Flower color inheritance in alfalfa has been studied more
extensively than any of its other qualitative characters. Alfalfa
flower color, because of its extensive variation, has been used
to identify variety, plant breeding and genetic materials, and to
distinguish between self-pollinated plants and those resulting
from cross-pollination (6, 8).1 According to Clement
(3) and Pedersen (11), flower
color is often an important factor in the attractiveness of
alfalfa clones to insect pollinators. The usefulness and
importance of alfalfa flower color has been established; but the
many hues, patterns, and intensities of purple, yellow, and
combinations of purple and yellow pigments have caused difficulty
in phenotypic classification.
This handbook briefly describes the genetics of alfalfa flower
color, discusses factors that influence alfalfa flower color
classification, presents a system for visually classifying flower
color, and presents color prints to illustrate the various flower
FLOWER COLOR INHERITANCE
Cultivated alfalfa is an autotetraploid. Difficulties in
working with tetraploid genetics have limited the number of
inheritance studies in alfalfa, especially on traits controlled
by two or more genes, such as flower color. Results from genetic
studies have demonstrated a close relationship between the
inheritance patterns for purple and yellow flower color in
diploid and tetraploid alfalfa. Therefore, in many instances,
diploid species of Medicago falcata L. and M. sativa
L. have been utilized to help interpret the more complicated
inheritance patterns. All available information on the
inheritance of alfalfa flower color was summarized and
reevaluated by Barnes (1). The data indicated
that flower color in alfalfa was conditioned by three types of
major gene effects and several types of modifying genes.
The major genes include one recessive gene (c) with tetrasomic
inheritance. The c gene is a basic color factor that, in the
homozygous recessive condition, produces white-flowered plants
that are devoid of all anthocyanin pigmentation in flowers,
seeds, stems, leaves, and roots. Purple flower color pigments are
expressed in the presence of one or more dominant C alleles.
Chemical analyses of diploid alfalfa indicated that purple flower
color is due to a group of three anthocyanin pigments identified
as malvidin, petunidin, and delphinidin diglucosides. These three
pigments are inherited as a unit and are expressed when one or
more dominant alleles of P gene are present. The
tetrasomically inherited P gene is present in purple-flowered M.
sativa plants. The homozygous recessive p genotype in
the presence of dominant C alleles produces a cream flower color
that does not interfere with anthocyanin pigmentation in floral
veins, seeds, leaves, stems, and roots.
Genes responsible for producing yellow pigments can be classified
as the third major type of alfalfa flower color pigmentation. The
origin of yellow flower color in alfalfa can be traced to M.
falcata. In diploid alfalfa the yellow pigments have been
identified as being primarily xanthophyll with a small amount of
þ carotene present. Diploid genetic data suggest that yellow
flower color is controlled by at least three and probably four
genes with accumulative effects. Only limited data are available
concerning the inheritance of yellow flower color pigments in
tetraploid alfalfa. However, a pattern of tetrasomic inheritance
for at least two genes (Y1 andY2) with accumulative
effects is hypothesized.2 The
homozygous recessive condition of both the P and Y
genes produces a cream-flowered plant. Joint pigmentation of the P
and Y genes produces phenotypes that have been commonly
referred to as variegated flowers. Variegated flower colors may
range from a very dark blue color to a green or yellow green.
Essentially no information is available regarding the inheritance
of color intensity. Data presented by Soudah3
suggested that the effects of the P gene were
accumulative and that the various intensities of purple could be
due to specific genotypes; that is, pppp = cream, Pppp
= light purple, PPpp = purple, PPPp =deep purple,
and PPPP = very deep purple.
1 Numbers in parentheses refer to Literature Cited
2 Several loci are responsible for
the expression of yellow flower color pigments, but for brevity
in the following text, only one representative gene will be used.
3 Soudah, R. E. TETRASOMIC
INHERITANCE OF FLOWER COLOR IN ALFALFA, Medicago sativa L.
(Unpublished Ph.D. thesis. Copy on file Dept. of Soils and Crops,
Rutgers Univ., New Brunswick, N. J.). 1962.
VISUAL CLASSIFICATION OF ALFALFA FLOWER COLOR
However, progeny tests needed to verify
or disprove the theory have not been conducted. Other evidence is
available that suggests accumulative effects of dominant alleles
do exist in alfalfa flower color; that is, crosses between cream
flowered plants and dark-orange-yellow plants produce progenies
with intermediate yellow flower color, and subsequent F:
segregations produce various intensities of yellow flower color
that can best be explained by assuming accumulative effects of
the Y genes. Modifying genes must be considered as another source
of factors that can affect flower color intensity.
