Abstract:
An inbred corn plant containing dominant genetic information that expresses high protein and increased lysine levels in corn kernels produced by the plant is provided. The genetic information in the plant can be used to produce novel inbred corn lines and hybrid corn lines containing high protein and lysine levels both by established plant breeding techniques and other techniques of genetic manipulation.

Description:
INTRODUCTION 
     1. Technical Field 
     This invention is related generally to the production of maize, commonly known in the United States as corn, and more particularly to maize containing genetic material capable of causing higher than normal production of protein and lysine, and to the unique genetic material itself. 
     2. Background 
     Single-cross hybrid corn is produced by crossing two homozygous inbred lines. Homozygosity in an inbred line is achieved by repeated inbreeding; in general, by the seventh or eighth generation the inbred line is considered genetically pure. Plant breeders basically have two sources of germplasm from which to develop new inbreds. They include adapted corn belt germplasm and exotic germplasm. The steady increase in corn yield in the United States over the past fifty years has been accomplished almost exclusively with adapted germplasm. 
     Industry&#39;s needs for corn are becoming increasingly segmented, opening new markets for corn that has specialized characteristics. The grain processing industry, especially the dry millers, are interested in new hybrids with grain that has an increased amount of hard endosperm and which can still be grown competititively against current commercial hybrids. The livestock industry, already the largest user of corn, has a potential for value-added, nutritionally improved corn. Globally, about two thirds of total maize production is used for livestock feed (Glover, D. V. and Mertz, E. T. (1987) Agronomy Monograph, 28: 183-336). Maize is thus an important food material which supplies 19 percent of the world&#39;s food calories. Corn also contributes 42 million tons of protein a year, which represents 15 percent of the world&#39;s annual production of food-crop protein. Understandably, high grain yields are the objectives of most corn improvement programs, and breeding for nutritional quality or special purposes has been of minor importance because of the lack of incentives, the lack of communication with the end-user for specific demands, and the lack of breeding efforts in using exotic germplasm which can offer some unique traits. 
     Accordingly, there remains a need for developing unique inbred lines of maize that offer genetic diversity and special traits such as increased amounts of hard endosperm in the grain and increased nutritive values such as high lysine. Additionally, there remains the need to develop the genetic material that can control the lysine content in a dominant and more stable manner than the previously known recessive opaque-2 (O2) system, the best known genetic source of high-lysine corn. 
     SUMMARY OF THE INVENTION 
     It is the object of the present invention to provide an inbred corn line that is significantly different genetically from commercially available adapted inbred corn lines. 
     It is also an object of this invention to demonstrate that the use of this inbred increases the protein and lysine content of resulting single-cross hybrids. 
     It is still a further object of this invention to demonstrate that the nutritive value of a resulting hybrid using this inbred is increased as measured by a significant increased rate of gain in livestock. 
     These and other objects of the invention have been accomplished by providing an inbred corn line (identified as WIL500), hybrid corn lines that have the WIL500 line as a parent, and other plant forms, such as cell lines and other inbred corn lines, that contain the genetic information of the WIL500 line that relates to high lysine and protein production. Characteristics of the genetic information are discussed in more detail below. 
     Seeds of the WIL500 inbred corn line plant have been deposited with the Plant Variety Protection Office, NAL Building, Room 500, 10301 Baltimore Boulevard, Beltsville, Md. 20705-2351 and have received CORN, FIELD, Application No. 8900156. Seeds of the WLL500 inbrid corn line plant have been deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 as ATCC No. 40844. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     WIL500 was derived as a self out in an exotic corn breeding experiment carried out at Wilson Hybrids, Ind., of Harlan, Iowa. This breeding program used corns primarily from tropical origins, especially subtropical white dent germplasm, to screen for useful genetic material capable of improving inbred corn lines used in producing hybrids for the North American corn belt and for other purposes. 
     A pedigree breeding method was used for the development of WIL500. In each of the six selfing generations during development, the line that eventually became WIL500 was selected for on a line-per-se basis for agronomic characteristics and specific traits that are discussed below. During the last four selfing generations, the line that became WIL500 was also evaluated in hybrid combination with other inbreds. In hybrid combination, the line was evaluated in replicated yield trials at different locations for grain yield, protein quantity, protein quality, hybrid agronomic traits, and stability of traits. 
     The initial multiplication of WIL500 was made by shelling all the seed from a single ear of corn selected by the selfing process as described above and planting these seeds in a nursery block. All of the plants that resulted were selfed by hand pollination, and at harvest the seeds from these ears were bulked and called WIL500. Using this initial seed bulk, subsequent seed multiplication was made in an isolation increase field. No variants were observed during the seed increase of WIL500. The inbred plants appeared stable and uniform in the seed increase fields. 
     WIL500 was compared to a public released corn inbred line called AR266 that is typical of U.S. corn belt inbred lines to demonstrate the particular characteristics of WIL500. Twenty to twenty-five measurements were made per trait on each of these inbreds. From these measurements, means, variances and t values were calculated. The t values were calculated by the following formula: ##EQU1## where: x 1 , x 2  =mean 1 and mean 2 respectively; s 2   1 , s 2   2  =variance 1 and variance 2 respectively; and n=number of measurements per mean. 
     Statistical differences between the means were determined by the t values for n-1 degrees of freedom at the 5% level of probability. Statistically significant differences between the means of traits of WIL500 and AR266 are marked as significant in Table 1. 
     
