WO1990001869A1

WO1990001869A1 - High lysine corn

Info

Publication number: WO1990001869A1
Application number: PCT/US1989/003534
Authority: WO
Inventors: Ronald L. Phillips; Michael S. Benner
Original assignee: Regents Of The University Of Minnesota
Priority date: 1988-08-19
Filing date: 1989-08-17
Publication date: 1990-03-08

Abstract

A corn plant and the seed therefrom is disclosed which carries the Zpr10/(22) gene so that wholekernel lysine and/or threonine levels are substantially elevated above the levels of lysine and/or threonine in the corresponding wild-type corn plant which does not contain said gene. Preferably, the gene is contributed by line BSSS53.

Description

HIGH LYSINE CORN

Background of the Invention Lysine, an amino acid essential in the diet of humans and monogastric animals, is among the three most limiting nutrients in most of the cereal crops. Conse¬ quently, grain-based diets, such as those based on corn, barley, wheat, rice, maize, millet, sorghum and the like, must be supplemented with more expensive synthetic lysine or with lysine-containing oilseed pro¬ tein meals. Unsupplemented corn feed is primarily limiting in lysine and tryptophan, while corn-legume mixtures often are deficient in methionine. Corn is deficient in methionine for some uses such as poultry rations. Therefore, the protein and amino acid content of corn has been the subject of numerous studies con¬ cerned with cereal quality. Increasing the lysine con¬ tent of these grains or of any of the feed component crops would result in significant added value. To date, attempts to elevate lysine levels in plants have relied on conventional breeding methods and, more recently, utagenesis and and cell culture technology.

Seed storage proteins have been defined as those proteins whose primary role is to store nitrogen and amino acids for later use by the developing seed¬ ling; no enzyme activity has been identified. These proteins are deposited in the developing seed. T. B. Osborne, in The Vegetable Proteins, Longmans, Green Co., London (1912), defined four classes of seed storage proteins entirely in terms of their solubilityi albumins (water-soluble), globulins (salt-soluble), prolamines (alcohol-soluble) and glutelins (alkali- soluble). While the prolamine class (zein) is the most abundant in corn, it is not nutritionally balanced. Zein is strongly deficient in essential amino acids, most notably lysine and tryptophan, while it contains large amounts of glutamic acid, leucine, proline and alanine.

Zein extracts contain -a^'mixture of polypep- tides with relative molecular weights of 27, 22, 19, 15 and 10 kD. The 22 and 19 kD proteins are extracted in alcohol alone (zein-1 e.xtract), whereas the 27, 15 and 10 kD proteins require the presence of a reducing agent (zein-2 extract). The 22 kD and 19 kD components con¬ tain similar levels of amino acids. The 15 kD and 10 kD components contain higher levels of methionine, cysteine, tyrosine and glycine.

In addition to the many structural zein genes, loci influencing the accumulation of entire classes of zein protein have been identified. These genes have been termed "regulatory," although the exact mechanisms underlying the protein changes have not been identi¬ fied. Mertz et al., in Science, 145, 279 (1964) recog¬ nized the opaque-2 endosperm mutant as the first maize variant with a modified protein composition in the ker- nels. The concentration of lysine in opaque-2 kernels is approximately double that found in near-isogenic counterparts. Additional endosperm mutants containing higher concentrations of nutritionally favorable albu¬ mins, globulins, and glutelins and lower concentrations of zeins have since been characterized by Misra et al., Cereal Chem., 56, 497 (1975): opaque-7, sugary-1, shrunken-1, shrunken-2, shrunken-4, floury-2 and brittle-1. In addition to increased levels of lysine, sugary-1 and floury-2 mutants contain 21-36% and 50-70SS more methionine in endosperm proteins, respectively. As discussed hereinabove, increased levels of methionine are also of significant interest. However, while nutritionally superior, these mutants are asso¬ ciated with reduced yields, disease susceptibility and poor grain quality, limiting their agronomic useful¬ ness. . In particular, the opaque-2 endosperm mutant has been found to yield less grain, to retain higher moisture at harvest, and to succumb to more fungal infections and storage insect infestations than do non- variant maizes. The opaque-2^' grain has a dull and chalky appearance, and the floury texture of its soft kernels make the grain difficult to store and mill. See National Research Council, "Quality-Protein Maize," National Academy Press, Washington, D.C., (1988), at vii-viii. Thus, in order to be agronomically useful, endosperm mutants such as opaque-2 require additional modifier genes to be incorporated prior to their use. Such modifications may significantly increase breeding time and effort. In higher plants (as in bacteria), lysine, threonine and methionine are synthesized in a branched pathway from aspartate (Figure 1). The first and third enzymes in this pathway, aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase, (EC 1.1.1.3) respectively, are known to be regulated by endproduct feedback inhi¬ bition. Lysine or the combination of lysine and thre¬ onine are known to inhibit the activity of aspartokin¬ ase, while threonine inhibits the activity of homoser¬ ine dehydrogenase. Feedback inhibition is a regulatory mechanism by which organisms, including plants, effi¬ ciently regulate the synthesis of cellular metabolites. Regulatory mutants, resistant to feedback inhibition or enzyme repression, have been well defined in pro- karyotes and in lower eukaryotes. Recently, tryptophan and methionine overproducers have been isolated from plant cell cultures. Feedback inhibition mutants result in overproduction of the pathway endproduct(s) due to altered regulatory sites which do not allow nor¬ mal inhibition of the enzyme. In crop plants, these mutants might provide a mechanism to increase the total synthesis of nutritionally limiting amino acids. Equi olar concentrations of lysine and thre¬ onine have been demonstrated to inhibit plant growth (Green and Phillips, Crop Sci. , 14, 827 (1974); Green and Donovan, Crop Sci., 20, 358 (1980)). Inhibition is relieved with the addition of methionine or one of its precursors, homocysteine or homoserine, indicating that the observed growth inhibition is a result of reduced methionine synthesis and subsequent methionine starva¬ tion. Green and Phillips, in Crop Sci., 14, 827

