Seedset on Synthetic Haploids of Durum Wheat

Cytological and Molecular Investigations

P.P. Jauhara, M. Dogramaci-Altuntepea, T.S. Petersona and A.B. Almouslemb

a
USDA-ARS, Northern Crop Science Lab., Fargo, ND 58105 USA
b
Dep. of Botany, Faculty of Sciences, Univ. of Aleppo, P.O. Box 12252, Aleppo, Syria

pjauhar@badlands.nodak.edu

ABSTRACT

Because of their great importance as cytogenetic and breeding tools, haploids have been
produced in several crop plants, including wheat (Triticum aestivum L.). Reports of
seedset on haploid plants are very rare. Earlier, we produced 142 haploids (2n = 2x = 14;
AB genomes) of seven commercial durum wheat (Triticum turgidum L.) cultivars
(Cappelli, Durox, Langdon, Lloyd, Medora, Monroe, and Renville) by crossing them with
maize (Zea mays L.). Of these, we studied 101 haploids. Some haploids from each of the
cultivars set seed without colchicine treatment or cross pollination. The cytological basis
of this interesting phenomenon was studied. Because all cultivars have the homoeologous
pairing suppresser Ph1, their haploids formed mostly univalents and had irregular
meiosis. Yet, viable seed was formed on some haploids. The seedset varied with the
genotype. Langdon, with a mean of 2.75 seeds per haploid, was the highest yielder. These
seeds gave rise to normal disomic (2n = 4x = 28; AABB) plants. The seeds had viable
embryos formed by fusion of unreduced male and female gametes with 14 chromosomes
each. The unreduced gametes were formed by two closely related first division restitution
mechanisms resulting in meiotic non-reduction: (i) complete failure of movement of
univalents at anaphase I, followed by normal second (equational) division, and (ii)
anaphase I movement of all univalents to one pole. Thus, formation of these gametes
bypassed the reductional division but occurred by normal equational division. It is
hypothesized that lack of pairing may be a prerequisite for the occurrence of meiotic
restitution and hence chromosome doubling. Fluorescent GISH (genomic in situ
hybridization) analyses of somatic and meiotic chromosomes of the haploid-derived
plants showed the complete duplication of both the A- and B-genome chromosomes.
Fertility of the derived disomics and the presence of two doses of the marker chromosome
involving the 4A·7B translocation, an evolutionary landmark of durum wheat, further
corroborated the precise duplication of all chromosomes. We found that the distal
segment translocated from chromosome 7B constitutes approximately 24% of the long
arm of 4A.
Abbreviations: DH, doubled haploid • FDR, first division restitution • FISH, fluorescent
in situ hybridization • FITC, fluorescein isothiocyanate • GISH, genomic in situ
hybridization • PI, propidium iodide • PMCs, pollen mother cells • SDR, second division
restitution

INTRODUCTION

HAPLOID PLANTS have half the somatic chromosome number and are powerful
cytogenetic and breeding tools. A large number of haploids have been produced in cereal
crops including wheat (Kasha et al., 1990; Jauhar et al., 1991; Riera-Lizarazu and
Mujeeb-Kazi, 1993). They can be employed in several areas of fundamental research in
genetics and cytogenetics, e.g., isolation of aneuploids (Sears, 1954) and elucidation of
genetic control of chromosome pairing (Sears, 1976; Jauhar et al., 1991, 1999). The
haploid-derived homozygous lines provide a rapid means of achieving homozygosity,
thereby accelerating breeding programs (Baenziger, 1996; Khush and Virmani, 1996). By
crossing durum wheat (T. turgidum L., 2n = 4x = 28; AABB) with maize, we produced
euhaploids (2n = 2x = 14; AB genomes) of seven commercial cultivars (Cappelli, Durox,
Langdon, Lloyd, Medora, Monroe, and Renville) of durum wheat (Almouslem et al.,
1998) and studied both inter- and intragenomic chromosome pairing (Jauhar et al., 1999).
Although the corresponding (homoeologous) chromosomes of the A and B genomes are
capable of pairing with one another, a homoeologous pairing suppresser gene, Ph1
(located in the long arm of chromosome 5B), suppresses homoeologous pairing (Sears,
1976). Consequently, only homologous partners pair in tetraploid durum wheat, which
ensures diploid-like pairing and disomic inheritance. Even when each chromosome in the
durum haploids is present in a single dose, Ph1 does not permit pairing among
homoeologues. However, in the absence of Ph1 extensive homoeologous pairing takes
place (Jauhar et al., 1999).

