WO2001067847A1

WO2001067847A1 - Method for increasing the yield of grain biomass per area

Info

Publication number: WO2001067847A1
Application number: PCT/EP2000/002134
Authority: WO
Inventors: Peter Stamp
Original assignee: Syngenta Participations Ag
Priority date: 2000-03-10
Filing date: 2000-03-10
Publication date: 2001-09-20
Also published as: AU3961600A

Abstract

The present invention relates to a method for increasing the yield of grain biomass per area, from a cross between hybrids of plants cultivated for their endosperm bearing grains, which comprises pollinating a high grain biomass male sterile hybrid AxB, with one or more high grains biomass yielding hybrids CxD, which are non-isogenic to hybrid AxB and have a positive pollinator ability for said hybrid AxB.

Description

METHOD FOR INCREASING THE YIELD OF GRAIN BIOMASS PER AREA

FIELD OF THE INVENTION

The present invention relates to a method of increasing the yield of grain biomass per area obtained from a cross between hybrids of plants cultivated for their endosperm bearing grains. The new method results in a product having a grain biomass per area above the level of conventional high-yielding hybrids. The invention also relates to a mixture of seeds of plants cultivated for their endosperm bearing grains giving rise to such increased grain biomass per area yield.

BACKGROUND OF THE INVENTION

Principles of traditional hybrid breedine

Most commercial plants are produced from hybrid seed. The production of hybrids require development of elite hybrid lines that have such attributes as good stalk strength, disease resistance, grain storage qualities, and drought tolerance. The development of hybrid lines for particular traits is time and labor extensive. One must select desirable plants in populations and self such plants through several generations in open-pollinated populations to produce homozygous inbred lines. Once the desired homozygous inbred lines are produced, typically two inbred lines are selected and crossed to generate hybrids. Preferably, the two inbred lines are little related (have different origin) and have good combining ability to give greater hybrid vigor to the resulting FI single cross hybrid. Seed from FI single cross hybrids is commercially grown on farmers' fields. By isogenic pollination under normal field conditions, F2 seeds are harvested from FI plants. Intermating sister plants in a field (sib-mating or isogenic pollination) has the same effect as selling. For this reason, the seed of FI plants has, genetically, the status of F2 plants and represents a first inbreeding generation. A FI hybrid can be further crossed with an inbred line to produce a three-way cross. A cross between a FI single cross hybrid and another FI single cross hybrid is called a double cross.

Male sterility

Fertile plants invest a considerable amount of water, energy, and nitrogen in the formation of fertile pollen. In fields where only one hybrid is planted, flowering synchronisation between male and female organs is not a problem under normal conditions. Therefore, the number of pollen grains is typically far beyond what is needed to assure a complete fertilization. It is known in the art that male sterility, which does not harm the plant, usually induces a yield increase even in high grain biomass yielding hybrids, (See Examples I and HI below).

Plants can be rendered male sterile by mechanical, chemical or genetic processes. Mechanical elimination of the pollen-bearing anthers is very time-consuming. Maize is the only species that allows the production of large amounts of hybrid seeds by such a mechanical method.

A chemical way to male sterility plants is possible by the application of gametocides which suppress the development of functional pollen. A large number of substances are known but most of them do not fulfil up to now the essential criteria of low toxicity and a reliable effect.

Genetic hybrid mechanisms have been used now for several decades. They interfere with anther and pollen development at different stages. The "cytoplasmic male sterility" (cms) is based on an interaction between mitochondrial genes and nuclear genes which leads to dysfunctional pollen. Certain nuclear genes can overcome this effect. They are called "restorer" because in crosses with cms-plants the male fertility of the offspring is restored. Such a system was commonly used for maize several decades ago because it offers a way to cheap seeds. Similar systems have been established for cereals like sorghum and rye.

A comparatively new way to pollen sterility is offered by nuclear male sterility (nms) which is based on nuclear genes coding for male sterility. Nuclear male sterile plants can be obtained in a manner known per se employing conventional plant transformation techniques. Nuclear male sterile plant systems are more complicated than cms type plant systems because crossing a nuclear male sterile mother leads to a segregation (i.e. a mixture) of male sterile and fertile offspring.