                                           TABLE 1__________________________________________________________________________Comparison of WIL500 to AR266        Mean     Variance   Obser./        WIL  AR  WIL  AR   CalcCharacter   inbred        500  266 500  266  t value__________________________________________________________________________Tassel  25    9.00             11.88                 8.42 8.28 3.524*branches/tasselTassel  25   16.80             34.60                 81.00                      133.17                           6.081*Branch AnglePeduncle   25    2.88              3.24                 1.86 1.27 1.017Length cmNo. of  25   14.00             13.32                 1.42 1.14 2.125*leaves/plantLeaf Angle   25   26.60             29.00                 84.82                      127.08                           0.824Ear Leaf   25   76.28             72.88                 36.88                      24.28                           2.174*Length cmEar Leaf   25    8.60              8.84                 0.33 0.47 1.342Width cmHusk Leaf   25    1.24              0.60                 0.19 0.58 3.647*Length cmPlant   25   129.88             143.92                 66.36                      636.33                           2.648*Height cmEar     25   37.84             48.00                 27.81                      106.67                           4.381*Height cmTop Ear 25    5.84             19.32                 11.72                      25.14                           11.102*InternodeLength cmHusk Ex-   25    7.56              7.72                 4.09 0.96 0.356tension cmShank   25   10.20             13.40                 1.75 7.50 5.261*Length cmShank Inter-   25    9.76             10.80                 1.36 1.17 3.269*nodesEar     20   14.05             12.15                 1.94 1.50 4.581*Length cmEar Weight gm   20   30.20             59.50                 145.75                      389.74                           5.662*Kernel Rows/   20   13.30             13.20                 2.22 2.27 0.211EarEar Diameter   20   35.75             35.60                 9.46 4.36 0.180mmCob Diameter   20   27.50             21.95                 7.84 1.31 8.205*mm100 Kernel   20   23.58             25.28                 12.37                      9.25 1.635Weight gm% Round 20   78.75             56.60                 315.25                      387.62                           3.736*KernelsKernel  20    4.35              4.90                 1.61 1.57 1.379Thickness mmKernel  20    7.60              7.30                 0.88 0.43 1.172Width mmKernel  20    6.90              8.20                 0.52 0.80 5.060*Length mmNo. of  25   0    0   0    0    0Tillers/Plant__________________________________________________________________________ *Significant 
    
