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II. The Gene(s) Controlling Apomixis
USE OF APOMIXIS
facultative apomixis (both sexual and apomictic reproduction can occur in a plant
concurrently at various frequencies). Despite difficulties encountered in studying
the genetics of apomixis, progress is being made in understanding the genetic
control of apomixis in various crops. Genetics of apomixis has been summarized
for a number of crops (Asker and Jerling, 1992; Nogler, 1984). It is generally
concluded that apomixis is controlled by qualitative inheritance. The simple genetic control of apomixis improves the potential for manipulating this reproductive mechanism and transferring it to other species.
The best source of the gene(s) controlling apomixis would be within the species
targeted for improvement. Unfortunately, genes controlling apomixis have not
been discovered in most of our major cultivated species. However, genes controlling apomixis can probably be found in the wild species of the genus or related
genera of most major cultivated crops in the world (Hanna and Bashaw, 1987).
Before genes controlling apomixis can be used, they have to be identified. Cytological, genetic, and morphological approaches to identifying and confirming
apomixis have been previously discussed (Bashaw, 1980b; Hanna and Bashaw,
Apomictic reproduction is mainly found in polyploid species. However, it appears that polyploidy is not necessary for the expression of apomictic reproduction. Nogler (1984) cited examples of apomixis in diploid species. Obligate
apomixis was reported in a polyhaploid of an interspecific Pennisetum hybrid
backcross derivative with between one and two sets of chromosomes from the
apomictic species, indicating that apomixis is possible in nonpolyploid plants
(Dujardin and Hanna, 1986).
Use of mutagens to produce mutant genes that cause plants to reproduce apomictically may be another source. Plants that reproduce by facultative apomixis
have been induced in P. gluucum (Hanna and Powell, 1973; Arthur et al., 1993)
and S. bicolor (Hanna et al., 1970). Apomixis is expressed at a variable but generally low level in these mutants. Apomictic reproduction in facultative types
should be at a high enough level to provide the uniformity needed and to preserve
the vigor of a particular genotype for its intended use.
Developments in molecular biological techniques should make it possible to
clone a desirable apomixis gene(s) and transfer it into any crop. Although the
previous statement sounds simple, much research is needed to locate molecular
markers, clone the gene(s), insert the gene(s) into a recipient species, and get the
gene(s) to express itself phenotypically. Progress is being made in developing molecular markers (Ozias-Akins et d., 1993; Miles et d.,1994).
W. W H A N N A
Phenotypic expression of a gene(s) controlling apomixis is important in both
the originating species and in plants of the recipient species, genera, or family if
the gene is transferred. First, it should be noted that technically there are few
obligate apomicts because offtypes can usually be found if large enough populations are observed in most apomictic species. For breeding and practical purposes,
this discussion assumes that plants producing more than 99% maternal types are
Obligate apomixis is preferable to facultative apomixis when sexual counterparts are available in a species for making hybrids and when the desirable genotype to be propagated is apomictic. Various levels of apomictic reproduction can
be found and selected in facultative species such as Kentucky bluegrass (Poa pratensis L.) (Bashaw and Funk, 1987).
Levels of apomixis can be increased by crossing selected facultative apomictic
types (Pepin and Funk, 1971). Apomictic reproduction is an advantage in species
like Kentucky bluegrass with high and diverse ploidy levels because it helps to
bypass sterility problems associated with unstable chromosome numbers. The
amount of facultative reproduction that can be tolerated in a cultivar depends on
its use. More offtype variation may be tolerated in a forage cultivar than in a grain
crop where uniformity in height and maturity is critical for mechanical harvesting.