Cross-pollination with non-isogenic hybrids

It is known from prior art that fertile father plants, i.e. the pollinator, can have a considerable influence on grain form, yield, grain colour and on grain quality traits such as oil content. The effect of pollen genes on kernel characteristics is referred to in the art as the "xenia effect." The work conducted thus far has been focused on the impact of the xenia effect on kernel or plant development, not whole field yield. Thus, there is an unmet need in the art to use pollinators to increase the grain yield per area.

It is therefore an object of the present invention to produce yields of grain biomass per area greater than the yields produced by today's conventional elite hybrids.

It is another object of the present invention to provide a method for increasing the yield of grain biomass per area, by crossing a high grain biomass male sterile hybrid with one or more high grain biomass yielding hybrid pollinators, which are non-isogenic to the male sterile hybrid.

It is still another object of the present invention to provide a mixture of seeds of a plant species cultivated for its endosperm bearing grain comprising a male sterile hybrid of such plant and one or more high grain biomass yielding hybrids of that same species which are non-isogenic to the male sterile hybrid and have a positive pollinator ability for the male sterile hybrid.

It is also an object of the present invention to provide a farming method for planting a field with a mixture of seed comprising the steps of selecting a hybrid seed mixture containing the seed of a high grain biomass male sterile hybrid and the seed of one or more high grain biomass yielding hybrids, which are non-isogenic to the male sterile hybrid and have a positive pollinator ability for the male sterile hybrid, and allowing for pollination of male sterile hybrid plants by the pollinator hybrid plants. It is yet another object of the present invention to provide a method for preparing a bag of high yielding seed comprising the steps of: a) choosing a male sterile hybrid seed for an additional yield increase caused by male sterility itself; b) choosing one or more high grain biomass yielding hybrid seed which are non-isogenic to the male sterile hybrid seed and which have a positive pollinator ability for the male sterile hybrid; and c) mixing the male sterile hybrid and the pollinator hybrid seeds together in the bag, thereby allowing for pollination of the plants of the male sterile hybrid by the plants of the pollinator hybrid.

SUMMARY OF THE INVENTION

The novel method of the present invention significantly increases the grain biomass per area beyond the level that is obtained with conventional common field cereal hybrids for the production of grain biomass for general food and fodder purposes. This is achieved by creating hybrid mixtures with two components. The first component of the hybrid mixture of the present invention is an elite hybrid that outyields in a male sterile form its fertile counterpart and is used in the mixture of the invention in its male sterile form. The second component of the hybrid mixture consists of one or more male fertile elite hybrids, which are non-isogenic to the male sterile hybrid (component 1) and have a positive pollinator ability for component (1). Fertile male hybrids (component 2) have the capability to further increase the yield of the said male sterile hybrid by non-isogenic pollination, a yield increase greater than the yield of either component 1 or component 2.

It is a surprising fact of the present invention that grain biomass yield itself and not just a quality component can be increased cumulatively in already high-yielding common field cereal hybrids by two independent mechanisms, a first time by male sterility, a second time by cross-pollination. Accordingly, the present invention is concerned with the choice of an already high-yielding common field cereal hybrid which reacts to male sterility by an additional yield increase, and further selecting one or more common field cereal hybrids (pollinators) that are non-isogenic, i.e. little related to the mother hybrid, which have a high grain biomass yield capacity themselves. Preferred plants cultivated for their endosperm bearing grains according to the method of the invention are rice, rye, wheat, triticale, sorghum and maize.

DEFINITIONS The following terms are defined:

• Breeding populations: this is a genetically heterogeneous collection of plants sharing largely a common genetic derivation. For the production of hybrids inbred lines must come from distinctly different breeding populations

• Common field cereals: cereal varieties which are cultivated extensively on large areas for the production of a grain biomass which is generally suitable for food and feeding purposes

• Cross pollination: the pollination of the ovules of a plant by another plant which is non-isogenic to it, see non- isogenic

• Grain biomass: this includes the dry matter, exclusive of water, of all kernels produced on a plant or on an area

• Hybrid: any offspring of a cross between two genetically unlike individuals. For common field cereal these are usually two inbred lines which are each homozygous.

