Plant Breeding 110, 96102 (1993)
1993 Paul Parey Scientific Publishers, Berlin and Hamburg
ISSN 0179-9541
Production of Doubled Haploids by Anther Culture
and Wheat X Maize Method in a Wheat Breeding Programme
N. S. K. K. NKONGOLO\ J. S. QUIGK^'^ and D. L. JOHNSON^
' Department of Agronomy, Colorado State University, Fort CoUins, CO 80523, USA; ^ National Agricul-
tural Research Centre, Park Road, Islamabad, Pakistan; ' Corresponding author.
With one figure and 2 tables
Received March 16, 1992 I Accepted August 6, 1992
Communicated by C. O. Qualset
Abstract
Utilization of the doubled haploid method of breed-
ing usually shortens the time to cultivar release, and
methods of haploid production need evaluation in a
breeding programme. Thirty-eight different three-
way crosses were tested for anther culture response.
On average 5.8 percent of the anthers cultured pro-
duced calli. Three crosses were found recalcitrant for
callus induction. Overall, the anther culture method
produced 0.6 plantlet per 100 anthers cultured. Five
crosses with an average of 5.8 and 2.8 percent of
anthers producing calli and plantlets, respectively,
were compared using anther culture and wheat X
maize crosses. Non-responsive genotypes for callus
induction and plantlet formation in the anther cul-
ture method proved to be good parental material in
wheat X maize crosses. The average percentages of
embryo formation and plantlet production in wheat
X maize crosses were 10.3 and 4.7, respectively.
Anther-derived plants were cytologically unstable,
whereas all the plants regenerated from wheat x
maize crosses were haploids (n = 21 chromosomes).
The chromosome numbers of the polyhaploids were
doubled with a colchicine treatment. Improvement
of the two haploid production methods to facilitate
their efficient use in a breeding programme is dis-
cussed.
Key words: Triticum aestivum anther culture
wheat X maize hybridization haploid production
chromosome stability breeding programme
The production of doubled haploids has been
an important development in wheat breeding
because the recovery of homozygous lines can
be achieved in a single generation. Methods for
U.S. Copyright Clearance Center Code Statement: 0 1 7 9 - 9 5 4 1 / 9 3 / 1 0 0 2 - 0 0 9 6 $ 0 2 . 5 0 / 0
haploid culture vary from seeking natural,
spontaneous haploids to anther culture, ovary
culture and chromosome elimination in in-
tergeneric crosses such as with Hordeum bul-
bosum.
Anther culture, a method for haploid induc-
tion, is widely used in wheat and many other
crops. Significant genotypic differences are
present for anther culture response in wheat
(ANDERSEN et al. 1987). Few responding geno-
types and low haploid recovery have been
major limitations with the anther culture
method. Thus, scientists are still seeking reli-
able techniques to overcome these problems.
Crossability of wheat genotypes with H. bul-
bosum is limited by compatibility with a series
of genes present in wheat and barley (FALK and
KASHA 1981, 1983). Recently, the use of maize
for hybridization with wheat and subsequent
haploid recovery has been reported (LAURIE
and BENNETT 1986, 1988). The production of
wheat haploids through wheat X maize crosses
has the advantage of being genotype independ-
ent. This system seems a potential way to
increase the haploid production in wheat.
Comparison between techniques for dou-
bled haploid production could help breeders to
decide either to use the most successful one, or
use them in parallel (DEVAUX 1987, 1988). In
this study, we evaluated three-way cross de-
rived lines for anther culture response and
compared this method with the wheat x maize
system for polyhaploid production. These
lines were initially selected for Russian wheat
aphid (RWA) resistance.
Production of Doubled Haploids by Anther Culture in Wheat
Materials and Methods
Production of three-way cross derived lines: The
wheat lines PI 372129, PI 294994, PI 262605, and PI
243781 previously identified as RWA resistant
(QUICK 1989) were crossed with four cultivars
Lamar', 'TAM 107', 'TAM 200', and Tuma'
selected for their agronomic performance and anther
culturability in vitro. The F, plants were topcrossed
to ten different wheats including 'Bronco',
CO850034, CO850260, CO850267, 'Hawk',
'Lamar', 'Mesa', 'TAM 107', 'TAM 200', and 'Yu-
ma'. The lines derived from these 38 different three-
way crosses were vernalized for eight weeks. The
vernalized seedlings were planted in greenhouse flats
and screened in the greenhouse for resistance to the
RWA according to the technique previously de-
scribed (NKONGOLO et al. 1991). The resistant lines
were transplanted into greenhouse soil beds and