     Additionally, the following differences, although not tested for statistical significance, where observed between WIL500 and AR266. as compared to AR266, WIL500 flowers two days later, forms kernel black layer five days later, reaches 25% kernel moisture two days later, has less pollen shed, has a lighter green leaf color, has fewer marginal leaf waves amd ore longitudinal creases. WIL500 has a white cob, wherereas AR266 has a red cob. 
     A number of the characteristics of WIL500 and its hybrids are of particular advantage because of requirements of the milling industry. Currently, some dry millers pay a premium for corn that meets certain standards in kernel characteristics. Acceptable corn must have the following characteristics: 
     1. A high percent of bright-yellow, hard-endosperm starch 
     2. A high percent of corn oil 
     3. Uniform, large-size kernels 
     4. An attractive, bright yellow color 
     Acceptable corn hybrids on the market today have a range of 56 to 58.8% hard endosperm (Table 2). Dry millers have expressed interest in seeing future hybrids developed that would have kernels that attained 62% hard endosperm and have yields within 10% of leading commercial hybrids. 
     
                       TABLE 2______________________________________Comparative Milling Characteristicof Previous Wilson Corn HybridsDescription    1500b   1700    1700b 1800b  2100 2300______________________________________Test Wt. 58.5    59.0    59.3  58.3   59.7 59.3lbs/bu% Small   3.4     5.6     7.7   5.8    2.4  4.1Kernels% Hard   57.0    57.3    58.8  56.0   58.8 57.7Endosperm% Floury 13.8    14.6    13.8  14.7   14.3 14.2Endosperm% Germ   14.9    14.9    13.5  14.7   13.8 13.9______________________________________ 
    
     A selective screening process was initiated on the testing phase of 1500 hybrids in the breeding program. The criteria consisted of the following: 
     1. Protein values to exceed 10%. 
     2. Test weight to exceed 59 lbs./bu. (normal=56 lbs./bu.) 
     3. Yield comparable to those of standard corn lines. 
     4. Plants tolerant to anthracnose. 
     5. Plants tolerant to stalk rot, especially fusarium. 
     6. Kernels to have a high percent of hard endosperm. 
     A sample of a hybrid having WIL500 as a parent, designated as T2021, was taken to a dry miller for analysis. The results (Table 3) indicated that this grain had a significant increase in the amount of hard endosperm. In addition, yield of the T2021 was comparable to leading Wilson hybrids. 
     
                       TABLE 3______________________________________Comparative Milling Characteristicsof Wilson Corn HybridsDescription  1700    1700b   2100  2300  T2021______________________________________Test Wt. lbs/bu        59.0    59.3    59.7  59.3  63.0% Small Kernels         5.6     7.7     2.4   4.1  0% Hard Endosperm        57.3    58.8    58.8  57.7  64.2% Floury Endosperm        14.6    13.8    14.3  14.2  14.0% Germ       14.9    13.5    13.8  13.9  10.0______________________________________ 
    
     Protein and Lysine Evaluation 
     During the selective screening process discussed above, it was discovered that T2021 not only had high levels of hard endosperm, it had significantly increased protein and lysine levels compared to regular corn (Table 4). All grain samples were obtained from a replicated yield trial, which means that the traits of higher protein and lysine are dominant since this corn had not been isolated during growth from other sources. Isolation of growing corn from other corn fields is required if a recessive trait is to be expressed since otherwise cross pollination from the other corn will mask the effects of the recessive trait. 
     
                       TABLE 4______________________________________Composition of Wilson T2021 vs. Regular Corn.sup.1Characteristic  Regular Corn Wilson T2021______________________________________Crude Protein   7.0 to 9.0   10.2 to 10.5Lysine (%)      0.23         0.38 (kernel)Oil (%)         3.3 to 4.0   4.0% Hard Endosperm           47 to 58     64.2% Floury Endosperm           14 to 15     14% Germ          14 to 19     10Test Wt. lbs/bu 55 to 60     63______________________________________ .sup.1 Range of values for commercial yellow dent corn were obtained from grain analysis completed by A and L Laboratories and by Lincoln Grain on hybrids that are representative of the majority of hybrids used in the midwest. Values are based on 12% moisture. The protein, lysine, and oil values are very representative of published values which are discussed in &#34;Corn: Chemistry and Technology,&#34; S. A. Watson and P. E. Ramstad, 1987, American Association of Cereal Chemists, Inc., St. Paul, Minnesota. 
    