In this study, we investigated 101 Ph1-haploids derived from seven commercial durum
cultivars. Although all haploids formed mostly 14 univalents, as expected, and showed
very irregular meiosis, at least some haploids in each of the seven durum cultivars
produced viable seed without colchicine treatment or cross pollination. Reports of seedset
on haploid plants are very rare. We investigated the meiotic basis of this interesting
phenomenon. Employing fluorescent in situ hybridization (FISH) or [fluorescent genomic
in situ hybridization (GISH)], we studied the chromosomal composition of the seed-
derived disomic plants. Cytological and molecular investigations on the causes of seedset
in synthetic durum haploids are described and the breeding implications of seedset are
briefly discussed.
 Materials and methods

Haploids of seven durum cultivars, viz., Cappelli, Durox, Langdon, Lloyd, Medora,
Monroe, and Renville, which we produced earlier via hybridization with maize
(Almouslem et al., 1998) were studied cytogenetically. During the course of these studies,
we observed seedset on some haploids of each of these cultivars. The seeds were
harvested for further studies.

Production of Plants from Seeds

Thirty-eight of the healthier looking seeds (Cappelli, 1; Durox, 6; Langdon, 7; Lloyd, 3;
Medora, 7; Monroe, 4; and Renville, 10) from the durum haploid plants were germinated
in petri dishes on moist filter paper. The seedlings were planted in a greenhouse [20–
23°C, with an 8-h dark period, a 16-h light period (supplemental lighting in conjunction
with natural lighting)] in 14-cm-diam pots containing Sunshine Mix No. 1 (Sun Gro
Horticulture, Bellevue, WA) and fertilized 7 d later with Osmocote Plus (15-9-12) (Scotts-
Sierra Hort. Prod., Marysville, OH) and raised to maturity.

Chromosome Studies by Conventional Techniques

Somatic chromosomes were studied from root-tips taken from the sprouted seed
according to the techniques described earlier (Jauhar, 1993; Almouslem et al., 1998). For
meiotic studies, immature spikes at the appropriate stage were fixed in Carnoy's fluid
containing 95% (v/v) ethanol:chloroform:glacial acetic acid (6:3:1). Anthers were
squashed and chromosomes stained with acetocarmine or carbol fuchsin according to
procedures described earlier (Jauhar and Almouslem, 1998).

Fluorescent In Situ Hybridization (FISH) Studies on Somatic and Meiotic

Chromosomes
Both somatic and meiotic chromosome spreads were obtained by squashing appropriately
fixed root-tips and anthers in 45% (v/v) acetic acid. The chromosome spreads were
covered with a glass cover slip and observed under a phase contrast microscope.
Chromosome numbers were recorded. Slides containing well-spread metaphase
chromosomes were transferred to a -80°C freezer and kept for up to one month, if
necessary.

FISH was conducted on well spread chromosome preparations with total genomic
Triticum urartu Tumanian DNA as the probe (100 ng/slide, labeled with biotin-14-dATP),
and Aegilops speltoides Tausch genomic DNA as the blocker (2000 ng/slide).
Hybridization protocols standardized earlier (Jauhar et al., 1999) were followed.
Propidium iodide (PI) was used as a counterstain and fluorescein isothiocyanate (FITC)-
conjugated avidin DCS was used to detect the probe.

Slides were observed under a Zeiss Axioskop epi-fluorescent microscope with a Zeiss
AttoArc 100 watt adjustable UV light source. To visualize the FITC signal of the labeled
probe a Zeiss set code 09 excitation filter with a Chroma D535/40 m (Chroma Technology
Corp., Brattleboro, VT) emission filter was used. For the PI counterstain a Zeiss set code
00 excitation filter with a Chroma D605/55 m emission filter was used. Images were
captured with a SPOT II digital camera (Diagnostic Instruments, Inc., Sterling Hts., MI)
connected to an IBM compatible computer using the camera software supplied by the
manufacturer. Images were oriented to the proper layout using Paint Shop Pro v. 5 (JASC
Software Inc., Minnetonka, MN). Final images were printed with a color printer.