grown under a 16-h photoperiod at 30/20 C day/
night temperatures.
Anther culture: Initially, one parental cultivar from
each cross was tested and gave three to six percent of
responding anthers. These were 'Lamar', 'TAM
200', 'TAM 107' and 'Yuma' (unpublished data). For
this study, only the RWA resistant plants derived
from the three-way crosses were evaluated. Since we
had an average of three tillers per plant, we used one
spike per plant for anther culture and five of the ten
selected Fi plants in each cross.
Spikes were removed from the leaf sheaths and
microspore developmental stage was examined under
a microscope. Anthers containing microspores in the
mid-to-late-uninucleate stage were selected. Col-
lected spikes were kept in the dark at 3 to 5 C for
one week for cold treatment. Anthers were excised
aseptically and cultured on potato-2 medium con-
sisting of 10 % potato extract, 1.5 mg L~' 2,4-D, 0.5
mg L""' kinetin, 9 % sucrose and 0.6 % agar (OUY-
ANG 1986). Twenty to thirty-five anthers were cul-
tured in 100 X 15 mm petri plates. Anthers from one
spike were cultured in one petri dish representing
one experimental unit. Petri plates with cultured
anthers were sealed with 'Parafilm' and kept in the
dark at 26 to 28 C. Five petri dishes were used per
cross combination. Callus induction was recorded 6
to 8 weeks after culturing. Calli were transferred
from induction medium to 190-2 regeneration
medium (ZHUANG and Xu 1983) supplemented with
0.5 mg/L kinetin and 0.5 mg/L NAA.
Wheat X maize crosses: This experiment was car-
ried out at the same time and under the same grow-
ing conditions as the anther culture experiment. Five
crosses selected for their different responses to
anther culture (Table 1) were crossed with maize in
the greenhouse. Emasculated spikes were pollinated
with fresh maize pollen. One spike per plant repre-
senting one experimental unit and five plants per
97
cross combinations were used. We selected the same
five plants used for the anther culture in each of the
five crosses used. The open-pollinated maize line
used as the pollen source was 'Crumpacker 47', a
Hopi Indian 'Bluecorn' landrace. After pollination
with maize, 5 ml of 20 ppm 2,4-D was injected into
the uppermost internode. The same treatment was
repeated on the day following pollination. The polli-
nated spikes were left on the plant for 12 to 14 days.
Embryos were rescued and transferred to vials con-
taining B5 medium (GAMBORG et al. 1968). Vials
were kept in the dark at 20 C until the embryos
germinated. After germination, embryos were trans-
ferred to a growth chamber under continuous fluo-
rescent light. After 10 to 15 days when shoot and
root development were sufficient, plantlets were
transferred to potting soil.
Cytology: Somatic chromosome number was ex-
amined in root-tip cells of the seedlings according to
TSUCHIYA (1971). At least four root tips from each
seedling per pot were cut to a length of 1 cm, kept in
distilled water at 4 C for 18 h and fixed in 3 : 1
ethanol : glacial acetic acid fixative solution. Root
tips were stained with 0.7 % acetocarmine solution
and chromosomes were observed after squashing in
45 % acetic acid.
Androgenic haploid plants as well as the plants
obtained by the wheat X maize method were treated
with colchicine. For this purpose, plants which had
developed four tillers were removed from the potting
soil and the roots and the tillers were washed and cut
back to about 2 cm. The plants were than immersed
to a depth of 5 cm in a 0.1 % colchicine solution
containing 2 % dimethyl sulphoxide (DMSO). They
were treated for 5 h at room temperature under
artificial light, then rinsed in running tap water for a
few minutes, potted and placed in a growth chamber
at 16 C (day) and 12 C (night) for a establishment
period of 14 days. They were then transferred to a
greenhouse and grown under a 16 h photoperiod at
30/20 C day/night temperature. Seed sets were re-
corded at maturity.
Statistical analysis: Callus induction ability was
measured as the number of calli induced per 100
anthers in anther culture. Embryo formation was
measured as the number of embryos formed per 100
pollinated florets in wheat X maize method. Plantlet
regeneration was the number of plantlets from 100
anthers or 100 pollinated florets, resgectively. All
data were transformed by arcsin V% to improve
their normality. The transformed data were analyzed
as completely randomized experiments, each experi-
mental unit containing five observations. Analysis of
variance and the LSD test were computed by
MSTAT-C statistical programs (MSTAT Develop-
ment Team, 1988).
98 KisANA, NKONGOLO, QUICK and JOHNSON