• Heterozygosity: a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes. Homozygosity describes the reverse condition that the same alleles reside at corresponding loci.

• Inbred line: an individual in a state of substantially complete homozygosity because of inbreeding

• Large strip test system: plots that are 32 rows by 500' long, harvesting only the 8 center rows.

• Male sterile: a plant without production of functional pollen because of mechanical castration (detasseling in the case of maize) or the chemical and genetic induction of sterility in general

• Non-isogenic: a state of genetic dissimilarity between individuals when their nuclear genomes possess less than 87% statistical similarity

• Pollinator ability: the performance of a pollinator concerning attributes like grain biomass on a non-isogenic pollinated plant. A positive specific pollinator ability for grain biomass indicates that a certain pollinator increases this attribute on a certain non-isogenic plant

• Small plot field system: plots that consist of two 17.5' rows of male sterile hybrid arranged within a block of a specific pollinator. Pollinator blocks are arranged to maximize pollination and πiinimize the chance of contamination by foreign pollen.

DETAILED DESCRIPTION OF THE INVENTION Maize (synonymous in North America with corn; Zea mays L.) wheat (Triticum aestivum L) and rice (Oryza sativa L.) are the three most important cereals worldwide, they provide more than 50% of mankind's direct energy need. Regionally, a large number of further cereals like rye (Secale Cereale L.) triticale (Triticosecale), Sorghum (Sorghum biocolor L.) play a considerable role because of specific tolerance to acid soils, drought or heat. They are all estimated as a comparatively low-prize commodity, which is easily storable. In industrialized countries, cereals are important, furthermore, because of their excellence as animal feed on a grain basis and, increasingly, as a raw material in industry. Because the grain is of paramount economic importance, it is highly desirable to grow cereals in a way that allows for a maximum conversion of natural resources and additional inputs into this main product. For a sexually derived organ, this means providing optimum conditions for the grain set and for the growth of kernels.

As common field cereal is a low price commodity, the principle aim in breeding for hybrids is a high grain biomass per area. In order to produce a high grain biomass per area of land for general food and fodder purposes, high yielding cereal hybrids are developed for all major growing areas. The composition of its two principal components of grain yield, the average single grain weight and the number of grains per area have been felt to be of secondary importance. Indeed, thus far it was generally believed that these two components are negatively correlated, i.e. a healthy field stand of cereal with a rather low number of grains set at flowering produces a relatively large average single grain weight and vice versa. However, maize hybrids can be chosen which produce a higher grain biomass per area when they are male sterile instead of male fertile. Thus the negative correlation between the two major components of grain yield can be minimized a first time.

Surprisingly, the negative correlation between the two major components of grain yield can be minimized a second time by choice of high-yielding fertile pollinator hybrids, which are non- isogenic to the aforesaid male sterile hybrids that have a positive yield reaction to male sterility itself, and which have a positive pollinator ability for those male sterile hybrids. Thus the invention provides a novel method for the production of a low price product like common cereal grain to achieve a higher grain biomass per area than it would be possible with conventional use of hybrids. For this novel production method it is highly essential that the non-isogenic fertile hybrids, the father hybrids, are contributing to the resulting synthetic hybrid a high grain biomass at the standard level of conventional common field cereal hybrids. Although the whole procedure, starting with the selection of single inbreds for the development of superior hybrids, will be prolonged by use of the method of the invention the main advantage of the method of the invention resides in the increased capacity of the thus obtained hybrid mixture for the production of a high grain biomass per area.

Such a hybrid mixture is beneficial, not only economically, but also ecologically. Because of the enhanced grain yield quantity in common field cereal the large input resources on large areas of arable land are utilized more efficiently and the genetic diversity in a cereal field is increased because of mixing non-isogenic cereal hybrids on the same area.