Results

Using the maize technique, we earlier produced 101 durum haploids (2n = 2x = 14) (Fig.
1A) with Ph1 (Almouslem et al., 1998) and studied their meiosis in pollen mother cells
(PMCs) (Jauhar et al., 1999). These Ph1-haploids formed mostly 14 univalents at
metaphase I (average of 1350 PMCs = 0.2 II + 13.6 I, Jauhar et al., 1999), and
consequently showed numerous abnormalities at anaphase I and telophase I. It is
remarkable that despite the meiotic abnormalities, at least some haploids of each of the
durum cultivars produced seed (Fig. 1B; Table 1) . We investigated the cytological basis
of this seedset on haploids. Using fluorescent in situ hybridization (FISH), we elucidated
the chromosomal constitution of the seed-derived disomic plants. Some interesting
meiotic abnormalities that led to seedset are listed below.

Fig. 1 (A) Parental durum plant cv. Durox (left); and its haploid
(right). (B) Normal durum seed (left); somewhat shriveled seed
from a haploid plant (right)

View larger
version (51K):
[in this window]
[in a new window]

View this table: Table 1 Seedset on haploid plants of seven durum cultivars
 [in this window]
[in a new window]

First Division Restitution (FDR): Meiotic Non-Reduction

Pollen mother cells of all haploids had mostly 14 univalents and showed FDR to varying
degrees (5–10% of the PMCs analyzed) (Fig. 2A–D) that led to the formation of
unreduced gametes. At metaphase I, the 14 univalents organized themselves on the
equatorial plate (Fig. 2A). As anaphase I approached, the univalents divided into
chromatids (Fig. 2B) but failed to move to the poles, resulting in restitution nuclei (Fig.
2C). Thus, the first meiotic (reductional) division was bypassed. These restitution nuclei
sometimes went through a normal second (equational) division and formed dyads (Fig.
2D) instead of tetrads. The dyads then produced apparently functional unreduced gametes
with 14 chromosomes.

Fig. 2 Meiotic stages in durum haploids leading to

first division restitution (FDR) nuclei and dyads.
(A) All 14 univalents arranged on metaphase plate.
(B) All univalents split into chromatids at meta-
anaphase I. (C) All divided univalents fail to move
to poles and undergo restitution. (D) An FDR
nucleus undivided (left), and second (equational)
division of an FDR nucleus producing a dyad (right)

View larger version (52K):

[in this window]
[in a new window]

Anaphase Movement of Chromosomes

During anaphase I in the haploids, varying numbers of chromosomes moved to the two
poles. Distributions of 7:7, 8:6, 9:5, 10:4, 11:3, 12:2, and 13:1 were observed (Fig. 3A–H)
. FISH analyses of PMCs at anaphase I showed the random movement of the A- and B-
genome chromosomes (Fig. 4A–D) . In extreme cases, all chromosomes moved to one
pole (Fig. 3H) which, in essence, resulted in nonreduction of chromosome number at the
first meiotic division (Fig. 3I). These nuclei underwent a normal second (equational)
division and gave rise to unreduced gametes with 14 chromosomes as shown in Fig. 2.
 Fig. 3 Unequal chromosome separation at anaphase
I in durum haploids. (A) 7:7 separation. (B) 6:8
separation. (C) 5:9 separation. (D) 4:10 separation.
(E) 3:11 separation. (F) 2:12 separation. (G) 1:13
separation. (H) 0:14 separation, where all
chromosomes move to one pole. (I) Telophase I
with all chromosomes on one pole

View larger version (82K):

[in this window]
[in a new window]

Fig. 4 Fluorescent in situ hybridization (FISH) study

on random anaphase movement of univalents to the
poles in durum haploids. (A) Propidium iodide (PI)-
stained chromosomes. (B) Same cell as in A after
FISH, showing 4:3 distribution of A-genome
chromosomes and 3:4 distribution of B-genome
chromosomes. (C) PI-stained chromosomes. (D)
Same cell as in C after FISH, showing 2:5
distribution of A-genome chromosomes and 5:2
distribution of B-genome chromosomes

View larger version (12K):

[in this window]
[in a new window]

Investigation of Seeds Produced by Durum Haploids

The synthetic haploid plants were vigorous and produced 5 to 12 tillers each. When
grown to maturity (Fig. 1A) they set seed in several spikes. There was variation among
haploids in their capacity to set seed (Table 1). The Langdon haploids, for example,
produced on an average 2.75 seeds per haploid.