Table L Percent callus induced and percent plantlets regenerated from anther culture of Russian wheat aphid
resistant wheat lines derived from 38 different three-way crosses
Calli* Plantlets''-
induced regenerated
Cross Anthers (%) (''/o)
(no.)

Lamar/PI 372129//CO850034 90 4.5 (12.3) 0.0 (0.0)

Lamar/PI 294994//TAM 200 87 2.3 (8.8) 0.0 (0.0)
Lamar/PI 294994//Mesa 110 8.2 (16.6) 0.0 (0.0)
Lamar/PI 262605//CO850260 113 4.1 (11.7) 0.0 (0.0)
Lamar/PI 243781//TAM 200 102 3.8 (11.3) 0.2 (2.5)
Lamar/PI 243781//CO850260 100 13.9 (21.9) 0.0 (0.0)
Lamar/PI 243781//Hawk 118 9.2 (17.7) 0.0 (0.0)
Lamar/PI 262605//Lamar 100 0.0 (0.0) 0.0 (0.0)
Lamar/PI 243781//Mesa 101 4.0 (11.5) 0.0 (0.0)
TAM 107/PI 372129//TAM 107 81 7.7 (16.1) 0.0 (0.0)
TAM 107/PI 372129//TAM 200 81 1.8 {7.7) 0.0 (0.0)
TAM 107/PI 294994//CO850267 77 2.8 (9.6) 0.0 (0.0)
TAM 107/PI 262605//TAM 107 102 2.9 (9.8) 0.0 (0.0)
TAM 107/PI 262605//CO850034 122 10.9 (19.3) 0.0 (0.0)
TAM 107/PI 262605//CO850267 96 2.6 (9.2) 0.0 (0.0)
TAM 107/PI 262605//Yuma 99 4.5 (12.2) 0.0 (0.0)
TAM 107/PI 243781//CO850260 83 15.6 (23.3) 3.4 (10.7)
TAM 200/PI 372129//Lamar 141 9.6 (18.6) 0.7 (4.9)
TAM 200/PI 294994//CO850060 111 5.4 (13.4) 3.3 (10.5)
TAM 200/PI 294994//CO850267 102 15.5 (23.2) 1.2 (6.2)
TAM 200/PI 262605//Bronco 94 4.5 (12.3) 0.2 (2.4)
TAM 200/PI 262605//TAM 107 100 0.6 (4.6) 0.0 (0.0)
TAM 200/PI 262605//CO850060 94 1.6 (7.2) 0.0 (0.0)
TAM 200/PI 262605//CO850034 84 0.0 (0.0) 0.0 (0.0)
TAM 200/PI 262605//Lamar 112 7.8 (16.2) 0.7 (4.8)
TAM 200/PI 243781//TAM 107 101 5.5 (13.5) 0.2 (2.5)
TAM 200/PI 243781//CO850060 101 19.8 (26.4) 11.9 (20.2)
Yuma/PI 372129//TAM 107 99 1.5 (7.1) 0.2 (2.3;
Yuma/PI 372129//CO850034 79 1.2 (6.2) 0.0 (0.0)
Yuma/PI 372129//CO850267 95 0.8 (5.0) 0.2 (2.3)
Yuma/PI 372129//TAM 200 83 0.0 (0.0) 0.0 (0.0)
Yuma/PI 294994//Mesa 92 8.6 (17.0) 0.0 (0.0)
Yuma/PI 294994//TAM 107 94 4.1 (11.7) 0.0 (0.0)
Yuma/PI 294994//Vona 84 0.7 (3.7) 0.0 (0.0)
Yuma/PI 262605//Yuma 83 6.4 (14.7) 1.0 (5.8)
Yuma/PI 262605//CO850267 98 8.3 (16.8) 0.0 (0.0)
Yuma/PI 243781//Lamar 98 14.1 (22.7) 0.0 (0.0)
Yuma/PI 262605//CO850260 95 4.5 (12.3) 0.6 (4.5)