(1974), proposed that feedback-inhibition-resistant mutants could be selected from maize callus cultures following the addition of lysine and threonine ("LT") to the culture medium. It was hypothesized that iso- lated mutants from this system might be insensitive to feedback inhibition^', and subsequently overproduce aspar- tate-derived amino acids. An alternate screening system utilizing germinating kernels rather than callus culture was also described (Phillips et al., Crop Sci. , 2_1, 601 (1981)).

Screening of inbred lines for LT resistance by germinating kernels on media supplemented with lysine- plus-threonine revealed a highly resistant strain from the Iowa Stiff Stalk Synthetic population, designated BSSS53 (Phillips et al., Crop Sci., 21, 601 (1981)). Germinated whole kernels of BSSS53 were LT resistant, whereas dissected embryos were inhibited, indicating that the endosperm was responsible for the seedling resistance. Amino acid analysis of BSSS53 kernels, along with kernels from inbreds differing in LT response, gave rise to the hypothesis that resistance is due to the relative concentrations of methionine and lysine in the kernel, with resistance being associated with a high methionine-to-lysine ratio (M/L ratio). Whole kernels of BSSS53 contain 21% more methionine than kernels of other inbreds analyzed. Phillips and McClure, Cereal Chem., 62, 213 (1985), observed that, while the distribution of pro¬ tein among the Osborne fractions varied only slightly, the methionine concentrations of the zein-2 fractions differed significantly among lines. Analysis of zein-2 fractions by SDS-PAGE indicated an increased proportion of 10 kD polypeptides in BSSS53 as compared to other inbreds. The 10 kD fraction differed not only in rela¬ tive amount but also in methionine composition, with 10 kD polypeptides from BSSS53 containing approximately 21 mol % methionine. Ten kD polypeptides from other inbreds tested contained 9 to 19 mol % methionine.

Overaccu ulation of the major 10 kD protein in BSSS53 is due to a single, semi-dominant allele at the Zprl0/(22) locus. A band detected by isoelectric focus¬ ing corresponding in position to Zp22/6 (formerly Zp6), showed linkage with the major 10 kD band, placing the gene near Gal. Separate tests showed linkage of the major 10 kD band with Gal, f!2 and ^ translocation 4-9g. M. S. Benner and R. L. Phillips, in Maize

Genetics Cooperation Newsletter, 60, 114 (1986), have proposed the gene symbol Zprl0/(22) , which will be used hereinafter. This gene is the putative regulator of the structural gene(s) which encodes the 10 kD zein polypeptide(s). See J. A. Kirihara et al., Mol. Gen. Genet., 211, 477 (1988).