The seed on haploid plants was generally shriveled, compared to the plump seed on
normal durum cultivars (Fig. 1B). Shriveling was due to poor development of endosperm,
presumably caused by the haploid nature of the plant; less nutrition was available for
endosperm formation. However, the seeds were viable. The 38 seeds selected from the
seven cultivars showed 100% germination in petri dishes under laboratory conditions. It
is possible that with double fertilization, the gametes that produced the endosperm might
not be balanced and may have led to shriveling.

Production of Disomic (2n = 28) Plants

Plants obtained from seed from haploids were disomic and normal, with a typical
phenotype of the parental durum cultivar from which they were derived. They were
vigorous and tillered as well as the parental cultivar and were fully fertile. No height or
leaf measurements were taken.

FISH Analysis of Somatic Chromosomes of Seed-Derived Plantlets

Chromosome counts from root-tips of seed-derived plantlets confirmed their disomic
status (Fig. 5A and C) . Fluorescent GISH, using total genomic probe of T. urartu (the A-
genome donor), showed the complete duplication of the A-genome and B-genome
chromosomes (Fig. 5B and D). The chromosomes involving the 4A·7B translocation, the
evolutionary signature of durum wheat, were easily observed (Fig. 5B and D). We found
that the distal segment translocated from chromosome 7B constitutes about 24% of the
long arm of 4A.

Fig. 5 Somatic and meiotic chromosomes of

disomic plants derived from seedset on durum
haploids. (A) 28 chromosomes counterstained with
propidium iodide (PI); (B) Fluorescent in situ
hybridization (FISH) analysis of the same cell
(hybridized with the A-genome probe) showing 14
brightly lit chromosomes of the A genome; note 14
chromosomes of the B genome faded into the
background. Note arrows showing the chromosome
View larger version (106K): 4A·7B translocation. (C) PI-stained 28
[in this window] chromosomes. (D) The same cell as in C after FISH,
[in a new window] showing 14 fluorescing A-genome chromosomes,
along with 14 faded B-genome chromosomes. Also
note arrows showing the chromosome 4A·7B
translocation. (E) 14 bivalents of seed-derived
disomic plants; note 13 ring II and 1 rod II. (F)
FISH of the same cell as in E (hybridized with the
A-genome probe) showing seven fluorescing
bivalents of the A genome, and seven bivalents of
the B genome faded into the background. This
further shows precise duplication of the A- and the
B-genome chromosomes

FISH Analysis of Meiotic Chromosomes of Seed-Derived Plants

At meiosis, all 38 haploid-derived disomic plants studied formed 14 bivalents as in
parental durum cultivars (Fig. 5E). FISH analysis of the meiotic chromosomes showed
that seven bivalents belonged to the A genome and seven to the B genome (Fig. 5F). This
confirmed the precise duplication of the A- and B-genome chromosomes in the seeds
obtained from haploid plants, as a result of functioning of unreduced gametes.

Discussion

Chromosome doubling during meiosis is believed to contribute significantly to the