     In addition to T2021, several other genetic combinations were made with the inbred line WIL500 and tested for grain protein levels in several experiments. Three WIL500 combinations were compared against a regular commercial corn for protein levels under different plant populations and different fertilizer rates. In this trial, all combinations of WIL500 crossed by any adapted foundation inbred line had significantly higher grain crude protein levels than commercial corn (adapted foundation line×adapted foundation line) under all population and fertilizer rates tested (Table 5). 
     
                       TABLE 5______________________________________Grain Protein Analysis of DifferentCombinations of WIL500 Comparedto Wilson 1700Pro-duct  Pedi-            Fert..sup.3                         %    Yield 0% H.sub.2 OI.D.  gree.sup.1          Pop..sup.2                  Rate   H.sub.2 O                              Bu/A  Prot.                                         Oil______________________________________T3138 WIL500   17,000  1x     15.1 158.95                                    12.5 5.1 ×  23,000  1x     16.0 179.77                                    12.3 4.6 F. Line  17,000  1x + 80                         16.4 152.24                                    12.7 4.9 A        23,000  1x + 80                         16.8 158.45                                    12.7 4.9T3166 WIL500   17,000  1x     21.0 136.70                                    13.3 4.2 ×  23,000  1x     20.4 164.56                                    12.8 4.6 F. Line  17,000  1x + 80                         21.2 159.00                                    13.7 4.5 B        23,000  1x + 80                         20.7 161.96                                    13.1 4.6T3127 WIL500   17,000  1x     16.6 152.32                                    12.4 4.5 ×  23,000  1x     16.9 153.90                                    12.3 5.2 F. Line  17,000  1x + 80                         15.7 138.18                                    12.9 4.5 C        23,000  1x + 80                         15.5 145.91                                    12.4 4.8Wilson F. Lines 17,000  1x     15.2 162.52                                    10.4 4.11700  G × H          23,000  1x     14.9 178.68                                     9.6 4.2          17,000  1x + 80                         14.1 166.85                                    10.9 4.3          23,000  1x + 80                         14.8 170.22                                    10.1 4.1______________________________________ L.S.D. = 0.34 (0.05) .sup.1 F. Line = Foundation inbred line .sup.2 Pop. = Plant population rate per acre .sup.3 Fert. Rate = Fertilizer rate; 1x = standard rate of 160 pounds of actual nitrogen; 1x + 80 = 160 pounds nitrogen plus 80 pounds of nitrogen 
    
     An additional trial was conducted in three different Iowa research locations to compare the crude protein content of T2021 to the standard commercial corn Wilson 1700 across a range of environmental conditions. In all locations, T2021 had significantly more grain protein than Wilson 1700 (Table 6). 
     
                       TABLE 6______________________________________Grain Protein Analysis of T2021(Wilson × F Line D) Compared toWilson 1700 Across Three ResearchLocations in Iowa       Harlan   Clarinda N. Sharon                                 Mean*       0% H.sub.2 O                0% H.sub.2 O                         0% H.sub.2 O                                 0% H.sub.2 OPedigree    Pro (%)  Pro (%)  Pro (%) Pro (%)______________________________________T2021 WIL500 ×           11.2     11.2   11.7    11.4 F Line D1700  LH119 ×            9.7      9.7   10.3     9.9 LH51______________________________________ *Mean of 7 replications 
    
     Seed of the hybrid T3166 was collected and analyzed (in comparison to generic corn) for nutrient composition. The results definitely indicated a significant increase in protein and lysine levels (Table 7). 
     