The gene Zprl0/(22) was identified by screen¬ ing for resistant plants from seeds germinated on LT supplemented medium. The Zprl0/(22) mutation could not have been selected in callus culture because its asso¬ ciated LT resistance is dependent on an interaction of the embryo and endosperm. The Zprl0/(22) mutation thus differs from previously reported LT resistant mutants, which were selected from callus cultures, in several ways. The first report by Hibberd et al., in Planta, 148, 183 (1980), identified a callus culture which possessed resistance to LT inhibition. The cells pro¬ duced an altered key enzyme, aspartokinase, and had elevated lysine in the free amino acid pool. No seed could be recovered. The second report (Hibberd and Green, Proc. Natl. Acad. Sci. U.S.A., 79, 559 (1982)) described an LT resistant callus culture and resistant regenerated plants. This mutant, designated Ltr*19, conferred high threonine levels in the free amino acid pool of kernels from resistant plants, but lysine levels were not elevated. A similar mutant, designated Ltr*20, has been identified by the same procedure (Diedrick, Ph.D. Thesis, University of Minnesota (1984)). At the present time, it is difficult to obtain these Ltr*19 and Ltr*20 mutations in homozygous form, due to negative associated traits such as defec¬ tive embryos, poor germination and the like. Therefore, a need exists to develop nutritionally-improved corn lines with increased con- centrations of amino acids such as lysine and/or threonine.

Brief Description of the Invention The present invention provides a corn plant which is homozygous for the Zprl0/(22) gene, and which exhibits whole kernel amino acid levels which are ele¬ vated over the levels present in the corresponding wild-type plant which does not comprise the Zprl0/(22) gene. The corn plant of the invention is produced by transferring the Zprl0/(22) gene from the high methi¬ onine strain, BSSS53, into other genetic backgrounds comprising the 10 kD zein protein structural gene by plant breeding procedures. The presence of the gene at each step is recognized by the overproduction of a specific protein (the 10 kD methionine-rich zein pro¬ tein), e.g., by using isoelectric focusing gels. Although the Zprl0/(22) gene was hypothesized to be responsible for increased zein methionine levels in donor line BSSS53 (genotype Zprl0/(22)/Zprl0/(22), sur¬ prisingly, substantially elevated levels of lysine were also found in the whole kernels of the Zprl0/(22)/- Zprl0/(22) lines derived from BSSS53. Furthermore, when inbred lines A619, A632, B73 or Mol7 were employed as the recurrent parent to produce an Fl hybrid, which was in turn backcrossed twice into the respective inbred line, and the heterozygous BC2 individuals selfed twice to yield homozygous plants, the average levels of asparagine, gluta ine, serine, histidine, glycine, arginine, alaπine, tyrosine, methionine, valine, phenylalanine, isoleucine and leucine, as well as lysine and threonine, were substantially (about 15-30%) elevated over the levels determined for the corresponding wild-type (+/+) plant.

Therefore, the present invention is directed to a corn plant, and the seed therefrom, which compri- ses and preferably is homozygous for the Zprl0/(22) gene, so that the whole-kernel levels of amino acids lysine, threonine or both lysine and threonine are substantially elevated above the levels of said amino acids in the corresponding wild-type hybrid corn plant which does not contain said gene. The corn plant of the invention can be produced by a process comprising:

(a) crossing a member of the Iowa Stiff Stalk Synthetic population, BSSS53, comprising the Zprl0/(22) gene, onto an inbred recurrent parent line which does not com¬ prise said Zprl0/(22) gene (e.g., A619, A632, B73 or Mol7 and the like), to yield an Fl hybrid; (b) twice backcrossing the Fl hybrid with the recurrent parent line;

(c) identifying BC2 progeny comprising said Zprl0/(22) gene; and

(d) selfing said progeny at least twice to produce a homozygous BC2, S2 line, wherein some ears contain only Zprl0/(22)/Zprl0/(22) kernels, so that the whole kernel amino acid levels of lysine, threonine or of both lysine and threonine of said kernels are substan¬ tially elevated above the levels of said amino acids in the corresponding BC2, S2 progeny which do not contain the Zprl0/(22) gene (see Figure 2).

The presence of the gene at each step is recognized by the overproduction of the 10 kD methionine-rich zein protein. Amino acid analyses demonstrate uniformly high total lysine and threonine in derived lines containing the Zprlθ/(22) gene. Selfing the progeny in step (d) at least 5-6 times yields an inbred corn plant as that term is understood by the art.

Brief Description of the Drawings Figure 1 is a diagrammatic representation of the L-lysine, L-threonine, and L-methionine biosynthe- tic pathway in higher plants.

