High oleic acid peanut

An Arachis hypogaea L. peanut seed, peanut plant, peanut line, peanut seed product and peanut oil having an oleic acid content of from about 80% to about 85% and a linoleic acid content of from about 1.5% to about 2.5%, each based upon the total fatty acid content of said seed and a ratio of the amount of oleic acid to linoleic acid in said seed from about 20:1 to about 58:1. The peanut seed, seed product, plant and line is of the genetic runner-type variety and has a low pod-splitting trait. `M2-225` seeds were deposited under the Budapest Treaty on Oct. 11, 1996, at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 USA, Accession number 97762.

FIELD OF INVENTION 
The present invention is directed to a high oleic acid peanut plant line 
and products derived therefrom. 
BACKGROUND OF THE INVENTION 
Peanuts (Arachis hypogaea L.) are grown worldwide in the tropic and 
temperate zones for seed oil and human foods such as peanut butter, 
roasted seed and confections. The final quality of edible peanuts is due 
principally to the chemical composition of the oil, protein and 
carbohydrate fractions of the seed. Since fatty acids make up the major 
portion of the weight of an oil molecule, the physical and chemical 
properties of the oil tend to be determined by the properties of the fatty 
acids which predominate in their makeup. Oils high in monounsaturates are 
desirable for both improved shelf life and potential health benefits. 
Depending upon the intended oil use, different fatty acid compositions are 
desired. Peanut breeders face the dilemma of satisfying both the 
requirement of the manufacturer, which is stability of the processed 
product, and the demand from consumers for an increased polyunsaturated to 
saturated (P/S) ratio. 
Oil stability and nutritional quality are both dependent on the relative 
proportions of the saturated and unsaturated fatty acids that constitute 
the oil. Moore, K. M. et al., J. Heredity 80 (3):252 (1989). Oxidative 
rancidity increases with increased levels of polyunsaturated fatty acids. 
Oxidation of the carbon double bonds of fatty acids produces acids, 
aldehydes, ketones, and other hydrocarbons that cause odors and flavors 
commonly associated with rancidity. St. Angelo, A. I. et. al., J. Am. 
Peanut Res. Educ. Assoc. 5: 128-133 (1973). The total amount of 
unsaturation, therefore, is inversely proportional to the keeping quality 
of the oil. The iodine value (IV) is a measure of oil chemical stability, 
with oils having higher IV being more unsaturated and chemically less 
stable. Low linoleic acid content ensures a product of high storage 
stability. Norden, A. J., WPI Accession No. 89-070645/10. 
The American Heart Association and the American Health Foundation have 
recommended diet modifications to achieve lower serum cholesterol levels 
in the population. These diet modifications include reducing consumption 
of saturated fatty acids and thereby increasing the polyunsaturated to 
saturated (P/S) ratio in the diet. Technical Committee, Food Fats and 
Oils, 5th ed. (1982). Edible peanut oils with a higher percentage of 
unsaturated fatty acids are desired for these cardio-vascular health 
reasons. Mattson, F. H. et al., J. Lipid Research 26: 194-202 (1985). 
High levels of the long-chain saturated fatty acids, arachidic and behenic 
are undesirable as they were suggested as being the responsible toxic 
element for enhancing atherosclerosis in rabbits fed diets utilizing 
peanut oil. Diets high in monounsaturates are able to lower serum 
cholesterol in a fashion similar to diets low in low fat. Grundy, S. M., 
New England J. Medicine 314(12): 745 (1986); Nutrition Foundation, Inc., 
Nutrition Review 30(3): 70-72 (1973). High levels of the monounsaturated 
oleic acid, which is present in olive oil, is as effective as the 
polyunsaturated linoleic acid in lowering the blood plasma cholesterol. 
Mattson, F. H., et al., J. Lipid Research 26: 194-202 (1985). 
Although as many as 12 fatty acids have been reported in peanuts, only 
three are present in amounts exceeding 5%: palmitic, oleic and linoleic. 
Ahmed, E. M. et al. in Peanut Science and Technology (1982 H. E. Pattec, 
et al., ed.). These three fatty acids comprise about 90% of the fatty acid 
composition of the oil, with oleic and linoleic comprising about 80%. The 
remainder of the fatty acids comprise about 10%, each ranging in 
concentration from 0.02% to 2.59%. 
Several factors affect the fatty acid composition of peanut oil: maturity, 
temperature, planting date, location, market grade, and peanut genotype. 
Moore, et al., supra; Cobb, W. Y. et al. in Peanuts-Culture and Uses 
(1973). Since 1970, studies on the genetic variability in the fatty acid 
composition of peanut genotypes have shown a range in the composition of 
the different acids. Norden, A. J., et al. Peanut Science 14:7-12 (1987). 
Peanut genotypes are known with as low as 21% oleic and as high as 43% 
linoleic acid. One investigator sub-divided 100 peanut genotypes into 
three maturity groups and into four U.S. market-types. Bovi, M. L. A. 
Ph.D. Dissertation, University of Florida (1982). A large variation in oil 
quality was found within each market-type and/or maturity group. A peanut 
line with 79.91% oleic acid and 2% linoleic acid has been reported. 
Norden, et al., supra. 
Fatty acid composition has been determined among seven U.S. runner-type 
peanut cultivars: `Florunner`, `Sunrunner`, `GK-7`, `Southern Runner`, 
`Sunbelt Runner`, and `Okrun`. Branch, W. D. et al., J. Am. Oil Chem. Soc. 
67(9: 591-593 (1990). Variety `GK-7` is described in Plant Variety 
Protection certificate 82001413. Significant year and cultivar differences 
are found within these fatty acid profiles. Southern Runner had the 
greatest oleic to linoleic ratio of 2.3 and iodine values of 90.5. 
`Florunner` and `Sunrunner` were the highest in unsaturated and lowest in 
saturated and long-chain fatty acids. `Florunner` exhibits 51.7% oleic 
acid and 29.8% linoleic acid, while `GK-7` exhibits 49.6% oleic acid and 
30.5% linoleic acid. `GK-7` and `Florunner` are the most widely cultivated 
peanut varieties in the United States. 
Major genes for fatty acid composition have been reported in three oilseed 
crop species: sunflower (Helianthus annuus L.), soybean (Glycine max L. 
Merr.), and rapeseed (Brassica napis L.) Urie, A. L., Crop Sci. 25:986-989 
(1985); Brunklaus-Jung E. et al., Plant Breeding 98:9-16 (1987); Erickson, 
E. A., et al., Crop Sci. 28: 644-646 (1988); Rennie, B. D. et al., Crop 
Sci. 28: 655-657 (1988). 
Artificial irradiation has been used to induce changes in peanut lines to 
produce Spanish improved groundnut mutants that produce high oleic acid 
and low linoleic levels. More specifically, a parental Spanish improved 
line with 39% oleic acid was irradiated to make mutants that produce 61% 
oleic acid. Sharma, N. D., et al., Qual. Plant Foods Hum. Nutr. 35: 3-8 
(1985). 
The variation in oil quality among diverse peanut genotypes has been 
determined. Norden et al., supra. The range in the percent of the 
saturated fatty acids found among peanut genotypes in the Florida breeding 
program is not widely different from the ranges reported in other lines. 
Id. Oleic acid (18:1) levels in the oil of cultivated peanut (Arachis 
hypogaea L.) have been reported as 36% to 81.4% of the total fatty acid 
composition. Moore, K. M., et al., J. Heredity 30(3) :252-253 (1989); 
Knauft, D. A., et al., Peanut Science 20: 74-76 (1993) and Norden, et al., 
supra. A Spanish type high-oleic-acid peanut line, designated F435, 
exhibits 79.91% oleic acid and 2% linoleic acid. Moore, K. M. et al., 
supra. The peanut line F435 produces peanut seeds with an oleic acid 
content about 74-79.91% and linoleic acid content about 2-8%, based on 
total fatty acids, and an oleic acid:linoleic acid ratio of 9-42:1. French 
Patent Application 2617675 (See Tableau 1). 
Initial genetic studies of the F435 peanut line showed that a single 
recessive gene controlled its fatty acid composition trait in two genetic 
backgrounds and a second recessive gene was necessary for expression in a 
third background. Moore, K. M. et al., supra. Further studies have shown 
that the high-oleic-acid trait in F435 is of monogenic inheritance in 12 
parental backgrounds and digenic inheritance in one background. Knauft, D. 
