Influence of nuclear and cytoplasmic factors in callus initiation in anther culture of wheat
by Philip William Becraft
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Agronomy
Montana State University
© Copyright by Philip William Becraft (1987)
Abstract:
Low success frequency limits the use of anther culture in crop breeding. Increasing the capacity of
crops to undergo the anther culture procedure is one approach to improving success rates. Nuclear and
cytoplasmic factors and cytoplasmic male sterility (cms) have been reported to affect tissue culture
response in wheat.
The objective of Part I was to assess the relative importance of nuclear and cytoplasmic factors and
determine if cms enhanced callus yields in anther culture of wheat. Three nuclear genotypes, each in
normal cytoplasm and two alien cytoplasms conferring cms, were evaluated for callus initiation
frequency. Cytoplasmic male sterility did not increase callus induction. The nuclear genotype of 'Chris'
outperformed 'Butte' and 'Coteau' across all cytoplasms. Overall, normal Triticum aestivum and
Triticum timopheevi cytoplasms outperformed Aeailops speltoides cytoplasm. Although nuclear
genotype had the greatest effect, cytoplasm and nucleus X cytoplasm interaction were also significant.
This suggests the best strategies for improving the capacity of wheat to initiate callus in anther culture
involve improving the nuclear capacity for callus initiation.
Heritability studies have suggested that variation in callus inducibility should respond to selection. The
objective of Part II was to determine if crosses designed for improving callus initiation would produce
segregation from which genotypes with improved callus inducibility could be selected. The winter
wheat cultivars 'Centurk 78', 'Norstar' and 'Norwin' were chosen as parents based on their high callus
initiation frequencies and diverse genetic backgrounds. F2 populations from crosses between each of
these parents were evaluated for callus initiation frequencies and callus vigor.
Heterosis was detected for callus initiation in the Norwin/Norstar cross and for callus vigor in all
crosses, but is believed to be of minor importance. Heritability estimates of .25 and .37 for callus
initiation in the Centurk 78/Norstar and Norwin/Norstar crosses, respectively, suggest substantial
genetic variation is available from these crosses for improving this trait. Heritability estimates for
callus vigor were less promising. INFLUENCE OF NUCLEAR AND CYTOPLASMIC FACTORS ON
CALLUS INITIATION IN ANTHER CULTURE OF WHEAT
by
Philip William Becraft
A thesis submitted in partial fulfillment
of the requirements for the degree
>
of
Master of Science
in
Agronomy
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1987
MAIN
l ib .
/V37?
Cop- oL
ii
APPROVAL
of a thesis submitted by
Philip William Becraft
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content , English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Date
Chairperson ,Graduate t/ommittee
Approved for the Major Department
Head, Major Department
Date
Approved for the College of Graduate Studies
-
Date
/7
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of
the requirements for a master's degree at Montana State
University, I agree that the Library shall make it
available to borrowers under rules of the Library. Brief
quotations from this thesis are allowable without special
permission, provided that accurate acknowledgment of source
is made.
Permission for extensive quotation from or
reproduction of this thesis may be granted by my major
professor, or in his absence, by the Director of Libraries
when, in the opinion of either, the proposed use of the
material is for scholarly purposes. Any copying or use of
the material in this thesis for financial gain shall not be
allowed without written permission.
Signature.
Date
iv
ACKNOWLEDGMENTS
Special thanks goes to Dr. G. Allan Taylor for his
friendship, support, guidance and wisdom while serving as
my major advisor.
I also wish to express sincere thanks to the
following:
Drs. J . M . Martin, T . K . Blake and E . R. Vyse for the
help and advice they offered me as friends and members of
my graduate committee..
Drs. S . S . Maan, K . A. Lucken
and D . J. Cox of North Dakota State University, Fargo, and
Dr. J. Gellner of South Dakota State University, Brookings,
for kindly providing seed for this research.
Drs. M . D . Lazar, T . H . H . Chen and The Alberta
Research Council for their hospitality and helpful
technical advice.