Gene expression becomes even more critical when genes controlling apomixis
are transferred from different species, genera, and/or families. Within an originating species, apomixis may be obligate. However, in a different genetic background (genotype or species), apomixis may be expressed differently. Sexual pearl
millet X obligate apomictic P. setacum (Forsk.) Chiov. hybrids are obligate apomicts but are highly male and female sterile (Hanna, 1979). High male sterility
and facultative apomixis were observed in sexual maize X Tripsacum dactyloides
(L.) L. (Savidan et al., 1993) and in sexual wheat X Elymus rectisetus (Nees in
Lehm.) (Carman and Wang, 1992) crosses. Crosses between sexual pearl millet
and obligate apomictic P. orientale result in partially male sterile facultative apomictic hybrids and derivatives (Dujardin and Hanna, 1987). Male fertile obligate
apomictic hybrids and derivatives were produced from crosses between sexual
pearl millet X obligate apomictic P. squamulatum Fresen crosses (Hanna et al.,
1992). Fortunately, several Pennisetum species reproduce by obligate apomixis,
and a gene(s) in a wild species that expressed obligate apomixis through the BC,
generation has been found (Dujardin and Hanna, 1989a). The highest level of
maternal types due to apomixis observed in the BC, generation was 90% (Hanna
et al., 1993). The reason for the reduction in apomictic reproduction from BC, to
BC, is unknown. It might be possible to recover obligate apomictic BC, plants if
larger populations of apomictic plants are screened.
USE OF APOMLXIS
Can the gene(s) conditioning obligate apomixis in the BC, generation of the
Pennisetum crossing project confer obligate apomixis in crops such as wheat, rice,
maize, sorghum, and soybean if it is isolated and inserted in the genomes of these
crops by molecular techniques? This question waits to be answered but it is reasonable to assume that the gene(s) would induce obligate apomixis in these and
Environmental conditions are able to influence apomictic expression in some
species (Asker and Jerling, 1992; Hussey e f al., 1991). Burton (1992) observed
no morphological variation in progeny established from seed of obligate apomictic Paspalum notatum Flugge plants grown at varying elevations and subjected to
water and nutrient stresses. Gounaris ef al. (1991) reported that application of
inorganic salts to the growing media changed the frequencies of sexual and apomictic embryo sacs in Cenchrus ciliaris L. (buffelgrass) plants but the study was
not documented with progeny tests. Artificial control of method of reproduction,
apomictic to sexual or sexual to apomictic (especially the latter), would be a
breakthrough for using apomixis in cultivar development.
A number of reports discuss present knowledge of the genetics of apomixis in
various crops (Asker and Jerling, 1992; Bashaw and Funk, 1987; Nogler, 1984).
Fortunately, apomixis appears to be qualitatively controlled by a single or, at most,
a few genes. Both recessive and dominant gene actions have been reported in the
same and different species. The potential for using apomixis in cultivar development and cloning the gene(s) for use in other species and genera is enhanced by
its qualitative inheritance. Obligate and facultative apomixis in the same species
and varying degrees of facultative apomixis in the same species indicate that apomixis may also be affected by modifying genes and/or genetic background.
Apomixis is only in the early stages of making contributions to cultivar development. Efforts in the past have been concentrated on identifying and studying
the various apomictic mechanisms and on the genetics of apomixis. More recently,
efforts have moved toward transferring the gene(s) controlling apomixis from wild
to cultivated species (Hanna et al., 1992; Ozias-Akins et al., 1992).
The discovery of a sexual plant in what was considered obligate apomictic buffelgrass provided an opportunity to manipulate apomixis in a breeding program
w. w. HANNA
(Bashaw, 1962). Bashaw (1980a) used the sexual plant as a female parent in
a breeding program with obligate apomicts to develop the improved cultivars:
‘Nueces’ and ‘Llano.’ Selection of highly apomictic clones in facultative apomictic Kentucky bluegrass has been the basis for improved cultivars in this species
(Bashaw and Funk, 1987). Apomixis is used in Citrus to produce uniform virusfree rootstock (Hearn et al., 1992).
It is yet to be determined if apomixis can be used to produce true-breeding
hybrids at the diploid level. This is an important consideration since many of our
major food crops are diploid and apomixis is usually found at a polyploid level. It
may be necessary to use apomixis at the tetraploid or a higher ploidy level. Although poor seed set is usually associated with autotetraploids, significant improvements in seed set have been made in sorghum (Doggett, 1964) and pearl
millet (Dujardin and Hanna, 1989b). Tetraploid pearl millet (2n = 4x = 28) has
been used in the apomixis gene transfer program in Pennisetum (Hanna et al.,
1992).Pearl millet plants (BC,) with 2n = 29 chromosomesare obligate apomicts
(Dujardin and Hanna, 1989a).