A., et al., Peanut Sci. 20(2):74-76 (1993). This suggested that either one 
of the two recessive genes may be common in the Spanish variety peanut 
germplasm, and that crosses could be expected to segregate in simple 
monogenic ratios. Knauft, et al., supra. More recently, the number of 
genes controlling inheritance of the high oleic acid trait in F435 has 
been determined. Isleib, T. G., et al., Crop Science 36(3): 556-558 
(1996). Segregation ratios of populations derived from crosses with NC-7, 
NC-9, NC-10C, and VA-C92R were consistent with a monogenic model and 
inconsistent with the digenic model. The activity of delta-12-desaturase, 
which catalyzes the conversion of oleate to linoleate, has also been shown 
to be greatly decreased in the F435 line. Ray, T. K. et al., Plant Sci. 
91(1):15-21 (1993). 
When the proportion of genes from F435 is reduced through backcrossing to 
less than 0.8%, fatty acid composition remains similar to the original 
F435 line. However, the concentration of oleic acid in F435 has never been 
shown to be greater than 79.91%. French Patent Application 2617675 (See 
Tableau 1); Brazil Patent Application 8803439; Japan Patent Application 
1091720 and China Patent Application 1030691. One backcross made between 
F435 and F519.9, with F519.9 as the recurrent parent, resulted in a 
backcross having an oleic acid composition of 81.4=/-0.4%. Knauft, D. A., 
et al., Peanut Sci. 20(2):74-76 (1993). Also, the concentration of 
linoleic acid in F435 has never been shown to be less than 2.14%. French 
Patent Application 2617675 (See Tableau 1). 
The high oleic acid trait of F435 has been transferred from the 
Spanish-type variety to one variety of runner-type to produce the 
commercially available runner-type peanut variety `SunOleic.RTM. 95R`. 
University of Florida Circular S 398 `SunOleic.RTM. 95R`. `SunOleic.RTM. 
95R` does not yield as well as other runner-type varieties and exhibits 
appreciable preharvest pod-splitting. Id. 
A need, therefore, remains for alternative sources of a high oleic acid 
characteristic that can be introgressed into diverse peanut backgrounds. 
In addition, a need exists for a high oleic acid characteristic peanut of 
the runner-type variety that has the additional characteristics of 
acceptable or high yield and negligible pod-splitting. 
SUMMARY OF THE INVENTION 
Embodiments of the present invention, therefore, provide a new peanut plant 
line of the runner-type variety that produces peanut seeds having a high 
oleic acid content that is at least 80% of the total fatty acids and a low 
linoleic acid content that is less than 2% of the total fatty acids. A 
further embodiment of the present invention provides a new peanut plant 
line that produces peanut seeds having a high oleic acid content greater 
than 80% and a low linoleic acid content less than 2%, in combination 
little or with no pod splitting. A further embodiment of the present 
invention provides a new peanut plant line that produces peanut seeds 
having a high oleic acid content greater than 80% and a low linoleic acid 
content less than 2%, in combination with acceptable or high yield. Yet a 
further embodiment of the present invention provides a new peanut plant 
line that produces peanut seeds having a high oleic acid content greater 
than 80% and a low linoleic acid content less than 2%, in combination with 
little or no pod splitting and acceptable or high yield. 
Another embodiment of the present invention provides a new peanut plant 
line that produces peanut seeds having a high oleic acid content greater 
than 80% and a low linoleic acid content less than 2%, in combination with 
little or no pod splitting, acceptable or high yield and acceptable 
milling characteristics. Acceptable milling characteristics include a 
grade of 75 and acceptable blanching. A grade of 75 means that 75% of the 
weight of the unshelled peanuts are comprised of nutmeats and seed coat 
while 25% of the weight is comprised of hulls and damaged kernels. 
Acceptable blanching means that the seed coat is removed easily without 
splitting the seeds in half. 
Yet another embodiment of the present invention provides a new peanut seed 
having an oleic acid content of from about 80% to about 85% and a linoleic 
acid content of from about 1.5% to about 2.5%, each based upon the total 
fatty acid content of the seed and a ratio of the amount of oleic acid to 
linoleic acid in the seed from about 7:1 to about 80:1, in combination 
with little or no pod splitting, acceptable or high yield and acceptable 
milling characteristics. 
Yet a further embodiment of the present invention provides a new peanut 
seed having an oleic acid content of from about 80% to about 85% and a 
linoleic acid content of from about 1.5% to about 2.5%, each based upon 
the total fatty acid content of the seed and a ratio of the amount of 
oleic acid to linoleic acid in the seed from about 7:1 to about 80:1, 
where the seed is the product of a peanut plant having the characteristics 
of a line designated `M2-225`. 
Yet another aspect of the present invention is to provide a new peanut seed 
having an oleic acid content of from about 80% to about 85% and a linoleic 
acid content of from about 1.5% to about 2.5%, each based upon the total 
fatty acid content of the seed and a ratio of the amount of oleic acid to 
linoleic acid in the seed from about 7:1 to about 80:1, where the seed is 
the product of a peanut plant of the runner-type genetic background. 
Another embodiment of this invention is a seed that it is a product of a 
peanut plant having the characteristic of low pod splitting. 
A further embodiment of the present invention provides a peanut plant which 
produces seeds having an oleic acid content from about 80% to about 85% 
and a linoleic acid content of from about 1.5% to about 2.5%, each based 
upon a total fatty acid content of the seed and a ratio of the amount of 
oleic acid to linoleic acid in the seed from about 7:1 to about 80:1, in 
combination with little or no pod splitting, acceptable or high yield and 
acceptable milling characteristics. 
Yet another embodiment of the present invention provides a peanut plant 
which produces seeds having an oleic acid content from about 80% to about 
85% and a linoleic acid content of from about 1.5% to about 2.5%, each 
based upon a total fatty acid content of the seed and a ratio of the 
amount of oleic acid to linoleic acid in the seed from about 7:1 to about 
80:1 where the plant has the characteristics of a line designated 
`M2-225`. A further embodiment of the present invention provides such a 
plant that is of the runner-type genetic background and/or has the 
characteristic of low pod splitting. 
A further embodiment of the present invention provides a Arachis hypogaea 
L. seed product consisting essentially of a substantially homogenous 
assemblage of peanut seeds having an oleic acid content of from about 80% 
to about 85% and a linoleic acid content of from about 1.5% to about 2.5%, 
each based upon the total fatty acid content of the seed and a ratio of 
the amount of oleic acid to linoleic acid in the seed from about 7:1 to 
about 80:1. 
An embodiment of the present invention provides such a seed product having 
an oleic acid content that is about 80-85%. A further embodiment of the 
present invention provides such a seed product having a linoleic acid 
content that is about 1.5-2.5%. Another embodiment of the present 
invention provides an Arachis hypogaea L. seed product consisting 
essentially of a substantially homogenous assemblage of peanut seeds 
having an oleic acid content of from about 80% to about 85% and a linoleic 
acid content of from about 1.5% to about 2.5%, each based upon the total 
fatty acid content of the seed and a ratio of the amount of oleic acid to 
linoleic acid in the seed from about 7:1 to about 80:1 in which the seed 
product is from a peanut plant having the characteristics of a line 
designated `M2-225`. 
Yet another embodiment of the present invention provides such a seed 
product in which the seed is the product of a peanut plant that is of the 
runner-type genetic background. A further embodiment of the present 
invention provides a seed product that has an oleic acid content that is 
about 80-85%. A further embodiment of the present invention provides such 
a seed product having a linoleic acid content that is about 1.5-2.5%. 
Another embodiment of the present invention provides such a seed product 
that is made form peanut seed that is the product of a peanut plant that 
has the characteristic of low pod splitting. 
Another embodiment of the present invention provides a peanut line 
consisting essentially of a substantially uniform population of Arachis 
hypogaea L. plants which produce seed having an oleic acid content of from 
about 80% to about 85% and a linoleic acid content of from about 1.5% to 
about 2.5%, each based upon the total fatty acid content of the seed and a 
ratio of the amount of oleic acid to linoleic acid in the seed from about 
7:1 to about 80:1. A further embodiment of the present invention provides 
such a peanut line where the oleic acid content is about 80-85%. Yet 
another embodiment of the present invention provides such a peanut line in 
which the linoleic acid content is about 1.5-2.5%. 
Another embodiment of the present invention provides a peanut line 
consisting essentially of a substantially uniform population of Arachis 
hypogaea L. plants which produce seed having an oleic acid content of from 
about 80% to about 85% and a linoleic acid content of from about 1.5% to 
about 2.5%, each based upon the total fatty acid content of the seed and a 
ratio of the amount of oleic acid to linoleic acid in the seed from about 
7:1 to about 80:1 in which the plants of the peanut line have the 
characteristics of a line designated `M2-225`. 