Total 3701
Mean 5.8 (13.9) 0.6 (4.4)
LSD 0.05 2.2 (8.5) 0.6 (4.5)

Means of calli induced and means of plantlets regenerated per 100 anthers. Angular transformed means in
parentheses.
Production of Doubled Haploids by Anther Culture in Wheat 99

Results Wheat X maize method

Anther culture method Since the seed set in wheat X maize crosses do
Of the 3701 anthers cultured, 214 (5.8 %) not always contain embryos, the term embryo
produced calli (Table 1) and only 22 calli formation was used instead of crossability.
(0.6 % of the cultured anthers) regenerated Table 2 shows the frequencies of embryo for-
plantlets. Eight of these 22 plantlets were al- mation in five crosses that were crossed with
binos. Thus, the overall result from all the maize. Differences among the five wheat cros-
material was 0.38 green plants per 100 cultured ses for embryo formation and plantlet produc-
anthers. Significant differences were observed tion (Table 2) were not significant. On average
among crosses for calli formation and plantlet 10.3 % (58 florets) of the 548 pollinated florets
regeneration. Most of the crosses showed rela- produced embryos. RIERA-LIZARARU and MU-
tively low frequencies of induced calli, but 11 JEEB-KAZI (1990) reported equal embryo recov-
were remarkably better with 8 to 20 % of the ery frequencies among genotypes when the
cultured anthers producing calli. uppermost internode of the emasculated wheat
spike was injected with 0.5 to 1 mL of a 100 mg
The highest calli induction was observed in L~^ 2,4-D aqueous solution. We found that
crosses involving TAM 200/PI 243781// plants recalcitrant to anther culture were good
CO850060 and TAM 107/PI 243781//
CO850060. Three cross combinations includ-
ing Yuma/PI 372129//TAM 200, Lamar/PI
262605//Lamar, and TAM 200/PI 262605//
CO850034 did not produce callus. In general,
it was observed in Table 1 that PI 243781
enhanced calli formation while PI 262605 and
PI 372129 reduced calli production in the cross
combinations studied. This indicates that
choice of genotypes for haploid production
with anther culture in wheat is important as
previously shown (ANDERSEN et al. 1987).
Only one cross (TAM 200/PI 243781//TAM
107) was remarkably better than others with
11.9 % of cultured anthers regenerating plant-
lets. Most of the crosses with high calli forma-
tion showed low plantlet regeneration. How-
ever, TAM 200/PI 294994//CO850060 had low
calli frequency with 61 % of the calli re-
generating plantlets. This confirms previous
reports (ANDERSEN et al. 1987, LAZAR et al.
1984) that calli formation and plantlet regener-
ation may be controlled by different genetic
mechanisms.
Chromosome numbers of the anther-de-
I #
rived plantlets were determined. Seven of the
14 regenerated plantlets were polyhaploids,
five were spontaneous doubled haploids and
two were aneuploids (2n = 36 and 39 chromo-
somes, respectively) (Fig. 1 b). Thus, only 12
5t
plants (0.3 % of cultured anthers) were useful
for practical breeding purposes. All polyha- Fig. 1. Somatic chromosomes in metaphase showing
ploid plants treated with colchicine to double a) 39 chromosomes in an aneuploid plant derived
the chromosome number survived and pro- from anther culture and b) 21 chromosomes in a
duced seed sets varying from 20 to 50 %. haploid plant derived from wheat x maize cross
100 KisANA, NKONGOLO, QUICK and JOHNSON