Some advantages of apomixis in a breeding program have been previously discussed (Hanna and Bashaw, 1987). In a commercial hybridization program where
male sterility is used, apomixis eliminates the need to develop and maintain Alines or cytoplasmic-nuclear male sterile systems, B-lines or male fertile maintainers of the A-lines, and R-lines or restorer lines for male fertility restoration of the
A-lines. The A-, B-, and R-lines require time and testing for their development
and space as well as isolation to maintain them. The development of A-lines rapidly narrows both the nuclear and cytoplasmic gene pools that can be used to
develop stable male sterility systems. Likewise, a search for R-lines to completely
restore the A-lines again narrows the gene pool. Gene pool vulnerability is further
discussed in Section 1II.D. The only requirement for producing apomictic hybrids
is that a cross-compatiblefemale with some degree of sexual reproduction is available for crossing with an apomictic pollinator. In apomictic species, the availability of a sexual female is the most limiting factor. When a gene@)for apomixis is
introduced in a sexual species, all germ plasm within a species has potential as a
parent of a new hybrid.
The genotype of every apomict is fixed in the F, generation and every apomictic
genotype from a cross has the potential of being a cultivar. Gene combinations
and vigor are not lost as in each segregating generation of sexual F, hybrids. Recessive genes controlling apomixis may be used to fix the genotypes of transgressive segregates in certain crosses (further discussed in Section 1II.C).
Planting true-breeding seeds from apomictic reproduction would have many
USE OF AF'OMIXIS
advantages over tuber propagation in crops such as potato, Solanum tuberosum L.
Seeds would reduce the propagation and spread of diseases and viruses which are
readily transmitted through the tubers (Asker and Jerling, 1992; Hermsen, 1980).
In addition, seed propagation by apomixis would greatly reduce the storage, shipping, and planting costs and volume compared to tuber propagation.
Apomixis would allow breeders to precisely engineer plants. It would allow one
to develop genotypes with characteristics such as quality, responses to management, and maturity that are highly reproducible from field to field and year to year.
At the same time, a number of apomictic genotypes could be mixed together in
various combinations to enhance genetic diversity to accomplish a specific goal.
Apomixis would change how commercial cultivars are produced and marketed.
Some may consider this a disadvantage but the author prefers to view it as an
opportunity for the future.
In summary, apomixis provides a unique opportunity to develop and maintain superior gene combinations in cultivars and it would simplify hybrid seed
It is necessary to determine the reproductive behavior of selected plants in a
breeding program involving apomixis. This can be done by progeny testing openpollinated seed from selected plants. Morphologically variable progeny from a
plant would indicate sexual origin. The frequency of uniform or maternal progeny
from a plant would indicate the level of apomictic reproduction. At least 20 to 25
progenies are needed to obtain a reliable estimate of a plant's reproductive behavior, especially if it reproduces by facultative apomixis. Fewer progeny may be
needed to identify sexual plants, but the same number of progenies should be
grown for all selected plants since the reproductive behavior may not be known
before they are progeny tested.
Cytological observations are more rapid than progeny testing for identifying
the method of reproduction. New ovule-clearing techniques (Young et al., 1979)
allow one to classify the reproductive behavior of a plant within 2 or 3 days after
collecting the ovaries. In Pennisetum, Paspalum, and Panicum, it is possible to
collect a few florets at the beginning of anthesis and to classify the reproductive
behavior of the plant before it completes anthesis. Apospory and adventitious embryony are the apomictic mechanisms easiest to identify at anthesis. Apospory
can be identified by the presence of multiple embryo sacs, lack of antipodal development, and/or shape and orientation of embryo sacs in the ovule. In adventitious embryony, the embryo develops as a bud-like structure through mitotic division of somatic cells of the ovule, integuments, or ovary wall. No embryo sac is
formed in which these embryos develop. However, a sexual embryo sac may de-
W. W. H A N N A
velop in the same ovule and is essential for endosperm development. Diplospory
is more difficult to identify and requires cytological observations at earlier ovule
development than the two previous mechanisms. Lack of meiosis or a linear tetrad
of megaspores is the best evidence for diplospory. Fertilization of the polar nuclei
by a sperm, pseudagamy, is necessary for endosperm development in apospory
and diplospory. Bashaw (1980b) provides a more detailed discussion on the apomictic mechanisms. It has been reported that the lack of fluorescing callose in
the walls of dyads, tetrads, and megaspore mother cells is also an indication for
diplospory (Carman and Wang, 1992).