Yet a further embodiment of the present invention provides a peanut line 
where the plants are of the runner-type genetic background. Another 
embodiment of the present invention provides a peanut line in which the 
plants have the characteristic of low pod splitting. 
Yet another embodiment of the present invention provides peanut oil derived 
from a seed having an oleic acid content of from about 80% to about 85% 
and a linoleic acid content of from about 1.5% to about 2.5%, each based 
upon the total fatty acid content of the seed and a ratio of the amount of 
oleic acid to linoleic acid in the seed from about 7:1 to about 80:1. A 
further embodiment of the present invention comprises a peanut oil in 
having an oleic acid content about 80-85%. A further embodiment provides a 
peanut oil in which the linoleic acid content is about 1.5-2.5%. Yet 
another embodiment of the present invention is a peanut oil derived from a 
seed having an oleic acid content of from about 80% to about 85% and a 
linoleic acid content of from about 1.5% to about 2.5%, each based upon 
the total fatty acid content of the seed and a ratio of the amount of 
oleic acid to linoleic acid in the seed from about 7:1 to about 80:1 in 
which the seed is the product of a peanut plant having the characteristics 
of a line designated `M2-225`. 
A further embodiment of the present invention provides a peanut oil in 
which the seed is the product of a peanut plant that is of the runner-type 
genetic background. Another embodiment of the present invention provides 
peanut oil derived from seed that is the product of a peanut plant that 
has the characteristic of low pod splitting. 
Yet another embodiment of the present invention provides a peanut seed in 
which the ratio of the amount of oleic acid to linoleic acid in the seed 
is from about 20:1 to about 58:1. A further embodiment of the present 
invention provides peanut seed where the seed is the product of a peanut 
plant having an acceptable milling characteristic. A further embodiment of 
the present invention provides a peanut seed that has acceptable milling 
characteristic that consist of a grade of at least about 75 and acceptable 
blanching. 
Another embodiment of the present invention provides a peanut plant with 
seeds having an acceptable milling characteristic combined with the high 
oleic acid trait. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
1. Definitions 
For the purposes of the present description, the terms "cultivar" and 
"variety" are used synonymously to refer to a group of plants (e.g., 
runner-type within a species (Arachis hypogaea L.) which share certain 
constant characters that separate them from the typical form and from 
other possible varieties within that species. While possessing at least 
one distinctive trait, a variety like `M2-225` is also characterized by a 
uniformity among individuals within the variety, based primarily on the 
Mendelian segregation of traits among the progeny of succeeding 
generations. 
A "line," as distinguished from a "variety," denotes a group of plants 
which display less variation between individuals, generally (although not 
exclusively) by virtue of several generations of self-pollination. In 
addition, a "line" is defined, for the purpose of the present invention, 
sufficiently broadly to include a group of plants vegetatively propagated 
from a single parent plant, using culture techniques. 
A variety or a line is considered "true-breeding" for a particular trait if 
it is genetically homozygous for that trait to the extent that when the 
true-breeding variety or line is self-pollinated, a significant amount of 
independent segregation of the trait among the progeny is not observed. 
The content of various fatty acids, such as oleic and linoleic, which is 
characteristic of oil from a given seed sample is commonly expressed as a 
percentage of the total fatty acid fraction in the oil. This convention 
will be followed for the following description, unless otherwise 
indicated. The ratios of oleic acid content to linoleic acid content are 
calculated by dividing the linoleic acid percentage of total fatty acids 
by the like percentage of oleic acid. 
The novel peanut of the present invention reproducibly expressed the high 
oleic acid trait of the runner-type cultivar against a phenotypic 
background of acceptable seed yield, and low pod-splitting, and other 
agronomic characteristics which are sufficiently consistent for commercial 
applications. In contrast, the original cultivar did not display the high 
oleic trait. 
Embodiments of the invention provide seeds having an oleic acid content of 
at least about 80% (e.g., at least 80%, including, e.g., 80%, 81%, 82%, 
83%, 84% and 85%), at least about 81% (e.g., at least 81%), at least about 
82% (e.g., at least 82%), at least about 83% (e.g., at least 83%), at 
least about 84% (e.g., at least 84%), and at least about 85% (e.g., at 
least 85%). 
The pod splitting characteristics of embodiments of the invention can be 
compared with any other peanut variety using yield trials, both within a 
particular location and between locations. The trials can be arranged in a 
Randomized Complete Block design with replications. Each plot size had 2 
rows that were each 20 feet long. Yield is determined as the pounds of 
unhulled peanuts per acre. To measure the pod splitting characteristic, a 
500 gram sample is taken from each plot and all split pods were removed 
and weighed. A percentage of the total plot weight was calculated for the 
split pod portion. When the number of split pods was less than two, the 
sample results were recorded as &lt;0.10%. The term "low or negligible 
pod-splitting" is defined as less than 1.0% of the total plot weight being 
comprised of a split pod portion. 
Peanuts of this invention exhibit pod-splitting in substantially less than 
1.0% of the pods. Advantageous embodiments of the present invention 
provide pods where splitting occurs in less than about or equal to about 
0.99% (e.g., less than or equal to about 0.99%), less than about 0.95% 
(e.g., less than 0.95%) less than about 0.9% (e.g., less than 0.9%), less 
than about 0.8% (e.g., less than 0.8%, less than about 0.7% (e.g., less 
than 0.7%), less than about 0.5% (e.g., less than 0.5%), less than about 
0.4% (e.g., less than 0.4%), less than about 0.3% (e.g., less than 0.3%), 
and less than about 0.2% (e.g., less than 0.2%). Particularly advantageous 
embodiments of the present invention provide low or negligible 
pod-splitting. The term "low or negligible pod-splitting" is defined as 
less than 1.0% of the total plot weight being comprised of a split pod 
portion. The frequency of pod-splitting found in `M2-225` is compared to 
that found in `AT-108` and `SunOleic.RTM. 95R` in Tables 3 and 4. The 
`SunOleic.RTM. 95R` produce a total plot weight comprised of 10% or 
greater split pod portion. 
Acceptable yield means yields that are, on a weight percent basis, at least 
about 70%, or preferably higher, of the yield of a commercially available 
variety in the same market class, when grown under average growing 
conditions in a replicated field trial comparison, using a randomized 
complete block design with four replications. Commercially available 
varieties in each market class include: (1) within the runner-type market 
class: `AT-108`, `GK-7`, `Florunner`; (2) within the Virginia market 
class: `GK-3`, (3) within the Peruvian market class: `Peruvian runner`, 
(4) within the Valencia market class: `Valencia`, and (5) within the 
Spanish market class: `Spanish`, and `F435`. 
A high yield for runner type market class varieties means, on a weight 
percent basis, a yield that is greater than a yield obtained with 
`SunOleic.RTM. 95R`, when grown under average growing conditions in a 
replicated field trial comparison, using a randomized complete block 
design with four replications. Such high yields may be 5%, 10%, 15%, 20% 
or greater than `SunOleic.RTM. 95R`. 
`M2-225` seeds were deposited under the Budapest Treaty on Oct. 11, 1996, 
at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, 
Md. 20852 USA, Accession number 97762. 
2. Mutagenesis 
The use of mutagenic agents to create genetic diversity in peanuts as well 
as useful peanut mutants has been described. Norden, A. J., et al., 
Breeding of the cultivated peanut in Peanut Science and Technology, the 
entirety of which is incorporated herein by reference. Peanut mutants may 
be induced by .gamma.-irradiation according to the method of Sharma, et 
al. Qual. Plant Foods Hum. Nutr. 35:3-8 (1985), the entirety of which is 
incorporated herein by reference. Methods for inducing mutations in peanut 
seeds chemically are also available. Ashri, A. Mutation Research, 
9:473-480 (1970). 
One single plant mutation experiment was conducted in North Carolina with 
peanuts. Starting with a uniform variety of "Virginia Bunch" peanuts, 
75,000 seeds were X-rayed with doses ranging from 10,000 to 18,500 r. From 
these, 84,213 M.sub.2 plants and about 250,000 M.sub.3 plants were grown. 
Intensive mutation research on peanuts was continued over a period of 30 
or more years. 