Several types of modifying factors are known to exist in alfalfa.
Nine anthoxanthin pigments have been chemically identified as
flower color modifying pigments in diploid alfalfa
(4). Six of the anthoxanthins were quercetin glycosides, and
three were kaempferol glycosides. None of these pigments appeared
to impart a phenotypically significant color of its own, but they
tended to act as modifying genes when copigmented with the P
or Y genes. No association or linkage has been shown
between any of the modifying genes (5).
Two additional types of modifying flower color characteristics
are purple bud color and floral vein color. Barnes
and Cleveland (2) reported that in diploids they had not
observed the purple bud color trait in unadulterated M.
falcata, but it was often observed in yellow- and
cream-flowered progeny from advanced generations of hybrids
between M. falcata and M. sativa. The trait was
shown to be conditioned by two complementary genes. The assumed
genotype of pure M. falcata was BfBf bsbs and the
assumed genotype of M. sativa was bfbf BsBs. Purple
bud color is produced by Bf __ Bs__genotypes. Barnes and Cleveland also established that the
presence of pigmented veins in the standard petals of
yellow-flowered diploid alfalfa was controlled by the presence of
one or more dominant alleles of two duplicate genes (Vs1and
Vs2). When both genes were homozygous recessive, no floral vein
pigmentation was observed. Barnes and Cleveland suggested
that vein color in the wing petals was conditioned by a separate
Except for the pure purple, yellow, cream and white flower
colors, most of the flower color phenotypes are caused by varying
concentrations of two or more pigments being present in the same
flower. The most distinctive differences in alfalfa are due to
the joint pigmentation of purple and yellow pigments. Lesins (9) suggested that in copigmentation the
purple pigments were in the epidermal layer of the flower, which
in turn was over a back-ground of yellow. However, Buker4
suggested that the yellow and purple pigments were both
present in the same cells and that the mosaic effect observed by
Lesins was caused by cell injury that occurred when the epidermal
cells were removed.
Extremely large numbers of copigmented genotypes are possible
when it is considered that as many as five genes with major
effects and numerous genes with modifying effects, all with
tetrasomic inheritance, exist. However, many of the differences
between phenotypes are very subtle and not obvious to the naked
eye. Therefore, when differences in joint pigmentation are
considered without the benefit of chemical analyses, similar
phenotypes can be placed into several relatively distinct groups.
These groups, generally, can be described as blue, blue green,
green, and yellow green.
Intensity of flower color varies considerably, regardless of
the type of pigments present in the flower. Purple pigmentation
varies among plants - from a very deep purple to a very light
purple. Similarly, yellow pigmentation can vary from a dark
orange yellow (typical of M. falcata) to a very light
yellow. The inheritance of flower color intensity is not fully
understood, but it has been suggested that flower color intensity
is influenced by the accumulative effects of the dominant alleles
of the P and Y genes. Sheridan and
McKee (13) used colorimetric measurements to determine
the effect of soil fertility, soil pH, and light on the intensity
of purple flower color. Only light appeared to be a significant
factor. Purple flowers from field-grown plants were generally
lighter than flowers grown in the greenhouse. Even though
environment can influence them, flower color intensities are
stable enough to be used in establishing subclasses within the
primary classes of purple, yellow, and variegated. However, color
intensity could be responsible for misclassification of flower
color if a plant has a very intense purple color that masks a
very low level of yellow pigmentation. Such a plant would be
classified as purple instead of variegated.
4Buker, R. J. FLOWER COLOR
INHERITANCE IN DIPLOID ALFALFA. (Unpublished M.S. thesis. Copy on
file Dept. of Agronomy, Purdue Univ., Lafayette, Ind.) 1960.
Purple bud color is not conditioned by the major purple flower
color gene (P); but to avoid misclassification, bud color
should be considered when flower colors are observed. Purple bud
color is characterized as having visible anthocyanin pigments at
the tip of the flower bud (pl. 1, A).
This pigmentation usually disappears as the flower opens. The
purple bud trait is masked in purple-flowered plants but is very
obvious on plants that have yellow or cream flower colors.
Because the pigments are faint and disappear rapidly, they do not
usually interfere with flower color classification. However,
cream- and yellow-flowered plants have been observed in which the
purple bud color is very intense and traces of purple pigments
are visible several days after the flowers have opened. If this
occurs, a decision must be made to determine whether the purple
pigment is caused by the bud color or by the basic flower color
gene. The best method for determining the cause is to observe the
distribution of the pigment in the flower. If the purple pigment
is uniformly distributed throughout the opened flower, it can be
assumed that the color is attributed to the P gene.