Molecular markers linked to the gene(s) controlling apomixis can be used to
identify apomictic plants in the seedling stage (Hanna et al., 1993; Ozias-Akins
et al., 1993). This eliminates the need to grow the plants in the field unless they
are needed in future hybridization studies. However, this molecular approach cannot be used to distinguish between obligate and facultative apomixis at this time.
In a breeding program, it must be remembered that obligate apomictic plants
can only be used as male parents in crosses. Microsporogenesis does function in
apomictics with resultant genetically recombined and chromosomally reduced
male gametes. An apomictic plant must have some pollen fertility if it is to be
used in a breeding program. The ideal apomictic mechanism in a breeding program would be one that is controlled by a dominant gene(s), is environmentally
stable, and reproduces only by obligate apomixis, especially when sexual counterparts are available for crossing with the apomicts.
Breeding procedures for utilizing apomixis have been previously described for
forage and turf grasses. Taliaferro and Bashaw (1966) and Bashaw and Funk
(1987) outlined a procedure for buffelgrass, based on the genetic control of apomixis in this species. Burson et al. (1984) described schemes for breeding obligate
and facultative apomictic species. Nakajima (1990) and Savidan (1981) discussed
the use of apomixis in breeding guinea grass (Panicum maximum Jacq.). Burton
and Forbes (1960) showed that by doubling the chromosome number of diploid
sexual ‘Pensacola’ Bahia grass (Paspalum notatum Flugge), it could be crossed
with tetraploid obligate apomictic ‘common’ Bahia grass to release the genetic
variability of this apomictic species. Others have pollinated obligate apomicts to
produce B,,, hybrids resulting from the fertilization of an unreduced apomictic egg
by a chromosomally reduced sperm from the pollen (Bashaw et al., 1992; Bashaw
and Funk, 1987). Production of B,,, hybrids allows one to develop hybrids in apomictic species where no sexual plants are available for use as female parents. New
gene combinations are developed by adding one or more whole genomes of the
species or an alien species.
USE OF APOMIXIS
In order to simplify the following discussion and figures, it will be assumed that
apomixis is controlled by a single gene in a diploid plant. The results from various
crosses would be modified and made more complex by modifier genes, genetic
background, ploidy, and if more than one gene controls apomictic reproduction.
1. Dominant Gene
Apomixis controlled by a dominant gene would be the easiest to use in a breeding program because all apomicts would be heterozygous for the method of reproduction. Therefore, sexual X apomictic crosses result in both sexual and apomictic F, progenies (Fig. 1). One would theoretically expect one-half of the F, plants
to be sexual and one-half of the plants to be apomictic if apornixis is controlled
by a single dominant gene. Sexual F, plants can be discarded or used in crosses
with other apomictic plants to produce new apomictic hybrids and sexual plants
with new gene combinations. Using improved sexual plants in crosses with improved apomictic plants from other crosses in each generation increases the likelihood of developing superior apomictic hybrids in succeeding generations. F,
apomicts with desirable agronomic traits produced from the cross in Fig. 1 can be
selected and immediately placed in replicated tests to evaluate desired traits.
Progeny tests for genotype stability are not necessary if the plants are obligate or
at least highly apomictic. Superior genotypes can be released as cultivars.