From the progeny of the irradiated peanuts both macro- and micromutants 
were identified. The visible macromutants included various morphological 
changes in leaf size, leaf shape, branching system; induced resistance to 
leaf spot and potato leafhopper; and other plant characteristics. Many of 
the macromutations are being maintained in the peanut germ plasm 
collections. Along with the visible or macromutations, some of which were 
simply inherited, quantitative changes in the background genotypes both 
injurious and beneficial were also induced. This suggested that selection 
for the beneficial changes could lead to improvements in the genotype. 
Hybridization between selections from different families led in some cases 
to apparent yield increases, presumably due to heterosis effects of 
mutated genes. The peanut variety "NC4X", selected from the irradiated 
material because it had less pod cracking, was released and grown in 
commercial production for a limited period. Several genotypes have been 
released in which desirable mutant characteristics have been incorporated 
by hybridization. 
The radiation experiment with peanuts is important to plant breeders 
because it focused attention on the small genetic changes in quantitative 
characteristics, such as yield or seed size, that may be induced, as well 
as the macromutations. Several of the induced mutations, potato leafhopper 
resistance for example, are being incorporated into conventional breeding 
programs. On the negative side, no commercial varieties of peanuts with 
improved yields were developed from this mutation breeding experiment. 
Mutagenesis of Arachis hypogaea L. is usually performed by treating the 
seed with the mutagen, letting the surviving seeds germinate, and then 
recovering the progeny for analysis. The plant generation that grows from 
the treated seeds is referred to as the M1; it should contain heterozygote 
chimeras for any given mutation. Progeny collected after selfing are 
referred to as the M2 generation, and should be segregating both 
heterozygotes and homozygotes for a given mutation. The existence of 
heterozygotes in the M2 is particularly important if the mutation of 
interest turns out to be sterile or lethal when homozygous. 
Treating seeds with a mutagen is not equivalent to treating a single cell. 
The seed after mutagenesis will be a chimeric individual, containing some 
nonmutant cells and a variety of cells that have mutations in their DNA. 
The seed will be chimeric not only for wild type versus mutant, but will 
also be chimeric for multiple mutations. The cells that matter are the 
ones that will contribute to the next generation, the M2. Mutations that 
are in cell lineages that do not lead to the germ line will be lost. Li 
and Redei have estimated that there are two precursor cells to the germ 
line in the peanut seed, based on patterns of genetic segregation in the 
progeny of just mutagenized seeds. Studies in tobacco and maize of the 
shoot apex using chimeral/clonal-analysis/fate-mapping suggest that the 
number of cells in the seed that give rise to the upper body of the shoot 
and the reproductive organs is quite small, with three cells being a 
reasonable approximation. Poethig, R. S., Clonal analysis of cell lineage 
patterns in the shoot apical meristem of the germinating maize embryo, Am. 
J. Bot., 74. 5814, (1987); McDaniel, C. N. et al., Cell lineage patterns 
in the shoot apical meristem of the germinating maize embryo, Planta. 175: 
13 (1988); Poethig, R. S., A non-cell autonomous mutation regulating 
juvenility in maize. Nature. 336: 82 (1988). In other words, the goal of 
seed mutagenesis is to target the roughly two to three cells that will 
give rise to the reproductive tissues later in development. A second 
consequence is that a given M1 plant may have several independent mutant 
cell lineages. Seeds collected from pods on one portion of the M1 shoot 
may be genetically different from those collected at another position on 
the same shoot. 
The variety of peanut chosen as the wild type starting material deserves 
some consideration. It is worth knowing the lineage of the seeds obtained; 
i.e., where did the investigator's source get the seeds, how many times 
have they been propagated, etc? 
a. Mutagenize with DES 
Diethyl sulphate (DES), a base alkylating agent, works well in seed 
mutagenesis of Arachis hypogaea L. Expressed in terms of killing versus 
mutagenesis, as described below, DES has a high induced mutation rate 
versus its toxicity. At optimum doses for total mutant recovery, more 
mutants are likely to be obtained from DES than other mutagens. However, 
there is considerable merit to other mutagens, in spite of the efficacy of 
DES. As already discussed, for proper interpretation of a mutational 
analysis, one would prefer to have a gene knockout/amorph. DES yields some 
of these, but it also gives many hypomorphs. DES induced mutations cannot 
be rapidly characterized at the DNA level; one usually must start with 
restriction fragment length polymorphisms (RFLPs) and then walk to the 
locus in order to clone the gene. 
b. Dose of Mutagen 
Usually mutagenesis is a balance between killing the treated cells versus 
increasing the yield of mutants with a higher dosage. After mutagenesis 
one needs also to have readily scorable markers to assess the induced 
mutant frequency. An additional consideration is the frequency with which 
multiple mutants can be expected to occur, and hence the probability that 
the phenomenally interesting phenotype just discovered is the product of 
twelve interacting loci, not one. 
c. Killing Seeds vs. Creating Mutants 
One rational way to choose a dose of a mutagen is to select the dose that 
will optimize the total yield of mutants. A very simple calculation 
suggests that the optimum mutagen dose can be calibrated initially based 
on the percent seed survival. 
N=number of mutants 
N=number of seeds, alive or dead 
l=fraction of seeds alive after mutagenesis 
m=fraction of seeds mutant among those alive 
d=mutagen dose 
k=exponential survival constant 
j=linear dose response constant 
The number of mutant seeds after mutagenesis will be 
EQU M=mlN 
Assume exponentially declining survival of seeds and linearly increasing 
fraction of mutants with dose of mutagen: 
EQU 1=e.sup.-kd and m=jd 
Substitute these assumptions into the basic equation: 
EQU M=jde.sup.-kd N 
To optimize the total yield of mutants, take the derivative of M with 
respect to d, set equal to O, and then solve for d: 
EQU dM/dd=O and d=l/k 
Substituting this dose back into l gives: 
EQU l=e.sup.-k/k =d.sup.-1 =0.368 
This simple calculation thus suggests that the optimum yield of mutants, 
when considering both increasing seed death and increasing mutant yield as 
a function of mutagen dose, should occur when the mutagenized seed 
survival, relative to wild type, is about 37%. 
d. Scoring the Increase in Mutant Frequency 
The ultimate measure of success is the production of mutants. A relatively 
simple and quick method for doing this is the use of embryonic-lethals, as 
pioneered by Meinke. Embryonic-lethal mutations are, by definition, 
expressed very quickly after fertilization. They can be scored directly in 
the siliques of the M1 plants, appearing as a 3:1 segregation of pale 
white embryos instead of the normal green. Since many loci can yield 
embryonic-lethals, the frequency of this phenotype can be relatively high. 
After a successful DES mutagenesis, the percentage of M1 plants 
segregating embryonic lethals is typically 5 to 10%. Many investigators 
will do a quick dose response curve for each new batch of mutagen, to make 
sure it has the desired efficacy. It is also possible to score the 
frequency of albinos in the M2, or the frequency of albino chimeras in the 
M1, as another measure of general mutagenesis. These frequencies will be 
on the order of 1 in 5000 to 1 in 250 after a successful DES mutagenesis. 
In the terms of the previous theoretical section, scoring mutant frequency 
as a function of surviving plants is a measurement of the dose-response 
curve, while scoring mutant frequency as a function of treated seeds 
measures the combination of both dose-response and seed kill. 
e. Sample Protocol 
An overview of mutagenesis and a sample DES mutagenesis protocol are given 
below. The basic strategies can be used with a variety of chemical 
mutagens, provided that the dose of mutagen is calibrated against seed 
survival and/or increase in mutant frequency. 
i. Use of M1 Seed Pools: Independence and Sterile or Lethal Mutants 
How does one collect the seeds from the M1 plants? One method for 
harvesting seeds from M1 plants is to harvest the plants and put all of 
the M2 seeds in one bag. Alternatively, seeds from each M1 plant are 
harvested individually. The seeds are then planted in soil for selection 
or screening. Mutagenized seeds from large seeded plants are collected on 
a plant by plant basis. 
ii. Outline of Arachis hypogaea L. Seed Mutagenesis Strategy 
1. Estimate needed M1 and M2 sizes based upon the desired number of mutants 
to isolate and the expected mutant frequency. 
2. Choose mutagen based on efficacy and need for specific types of 
mutations, such as deletions, for further experiments. Find out how to 
neutralize or detoxify the mutagen if it is a chemical. 
3. Do a survival curve for the mutagen, comparing survival and germination 
frequency after treatment at a variety of doses. Pick a dose that gives 35 
to 40% survival compared to the zero dose control. 
4. Choose the pool size of M1 plants. A useful number is the square root of 
the projected total M1 population. 