In accordance with the method of the invention, an increase in the grain biomass per area of common field maize grain is actually possible by non-isogenic pollination of a male sterile mother hybrid, even when the grain biomass yield of that sterile mother line had already increased due to male sterility of the mother hybrid. The invention accordingly provides a method for increasing the yield of grain biomass per area, from a cross between hybrids of plants cultivated for their endosperm bearing grains, which comprises pollinating a high grain biomass male sterile hybrid AxB, with one or more high grain biomass yielding hybrids (hereinafter hybrids CxD), which are non-isogenic to hybrid AxB and have a positive pollinator ability for said hybrid AxB, such method comprising the steps of:

a) choosing hybrid AxB, which hybrid is either male sterile or rendered male sterile prior to its pollination in step c hereinafter for an additional yield increase caused by male sterility itself,

b) choosing one or more hybrids (hereinafter hybrids CxD), which are non-isogenic to hybrid AxB and which have a positive pollinator ability for said hybrid AxB, and c) sowing seeds of hybrids AxB and CxD in a mixture and allowing pollination of male sterile hybrid AxB by hybrid CxD.

The grain biomass produced according the method of the invention has an increased grain biomass yield per area because of the superior yield of the elite male sterile hybrid AxB combined with the high conventional yields of the elite hybrids CxD.

The invention also provides a mixture of seeds of a plant species cultivated for its endosperm bearing grain comprising a male sterile hybrid of such plant and one or more high grain biomass yielding hybrids of that same species which are non-isogenic to the male sterile hybrid and have a positive pollinator ability for said male sterile hybrid.

It will be appreciated that the choice of appropriate starting materials can be made based on standard tests; choosing the appropriate male sterile hybrids which outyield in male sterile form their fertile counterpart (component 1), and male fertile hybrids which are non-isogenic to component (1) and have a positive pollinator ability for said component (1).

The following non-limitative examples illustrate the invention:

EXAMPLE I Influence of male sterility on yield expression in maize

Due to the disease-susceptibility that was transmitted by Texas cms, for a long time commercial hybrids were produced on the basis of cms on a small scale only. Although different male sterile cytoplasms may act in a slightly different way on yield expression, any male sterility which does not harm the plant, inducing careful detasselling without losses of leaves, can induce a yield increase dependent on the genetic constitution of the plant. The following two maize varieties with isogenic cms counterparts were used:

1. Suwan2: an open-pollinated variety bred by the Kasetsart University in Thailand. 2. Corso: a hybrid released by the Swiss Federal Research Station at Reckenholz.

The Swiss hybrid was tested in field experiments at the experimental station of the Institute of Plant Sciences, Swiss Federal Institute of Technology, near to Zurich in Switzerland under various high and low input conditions in 1994 and 1995; the same was done with the Thai variety at the Corn and Sorghum Research Center of the Kasetsart University Thailand in 1995/1996.

The optimum local practices for protecting against weeds, insects and fungi and for applying nutrients were maintained. The Swiss hybrid was planted on 7 May 1994 and 5 May 1995, the Thai variety on 18 December 1995.

Plant densities were 9 (recommended) and 12 plants/m² in Switzerland and 5.5 (recommended), 8 and 10.5 plants/m² in Thailand. One plot consisted of 8 rows, 75 cm apart and 5 m long, in a randomized block design with four replications in Switzerland and six replications in Thailand. The four middle rows were sown with male sterile or fertile versions, the other rows with the fertile versions of a variety.

The increase of grain biomass yield by male sterility was confirmed for the Swiss hybrid, Corso (Table 1), and the variety Suwan (Table 2) where the hybrid and the open pollinated variety were bred for a cool temperate and a tropical climate, respectively, indicating that the present invention is applicable to a wide range of climatic zones.

Table 1: Grain biomass yield (g x m^'2) of the male sterile cms Swiss hybrid, Corso in 1994 and 1995 at two plant densities. Changes (%) in relation to the fertile version are presented in brackets.

Table 2: Yield (g x m^"2 ) of the male sterile cms Thai variety Suwan 2 at three plant densities. Changes (%) in relation to the fertile version are presented in brackets.

EXAMPLE π Testing the combined efficiency of male sterility by detasseling and non-isogenic pollination on yield expression.