                       TABLE 7______________________________________Nutrient Composition of Grains Involved in theHigh Protein Corn Evaluation Study.sup.1                     Exper. high          Generic    protein cornComponent      Yellow Corn.sup.2                     (T3166)______________________________________MetabolizableenergyKCal/lb        1530       1530KCal/kg        3370       3370Fat %          3.8        3.8Linoleic acid %          1.9        1.9Crude Protein %          8.9        11.2Lysine %       .24        .32______________________________________ .sup.1 Nutrient composition values are based on 12% moisture .sup.2 Generic corn is from the 1987 crop of commercial yellow dent corn representative of corn used in Nebraska for feeding livestock 
    
     the grain of the inbred line WIL500 was also tested for crude protein and lysine levels and compared to other inbred lines (Table 8). The results clearly indicated that the lysine level of WIL500 was similar to that of inbred lines that possessed the recessive opaque-2 gene, and far superior to standard foundation inbred lines. The previous analysis (Table 7) clearly indicated that hybrids with WIL500 did have a significantly higher lysine content. An additional important fact is that the increase in protein and lysine in the hybrid was obtained without isolating the field. This is a significant advantage over use of the opaque-2 system, as the recessive opaque-2 system (O2) requires field isolation. 
     
                       TABLE 8______________________________________Protein and Lysine Analysis of the Grain ofVarious Inbreds and of Hybrid CombinationsProduct I.D.      Protein (%)                        Lysine (%)______________________________________WIL500 (Harlan Nursery)             10.38      0.44Tuxpeno QPM 02    10.06      0.46IPTT42 QPM 02     9.76       0.39Mo17 02           11.18      0.47Foundation Line A 9.08       0.31Foundation Line B 9.03       0.29Foundation Line C 9.28       0.31______________________________________ 
    
     To determine if the increased protein levels of the experimental corn T3166 (WIL500×adapted inbred B) also increase nutritive value, two feeding trials were conducted. The first used grains in diets considered low in protein for starting chicks (18.4% protein). The rationale for this was that if the experimental corn was truly higher in protein, chicks would gain more weight. In trial two, recommended (NRX, 1984) protein levels (22% in the starter, 19% in the grower) were used, and the birds were fed to 42 days of age. In each trial, body weight gain and feed efficiency were used as response criteria. 
     Results of feeding trials one and two are shown in Table 9. In the first trial, chicks receiving the experimental high protein corn diet gained about 40 grams (about 0.10 pound) more weight than chicks fed diets with the other grain sources. Therefore, this cultivar of corn can be called high protein since the protein level is great enough to end the need for soybean meal, and chicks still grow at a fast rate. In trial two, there were no significant differences in 21-day weight gains (these diets contained recommend instead of low protein levels). However, at 42 days the birds fed diets containing high protein corn had gained 117 grams and 70 grams more (about 0.25 and 0.15 pound) than birds fed generic corn and grain sorghum diets, respectively. 
     
                       TABLE 9______________________________________Performance of Chicks Fed High Protein Corn,Generic Corn, and Grain SorghumTrial 1         Trial 2Body wt     Feed/   Body wt    Feed/gain        gain    gain       gain    Shank(grama).sup.1       ratio   (grama).sup.2                          ratio   colorTreat- Day      Day     Day  Day   Day  Day  Dayment   21       21      21   42    21   42   42______________________________________Generic  585a     1.68b   571a 1633a 1.64b                                   2.00b                                        7.3cCornExperi-  627b     1.57a   590a 1750b 1.57a                                   1.96a                                        6.5bmentalHighProteinCorn(#3186)Sorghum  588a      1.61ab 596a 1680a 1.54a                                   1.94a                                        1.0a(NC +271)______________________________________ .sup.1 Each value is the average of 5 replicate pens containing 6 Vantres X Arbor Acre chicks per pen. .sup.2 Each value is the average of 5 replicate pens of males and 5 replicate pens of females containing 6 Vantress X Arbor Acre chicks per pen.  a, b, c Values within the same column which are followed by different subscripts are statistically different (P &lt; .05). 
    