5. Begin mutagenizing the seeds and plant the M1 generation. It may be 
convenient to plant all the M1 plants for a given pool in greenhouse flats 
and transplant sprouted seed to field plots. 
6. Collect M1 seeds and keep separated by plant. 
7. Plant sufficient M2 seeds to get a 95% representation of each M1 plant. 
8. Select or screen mutants as appropriate. 
9. If the phenotype of the mutant is a recessive lethal or sterile, replant 
the critical M1 plant seeds in an organized manner, so that heterozygotes 
segregating for the mutant can be identified. 
iii. Mutant Characterization 
1. If more than one mutant of similar phenotypes develops, choose only one 
mutant from each M1 plant to characterize. 
2. Backcross the mutant to wild type at least five times. This process can 
be accelerated by crossing presumptive heterozygotes and then using 
progeny analysis to check the phenotype of the heterozygotes. 
3. Begin mapping the mutant to chromosomes using standard visible, RFLP, 
selectable, or other markers. 
4. Cross independent mutants from different M1 pools with each other for a 
complementation test to determine the number of loci identified or the 
number of alleles per locus. 
5. Cross new mutants with other similar mutants to test for interactions 
between loci. 
6. Determine the developmental stage at which the mutant phenotype diverges 
from the wild type phenotype. 
f. Typical DES Mutagenesis Protocol 
Use 1.5% v/v DES (diethyl sulfate). Safety Precautions: wear gloves, work 
in a fume hood, and neutralize DES solutions, glassware, gloves, etc. with 
1M NaOH. 
0. Before doing this protocol on a large scale, estimate dose versus the 
percent survival and germination under the given conditions and current 
batch of DES. Also, check for the frequency of embryonic lethals or 
albinos induced by each dose of the mutagen. 
1. Place dry seeds in a beaker that is large relative to the volume of 
seeds. Add the DES solution, and incubate 20 min with occasional stirring. 
2. Wash seeds with distilled water for 1 h. Change the wash every 10 to 15 
min. About halfway through the washes, transfer seeds to a fresh beaker. 
3. Plant seeds in soil. Thoroughly soak the soil ahead of time and then sow 
the seeds in an even distribution across the soil. 
4. Keep careful track of the survival rate and actual number of plants in 
the M1 generation. Collect seeds from the M1. 
5. Plant sufficient M2 seeds to obtain a 99% representation of each M1 
plant. Screen or select for mutants. Keep good records of the origin of 
each batch of seeds, so that it can be determined whether similar mutants 
are independent or not. 
Obviously, collecting seeds in one pool is easier. The process of 
collecting seeds from each individual plant and then planting individually 
has multiple advantages. First, if two mutants are identified with the 
same phenotype, one can be sure they are of independent origin; if they 
fail to complement each other, then two alleles have been isolated at the 
same locus. Second, if the phenotype entails sterility or lethality, then 
the mutation can be recovered from its heterozygous sibling seeds. The 
sectors of the M1 plant containing the mutation are heterozygous, so their 
selfed progeny are naturally segregating heterozygotes for the mutation as 
well as homozygotes. The phenotypically normal progeny from the same plant 
will contain some +/m heterozygotes, which can be identified in turn based 
on analysis of their progeny. Careful bookkeeping and collecting of seeds 
from each M1 plant individually thus allow one to recover sterile or 
lethal mutants, as well as to be absolutely sure that all mutants of the 
same phenotype are independent. 
g. Biochemical Mutant Selection 
Mutants can be identified by selection. The metabolic pathways of plants 
are not as well defined as those in bacteria, yeast, or mammalian systems. 
Sometimes plants have different pathways than the textbook paradigms; 
sometimes plants have alternative pathways. The metabolic diversity of 
plants is extraordinary. For these reasons, a mutational analysis of a 
plant metabolic pathway will often help define the biochemical steps in 
that pathway. Characterization of a biochemical mutant will usually be 
more complex than simply looking up the pathway and rapidly deducing the 
afflicted gene product. To identify oil chemistry variants, oil is 
extracted from M.sub.2 seeds and analyzed to determine their fatty acid 
profile using gas chromatography. Mutants are identified based on the 
absence or superabundance of a particular chromatography peak. The success 
of this mutational analysis allowed the investigators to define the fatty 
acid biosynthetic pathway in Arabidopsis. Direct biochemical screening 
such as this can be successful if the assay is relatively quick and if a 
labor force exists to perform several thousand assays. Since multiple 
fatty acids could be monitored on one chromatogram, the screening method 
simultaneously searches for mutants in more than one gene, thereby 
increasing the probability of finding any one mutation. The delineation of 
fatty acid biosynthesis in Arabidopsis this way is an achievement that can 
serve as a useful model for peanut. 
3. Breeding Selection 
Methods for producing novel peanut hybrids through selection are known in 
the art. Each of the following references is incorporated in its entirety, 
herein, by reference: Moore, K. M. et al., J. Heredity 80(3): 252 (1989); 
Norden, A. J., Peanuts, Culture and Uses. Am. Peanut Res. and Educ. Soc., 
Stillwater, Okla. (C. T.Wilson ed. 1973); Norden, A. J. in Hybridization 
of Crop Plants (H. H. Hadley ed. 1980); Norden, A. J., et al., Breeding of 
the cultivated peanut in Peanut Science and Technology, (H. E. Pattee ed. 
1992); Norden, A. J. et al. Florida Agr. Res. 3:16-18 (1984). 
Single plants are selected through early generations of a cross. Different 
plants are combined only when they approach genetic uniformity. 
To produce the novel peanut of the present invention, runner-derived parent 
lines and varieties possessing the desired agronomic characteristics may 
be used to advantage, although runner-type germplasm can be used as 
starting material. In any case, a preferred line can be obtained, 
following conventional peanut breeding by self-pollination for a number of 
generations, to usually three or more, of runner-type progeny or of 
crosses of runner-type with other lines or varieties, selected for high 
oleic content. 
After inbreeding has progressed to the point where progeny are 
true-breeding for oleic acid content, the runner-derived starting material 
may be introgressed into diverse peanut backgrounds in the same, or 
different market classes by breeding methods known in the art. Parent 
lines and varieties meeting the requirements of the present invention, as 
set out in greater detail elsewhere herein, can be produced by 
manipulation of existing peanut materials, using other conventional 
methods, based on successive selection and inbreeding, or newly developed 
molecular approaches to altering the genetic content of plants. The 
production of suitable parent lines and varieties in accordance with the 
present invention entails the elimination of a certain amount of 
variability, at least to the extent that an appreciable number of progeny 
derived from self-pollinating at least one of the parents produce seed 
having a high oleic acid content. 
The high oleic acid trait of `M2-225` can be introgressed into diverse 
peanut backgrounds in the same, or different market classes. The high 
oleic acid trait can be introgressed into other varieties in the 
runner-type market class (A. hypogaea subsp. hypogaea var. hypogaea 
botanical type Virginia) as well as the Virginia (A. hypogaea subsp. 
hypogaea var. hypogaea botanical type Virginia), Peruvian (A. hypogaea 
subsp. hypogaea var. hypogaea botanical type Peruvian runner), Valencia 
(A. hypogaea subsp. fastigata var. fastigata botanical type Valencia) and 
Spanish (A. hypogaea subsp. fastigata var. vulgaris botanical type 
Spanish) market classes. Peanuts in the runner-type market class are the 
most commonly used varieties and are found in diverse products such as 
peanut butter, salted nuts and confectionery products. On the other hand, 
peanut varieties in the Virginia market class are largely used as salted 
nuts and in-shell market. The Valencia is largely used in peanut butter 
while the Spanish type is used in certain niche markets where small round 
peanuts are needed such as confectionery products and red skin peanuts. 
Finally, the Peruvian runner market class is grown in certain regions of 
Mexico. 
The high oleic acid trait from `M2-225` is introgressed into different 
peanut backgrounds by conventional methods well know to the skilled 
artisan in the field of peanut breeding. More specifically, crosses are 
made according to methods described by Norden, A. J., Peanuts, Culture and 
Uses, supra.. Am. Peanut Res. and Educ. Soc., Stillwater, Okla. (C. 
T.Wilson ed. 1973); Norden, A. J. in Hybridization of Crop Plants (H. H. 
Hadley ed. 1980); Norden, A. J., et al., Breeding of the cultivated peanut 
in Peanut Science and Technology, (H. E. Pattee ed. 1992); Norden, A. J. 
et al. Florida Agr. Res. 3:16-18 (1984), the entirety of each is 
incorporated by reference. Introgression of the high oleic characteristic 
has been proceeding using the traditional plant breeding cross pollination 
techniques. Crosses have been made between `M2-225` and the following 
common peanut varieties: AT 108, GK-7, VC-1, Florunner, and AT 120. 