widespread occurrence of polyploids in nature (Harlan and de Wet, 1975; Veilleux, 1985;
Jauhar, 1993), even though mitotic chromosome doubling may also occur spontaneously
(Jauhar and Singh, 1969). Meiotic nonreduction and functioning of unreduced male and
female gametes leads to chromosome doubling in intergeneric hybrids of grasses (see for
reference, Xu and Joppa, 1995). The unreduced gametes generally arise through two types
of meiotic restitutions: the first division restitution (FDR), or second division restitution
(SDR) (Peloquin et al., 1989a,b; Tai, 1994). Haploids have been produced in numerous
species of grasses including cereals: rice (Oryza sativa L., Gosal et al., 1997), wheat (e.g.,
Jauhar et al., 1991; Riera-Lizarazu and Mujeeb-Kazi, 1993; Baenziger, 1996; Hu, 1997),
maize (Büter, 1997), barley (Hordeum vulgare L., Kasha et al., 1990; Pickering and
Devaux, 1992; Forster and Powell, 1997), oat (Avena sativa L., Rines et al., 1997), and
rye (Secale cereale L., Deimling and Flehinghaus-Roux, 1997). However, reports of
seedset on haploids are extremely rare. We observed seedset on haploids of all seven
commercial durum wheat cultivars and investigated the cytological basis of this
interesting phenomenon. We discovered that two types of meiotic abnormalities in our
durum haploids played a major role in producing fertile seeds: (i) FDR, and (ii) anaphase
I movement of all chromosomes to one pole.

FDR in pollen mother cells resulted in the inclusion of all 14 univalents (or 28
chromatids) in one nucleus, which on equational division led to the production of
unreduced, functional gametes. It is reasonable to assume that this phenomenon also
occurred during megasporogenesis and produced unreduced female gametes. And the
fusion of unreduced male and female gametes produced normal seed which gave rise to
disomic durum plants. FISH analysis showed the complete duplication of the A- and B-
genome chromosomes. Fertility of the derived disomics and the presence of two of the
marker chromosomes involving the 4A·7B translocation, a typical evolutionary signature
of durum wheat, further testified to the precise duplication of all chromosomes. Meiotic
nonreduction is of common occurrence in nature. As early as 1930, Aase stated:
"Haploidy may be changed to diploidy in the meiotic division through nonreduction of
univalents." Meiotic restitution is known to induce chromosome doubling and hence
fertility in several interspecific and intergeneric hybrids of grasses (Maan and Sasakuma,
1977; Jauhar, 1993; Xu and Joppa, 1995). It is important to note that in all these wide
hybrids, there is very little chromosome pairing, if any. We observed this phenomenon
mostly in our durum haploids which had Ph1 and hence no pairing. It is possible that lack
of pairing may be a prerequisite for the occurrence of meiotic restitution and hence
chromosome doubling.

Seedset was observed on maize and oat haploids; both sets of haploids had little or no
chromosome pairing. In a population of 282 maize haploids (monoploids), Chase (1949)
found that 139 shed pollen, 68 formed kernels, and 34 yielded self-pollinated progeny.
This self-fertility was attributed to spontaneous chromosome doubling in cells giving rise
to reproductive tissue (Chase, 1949). Rines and Dahleen (1990) found that haploid oat
plants recovered from oat x maize crosses were partially self-fertile with up to 23%
seedset. However, they found that many of the plants grown from this seed were
aneuploids.

Another interesting meiotic abnormality observed in durum haploids was the movement
of all 14 univalents to one pole at anaphase I. This phenomenon has essentially the same
consequences as the FDR described above. When all univalents move to one pole, they, in
essence, bypass the reductional division of meiosis and then, on equational division,
produce unreduced gametes. In spontaneous haploids of pearl millet, Jauhar (1970) and
Powell et al. (1975) observed that all seven univalents moved to one pole in some PMCs
and possibly contributed to the development of unreduced gametes.

The phenomena described above could have breeding implications. In both phenomena,
the diploid chromosome complement is first restored and then the diploid complement
undergoes mitotic (equational) division resulting in unreduced gametes (microspores
during male meiosis and megaspores during female meiosis). During the equational
division, sister chromatids of each chromosome (univalent of durum haploids) move to
opposite poles, and therefore the two resultant nuclei are essentially similar to each other
and to the parental meiocyte. The haploid-derived homozygous lines or doubled haploids
(DH) may prove useful in basic cytogenetic studies, in mapping, and in practical plant
breeding. Thus, DH can be employed in developing comprehensive maps and analysis of
quantitative trait loci (Hayes et al., 1996; Cadalen et al., 1998). The instant homozygosity
derived through chromosome doubling of haploids can accelerate breeding programs
(Baenziger, 1996; Khush and Virmani, 1996). To be useful, however, an efficient method
of DH production is necessary. Our durum haploids produced a low frequency of DH.
However, there was a genotypic variation for DH production in our durum cultivars and it
may or may not be possible to select for this trait. It is nevertheless significant that we
obtained doubled haploids in all seven commercial cultivars of durum wheat without
colchicine treatment or cross pollination.Aase 1930

NOTES

Mention of a trademark or proprietary product does not constitute a guarantee or warranty

of the product by the USDA or imply approval to the exclusion of other products that also
may be suitable.