Table 2. Percent of embryos rescued and percent plantlets produced from wheat X maize crosses and percent
of calli induced and plantlets regenerated in five cross combinations
Wheat X maize cross Anther culture
Wheat lines Pollinated Embryos* Plantlets'"^ Plantlets*'^
florets produced produced induced regenerated

Lamar/PI 372129//CO850034 104 9.7 (18.1) 5.7 (13.7) 4.5 0.0

Lamar/PI 262605//Lamar 102 12.9 (21.8) 4.2 (11.8) 0.0 0.0
TAM 200/PI 243781//CO850060 100 5.1 (13.1) 2.7 (9.4) 19.8 11.9
Yuma/PI 294994//TAM 107 132 16.6 (24.0) 6.3 (14.5) 4.1 0.0
Yuma/PI 294994//Vona 110 8.2 (16.6) 6.3 (14.5) 0.7 0.0

Total 548
Means 10.3 (18.7) 4.7 (12.5) 5.8 2.4

Means of embryos and plantlets produced per 100 pollinated florets. Angular transformed means in
parentheses. No significant differences among means at P < 0.05.
Means of calli induced and plantlets regenerated per 100 anthers.

parental material in the wheat X maize Discussion

method. Embryo size varied greatly (0.5 to
3 mm). Several caryopses with endosperm but
without embryos were found. This parallels
findings of LAURIE and BENNETT (1988) who
reported that at 48 h after pollination, florets
would contain only an embryo, only an endo-
sperm, or an embryo and an endosperm.
In total, 27 of the 58 embryos produced
(4.8 % of pollinated florets) produced plant-
lets. The frequencies are within the range of
variation reported elsewhere (RIERA-LIZARAZU
and MUJEEB-KAZI 1990, INAGAKI and TAHIR
1990, LAURIE and BENNETT 1988). Most of the
embryos that failed to develop plantlets were
small. Leaving them longer than 14 d on the
spikes did not seem to improve their develop-
ment in vivo. Eighteen regenerated plantlets
died ex vitro because they failed to develop
roots.
Eight wheat X maize derived plantlets
(1.4 % of the pollinated florets) were haploids
and two were doubled haploids. There was no
cytological evidence of retention of entire
maize chromosomes (Fig. 1 a). The two di-
ploid plants (42 chromosomes) obtained ap-
peared to be derived from accidental selfings
since their embryo and endosperm structure
and size were normal. The eight wheat X
maize derived plantlets survived after col-
chicine treatment and produced seed sets simi-
lar to anther-derived plants (20 to 50 % ) .
In general, the wheat X maize system gave the
highest yield of green plants (4.8 plants per 100
florets pollinated) compared to that achieved
from anther culture (2.8 plants per 100 anthers)
for the five crosses studied. The influence of
genotypes in anther culture appeared to be a
major practical disadvantage. PAUK et al. (1991)
showed that the frequency of plantlet regener-
ation can be enhanced in unresponsive calli,
but the efficacy of this technique varied with
wheat cultivars. In the wheat X maize system,
it is possible to obtain high embryo recovery
using improved hormone treatment and tech-
nique (KiSANA et al. 1991, LAURIE and BENNETT
1988, RiERA-LiZARAZU and MUJEEB-KAZI 1990).
Plantlet production and survival in in and ex
vitro could have been increased by using im-
proved medium for small embryos that failed
to regenerate, and by subculturing the plantlets
produced in a rooting medium prior to trans-
planting in potting soil.
Another criterion for comparing doubled
haploid systems is the stability of the derived
plants ( D E BUYSER et al. 1985). Doubled ha-
ploids should be cytologically stable to be
successfully used in a breeding programme.
Aneuploids and completely sterile plants were
observed in anther-derived plants. Usually,
these chromosome variations originate in the
anther of donor plants or during the process of
Production of Doubled Haploids by Anther Culture in Wheat
anther culture ( D E BUYSER et al. 1985). Conse-
quently, the frequency of aneuploids in anther
culture could be reduced if the parents are
cytologically stable. In wheat X maize-derived
plants, aneuploids or gross chromosomal ab-
normalities were not observed. This confirms
that chromosome variations are not common
in wheat X maize crosses (LAURIE and BENNETT