However, if the color is concentrated only at the tip and edges
of the standard petal and wings, the color can be attributed to
the bud color genes.
The life of an untripped alfalfa flower ranges from 8 to 15
days, according to Hanson (7). During this
period, the flower color of each floret is continually changing.
Many, if not all, of the purple pigments are very sensitive to
sunlight and will begin to fade soon after the flower opens. The
yellow pigments appear to be more stable than the purple
pigments. Because of the aging process in alfalfa flowers, the
same age flowers must be used for all flower color
classification. Flower age can be readily established by the
position of a floret on a raceme (pl. 1, C).
One-day old flowers are the best age for flower color
classification in the field. In the greenhouse, flower colors may
remain stable for 2 or 3 days, depending on the conditions. Even
though 1- or 2-day old flowers are used for classification, it is
a good practice to observe several faded flowers on the same
plant for traces of yellow pigments. This essentially eliminates
the misclassification of plants that may appear to be purple at
first glance, but are actually variegated (joint pigmentation of
purple and yellow pigments).
Irregular Petal Color Patterns
Alfalfa flowers are formed by five petals: two wing petals,
two keel petals, and a standard petal. All of the petals are
uniformly colored in most alfalfa flowers. However, irregular
color patterns can be observed in some purple pigmented flowers.
The most frequently observed irregularity is in keel petals whose
tops are more or less intensely pigmented than the rest of the
flower (pl. 1, B). An unusual
mutant, characterized by darkly pigmented wing petals, cream
colored keel, and standard petals, is shown in plate 2, A. Partial pigmentation of the
standard petal (pl. 2, B) is
another unusual color pattern. None of these traits can be
related to known flower color patterns, because their inheritance
has not been studied. Practical judgment must be used when
classifying unusual flower colors. The flowers in plates 2, A and B were classified as
having purple flower color, because they lacked all traces of
yellow pigments and could not be classified as either cream or
Most alfalfa flowers have pigmented veins in
the standard petals, and a few plants have pigmented veins in the
wing petals. In yellow and cream-colored flowers the veins are
usually brown (pl. 2, C), while
in purple pigmented flowers the veins are purple (pl. 2, D). Vein color usually does
not contribute a great deal to the total flower color, but the
presence or absence of pigmented veins or degree of vein
pigmentation can be very useful identification characteristics.
The presence of brown pigmented floral veins in an otherwise
'white' flower denotes cream flower color. The presence of purple
pigmented floral veins in an otherwise 'white' or cream flower
generally denotes that the flower color should be classified as a
very light purple (pl. 2, D).
However, it should be pointed out that even though purple vein
color and purple flower color are closely associated, no studies
have yet been conducted to demonstrate pleiotropy.
SYSTEMS FOR FLOWER
Previous Classification Systems
A number of methods have been used for scoring alfalfa flower
color, such as the British Color Council
Horticultural Color Chart (5), Nickerson
Color Fan (10), and the Munsel
Color Key (3). However, such systems based on solid colors
have usually proved difficult to use, because age of flowers,
variable color patterns, and vein pigmentation tend to influence
the results and make classification very confusing.
Arbitrary systems of visual classification have often been
originated by researchers for special studies. Three of these
systems are presented in table 1. Several additional schemes no
doubt can be found in the literature. In all of the
classification systems described, flower colors have been
arranged according to shade and intensity of purple, yellow, and
variegation (joint pigmentation of yellow and purple pigments).
Each Scheme phenotypically classified plants, but the numerical
scores usually did not reflect any genotypic relationship among
the various colors. Another difficulty with the arbitrary
classification systems is the discrepancy in descriptions and
interpretations of colors among workers. What is called blue by
one worker may be considered as purple by another.
New Classification System
Before attempting to design a new system for classifying
alfalfa flower color, I asked alfalfa scientists what type
systems would be most useful to them. The consensus of opinion
was that we needed a system of only a few phenotypic classes,
TABLE 1.-- Three previously proposed systems of flower
color rating for visually classifying alfalfa flower color1
||Deep reddish purple
|| Much variegated
||White or cream
||Strong yellow green
||Strong greenish yellow
|| Light purple
1, System proposed by Nittler, McKee,
and Newcomer (10); 2, system used by North Central Regional
Plant Introduction Station, Ames, Iowa; 3, unpublished system
used by D. K. Barnes, L. J. Elling, and A. G. Peterson in studies
on the attractiveness of alfalfa clones to pollinating insects,
Univ. of Minnesota.