The gene or genes responsible for the specific high oleic acid trait in 
`M2-225` can be identified using transposon mutagenesis. Methodologies for 
transposon mutagenesis are known in the art. The following references are 
incorporated by reference, in their entirety: Walbot, V., Annual Rev. 
Plant Phys. & Plant Mol. Biol. 43:49-82 (1992) Strategies for mutagenesis 
and gene cloning using transposon tagging and T-DNA insertional 
mutagenesis; Koncz, N. K. et al., Plant Mol. Biol. 20(5):963-76 (1992) 
T-DNA insertional mutagenesis in Arabidopsis--T-DNA tagging, gene tagging 
and high-frequency transformation, a review; Baker et al., PNAS 83: 
4844-4848 (1986); Yoder et al., Mol. Gen. Genet. 213: 291-296 (1988); 
Federoff, N. V., Maize transposable elements. In: Mobile DNA, Berg, D. E. 
and Howe, M. M. (Eds.) American Society for Microbiology, Washington, D.C. 
(1989); Belzile et al., Genetics 123: 181-189 (1989); Lassner et al., Mol. 
Gen. Genet. 218: 181-189 (1989). 
A peanut line is transformed using a transposon known to insert itself into 
the peanut genome. The presumptive mutants are screened for loss of the 
high oleic acid trait. Molecular methods are used to analyze and identify 
the particular locus of the insertional event. Such molecular methods 
include restriction enzyme digestion and Southern blot analysis using a 
labelled DNA fragment from the transposon as the hybridization probe to 
identify the particular DNA fragment containing the "knock-out" insertion. 
Once a DNA fragment associated with the trait is identified by transposon 
mutagenesis, the wild-type sequence is cloned from a cDNA or genomic 
library by methods well known to the skilled artisan. See Gruber, M. Y., 
et al. in Methods in Plant Molecular Biology and Biotechnology (B. R. 
Glick 1993). 
4. Fatty Acid Determinations 
Fatty acid distributions are determined by a standard procedure designated 
"CE 1-62" in OFFICIAL AND TENTATIVE METHODS, Volume 2 (1980), American Oil 
Chemists Society (AOCS). Alternatively, fatty acid analysis is performed 
on an HP 5390 gas chromatograph. 
The oil derived from the peanut seed of the present invention is of unique 
character, particularly with regard to the concentration of oleic and 
linoleic acid. A composite sample of seed from a number of plants may be 
processed according to methods well known in the art. For example, 
McWatters, K. H. et al., Potential Food Uses of Peanut Seed Proteins. ch. 
18, 689-736 in Peanut Science and Technology (H. E. Pattee, ed. 1982) and 
Fick, G. N. in U.S. Pat. No. 4,627,192, the entirety of each of which is 
hereby incorporated by reference. 
5. Genetic Engineering of Peanuts to Produce High Oleic Acid Varieties 
One means to obtain peanut oil with a higher percentage of unsaturated 
fatty acids is through the genetic engineering of plants, such as that 
described in the U.S. Pat. No. 5,510,255 to Knauf, et al. 1996. Methods 
for transformation and regeneration of peanut cells are known. Ozias, P. 
et al., Plant Science 93:185-194 (1993); Norden, A. J., et al., chapter 4, 
supra. In addition, specific genes associated with improved peanut traits 
may be introduced using peanut transformation methods known in the art. 
Ozias-Akins, P. et al., Plant Science 93:185-194 (1993). 
To genetically engineer a plant a means to transfer genetic material to the 
plant in a stable and heritable manner is required along with the nucleic 
acid sequences capable of producing the desired phenotypic result. To 
produce the desired high oleic acid/low linoleic acid phenotype, requires 
the Fatty Acid Synthetase (FAS) pathway of the peanut plant is modified to 
the extent that the ratios of reactants are modulated or changed. 
Higher plants appear to synthesize fatty acids via a common metabolic 
pathway. In developing seeds, where fatty acids attached to triglycerides 
are stored as a source of energy for further germination, the FAS pathway 
is located in the proplastids. The first step is the formation of 
acetyl-ACP (acyl carrier protein) from acetyl-CoA and ACP catalyzed by the 
enzyme, acetyl-CoA:ACP transacylase (ATA). Elongation of acetyl-ACP to 16- 
and 18-carbon fatty acids involves the cyclical action of the following 
sequence of reactions: condensation with a two-carbon unit from 
malonyl-ACP to form a beta-ketoacyl-ACP (beta-ketoayl-ACP synthase), 
reduction of the keto-function to an alcohol (beta-ketoacyl-ACP 
reductase), dehydration to form an enoyl-ACP (beta-hydroxyacyl-ACP 
dehydrase), and finally reduction of the enoyl-ACP to form the elongated 
saturated acyl-ACP (enoyl-ACP reductase), beta -ketoacyl-ACP synthase I, 
catalyzes elongation up to palmitoyl-ACP (C16:0), whereas 
beta-ketoacyl-ACP synthase II catalyzes the final elongation to 
stearoyl-ACP (C18:0). 
Common plant unsaturated fatty acids, such as oleic, linoleic and 
alpha-linolenic acids found in storage triglycerides, originate from the 
desaturation of stearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction 
catalyzed by a soluble plastid DELTA-9 desaturase (also often referred to 
as "stearoyl-ACP desaturase"). Molecular oxygen is required for 
desaturation in which reduced ferredoxin serves as an electron co-donor. 
Additional desaturation is effected sequentially by the actions of 
membrane bound DELTA-12 desaturase and DELTA -15 desaturase. These 
"desaturases" thus create mono- or polyunsaturated fatty acids 
respectively. 
Obtaining nucleic acid sequences capable of producing a phenotypic result 
in FAS, desaturation and/or incorporation of fatty acids into a glycerol 
backbone to produce an oil is subject to various obstacles including but 
not limited to the identification of metabolic factors of interest, choice 
and characterization of a protein source with useful kinetic properties, 
purification of the protein of interest to a level which will allow for 
its amino acid sequencing, utilizing amino acid sequence data to obtain a 
nucleic acid sequence capable of use as a probe to retrieve the desired 
DNA sequence, and the preparation of constructs, transformation and 
analysis of the resulting plants. 
Thus, the identification of enzyme targets and useful plant sources for 
nucleic acid sequences of such enzyme targets capable of modifying fatty 
acid compositions are needed. Ideally an enzyme target will be amenable to 
one or more applications alone or in combination with other nucleic acid 
sequences, relating to increased oleic acid and/or decreased linoleic acid 
production. Once enzyme target(s) are identified and qualified, quantities 
of protein and purification protocols are needed for sequencing. 
Ultimately, useful nucleic acid constructs having the necessary elements 
to provide a phenotypic modification and plants containing such constructs 
are needed. Battey, et al., "Genetic engineering for plant oils: potential 
and limitations," Trends in Biotech (1989) 7:122-126; Knauf, "The 
Application of Genetic Engineering to Oilseed Crops", Trends in Biotech. 
(1987) 5:40-47.

Other objects, features and advantages of the present invention will become 
apparent from the following detailed description. It should be understood, 
however, that the detailed description and the specific examples, while 
indicating preferred embodiments of the invention, are given by way of 
illustration only. Indeed, various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description. 
EXAMPLE 1 
Origin and Breeding 
A high oleic acid peanut variety is developed by chemical mutagenesis of a 
peanut runner-type variety. Currently available runner-type peanut 
varieties include `Florunner`, `Sunrunner`, `GK-7`, `Southern Runner`, 
`Sunbelt Runner`, `Okrun` and `AT-108`. Branch, W. D. et al., supra.; 
`GK-7`, supra. 
One parental variety, `AT-108`, was developed from an intervarietal cross 
between `GK-7` and `GK-3`. `GK-3` is described in Plant Variety Protection 
certificate 73000094 and `GK-7` is described in Plant Variety Protection 
certificate 82001413. `GK-3` is a very productive Virginia market-type 
peanut with spreading habit of plant growth and dense foliage. `GK-7` is a 
commercial runner-type variety with good productivity and some tolerance 
to tomato spotted wilt virus. `GK-7` and `GK-3` are cross pollinated. F1 
seed is planted in the spring and single plant selections are made and 
planted in the subsequent generations. Selection criteria include 
runner-type seed and pod size, uniformity, and yield potential. 