Received for publication March 1, 2000.

REFERENCES

 Aase H.C. Cytology of Triticum, Secale, and Aegilops hybrids with reference to
phylogeny. Res. Studies, State Coll. Wash. 1930;2:5-60.
 Almouslem A.B., Jauhar P.P., Peterson T.S., Bommineni V.R., Rao M.B. Haploid
durum wheat production via hybridization with maize. Crop Sci. 1998;38:1080-
1087.[Abstract/Free Full Text]
 Baenziger P.S. Reflections on doubled haploids in plant breeding. In: Mohan Jain
S., et al. , ed. In vitro haploid production in higher plants. Volume 1: Fundamental
aspects and methods. Dordrecht, the Netherlands: Kluwer Academic Publishers,
1996:35-48.
 Büter B. In vitro haploid production in maize. In: Mohan Jain S., et al. , ed. In
vitro haploid production in higher plants. Volume 4: Cereals. Dordrecht, the
Netherlands: Kluwer Academic Publishers, 1997:37-71.
 Cadalen T., Sourdille P., Charmet G., Tixier M.H., Gay G., Boeuf C., Bernard S.,
Leroy P., Bernard M. Molecular markers linked to genes affecting plant height in
wheat using a doubled-haploid population. Theor. Appl. Genet. 1998;96:933-940.
 Chase S.S. Spontaneous doubling of the chromosome complement in monoploid
sporophytes of maize. Iowa Acad. Sci. Proc. 1949;56:113-115.
 Deimling S., Flehinghaus-Roux T. Haploidy in rye. In: Mohan Jain S., et al. , ed.
In vitro haploid production in higher plants. Volume 4: Cereals. Dordrecht, the
Netherlands: Kluwer Academic Publishers, 1997:181-204.
 Forster B.P., Powell W. Haploidy in barley. In: Mohan Jain S., et al. , ed. In vitro
haploid production in higher plants. Volume 4: Cereals. Dordrecht, the
Netherlands: Kluwer Academic Publishers, 1997:99-115.
 Gosal S.S., Sindhu A.S., Sandhu J.S., Sandhu-Gill R., Singh B., Khehra G.S.,
Sidhu G.S., Dhaliwal H.S. Haploidy in rice. In: Mohan Jain S., et al. , ed. In vitro
haploid production in higher plants. Volume 4: Cereals. Dordrecht, the
Netherlands: Kluwer Academic Publishers, 1997:1-35.
 Harlan J.R., de Wet J.M.J. On Ö. Winge and a prayer: the origins of polyploidy.
Bot. Rev. 1975;41:361-390.
 Hayes P.M., Chen F.Q., Kleinhofs A., Kilian A., Mather D.E. Barley genome
mapping and its applications. In: Jauhar P.P., ed. Methods of genome analysis in
plants. Boca Raton, FL: CRC Press, 1996:229-249.
 Hu H. In vitro induced haploids in wheat. In: Mohan Jain S., et al. , ed. In vitro
haploid production in higher plants. Volume 4: Cereals. Dordrecht, the
Netherlands: Kluwer Academic Publishers, 1997:73-97.
 Jauhar P.P. Haploid meiosis and its bearing on the phylogeny of pearl millet,
Pennisetum typhoides Stapf et Hubb. Genetica 1970;41:532-540.
 Jauhar P.P. Cytogenetics of the Festuca-Lolium complex: Relevance to breeding.
Heidelberg, Germany: Springer-Verlag, 1993.
 Jauhar, P.P., and A.B. Almouslem. 1998. Production and meiotic analyses of
intergeneric hybrids between durum wheat and Thinopyrum species. p. 119–126.
In Proc. 3rd International Triticeae Symposium, Aleppo, Syria. 4–8 May 1997.
Science Publishers, New Hampshire.
