resistance alone being attributable to a homozygous dominant core marker gene and the resistance gene not being present in the female parent plants (rr);
• and that the process comprises a further step,
• (d) treating the harvested seeds (rr and Rr) with the phytotoxic chemical to produce true F₁ hybrid plants (Rr).

2. The method according to claim 1, characterized in that both parents are self-compatible. 3. The method according to claim 1, characterized in that a parent is only self-incompatible. 4. The method according to any one of the preceding claims, characterized characterized that the phytotoxic chemical is a herbicide. 5. The method according to any one of claims 1 to 3, characterized in that the phytotoxic chemical is an antibiotic. 6. The method according to claim 5, characterized in that the antibiotic is kanamycin. 7. A method of producing an essentially pure F₁ hybrid population of plants in which both parents are self-incompatible, which method includes:

• (a) planting either alternating plots or an arbitrary mix of first and second parent plants;
• (b) fertilizing the first parent plants with pollen from the second plants and fertilizing the second parent plants with pollen from the first parent plants;
• (c) harvesting fertilized seeds from the first parent plants and the second parent plants,

characterized in that

• the first parent plants (AAbb) are resistant to a phytotoxic chemical A and the second parent plants (aaBB) are resistant to a phytotoxic chemical B, the resistance to the phytotoxic chemicals A or B in each plant alone being a homozygous dominant marker Gene is attributable and the gene resistant to chemical A is not present in the second parent plants (aaBB) and the gene resistant to chemical B is not present in the first parent plants (AAbb),
• - and that the process contains a further step,
• (d) treating the harvested seeds (Aabb, aaBb, AaBb) with both phytotoxic chemicals A and B to produce pure F1 hybrid plants (AaBb).

8. The method according to claim 7, characterized in that the phytotoxic chemicals are herbicides. 9. The method according to claim 7, characterized in that the phytotoxic chemicals are antibiotics. 10. The method according to claim 9, characterized in that the antibiotic is kanamycin. 11. The method according to any one of the preceding claims, characterized characterized in that step (d) before planting the seed is carried out.   12. The method according to any one of claims 1 to 10, characterized characterized in that step (d) after the growth of the Seedlings is carried out from the seeds. 13. A method for testing the purity of F₁ hybrid population of plants, the method containing:

• (a) planting alternate plots of male and female plants;
• (b) fertilizing the female plants with pollen from the male plants;
• (c) harvesting fertilized seeds only from the plots of the female plants;
• (d) planting a small amount of seeds as a sample,

characterized in that

• - the male parent plants (RR) are resistant to a phytotoxic chemical, the resistance alone being attributable to a homozygous dominant core marker gene and the resistance gene not being present in the female parent plants (rr);
• - and that this procedure contains further steps,
• (e) treating the seedlings (rr and rR) after outgrowth with the phytotoxic chemical; and
• (f) determining the percentage of seedlings (Rr) that are resistant to the phytotoxic chemical.

14. A method for testing the purity of F₁ hybrid populations of plants in which both parents are self-incompatible, which method includes:

• (a) planting either alternating plots or an arbitrary mix of first and second parent plants;
• (b) fertilizing the first parent plants with pollen from the second plants and fertilizing the second parent plants with pollen from the first parent plants;
• (c) harvesting fertilized seeds from the first parent plants and the second parent plants;
• (d) planting a small amount of these seeds as a sample;

characterized in that

• the first parent plants (AAbb) are resistant to a phytotoxic chemical A and the second parent plants (aaBB) are resistant to a phytotoxic chemical B, the resistance to the phytotoxic chemicals A or B in each plant alone being a homozygous dominant marker Gene is attributable and the chemical A-resistant gene is not present in the second parent plants (aaBB) and the chemical B-resistant gene is not present in the first parent plants (AAbb);
• - and that the process contains further steps,
• (e) treating the seedlings (Aabb, AaBb, aaBb) after growth with phytotoxic chemicals A and B; and
• (f) determining the percentage of seedlings (AaBb) that are resistant to the phytotoxic chemicals A and B.

15. The method according to any one of the preceding claims, characterized characterized in that the core marker gene by a Plant transformation technique is introduced.   16. The method according to claim 15, characterized in that the plant transformation technique a transfer with Is help of agrobacterium. 17. The method according to claim 15, characterized in that the plant transformation technique a transformation with direct DNA uptake. 18. The method according to any one of claims 1 to 14, characterized characterized in that the core marker gene in a somatic Cell culture is selected. 19. The method according to any one of claims 1 to 14, characterized characterized in that the core marker gene in a protoplast fusion transfer is selected. 20. The method according to any one of claims 1 to 14, characterized characterized in that the core marker gene after induction a plant mutagenesis is selected. 21. The method according to any one of claims 1 to 14, characterized characterized that the core marker gene by selection a parent plant that has the desired gene, is present. 22. Hybrid seeds obtainable by the method of one of the previous claims.