Extensive testing was carried out by choosing a group of six commercial hybrids from each of the contrasting regions in Thailand and Switzerland with a similar flowering time for each group. For these hybrids, no isogenic cms counterparts existed. Therefore, four of the Swiss hybrids and four of the Thai hybrids with similarly high yield potentials into male steriles by careful detasselling. Some physical damage cannot be avoided completely. Thus, it is possible that cytoplasmic male sterility would have a more positive effect on yield than detasseling. Since controlled hand-pollinations are not possible to this extent and in order to facilitate a statistically correct comparison, a new testing design was developed which puts all mother hybrids together into a unit, intersected and surrounded by a pollinator hybrid. Thus, maximum non-isogenic pollination and manageable small single plots were made possible. Trials were carried out in a randomized block design with irrigation for the Thai hybrids at the Com and Sorghum Research Centre of the Kasetsart University, Thailand, in the dry season of 1996/97 and for the Swiss hybrids at the Experimental Station of the Institute of Plant Sciences, Swiss Federal Research Institute in Eschikon near Zurich in summer 1997.

Testing revealed an increased grain biomass yield by the combined effects of detasseling (male sterility) and cross-pollination in almost all cases. Furthermore, pollinator abilities were found in combinations of a male sterile mother and a pollinator that yielded significantly more than the isogenic pollinated male sterile mother even though yield was already increased by male sterility. Thus, the method of the present invention combines the positive effects on grain biomass yield due to male sterility with the positive pollinator yield effect to generate an increase in grain biomass per area when compared to either the isogenic pollinated fertile mother or the pollinator as shown in the following Tables 3 and 4.

Table 3: Grain biomass yield (g x m^"2) of isogenic pollinated fertile and detasselled (male sterile) maize hybrids, and cross-pollinated male sterile maize hybrids in Thailand, winter season 1996/1997. Yield changes in comparison to the isogenic pollinated fertile mother hybrid are given in brackets as percentages. Pioneer 3011, Ciba G-5445 A, Dekalb 999, Cargill 993 were used as mother-hybrids and pollinator hybrids. Pacific 300, Suwan 3601 were used exclusively as pollinator hybrids.

Table 4: Grain biomass yield (g x m^'2) of isogenic pollinated fertile and detasselled (male sterile) maize hybrids in Switzerland 1997. Yield changes in comparison to the isogenic pollinated fertile mother hybrid are given in brackets as percentages. Banguy Fanion, Magister, Pactol were used as mother and pollinator hybrids. T-90649 and Delval were used exclusively as pollinator hybrids.

EXAMPLE m Testing the combined efficiency of cytoplasmic male sterility and non-isogenic pollination: 1995,

Switzerland The effect of non-isogenic pollination was investigated on the Swiss hybrid for which a cms version exists. In a system similar to that described above, Corso was grown in a field experiment at the Experimental Station of the Institute of Plant Sciences in Eschikon in 1995. Isogenic pollination and non-isogenic pollination by the Swiss hybrid Silex were compared at normal and elevated plant densities as shown in Table 5 immediately below.

Table 5: Grain biomass yield (g x m^"2) of the sib-pollinated fertile (f) and cytoplasmic male sterile (cms) hybrid Corso, in comparison to cms Corso cross-pollinated with the hybrid Silex (cms +), at low and high plant density near to Zurich, in 1995. The changes induced by cms in comparison to f, and by cms+ in comparison to cms, are given in brackets as percentages.

The results of this trial show that a very high increase in grain yield quantity by cms is further increased by non-isogenic pollination especially at elevated plant densities.

EXAMPLE IV Testing the combined efficiency of cytoplasmic male sterility and non-isogenic pollination: 1998,

Switzerland

In 1998, six field trials were conducted at three locations in Switzerland: Eschikon, Rickenback, and St. Aubin.

Exact Trials

Eight pollinators were included to investigate the cross-pollination effects. Table 6 shows the effect of pollinators on yields across the 5 ms hybrids T- 19314, Corsco, Deprim, Silpro, Silterzo.