After continuous selection over several generations, the line is stabilized 
as a uniform productive commercial runner-type peanut line. Multi-location 
yield trials are conducted over several years, to determine if the 
resulting line has a yield advantage over other available runner-type 
peanut varieties. 
Mutagenesis 
Seed of a runner-type peanut variety is treated with diethylsulfate (DES) 
or other known seed mutagen. The seeds are shaken for 15 minutes in a 
suspension of DES or other mutagen. The seed is rinsed until no odor of 
the mutagen can be detected. The mutagen-treated seed is placed in a 
germination chamber to determine the percentage of seed that germinate. 
The concentration of mutagen used is determined by that amount that 
permits approximately 30-60% of the seed to germinate. A 1.5% suspension 
of DES (i.e., 30 ml of DES in 2 liters of water) permitted approximately 
50% of the seed to germinate. The sprouted seeds are moved to the field 
and planted in rows in late Spring/early Summer. In late summer/early 
fall, individual plants are harvested. 
The seed of each individual plant is kept separate for analysis. Three 
seeds from each plant are analyzed to determine the fatty acid composition 
of their oil. The tip of the seed is removed before the oil is extracted 
for analysis. Oil is extracted by solvent extraction. Metcalfe, L. D., et 
al., Analytical Chemistry 33(3):363-364 (1961). The fatty acid analysis is 
performed on an HP 5390 gas chromatograph. The remaining embryo ends of 
the seeds are saved for planting. 
Sampled seed containing 80% oleic acid, as a percentage of the total fatty 
acid composition in its oil, are pursued further as expressing the high 
oleic acid trait. Additional seed samples from plants producing seed with 
the high oleic acid trait also have their fatty acid profiles analyzed. 
The remaining seeds are planted in the field in the spring. The seeds 
harvested in the fall are then analyzed for fatty acid composition and all 
high oleic seeds are saved for a winter seed increase. Seed harvested in 
the spring are then analyzed to determine if they are true breeding for 
the high oleate character. 
Phenotype 
The variety is analyzed for its phenotype, as compared to other runner-type 
varieties. The traits analyzed include: color, direction of its mainstem, 
height, pod size, pod shape, seed size, seed shape, yield, pod splitting, 
fruiting habit, leaflet size and fatty acid profile. The fatty acid 
composition of oil from the stabilized variety is compared to other 
runner-type varieties. The distribution of the following fatty acids is 
determined: oleic acid, linoleic acid, palmitic acid, stearic acid, 
arachidic acid, eicosoenic acid, behenic acid, and lignoceric acid. Also, 
the variation in oleic acid and linoleic acid found in the oil from the 
stabilized variety is determined. 
EXAMPLE 2 
Origin and Breeding of `M2-225` 
Definitions 
The term "oleic acid content" means the quantity of oleic acid compared to 
the total fatty acid content of the seed. The term "linoleic acid content" 
means the quantity of linoleic acid compared to the total fatty acid 
content of the seed. The term "total fatty acid content of the seed" means 
that amount of fatty acid extracted from a seed by the method of Metcalf 
et al., supra. The term "ratio of the amount of oleic acid to linoleic 
acid in the seed" means a comparison of the oleic acid content to the 
linoleic acid content. The term "peanut plant having the characteristics 
of a line designated `M2-225`" means a peanut plant that produces a 
runner-type market-type peanut in which the peanut seeds have a high oleic 
acid content of at least 80%, negligible to no preharvest pod splitting 
and acceptable or high yield. 
The term "peanut plant of the runner-type genetic background" means a 
peanut plant that was derived from runner market-type parental lines 
(i.e., varieties in the runner-type market class (A. hypogaea subsp. 
hypogaea var. hypogaea botanical type Virginia). Examples of runner 
market-type parental lines include `AT-108`, `Florunner`, `Sunrunner`, 
`GK-7`, `Southern Runner`, `Sunbelt Runner` and `Okrun`. The term "low or 
negligible pod splitting" means no pod-splitting or less than 1.0% of the 
total plot weight being comprised of a split pod portion, where the 
percentage of the total plot weight was calculated for the split pod 
portion on a weight basis. 
The peanut variety, `M2-225` was developed by chemical mutagenesis of the 
runner-type variety `AT-108`. The parental variety, `AT-108`, was 
developed from an intervarietal cross between `GK-7` and `GK-3`. `GK-3` is 
a very productive Virginia market-type peanut with spreading habit of 
plant growth and dense foliage. `GK-7` is a commercial runner-type variety 
with good productivity and some tolerance to tomato spotted wilt virus. 
Cross pollination of `GK-3` and `GK-7` yielded F1 seed. F1 seed was planted 
in the spring and single plant selections were made and planted in the 
subsequent generations. Selection criteria were runner-type seed and pod 
size, uniformity, and yield potential. After continuous selection over 
nine generations, the line was stabilized as a uniform productive 
commercial runner-type peanut line. Over five years of multi-location 
yield trials, the results indicate a yield advantage over currently 
available runner-type peanut varieties. 
Mutagenesis 
Seed of `AT-108` was treated in the spring with diethylsulfate (DES). The 
seeds were shaken for 15 minutes in a suspension of 30 ml of DES in 2 
liters of water. The seed was then rinsed until no odor of DES could be 
detected. This seed was placed in a germination chamber and approximately 
50% of the seed germinated. The sprouted seeds were moved to the field and 
planted in rows in June. In September, approximately 1700 individual 
plants were harvested. The seed of each individual plant was kept separate 
for analysis. Three seed from each plant were analyzed for fatty acid 
composition of the oil. This was done by removing the tip of the seed and 
extracting the oil for analysis. The fatty acid analysis was performed on 
an HP 5390 gas chromatograph. The remaining embryo ends of the seeds were 
saved for planting. 
Three seeds from the plant number 225 were analyzed. It was found that one 
of the three seeds sampled contained 80% oleic acid as a portion of the 
total fatty acid composition of its oil. Additional seed samples from 
plant number 225 contained only wild type fatty acid profiles. The 
remaining seeds were planted in the field in the Spring. The seeds 
harvested in the fall were analyzed for fatty acid composition and all 
high oleic seeds were saved for winter seed increase. Seed harvested in 
the spring was analyzed and found to be true breeding for the high oleate 
character. 
The peanut variety, `M2-225` is phenotypically most similar to `AT-108`. 
`M2-225` is a commercial runner-type market-type peanut. Variety `M2-225` 
has an alternate fruiting habit as with other Virginia botanical types. It 
is dark green in color and similar to `GK-7` and `AT-108`. It has an erect 
mainstem but not as tall as `GK-7`. The mainstem but averages about 22.1 
cm in height as compared to 39.6 for `GK-7`. Pods and seeds are similar in 
size and shape to `AT-108`. There is no fruiting on the mainstem and 
fruiting occurs at alternate nodes. The leaflet averages 2.23 cm in width 
and 4.83 cm in length. This is smaller than `GK-7` which has leaflet width 
of 2.7 cm by 6.65 cm in length. 
In three generations of observations, there has been low or negligible pod 
splitting. Seed and pod size is uniform and similar to `AT-108`. Yield 
appears to be similar to `AT-108`, which is an average of 5 to 8% greater 
than `GK-7`. The most distinctive characteristic of `M2-225` is its fatty 
acid profile, particularly the high oleic acid level, which is 82% in the 
oil. The `M2-225` variety also has a acceptable or high yield and no pod 
splitting. 
Table 1 compares the fatty acid composition of oil from `M2-225` and its 
parental line `AT-108`. As a percentage of total fatty acids, `M2-225` 
exhibits the following distribution of fatty acids: 81.65% oleic acid, 
2.45% linoleic acid, 5.6% palmitic acid, 2.39% stearic acid, 1.66% 
arachidic acid, 2.09% eicosoenic acid, 2.54% behenic acid, and 2.25% 
lignoceric acid. By contrast, the parental variety `AT-108` exhibits the 
following distribution of fatty acids: 52.78% oleic acid, 25.32% linoleic 
acid, 9.80% palmitic acid, 3.25% stearic acid, 2.05% arachidic acid, 1.92% 
eicosoenic acid, 2.75% behenic acid, and 2.25% lignoceric acid. 