 Jauhar P.P., Singh U. Amphidiploidization induced by decapitation in an
interspecific hybrid of Pennisetum. Curr. Sci. 1969;38:420-421.
 Jauhar P.P., Riera-Lizarazu O., Dewey W.G., Gill B.S., Crane C.F., Bennett J.H.
Chromosome pairing relationships among the A, B, and D genomes of bread
wheat. Theor. Appl. Genet. 1991;82:441-449.[ISI]
 Jauhar P.P., Almouslem A.B., Peterson T.S., Joppa L.R. Inter- and intragenomic
chromosome pairing in haploids of durum wheat. J. Hered. 1999;90:437-445.
[Abstract/Free Full Text]
 Kasha, K.J., A. Ziauddin, and U.-H. Cho. 1990. Haploids in cereal improvement:
Anther and microspore culture. p. 213–235. In J.P. Gustafson (ed.) Gene
manipulation in plant improvement II. 19th Stadler Genetics Symposium, Plenum
Press, New York.
 Khush G.S., Virmani S.S. Haploids in plant breeding. In: Mohan Jain S., et al. ,
ed. In vitro haploid production in higher plants. Volume 1: Fundamental aspects
and methods. Dordrecht, the Netherlands: Kluwer Academic Publishers, 1996:11-
33.
 Maan S.S., Sasakuma T. Fertility of amphihaploids in Triticinae. J. Hered.
1977;68:87-94.[Free Full Text]
 Peloquin S.J., Yerk G.L., Werner J.E. Ploidy manipulations in the potato. In:
Adolph K.W., ed. Chromosomes: Eukaryotic, prokaryotic, and viral. Volume 2.
Boca Raton, FL: CRC Press, 1989:167-178 a.
 Peloquin S.J., Yerk G.L., Werner J.E., Darmo E. Potato breeding with haploids
and 2n gametes. Genome 1989;31:1000-1004 b.
 Pickering R.A., Devaux P. Haploid production: Approaches and uses in plant
breeding. In: Shewry P.R., ed. Barley: genetics, molecular biology and
biotechnology. Wallingford, UK: CAB Int, 1992:519-547.
 Powell J.B., Hanna W.W., Burton G.W. Origin, cytology, and reproductive
characteristics of haploids in pearl millet. Crop Sci. 1975;15:389-392.
[Abstract/Free Full Text]
 Riera-Lizarazu O., Mujeeb-Kazi A. Polyhaploid production in the Triticeae:
Wheat x Tripsacum crosses. Crop Sci. 1993;33:973-976.[Abstract/Free Full Text]
 Rines H.W., Dahleen L.S. Haploid oat plants produced by application of maize
pollen to emasculated oat florets. Crop Sci. 1990;30:1073-1078.
[Abstract/Free Full Text]
 Rines H.W., Riera-Lizarazu O., Nunez V.M., Davis D.W., Phillips R.L. Oat
haploids from anther culture and from wide hybridizations. In: Mohan Jain S., et
al. , ed. In vitro haploid production in higher plants. Dordrecht, the Netherlands:
Volume 4: Cereals. Kluwer Academic Publishers, 1997:205-221.
 Sears, E.R. 1954. The aneuploids of common wheat. Missouri Agric. Exp. Stn.
Bull. 572.
 Sears E.R. Genetic control of chromosome pairing in wheat. Annu. Rev. Genet.
1976;10:31-51.[ISI][Medline]
 Tai G.C.C. Use of 2n gametes. In: Bradshaw J.E., Mackay G.R., eds. Potato
genetics. Wallingford, UK: CAB Int, 1994:109-132.
 Veilleux R. Diploid and polyploid gametes in crop plants: Mechanisms of
formation and utilization in plant breeding. Plant Breed. Rev. 1985;3:253-288.
 Xu S.J., Joppa L.R. Mechanisms and inheritance of first division restitution in
hybrids of wheat, rye, and Aegilops squarrosa. Genome 1995;38:607-615.