representing specific genotypes arranged in order of their
genetic relationship. The system should easily and accurately
classify the flower color of individual alfalfa plants or plant
populations by a numerical rating so that data could be arrayed
according to percent of plants in each flower color class;
population means could be obtained and analyzed statistically; or
flower color data could be stored in a data retrieval system for
After considering the proposed requirements for a flower color
classification system and the problems involved with phenotypic
classification of alfalfa flower color, the writer designed the
visual classification system in table 2. The rating scale is
similar in some respects to those of previous systems; however,
primary emphasis is given to the effects of major genes and less
emphasis to effects of modifying genes. The proposed system is
intended to be more meaningful than previous systems for
comparing or averaging flower color scores of individual plants
of a variety or plant introduction. The proposed system deals
more with gene frequency of several major loci than with various
allelic interactions at any one locus. Also, the system is
flexible because it can be used either as a five-class system for
general studies; or, in cases of clonal identification and
critical genetic studies, the secondary classes can be used.
As mentioned earlier, word descriptions of color can sometimes be
misleading because all people do not associate colors in the same
manner. In this handbook, photographs of each flower color
subclass serve as color guides. Variations for color hue and
color patterns occur within nearly all subclasses; therefore, it
was postulated that groups of racemes illustrated the normal
variability within each subclass better than could be done by
using only one typical raceme.
The theory for arranging the order of the primary flower color
groups as presented in table 2 was based on the fact that pure M.
sativa and pure M. falcata have dark purple and dark
yellow flowers, respectively. Variegated colors result from
intercrosses of purple and yellow flower colors. Therefore,
purple and yellow flower color classes should be located on
opposite ends of any color scale with the variegated types in the
center. The next consideration was where to place white- and
cream-flowered plants. Usually these plants make up less than 1
percent of a population, but they each represent distinct
genotypes and must be given a place in any flower color
TABLE 2.--Proposed scale for visually scoring alfalfa flower
color. Scale is based on genotype as well as phenotype
characteristics of the alfalfa plant
||Flower color class and subclass
||Purple or violet
||C___ P___ Y___
||Dark purple variegated
||Dark yellow green
||Light yellow green
||C___ pppp yyyy
||C___ pppp Y___
||cccc P___ yyyy
White flower color is produced by the homozygous recessive
genotype of the basic color factor (c) which blocks the
expression of all purple and yellow flower color pigments. Since
white flower color does not give any identity of the genotypes
for either the P or Y genes, it should be placed at
one end of the scale to denote a special genotype. All reported
cases of naturally occurring white flower color have been found
in M. sativa. For this reason, the white-flowered class
could be placed at the purple end of the color scale. However, it
has been the consensus of a number of plant breeders that they
would prefer purple to be the number I class, because of prior
usage. Therefore, white flower color was placed at the other end
of the scale in class 5. The cream-flowered class was placed
between the variegated and yellow-flowered groups because of its
assumed hybrid origin and similarity to the very light yellow
flower color class.
The only primary flower color classes used in table 2 that may be
troublesome to correctly identify are the creams (pl. 3, A) and the whites (pl. 3, B). White-flowered plants
lack the basic color factor and so are devoid of anthocyanin
pigmentation in all tissues.
The genotype of the cream-flowered plants does not affect
color production of any organs of the plant except the flower. In
direct comparison of white and cream flowers, the cream flowers
usually will be more ivory colored than true white. However, in
cases when white flowers are not available for comparisons, the
presence of pigmented floral veins or purple bud color can be
used as evidence of cream flower color. White flowered plants
occur very seldom in natural populations, so as a general rule,
plants lacking purple and yellow pigments are usually cream
rather than white. If white flowered types are found that lack
pigmented floral veins, positive identification can be made by
producing a few seed to check for white seed color or by testing
stem tissue for the presence of anthocyanin.
Most of the flower color subclasses used in table 2 are
relatively easy to identify. The four purple subclasses (pl. 3, C, D, E, and F) are differentiated
according to color intensity variations; differences in hues of
purple or violet are not considered. The variegated class
consists of nine heterogeneous subclasses, all of which are
characterized by varying degrees of purple and yellow copigmentation. Flowers in the dark purple variegated subclass (pl. 4, A) are usually dark purple when
they first open, but the purple pigments fade with age and the
flowers become a smudgy purple with faint traces of yellow
pigmentation. The maroon class (pl. 4, B) occurs
rather infrequently and is characterized by obvious joint purple
and yellow pigmentation that lacks any trace of blue or green
color. The two blue subclasses (pl. 4, C
and D) are differentiated from each other on the basis of
color intensity. The blue-green subclasses (pl.