Table 6: Effects of pollinators on yields (15% H₂O-content)

SNK Grouping Mean [dt/ha] N Pollinator

A 119 74 Delprim

B 115 74 Banguy

B 113 75 Pactol

C B 112 75 Silterzo

C B D 111 74 Corso

C D 108 75 16391A6

D 106 73 Silpro D 106 74 T7042 ^υ SNK = Student-Newman-Keuls test. Means with the same letter are not significantly different at the 5% level.

From the set of 8 different non-related hybrids used as pollinators for the ms-hybrids, some could be identified to cause significantly higher yield at all three locations. The differences in yield according to a pollinator are apparent when comparing the relative yields on one ms-hybrid at one location pollinated with the different genotypes as illustrated in Figure 1. In Figure 1, the yield gain in the second column depicts the male sterility effect, whereas the yield gains in the next 7 columns represent the pollinator effect.

Among the set of chosen pollinators, there are some that can cause a higher yield on most of the ms-hybrids included in the study. The fact that some ms-hybrids had different yields according to the pollinator can be considered as the pure effect of the foreign pollen. Regarding the yield components, the yield gain caused by the pollinator, Depprim, is related to both a higher thousand-kernel weight and a higher kernel number.

EXAMPLE VI Testing the combined efficiency of cytoplasmic male sterility and non-isogenic pollination:

1998, 1999 Switzerland

Tests conducted in Switzerland in 1999 confirmed previous year results. The following Tables 7 and 8 are summary tables that illustrate the effect of pollinators on yield in both 1998 and 1999 in Switzerland.

Table 7: Effect of pollinators on the yield of the 3 cms-hybrids tested in Exact trials at three locations 1998 and 1999 in Switzerland. Number of measurements =n.

Pollinator n Yield relative SNK dt/ha Yield Grouping

15% (% of (refers to

H₂0 fertile Yield) control) sterile 1998 Delprim 44 122.4 113.1 A

Banguy 44 118.3 108.9 B A

Pactol 45 113.7 105.6 B C

Corso 44 113.6 104.5 B C

Silpro 43 108.9 101.2 D C

T7042 44 106.3 98.3 D

1999 Delprim 45 125.1 115.0 A

Banguy 45 123.7 113.5 B A

Pactol 45 120.9 111.2 B A C

Corso 45 117.4 107.6 B D C

T7042 43 114.5 104.6 D C

Silpro 44 113.5 104.4 D

1998/99 Delprim 89 123.8 114.1 A

Banguy 89 121.0 111.3 A

Pactol 90 117.3 108.4 B

Corso 89 115.5 106.1 B

Silpro 87 111.2 102.9 C

T7042 87 110.4 101.5 C

^1} SNK = Student-Newman-Keuls test. Means with the same letter are not significantly different at the 5% level.

Table 8: Yields of the 3 non related cms-hybrids tested in Exact trials at three locations 1998 and 1999 in Switzerland. Number of measurements =n.

Year Hybrid iϊ Yield SNK dt/ha Grouping

15%

H₂O sterile

1998 Silpro ms 88 122.2 A

Delprim ms 89 113.0 B

Corso ms 87 106.4 C 1999 Silpro ms 90 124.8 A

Corso ms 90 116.9 B

Delprim ms 87 115.9 B

1998 & 1999 Silpro ms 178 123.5 A

Delprim ms 176 114.4 B Corso ms 177 111.7 C ^l) SNK = Student-Newman-Keuls test. Means with the same letter are not significantly different at the 5% level.

The order of pollinators, which appears in the SNK-Grouping for yields, is nearly consistent over the two years. The pollinators Delprim and Banguy caused an increase in yield of the three cms- hybrids tested in the two years by 14% relative to the fertile control and 11% respectively. Pactol and Corso raised yields relative to fertile control 8% and 6% respectively. Silpro and T7042 raised relative yields to a lesser extent.