Figure 2 is a diagrammatic representation of the crossing path employed to produce Zprl0/(22)/- Zprl0/(22) and +/+ corn ears for amino acid compari- sons. • Figures 3a and 3b present a diagrammatic representation of the crossing path which can be employed to recover near-isogenic lines producing ears containing all Zpr10/(22)/Zpr10/(22) kernels, from the BC2 genotype shown in Figure 2.

Detailed Description of the Invention The general goals and techniques of hybrid corn breeding are disclosed in U.S. Patent No. 4,731,499, the disclosure of which is incorporated by reference herein. Specifically, the goal of plant breeding is to combine in a single variety/hybrid various desirable traits of the parental lines. For field crops, these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield and better agronomic quality.

Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinating if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.

Plants that have been self-pollinated and selected for type for many generations become homozy¬ gous at almost all gen-e loci and produce a uniform population of true breeding progeny. A cross between two homozygous plants from differing backgrounds or two homozygous lines produce a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants that are each heterozygous at. a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform. Corn plants (Zea mays L_. ) can be bred by both self-pollination and cross-pollination techniques. Corn has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears. The development of corn hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedi¬ gree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding popula- tions. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired pheno- types. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential.

Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complement the other. If the two original parents do not provide all of the desired characteris¬ tics, other sources can be included in the breeding population. In the pedigree method, superior plants are sεlfed and selected in successive generations. In the succeeding generations, the heterozygous condition gives way to homogeneous lines as a result of self- pollination and selection. Typically, in the pedigree method of breeding, five or more generations of selfing and selection is practiced. f^rι+F2; ^ *^ _* ^y* k _> F4→-F5, etc.

Backcrossing can be used to improve an inbred line. Backcrossing transfers a specific desirable trait from one inbred or source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate gene(s) for the trait in question. The progeny of this cross is then mated back to the superior recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny will be heterozygous for loci controlling the^• characteristic being transferred, but will be like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give pure breeding progeny for the gene(s) being transferred.

A hybrid corn variety is the cross of two inbred lines, each of which may have one or more desir¬ able characteristics lacked by the other or which com¬ plement the other. The hybrid progeny of the first generation is designated F_j_. In the development of hybrids, only the Fj_ hybrid plants are sought. The Fi hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.

The development of a hybrid corn variety involves three steps: (1) the selection of superior plants from various germplasm pools; (2) the selfing of the superior plants for several generations to produce a series of inbred lines which, although different from each other, each breed true and are highly uniform; and (3) crossing the selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F^*L). During the inbreeding process, the vigor of the lines decreases. Vigor is restored when two unrelated inbred lines are crossed to produce the hybrid progeny (Fj_). An important consequence of the homozygosity and homo¬ geneity of the inbred lines is that the hybrid between any two inbreds will always be the same. Once the inbreds that give the best hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parent is maintained. A single cross hybrid is produced when two inbred lines are crossed to produce the Fj_ progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (AXB and CXD) and then the two Fι_ hybrids are crossed again (AXB)X(CXD). Much of the hybrid vigor exhibited by F_]_ hybrids is lost in the next generation (F2). Consequently, seed from hybrid varieties is not used for planting stock.

Hybrid corn seed can be produced by manual detasseling. Alternative strips of two inbred variet- ies of corn are planted in a field, and the pollen- bearing tassels are removed from one of the inbreds. Providng that there is sufficient isolation from sour¬ ces of foreign corn pollen, the ears of the detasseled inbred (female) will be fertilized only by pollen from the other inbred (male), and the resulting seed is therefore hybrid and will form hybrid plants.

The laborious detasseling process can be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMS inbred are fertilized with pollen from another inbred that is not male-sterile.

Pollen from the second inbred can contribute genes that make the hybrid plants male-fertile. Usually seed from detasseled normal corn and CMS produced of the same hybrid seed is blended -to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown.

The invention will be further described by reference to the following detailed example. Example I - Production of Homozygous Zprl0/(22)/Zprl0/(22) Corn Plants A. Crossing BSS553 with selected inbred lines.

