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II. The Gene(s) Controlling Apomixis

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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.



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

other species.

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


3 39

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-

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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.



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




(Dominant gene)



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



Figure 1. The breeding procedure when obligate apomixis is controlled by a dominant gene(s).

3 42




(Recessive gene)






(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

Fig. 1)

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.


(Recessive gene)



(Recessive gene)

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

apomictic crosses).


(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.









(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




(Recessive gene)



(Follow same procedure as for F,

sexual (H) selection in Fig. 3)


(Follow same procedures as

for apomictic selections in

Fig. 1)

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



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.


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.

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