TABLE 1 
______________________________________ 
A Comparison of Fatty Acid Composition of 
Oil From `M2-255` and its Parental Variety 
% Fatty Acids 
Pal- Ste- Lin- Ara- Eico- Be- Ligno- 
Variety 
mitic aric Oleic 
oleic 
chidic 
soenic 
henic 
ceric 
______________________________________ 
`M2- 5.4 2.36 81.65 
2.05 1.66 2.09 2.54 2.25 
225` 
AT-108 
9.80 3.25 52.78 
25.32 
2.05 1.92 2.75 2.13 
______________________________________ 
Table 2 compares the variation in oleic acid and linoleic acid found in the 
oil from `M2-225` and its parental line `AT-108`. As a percentage of total 
fatty acids, `M2-225` exhibits from about 77.25% to 86.23% oleic acid as 
compared to the range of variation found in `AT-108` from about 48.25% to 
about 54.53% oleic acid. `M2-225` exhibits a lower concentration of 
linoleic acid from about 1.75% to 3.1% as compared to the range of 
linoleic acid concentration found in `AT-108` from about 32.22% to 22.34%. 
TABLE 2 
______________________________________ 
A Comparison of the Variation in Oleic Acid and 
Linoleic Acid Composition of `M2-225` and `AT-108` 
Varieties Oleic Acid Linoleic Acid 
______________________________________ 
`M2-225` 77.25 to 86.23% 
1.75 to 3.1% 
`AT-108` 48.25 to 54.53% 
32.3 
______________________________________ 
The pod splitting characteristics of the `M2-225` variety was compared to 
the "SunOleic.RTM. 95R" variety. Yield trials for the `M2-225`, AT 108 and 
"SunOleic.RTM. 95R" varieties at three locations provided the source of 
samples for pod splitting evaluation. The trials were arranged in a 
Randomized Complete Block design with four replications. Each plot size 
had 2 rows that were each 20 feet long. A 500 gram sample was taken from 
each plot and all split pods were removed and weighed. A percentage of the 
total plot weight was calculated for the split pod portion. When the 
number of split pods was less than two, the sample results were recorded 
as &lt;0.10%. The data is presented in Tables 3 and 4. `M2-225` has less than 
0.1-0.3% pod splitting, while SunOleic.RTM. 95R has 1.0-7.0% pod 
splitting. The `M2-225` variety has a significant and advantageous trait 
due to its significantly lower pod splitting characteristic. The frequency 
of pod-splitting found in `M2-225` is compared to that found in `AT-108` 
and `SunOleic.RTM. 95R` in Tables 3 and 4. 
The Randomized Complete Block design with four replications at each of 
three locations provided the yield data for the `M2-225`, AT 108 and 
"SunOleic.RTM. 95R" varieties. The yields for each of these varieties was 
recorded in pounds of unhulled peanuts per acre, as presented in Table 5. 
The `M2-225` variety is significantly higher yielding than the 
"SunOleic.RTM. 95R" variety. `M2-225` does not have a significantly 
different yield from the source variety, AT108. 
TABLE 3 
__________________________________________________________________________ 
A Comparison of the Objective Characteristics of `M2- 
225` to Other Runner-type Peanut Seeds and Plants 
Oleic/ Main 
% % linoleic 
Iodine 
Shelling 
5 mk 
EIK 
stem 
% Pod 
Variety Oil 
Protein 
ratio 
number 
% % % height 
splitting 
__________________________________________________________________________ 
`M2-225` 
52.0 
26 50 74 78 76 -- 21 &lt;0.10% 
`AT 108` 
52. 
26 1.9 94 78 76 -- 21 &lt;0.10% 
`Sun Oleic 95 R` 
47.0 
29 23 77 78 74 -- 49 1.00-7.00% 
__________________________________________________________________________ 
TABLE 4 
______________________________________ 
A comparison of the pod splitting characteristic of 
`M2-225` and `SunOleic .RTM. 95R` 
% Pod Splitting 
Location Replication `M2-225` SunOleic 
______________________________________ 
Ashburn, GA 
1 &lt;0.10% 3.00% 
2 &lt;0.10% 4.00% 
3 &lt;0.10% 5.00% 
4 &lt;0.10% 7.00% 
Statesboro, GA 
1 &lt;0.10% 1.00% 
2 &lt;0.10% 2.00% 
3 &lt;0.10% 3.00% 
4 &lt;0.10% 5.00% 
Pleasanton, TX 
1 &lt;0.10% 6.00% 
2 &lt;0.10% 2.00% 
3 &lt;0.10% 3.00% 
4 &lt;0.10% 4.00% 
______________________________________ 
TABLE 5 
______________________________________ 
Yield comparision of `M2-225`, `AT 108` and `Sunoleic .RTM. 95R` 
(pounds of unhulled peanuts per acre) 
Location `M2-225` `AT 1-8` `SunOleic .RTM. 95R` 
______________________________________ 
Ashburn, GA 
3539 3655 3230 
Statesboro, GA 
3856 3745 3059 
Pleasanton, TX 
4590 4325 4120 
______________________________________ 
Milling characteristics were evaluated on `M2-225`, AT 108, and 
`SunOleic.RTM. 95R` to determine the percentage of total seed that 
comprised of: (1) Sound Mature Kernels (SMK); (2) Sound Splits (SS); (3) 
Total Sound Mature Kernels (TSMK) and (4) Other Kernels (OK). This milling 
characteristics are shown in Table 6. Blanchability was determined by 
running a 500 g sample through a pilot plant blancher and the percentage 
of kernels retaining seed coat was calculated. Grades and kernel sizing 
were determined by the standard USDA peanut grading and sizing procedure. 
The blanchability and grades are shown in Table 7. 
TABLE 6 
__________________________________________________________________________ 
Milling Characteristics 
% % 
% % % % % % % Medium 
Jumbo 
Hulls 
SMK 
SS 
TSMK 
OK Damage 
No. 1's 
Runners 
Runners 
__________________________________________________________________________ 
Entry 23.0 
71.0 
3.0 
74.0 
3.0 
4.0 8.0 34.0 
28.0 
`M2-225` 
26.0 
70.0 
2.0 
72.0 
5.0 
3.0 8.0 33.0 
25.0 
`Sunoleic .RTM. 95R` 
24.0 
69.0 
1.0 
70.0 
5.0 
3.0 10.0 
34.0 
25.0 
__________________________________________________________________________ 
TABLE 7 
______________________________________ 
Blanchability 
Entry % Unblanched Kernels 
______________________________________ 
`M2-225` 0% 
SunOleic .RTM. 95R 
0.10% 
AT 108 0% 
______________________________________ 
EXAMPLE 3 
Mode of Inheritance and Breeding of New High Oleic Peanut Varieties 
Cross pollinations were made between the mutant `M2-225` line and the 
parental variety AT 108. Both reciprocal crosses were made. The F2 seed 
from that cross was recovered and fatty acid analysis was performed using 
an HP 5890 gas chromatograph. The results are summarized in Table 8. The 
X2 values indicate the segregating ratios to be 3:1. The data therefore 
show that the `M2-225` high oleic characteristic fits a simple Mendelian 
model of a single recessive gene that must be homozygous for expression. 
In view of the genetics of the high oleic trait characterized in variety 
`M2-225`, new high oleic varieties of peanut can be easily made. The high 
oleic trait from `M2-225,` which is controlled by a single recessive gene, 
can be predictably and reproducibly introgressed into diverse genetic 
backgrounds of peanut using methods well know to the skilled artisan. For 
example, `M2-225` is crossed as the male or female parent to another 
peanut line with desired agronomic characteristics. Progeny are selected 
and selfed to produce lines that are homozygous recessive for the high 
oleic trait combined with other desired traits. Alternatively, the progeny 
are backcrossed to one of the parents over one or more generations prior 
to the step of selfing. The skilled artisan can envision many other 
breeding strategies in which the high oleic trait is combined with other 
agronomic characteristics to produce new peanut cultures. 
TABLE 8 
__________________________________________________________________________ 
High Oleic 
Parents Low Oleic 
High Oleic 
Low Oleic 
Total 
Female 
Male Observed 
Observed 
Expected 
Expected 
X2 
__________________________________________________________________________ 
AT 108 
`M2-225` 
1 40 10 37.5 12.6 50 0.4142162 
2 4 3 5.25 1.75 7 0.2752338 
3 23 9 24 8 32 0.6830914 
`M2-225` 
AT 108 
1 27 13 30 10 40 0.2733219 
2 89 25 85.5 28.5 114 0.4490299 
3 19 6 16.5 5.5 22 0.5148277 
__________________________________________________________________________ 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those in the art to which the 
invention pertains. All publications and patent applications are herein 
incorporated by reference to the same extent as if each individual 
publication or patent application were specifically and individually 
indicated to be incorporated by reference in its entirety.