Inbred lines A619, A632, B73 and Mol7 were chosen as recurrent parents in the backcrossing of Zprl0/(22) into elite materials (Figure 2). These lines were obtained from J. L. Geadelmann, University of Minnesota, and are widely available, for example, from Illinois Foundation Seeds, Inc., Box 722, Champaign, Illinois, 61820. The above lines were crossed as female with BSSS53, a random line isolate from the Iowa Stiff Stalk Synthetic population; the present line being descended from material provided to J. L. Geadelmann, University of Minnesota by A. Hallauer, USDA Agricultural Research Service, Iowa

State University, Ames, Iowa. Each Fl was subsequently backcrossed twice as female to the respective male recurrent parent. The presence of Zprl0/(22) was detected in heterozygous BC2 progeny due to its ability to cause overaccumulation of the major 10 kD zein pro¬ tein, as visualized on isoelectric focusing gels (des¬ cribed below). Heterozygous BC2 individuals were selfed to produce segregating seed, which were again selfed. Ears from homozygous Zprl0/(22) (Zprl0/(22)/- Zprl0/(22)) and wild-type (+/+) plants were identified by their failure to produce segregating seed. Homo¬ zygous Zprl0/(22) kernels contained elevated 10 kD pro¬ tein.

B. Protein Extraction and Isoelectric Focusing

Whole kernels or portions thereof were ground in a mill similar to that described by Paulis and Wall, in Cereal Chem., 56, 497 (1979). The zein-1 fraction was extracted twice from 50 mg of meal with 0.5 ml 70% ethanol for 30 min at room temperature. The zein-2 -In_¬

fraction was subsequently removed with one ml ethanol containing 1% 2-mercaptoethanol for 30 min at room temperature. In preparation for -isoelectric focusing, 75 μl of zein-2 extract was dried ^ vacuo and resus- pended in 10 μl 70% ethanol, 1% 2-mercaptoethanol.

Proteins were loaded onto gels consisting of: 5% acry¬ lamide, 6.4 M urea, 2% carrier ampholytes (Servalyte 5-8), and 0.3 ml 10% ammonium persulfate per 60-ml gel to initiate polymerization as disclosed by Kirihara et al., Mol. Gen. Genet., 211, 477 (1988). Isoelectric focusing was performed for 3 hr at 10°C with 25 W con¬ stant power. Proteins were visualized by precipitation with 10% trichloroacetic acid.

C. Amino Acid Analysis

Fifty kernels were removed from one each of the following ears, identified as described herein- above: ZprlO/(22)A619, wild-type A619, ZprlO/(22)A632, wild-type A632, Zprl0/(22)B73, wild-type B73, ZprlO/(22)Mol7 and wild-type Mol7. Samples were ground to a fine powder in a Cyclone mill and defatted twice with petroleum ether (15 ml per gm meal). Thirty-five mg of defatted meal were hydrolyzed with 2 ml of 6M HCl at 110°C for 24 hr. Vials were flooded with nitrogen prior to hydrolysis to prevent oxidation. Hydrolyzed samples were dried iri_^ vacuo and amino acids redissolved in 0.2M sodium borate buffer, pH 9.5. Samples were diluted 1 part in 48 with sodium borate prior to analy¬ sis on a Waters HPLC analyzer.

D. Results

Elevation of whole-kernel amino acid levels are summarized in Table 1, below.

Average 49.98 57.08 14

Average 162.60 186.15 15

Serine nM ser/mg defatted meal

Line Wild-type Zpr % increase

A619 24.03 35.12 46 A632 30.90 35.50 15

B73 27.06 29.02 7

Mθl7 17.86 20.96 17

Average 25.77 30.57 19

Histidine nM his/mg defatted meal

Line Wild-type Zp_r % increase

A619 14.18 24.84 75

A632 23.74 30.06 27 B73 20.76 22.45 8

Mol7 10.57 15.04 42

Average . 18.30 23.73 30 Glycine nM gly/mg defatted meal

Line Wild-type Zpr % increase

A619 29.64 47.87 62 A632 37.90 44.43 17 B73 34.00 37.25 10 Mo17 27.52 29.55 7

Average 33.00 39.99 21

Threonine nM thr/mg defatted meal

Line Wild-type Zpr % increase

A619 21.17 30.58 45 A632 28.01 33.57 20 B73 24.62 25.90 5 Mo17 17.32 20.25 17

Average 23.49 28.00 19

Arginine nM arg/mg defatted meal

Line Wild-type Zpr % increase

A619 16.54 27.22 65 A632 24.08 28.44 18 B73 19.44 20.48 5 Mol7 16.24 18.48 14

Average 19.61 23.81 21

Alanine nM ala/mq defatted meal Line Wild-type - Zpr % increase

A619 79.47 115.40 45 A632 98.81 113.92 15 B73 90.95 91.75 1 Mol7 58.31 72.55 24 Average 84.48 99.29 18 Tyrosine nM tyr/mg defatted meal Line Wild-type Zp_r % increase