2. Recessive Gene
All sexual plants are heterozygous for a recessive gene controlling apomixis in
crosses between sexual plants homozygous for method of reproduction and apomictic plants in which apomixis is controlled by a recessive gene (Fig. 2). Compared to the cross in Fig. 1, this cross requires selfing the F,, a loss of vigor in
1. Use s e l e c t e d p l a n t s i n
1. S e l e c t best phenotypes
crosses w i t h o t h e r
2 . Discard unsel ected p l a n t s
2. P l a n t i n r e p l i c a t e d t e s t s
APOMICTIC CULTIVAR RELEASE
Figure 1. The breeding procedure when obligate apomixis is controlled by a dominant gene(s).
W. W. HANNA4
(Follow procedure as with
sexual F,s in Fig. 1 i f plants a r e
superior - otherwise discard)
(Follow procedure as with
apomictic F, selections in
Figure 2. The breeding procedure when obligate apomixis is controlled by a recessive gene(s)
and when sexual plants are homozygous for the method of reproduction.
progenies due to selfing results, and only one-fourth of the F2 progenies are apomictic. However, it is possible to select superior apomictic transgressive segregates in the F, generation that are superior to the F,. Selection and testing of
apomictic plants and release of apomictic cultivars would be similar to the procedure followed for the cross in Fig. 1.
Crosses between two sexual plants, both heterozygous for method of reproduction, results in F, plants that (1) breed true for sexuality, (2) are sexual but heterozygous for apomixis, and (3) breed true for apomixis (Fig. 3). This procedure can
capture heterosis in apomictic plants in a similar way to the apomictic plants produced in the cross in Fig. 1. Only 25% of the F, plants can be apomictic in this
cross whereas 50% of the plants in the cross in Fig. 1 can be apomictic. Sexual F,
plants heterozygous for the gene controlling apomixis could be handled similarly
to the F, sexual plants in Fig. 2. True-breeding sexual plants should be handled
similarly to the sexual F, plants in Fig. 2.
t i ' -
(Follow procedure as
(Follow procedure as with
with sexual F, p l a n t s F, sexual plants in Fig. 2
in Fig. 2 ) .
o r use in crosses with
other sexual (H) or
(Follow procedure as
with apomictic F,
selections in Fig. 1).
Figure 3. The breeding procedure when obligate apomixis is controlled by a recessive gene and
when both parents are heterozygous (H)for the method of reproduction.
USE OF APOMIXIS
(Follow the same procedure as for the F, sexual and sexual (H)
plants in Fig. 3).
Figure 4. The breeding procedure when obligate apomixis is controlled by a recessive gene and
when one of the parents of a cross is heterozygous (H)for the gene controlling apomixis. A reciprocal
cross would produce the same results.
A cross between a plant homozygous for sexuality and a sexual plant heterozygous for the method of reproduction is probably the most inefficient cross to
make for producing superior apomictic plants (Fig. 4). About 50% of the progeny
of this cross should be sexual and heterozygous for the gene controlling apomixis
as in the Fig. 3 cross, but no apomictic F, progeny are produced.
A sexual female plant heterozygous for the gene controlling apomixis pollinated with an obligate apomict is the most efficient way to develop superior apomictic cultivars when apomixis is controlled by a recessive gene (Fig. 5 ) . The
outcome of the cross and the selection and testing of apomictic plants are similar
to that for the cross in Fig. 1. Sexual plants from the cross in Fig. 5 are heterozygous for genes controlling apomixis whereas sexual plants in Fig. 1 are homozygous for sexuality because no other genotype is possible for sexuality when the
gene controlling apomixis is dominant.
Population breeding methods may also be applied to improving apomictic species. Five cycles of recurrent restricted phenotypic selection (RRPS) increased
yields of diploid Pensacola Bahia grass, Puspalurn notutum, but failed to produce
(Follow same procedure as for F,
sexual (H) selection in Fig. 3)
(Follow same procedures as
for apomictic selections in
Figure 5. The breeding procedure when obligate apomixis is controlled by a recessive gene and
when the sexual parent is heterozygous (H)for the method of reproduction.
w. w. H A N N A
high-yielding obligate apomictic plants in a population of tetraploid Bahia grass
with the recessive gene for apomixis (Burton and Forbes, 1960; Burton, 1992).