EXAMPLE V Testing the combined efficiency of cytoplasmic male sterility and non-isogenic pollination:

1998, United States

Large strip mixture trials

In 1998, 12 trials at 11 locations were planted with large strip mixture trials. Large strip mixture trials tested 85/15 (±10%) mixtures of ms-hybrids with suitable pollinators (.25 ha per plot). Due to water damage, green snap or poor emergence, data from 4 locations had to be discarded. Despite these losses, and although only three hybrids with the cms-version were available, results from these trials showed significant increase in grain biomass yield. AU tested combinations with cross-pollination yielded more than the fertUe control as shown in the following Table 9.

Table 9: Yield gain in % from large strip mixture trials compared to the self pollinated_.fertile control

Mixture Locations Plot- Yield [dt/ha, 15%H₂O] Yield gain

T2422 Fertile 3 98 T2422ms/T2422 3 100 2%

T2422ms/5228 3 102 6%

T2422ms/3728 3 108 10%

T3244 Fertile 2 84

T3244ms/T3244 2 93 14%

T3244ms/T6050 2 103 24%

T3244ms/9423 2 101 22%

T7042 Fertile 3 94

T7042ms/T7042 3 98 4%

T7042ms/T3422 3 103 11%

T7042ms/5331 3 KM 11%

EXAMPLE Vπ Testing the combined efficiency of cytoplasmic male sterility and non-isogenic pollination:

1999, United States

Exact trials with four cms-hybrids and seven different pollinators were established at 9 different locations in the United States during 1999. The foUowing Table 10 shows that three of four cms- hybrids reacted positively to male steriUty.

Table 10: Effect of male steriUty on yields and relative yields of the 4 cms- hybrids tested in Exact trials at eight locations 1999 in the U.S.

Hybrid Yield relative Yield bushels/acre (% of fertile

15% H₂O control)

T3244 168.79 T3244ms 172.74 102.3

T7832 156.22 T7832 ms 164.04 105.0 T3228 156.22

T5020 ms (T3228 ms) 162.18 103.8

T8624 169.18 T8624 ms 166.30 98.3

Table 11 : Combined effect of male steriUty and cross pollination on yields and relative yields of the 4 cms-hybrids tested in Exact trials at eight locations in 1999 in the United States.

hybrid pollinator Yield relative Yield bushels/ac (% of fertUe re control)

15% H₂O

T3244 168.79 100.0

T3244 ms T3244 172.74 102.3

T3244 ms T3228 174.55 103.4

T3244 ms T7832 164.54 97.5

T3244 ms T6050 167.53 99.3

T3244 ms T8624 164.48 97.4

T3244 ms T4920 159.65 94.6

T3244 ms 9423 164.15 97.3

T7832 156.22 100.0

T7832 ms T7832 164.04 105.0

T7832 ms T3228 166.27 106.4

T7832 ms T3244 163.62 104.7

T7832 ms T6050 164.99 105.6

T7832 ms T8624 168.78 108.0

T7832 ms T4920 171.41 109.7

T7832 ms 9423 173.75 111.2

T3228 156.22 100.0

T5020 ms T3228 162.18 103.8

(T3228 ms)

T5020 ms T7832 164.35 105.2

(T3228 ms)

T5020 ms T3244 165.43 105.9

(T3228 ms)

T5020 ms T6050 171.19 109.6

(T3228 ms)

T5020 ms T8624 161.47 103.4 (T3228 ms)

T5020 ms T4920 157.86 101.0

(T3228 ms)

T5020 ms 9423 170.84 109.4

(T3228 ms)

T8624 169.18 100.0

T8624 ms T8624 166.30 98.3

T8624 ms T7832 172.89 102.2

T8624 ms T3244 171.68 101.5

T8624 ms T3228 171.14 101.2

T8624 ms T6050 164.38 97.2

T8624 ms T4920 166.18 98.2

T8624 ms 9423 171.63 101.4

In summary, the above Examples of tests conducted in the United States and in Switzerland over multiple years show that the present invention provides a unique method for increasing the grain biomass per area of a hybrid mixture containing ms-hybrids and pollinators. Further, the Examples show that the hybrid mixture of the present invention exhibit a genetic effect (G).

Testing in both Europe and the United States showed that hybrid mixtures of the invention exhibit significant environmental effect (E) and or G x E effect. This effect is most evident when contrasting 1998 and 1999 yield results from large-plot trials. The yield differences were more consistently positive in the central and eastern Midwest and in yield environments below 200 bu/a.