1986) since all the maize chromosomes are
eliminated during cell divisions.
Overall data indicate that the maize cross
system gave higher success rate than the anther
culture technique for the five crosses studied.
In addition to the high frequency of haploid
regeneration in the maize crosses, this tech-
nique seems to extend the haploid induction to
genotypes which otherwise might not respond
to anther culture. The wheat X maize tech-
nique can also save four to six weeks to obtain
the same age plantlet compared to anther cul-
ture. The time needed for processing a single
spike, from tiller removal or emasculation to
the transfer of a viable green plant to soil, was
similar. To be considered as a reliable system
of doubled haploid production, the maize
technique requires further enhancement of em-
bryo recovery and plantlet production.
Practically, the probability of obtaining de-
sirabel homozygous genotypes resistant to the
RWA was very low due to the small haploid
population size regenerated from both anther
culture and the wheat X maize system. Since
the materials used were at the F] level, few
recombinations and high frequency of
chromosomal abnormalities were expected (DE
BUYSER et al. 1985). Furthermore, a large
number of haploids must be produced because
only few genotypes are retained in plant breed-
ing. The line derived from TAM 200/PI 243781/
/CO850060 which had high calli induction and
plantlet regeneration (Table 1), may regenerate
a sufficient population size to obtain desirable
genotypes if more anthers are cultured. Any of
the crosses could be used in the maize cross
system to produce a larger population by cul-
turing more embryos and improving the em-
bryo culture technique as previously sug-
gested.
Given the small size of the doubled haploid
populations obtained with both techniques, it
might be appropriate to use material having
undergone a few generations of selfing and
101
selection as suggested by D E BUYSER et al.
(1985). The number of, required plants will
then be reduced, but savings in cultivar de-
velopment time will also be reduced.
Zusammenfassung
Die Erzeugung Doppelt-Haploider uber An-
therenkultur und tiber Weizen X Mais-
Kreuzungen in der Weizenziichtung
Da die Anwendung von Methoden zur Her-
stellung von Doppelt-Haploiden in der Regel
die Zeit bis zur Fertigstellung einer Sorte ver-
kiirzt, erscheint es notwendig, die Eignung
solcher Methoden fiir die Pflanzenzuchtung zu
priifen. Es wurde das Verhalten der Nachkom-
men von 38 verschiedenen Dreiweg-Kreuzun-
gen in der Antherenkultur untersucht. Im
Durchschnitt bildeten 5,8 % der aufgelegten
Antheren Kalli aus. Bei 3 Kreuzungen wurde
keine Kallusbildung beobachtet. Insgesamt
wurden uber Antherenkultur 0,6 Pflanzchen je
100 Antheren erhalten. Fiinf Genotypen, die
eine Kallusbildung und Pflanzenregeneration
von durchschnittlich 5,8 bzw. 2,8 % aufwie-
sen, wurden mit Mais gekreuzt. Die Ergeb-
nisse dieser Kreuzungen wurden mit den Be-
funden aus der Antherenkultur verglichen.
Genotypen mit geringer Neigung zur Bildung
von Kalli und zur Regeneration von Pflanzen
erwiesen sich als geeignete Eltern fiir Weizen
X Mais-Kreuzungen. Im Durchschnitt wurde
bei solchen Kreuzungen eine Embryonen- und
Pflanzenausbeute von 10,3 bzw. 4,7 % er-
reicht. Die aus Antherenkultur erhaltenen
Pflanzen waren zytologisch nicht stabil. Dage-
gen waren alle aus Weizen X Mais-Kreuzun-
gen entwickelten Pflanzen haploid (n = 21
Chromosomen). Die Chromosomenzahlen der
Polyhaploiden wurden durch eine Colchizin-
behandlung verdoppelt. Es wird diskutiert, in
welchem Mafie sich die beiden Verfahren zur
Gewinnung von Haploiden verbessern lassen,
um ihre Anwendung in der Plfanzenziichtung
noch wirkungsvoUer zu machen.
We are grateful to Dr. S. WANG for useful comments
during this study. We thank JEFF RUDOLPH and
GERRY ELLIS for their skillful assistance with aphid
rearing, screening, crossing and transplanting.
102 KiSANA et al.. Production of Doubled Haploids by Anther Culture in Wheat
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