     In summary, an experimental high protein corn with increased lysine levels improved performance of broiler chicks receiving both low and recommended or standard protein levels in their diets. High protein corn has the potential to reduce the need for soybean meal in poultry diets. 
     Restriction Fragment Length Polymorphism 
     Restriction fragment length polymorphism (RFLP) has been extensively used in human and plant genetics for varietal and parental lineage identification. A complete description of this technology is described in Soller, M. and J. S. Bockmann, Genetic polymorphism in varietal identification and genetic improvment, (1988) Theor. Appl. Genet. 67: 25-33 and Heleutjaris, T., A genetic linkage map of maize based on RFLPs (1987) TIG 3: 217-221. In this technology DNA is isolated from the seedlings obtained after planting seed of the corn line to be tested, using published techniques. After restriction with HIND III, a commercially available restriction enzyme, the DNA is tested with DNA probes. In the case reported here UMC probes and BNL probes were obtained from Dr. D. Hoisington, 303 Curtis Hall, University of Mo., Columbia, Mo. 65211 and Dr. F. A. Burr, Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973. In addition to WIL500 line, DNA was also extracted from MO17, B73 and LH123 corn homozygous lines. The size of the DNA fragments reacting with the probes (Table 10) indicate a specific pattern of the WIL500 line which identify the genetic material of this corn line. 
     
                       TABLE 10______________________________________Characterization of the Line WIL500    Length of Restriction Fragment    in KbProbe      WIL500   LH123     MO17    B73______________________________________UMC83      10       3.9       9.8     2.7BNL12.06   4.1      4.1       18.4    3.7UMC5       2.7      4.2       2.6     2.6UMC34      7.5      18.0      4.8     5.6UMC131     3.8      3.8       --      --UMC139     5.3      5.3       6.4     4.5BNL8.45    11.0     11.0      8.6     8.6UMC10      5.3      7.3       7.2     5.2UMC102     6.7      6.7       5.7     5.5UMC19      3.9      2.3       --      --UMC56      6.2      6.2       2.3     2.3UMC151     2.3      4.4       2.7     2.7BNL14.07   10.0     10.0      --      --BNL15.21   7.0      20.0      --      --______________________________________ 
    
     Characteristics of WIL500 are as follows: 
     Plant color--dark green 
     Emergence--Average to good (similar to B73) 
     Vigor--Average to good (similar to B73) 
     Stalk 
     Height (cm)--129.88 
     Ear Height (cm)--37.84 
     Anthycyanin--Stalks are green with no visual evidence of anthocyanin pigment 
     Tillers/Plant--0 
     Brace roots--Good brace root development at the base. Color=green 
     Leaves 
     Angle--26.60 
     Number/Plant--1400 
     Leaf Sheath 
     Anthocyanin--Leaf sheath is dark green with no visual evidence of anthocyanin pigment 
     Pubesence--Heavy (similar to OH26) 
     Width (cm)--8.60 
     Ear leaf length (cm)--76.28 
     Tassel 
     Length (cm)--23.0 
     Branch angle (cm)--16.80 
     Branching--9.00 
     Anther color--Green 
     Peducle length (cm)--2.88 
     Ear 
     Silk color--Green 
     Husk bracts--Buff in color 
     Ear/Stalk--one 
     Length (cm)--14.05 
     Weight (gm)--30.20 
     Shape--Conical 
     Diameter (mm)--35.75 
     Top ear internode length (cm)--5.84 
     Kernel Rows/Ear--13.30 
     Shank length (cm)--10.20 
     Shank internodes--9.76 
     Husk number --9-11 
     Husk extension (cm)--7.56 
     Husk leaf length (cm)--1.24 
     Husk width (cm) --5 
     Cob color--White 
     Cob diameter (cm)--27.50 
     Kernel 
     Type--Flint 
     Color--Yellow 
     Cap--Medium yellow 
     Sides--Dark yellow 
     Thickness (mm)--4.35 
     Form--78.75% round kernels 
     Width (mm)--7.60 
     Length (mm)--6.90 
     Weight 100 Kernels (gm)--23.58 
     Texture--&gt;58% of a very hard endosperm with very little white floury starch present 
     Pollen Shed 
     Days--75 
     Degree Days--1745 
     50% Silk 
     Days--79 
     Degree days--1765 
     Shank diameter (cm)--1.5 
     Glume Color--Green 
     Glume band--Red 
     Tassel--branch angle --16.8° 
     Brace root color--green 
     Days to flower--75-79 
     Days to black layer--148 (from emergence) 
     Days to 25% kernel moisture--158 (from emergence) 
     All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.