European hybrid combinations of the invention produced more consistent yield advantages across the two years tested in the U.S. Several factors are likely to have contributed to this difference. Germplasm type used in central and northern Europe is highly divergent, encompassing both dent and flint types. Dent x flint hybrid combinations provided greater yield than dent x dent hybrid combinations.

Claims

1. A farming method for planting a field with a mixture of seed comprising the steps of selecting a hybrid seed mixture containing the seed of a high grain biomass male sterile hybrid AxB (hereafter AxB) and the seed of one or more high grain biomass yielding hybrids CxD (hereafter CxD), which are non-isogenic to hybrid AxB and have a positive pollinator abiUty for said hybrid AxB, and sowing said seeds of hybrids AxB and CxD thereby aUowing for pollination of male sterile hybrid AxB plants by hybrid CxD plants.

2) A method according to claiml, wherein the ratio of AxB:CxD is approximately 80:20.

3) A method according to claim 1, wherein the ratio of AxB:CxD is approximately 85: 15.

4) A farming method for planting a field with a seed mixture comprising the steps of: a) planting within a field: i) male sterUe hybrid seeds AxB (hereinafter hybrid AxB) to produce male sterile female plants that contribute an additional yield increase caused by male steriUty itself; ϋ) the seeds of one or more hybrids CxD (hereinafter hybrid CxD) which are non- isogenic to said hybrid AxB and which plants produced therefrom have a positive pollinator ability for the plants produced from said hybrid AxB; b) permitting male plants of said hybrids CxD to pollinate said hybrid AxB; and c) harvesting the resulting grain of said CxD and AxB, wherein the yield of grain biomass per area harvested is greater than the yield of grain biomass per area that would result from the planting of either said hybrid CxD alone or said hybrid AxB alone.

5) A method for preparing a bag of high yielding seed comprising the steps of: a) choosing a male sterile hybrid seed AxB (hereinafter hybrid AxB) for an additional yield increase caused by male steriUty itself; b) choosing one or more high grain biomass yielding hybrid seed CxD (hereinafter hybrid CxD) which are non-isogenic to said hybrid AxB and which have a positive pollinator abiUty for said hybrid AxB; and c) mixing said hybrid AxB and said hybrid CxD together in said bag in a quantity of hybrid CxD that is less than the quantity of hybrid AxB in said bag, yet still aUowing for pollination of the plants of said hybrid AxB by the plants of said hybrid CxD.

6) A method according to claim 5, wherein the ratio of AxB:CxD is approximately 80:20.

7) A method according to claim 5, wherein the ratio of AxB:CxD is approximately 85:15.

8) A method according to claim 1 wherein the plants cultivated for their endosperm bearing grains are rice, rye, wheat, triticale and sorghum.

9) A method according to claim 1 wherein the plant cultivated for its endosperm bearing grains is common field maize.

10) A method according to claim 4 wherein the plants cultivated for their endosperm bearing grains are rice, rye, wheat, triticale and sorghum

11) A method according to claim 4 wherein the plants cultivated for their endosperm bearing grains is common field maize.

12) A method of claim 1, wherein pollination of said hybrid AxB with said hybrids CxD produces a yield of grain biomass per area that is greater than the yield of grain biomass per area that would result from planting of either said CxD alone or said hybrid AxB alone.

13) A method according to claim 5, wherein said seeds are rice, rye, wheat, triticale and sorghum. 14) A method according to claim 5, wherein said seed is common field maize.

15) A mixture of seeds of a plant species cultivated for its endosperm bearing grain comprising a male sterUe hybrid of such plant and one or more high grain biomass yielding hybrids of that same species which are non-isogenic to the male sterUe hybrid and have a positive pollinator abiUty for said male sterUe hybrid.

16) A mixture of seeds of a plant species cultivated for its endosperm bearing gram according to claim 15, wherein said mixture comprises approximately 80% of said male sterUe hybrids and approximately 20% of said high grain biomass yielding hybrids non-isogenic to the male sterUe hybrid.