A619 18.16 30.85 70 A632 23.69 28.43 20 B73 20.64 20.67 0 Mol7 14.50 17.15 18

Average 19.83 24.33 23

Average 7.76 9.41 21

Valine nM val/mq defatted meal

Line Wild-type Z£_r % increase

A619 33.77 50.56 50

A632 44.75 53.41 19

B73 39.30 40.84 4 Mol7 27.71 34.22 24

Average 37.51 45.23 21

Phenylalanine nM phe/mg defatted meal Line Wild-type Zβr % increase

A619 29.00 43.78 51 A632 33.96 38.89 15

B73 32.11 34.01 6

Mol7 20.93 24.49 17 Average 29.81 35.52 19

Average 27.32 31.72 23

Average 13.92 17.16 23

As demonstrated by the results shown in Table 1, the average levels of all 15 amino acids extracted from kernels of the BC2, S2 Zprl0/(22)/-

Zprl0/(22) lines were substantially elevated over the amino acid levels measured in the corresponding wild- type +/+ kernels. Example II - Recovery of High Amino Acid Zprl0/(22)/Zprl0/(22) Lines A breeding strategy which can be used to obtain near-isogenic high amino acid lines is summar- ized in Figures 3a and 3b.

In accord with Figures 3a and 3b, the BC2 strain shown in Figure 2 is backcrossed twice with an inbred recurrent parent line (+/+). The BC strains are screened for Zprl0/(22) kernels by extracting zein-2 protein from small pieces of individual kernels and screening for elevated levels of the 10 kD zein protein on IEF gels. Zprl0/(22) individuals are grown and are crossed to the recurrent parent to ultimately yield strain BC6. Zprlθ/(22) individuals are grown and selfed twice to yield the BC6, S2 line which is near isogenic to the recurrent parent. The ears containing all Zpr10/(22)/Zpr10/(22) are identified as described above by their consistently high accumulation of the zein 10 kD protein. Therefore, the present invention is also directed to useful inbred corn plants, hybrids derived therefrom and the seeds therefrom, which share the desirable phenotypic characteristics of the BC2, S2 Zpr10/(22)/Zpr10/(22) line shown in Figure 2.

The present invention also includes cultures from which corn plants can be regenerated, including cultures of the cells, protoplasts, tissue or calli of the present plants.

The invention has been described with refer¬ ence to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remain¬ ing within the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A corn plant which comprises ^'the Zprl0/(22) gene so that the whole-kernel amino acid levels of lysine, threonine or both lysine and threonine are substan¬ tially elevated above the levels of said amino acids in the corresponding. ild-type corn plant which does not contain said gene.

2. The corn plant of claim 1 wherein the Zprl0/(22) gene is contributed by a member of the Iowa Stiff Stalk Synthetic population, BSSS53.

3. The corn plant of claim 1 wherein the recurrent parent is an inbred line selected from the group consisting of A619, A632, B73 and Mol7.

4. A corn plant produced by a process comprising:

(a) crossing a member of the Iowa Stiff Stalk Synthetic population, BSSS53, comprising the Zprl0/(22) gene with an inbred recurrent parent line which does not comprise said Zprl0/(22) gene, to yield an Fl hybrid;

(b) twice backcrossing the Fl hybrid with the recurrent parent line;

(c) identifying BC2 progeny comprising said Zprl0/(22) gene;^" and

(d) selfing said progeny at least twice to produce a homozygous BC2, S2 line, wherein some ears contain only Zpr10/(22)/Zpr10/(22) kernels, so that the whole-kernel levels of lysine, threonine or of both lysine and threonine in said Zprl0/(22)/Zprl0/(22) kernels are sub¬ stantially elevated above the levels Of said amino acids in the corresponding BC2, S2 pro¬ geny which does not contain said gene.

5. The corn plant of claim 4 wherein the inbred recurrent parent is selected, from the group con¬ sisting of A619, A632, B73 and Mol7.

6. The corn plant of claim 5 wherein the inbred recurrent parent is crossed as female with BSSS53.

7. The corn plant of claim 6 wherein the Fl hybrid is backcrossed twice as female to the male recurrent parent to yield the BC2 progeny.

8. The corn seed of the corn plants of claims 1 or 4.

9. A culture derived from cells, protoplasts, tissue or calli of the corn plants of claims 1 or 4.

10. An inbred corn plant with the phenotypic character¬ istics of the corn plant of claims 1 or 4.

11. A hybrid corn plant derived from the corn plants of claims 1 or 4.

12. A hybrid corn plant derived from the inbred corn plant of claim 10.