4, E and F) are quite variable in color patterns, but they
have a decided green color over a blue background.
The blue-green flower colors are also separated by color
intensity into dark and light subclasses. Plate
5, A, illustrates the green subclass. The green color is
typical of F1 hybrids between M. sativa and M. falcata.
The yellow-green subclasses (pl. 5, B
and C) are predominantly yellow pigmented flowers with
varying degrees of purple and green pigmentation. Intensity of
yellow color separates the dark and light yellow green
subclasses. The four yellow subclasses (pl.
6, A, B, C, and D), like the purple subclasses, are
differentiated only according to color intensity. The
orange-yellow subclass is characteristic of unadulterated M. falcata
Differences in scores among observers occurred infrequently
during trial runs with the new Classification system. No
differences appeared when the five-class primary scale was used,
and usually only differences of 0.1were observed when the
secondary scale was used. The system was adopted by the
Twenty-first Alfalfa Improvement Conference5. It is
presently being used by the North Central Regional Plant
Introduction Station of the U. S. Department of Agriculture in
their uniform recording and retrieval system for use in
evaluating alfalfa. The system was successfully used by the NC-83
technical committee in a study to determine whether associations
exist between seed yield in the North Central States, California,
5 W. H. Skrdla and others. Report
of the committee on development of a coordinated system of data
retrieval for alfalfa introductions. Report of the Twenty-first
Alfalfa Improvement Conference. July 9-11, 1968, Reno, Nev.
||Barnes, D. K. 1966. FLOWER COLOR INHERITANCE IN
DIPLOID AND TETRAPLOID ALFALFA: A REEVALUATION. U.S.
Dept. Agr. Tech. Bull. 1353, 26 pp.
||__________, and Cleveland, R. W. 1964.
FLORAL-BUD-COLOR AND VEIN-COLOR INHERITANCE IN DIPLOID
ALFALFA. Crop. Sci. 4: 174-177.
||Clement, W. M., Jr. 1965. FLOWER COLOR, A FACTOR IN
ATTRACTIVENESS OF ALFALFA CLONES FOR HONEY BEES. Crop
Sci. 5: 267-268.
||Cooper, R. L., and Elliott, F. C. 1964. FLOWER COLOR
PIGMENTS IN DIPLOID ALFALFA. Crop Sci. 4: 367-371.
||____________1965. INHERITANCE OF FLOWER PIGMENTS IN
DIPLOID ALFALFA AND THEIR RELATIONSHIP TO FLOWER COLOR
INHERITANCE. Crop Sci. 5: 63-69.
||Gartner, Alvro, and Davis, R.L. 1965. EFFECTS OF
SELF-COMPATIBILITY ON CHANCE CROSSING IN Medicago
sativa L. Agron. Abstr. p. 12
||Hanson,C.H. 1961. LONGEVITY OF POLLEN AND OVARIES OF
ALFALFA. Crop Sci. 1: 114-116.
||Hanson, C. H., Graumann, H. 0., Elling, L. J., and
others. 1964. PERFORMANCE OF TWO-CLONE CROSSES IN ALFALFA
AND AN UNANTICIPATED SELF-POLLINATION PROBLEM. U.S. Dept.
Agr. Tech. Bull. 1300. 46 pp.
||Lesins, K. 1956. SOMATIC FLOWER COLOR MUTATIONS IN
ALFALFA. J. Hered. 47: 171 179.
||Nittier, L. W., McKee, G. W., and Newcomer, J. L.
1964. PRINCIPLES AND METHODS OF TESTING ALFALFA SEED FOR
VARIETAL PURITY. New York State Agr. Expt. Sra. Bull.
807. 46 pp.
||Pedersen, M. W. 1967. CROSS-POLLINATION STUDIES
INVOLVING THREE PURPLE FLOWERED ALFALFAS, ONE
WHITE-FLOWERED LINE AND TWO POLLINATOR SPECIES. Crop Sci.
||Rumbaugh, M. D., Kehr, W. R., Axtell, J. D., and
others. 1971. PREDICTING SEED YIELD OF ALFALFA CLONES.
South Dakota Expt. Sta. Bull. 38, 48 pp.
||Sheridan, Kevin P., and McKee, Guy W. 1970.
COLORIMETRIC MEASUREMENTS OF PURPLE FLOWER COLOR IN
ALFALFA AS AFFECTED BY VARIETY, SOIL PH, SOIL FERTILITY,
LIGHT, AND SEED SOURCE. Crop Sci. 10: 323-326.