Apomictic plants homozygous for the recessive gene controlling apomixis occurred at a low frequency, less than the 1 in 36 expected in tetraploid material, and
failed to yield as well as the sexual plants in the population. Burton (1992) developed another P. notutum population of apomictic and sexual plants with apomixis
controlled by a dominant gene. After three cycles of RRPS, the best apomictic
plants yielded more dry matter than Argentine Bahia grass.
3. Facultative Apomixis
Facultative apomixis is useful in species where obligate sexual plants are not
available for crossing with apomictic pollinators and where the frequency of apomixis can be increased by crossing diverse facultative types (Bashaw and Funk,
1987). It can be a disadvantage because it can complicate and make the breeding
process unpredictable. The same procedures could be used for breeding facultative
apomicts as for obligate apomicts, except that more progeny testing would be
required to establish the stability and frequency of apomixis of various apomictic
4. Interspecific Hybrids
Interspecific hybridization between a sexual and apomictic species can be used
to release the genetic variability in apomictic species. Lutts et ul. (1991) crossed
induced tetraploids of sexual Bruchiuriu ruziziensis with apomictic B. decumbens
which released the genetic variation of the apomictic species. Burson (1989) identified sexual progenitors of obligate apomictic pentaploid Puspalum dilututum
Poir for use as a female parent to release the genetic variability of the apomictic
species. Hanna and Dujardin (1990) developed apomictic interspecific hybrids
with over 20 different chromosome and/or genome combinations from crosses
among five Pennisetum species. A number of the interspecific hybrids produced
high yields of high quality forage (Hanna et al., 1989).
5. Chemical Control
Chemical control of the apomictic mechanisms by turning them “on” or “off’
at will would have a major impact on using them to produce hybrids. Presently,
little information is available on this subject. The ability to turn apomixis “off’
would make sexual plants in apomictic species available and allow one to release
at will the genetic variability of any genotype that reproduces by apomixis. If
apomixis could be turned “on” at will with a chemical, a sexual hybrid could be
USE OF APOMIXIS
made temporarily apomictic in a commercial production field to increase seed.
The hybrid would be sexual in the farmer’s field if not treated with the chemical.
Chemical control of the apomictic mechanisms presents a challenge to biochemists and genetic engineers in the future.
A major concern for using apomixis in cultivar development is that a few superior cultivars would occupy most of the area planted to a particular crop. A
report published by the National Academy of Science (1972) showed that the area
commercially planted to most of the major sexually reproducing agronomic and
horticultural crops in the United States is already represented by a limited number
of cultivars for each crop. The impact of the corn blight in 1970 due to susceptibility of the major male sterility-inducing cytoplasm used to produce hybrid maize
was discussed in the same report. Sorghum uses the milo cytoplasm in most of its
commercial hybrid production (Bosques-Vega et al., 1989; Schertz and Pring,
Use of apomixis in cultivar development could actually enhance genetic diversity. Each apomictic plant from a sexual X apomictic cross is potentially a unique
cultivar regardless of the heterozygosity or homozygosity of its parents. Apomixis
would allow breeders to build and fix unique genotypes that would not be possible or at least very difficult with sexual reproduction. Vulnerability due to cytoplasm would virtually be eliminated because a specific cytoplasmic-nuclear male
sterility-inducing cytoplasm would not be needed to commercially increase a hybrid. There could be as many different cytoplasms as there are commercial hybrids
if apomixis is used in cultivar development.
IV. IMPACT ON SEED INDUSTRY
Apomixis would no doubt have an impact on the way commercial cultivars are
produced and increased. production practices would be radically changed and at
the same time greatly simplified. The need to maintain and increase parental lines
(except for breeding) and the need to be concerned about isolation to prevent
outcrossing would be eliminated. The major concern in seed production would be
to prevent mechanical mixtures. Outcrossing would only be a problem when a
cultivar reproduced by some degree of facultative apomixis. Offtypes in facultative apomictic cultivars would need to be rogued. The land needed to produce
hybrid seed would be significantly reduced.