USDA, which supported this research through a grant.
Special thanks also goes to Diane Luth, soon to be my
wife, whose strength, understanding and encouragement made
graduate school more tolerable.
V
TABLE OF CONTENTS
Page
APPROVAL...............
STATEMENT OF PERMISSION TO USE.......................
ACKNOWLEDGMENTS..........
TABLE OF CONTENTS...........................
LIST OF TABLES....................
LIST OF FIGURES...........................
ABSTRACT..........................
INTRODUCTION............
PART I .
EFFECTS OF NUCLEUS, CYTOPLASM AND CYTOPLASMIC
MALE STERILITY ON CALLUS INITIATION IN ANTHER
CULTURE OF WHEAT.......... ........ *.......
ii
iii
iv
V
vi
vii
Viii
I
4
INTRODUCTION................
MATERIALS AND METHODS...........................
RESULTS.........
DISCUSSION.........
5
8
10
14
PART II. GENETIC VARIATION FOR ANTHER CALLUS
INDUCIBILITY IN CROSSES OF HIGHLY CULTURABLE
WINTER WHEATS ................................
19
INTRODUCTION. ... ...........................
MATERIALS AND METHODS.............
RESULTS.........................................
DISCUSSION......................................
20
22
25
32
SUMMARY........
36
REFERENCES,..........................................
39
.vi
LIST OF TABLES '
Table
1.1.
Page
Mean callus induction percentages for
environments, nuclear genotypes, and cytoplasms.
11
Analysis of variance for nuclear and cytoplasmic
effects with data pooled across environments....
12
1.3.
Results of Nucleus-Cytoplasm combinations......
12
11.1.
Mean callus initiation frequencies of winter
wheat cultivars tested for use as parents.... ..
25
Mean callus initiation frequencies and callus
vigor ratings of the parent .and F2 populations
over all environments.... .........
26
Estimates of heterosis for callus initiation and
callus vigor in the F2 populations..........
27
Broad sense heritability estimates for callus
initiation frequency and callus vigor...........
28
1.2.
11.2.
11.3.
11.4.
vii
LIST OF FIGURES
Figure
Page
I.I Performance of nuclear genotypes across
cytoplasms for callus initiation...............
11.1
11.2
11.3
13
Percentage distribution of callus initiation
frequencies in the parent and F2 populations of
the Centurk78/Norwin cross....... ...............
29
Percentage distribution of callus initiation
frequencies in the parent and F2 populations of
the Centufk78/Norstar cross... .................
30
Percentage distribution of callus initiation
frequencies in the parent and F2 populations of
the Norwin/Norstar cross...... ...... -..........
31
viii
ABSTRACT
Low success frequency limits the use of anther culture
in crop breeding. Increasing the capacity of crops to
undergo the anther culture procedure is one approach to
improving success rates. Nuclear and cytoplasmic factors
and cytoplasmic male sterility (cms) have been reported to
affect tissue culture response in wheat.
The objective of Part I was to assess the relative
importance of nuclear and cytoplasmic factors and determine
if cms enhanced callus yields in anther culture of wheat.
Three nuclear genotypes, each in normal cytoplasm and two
alien cytoplasms conferring cms, were evaluated for callus
initiation frequency. Cytoplasmic male sterility did not
increase callus induction. The nuclear genotype of 'Chris'
outperformed 'Butte' and 'Coteau' across all cytoplasms.
Overall, normal Triticum aestivum and Triticum timopheevi
cytoplasms outperformed Aeailops speltoides cytoplasm.
Although nuclear genotype had the greatest effect,
cytoplasm and nucleus X cytoplasm interaction were also
significant. This suggests the best strategies for
improving the capacity of wheat to initiate callus in
anther culture involve improving the nuclear capacity for
callus initiation.
Heritability studies have suggested that variation in
callus inducibility should respond to selection. The
objective of Part II was to determine ii: crosses designed
for improving callus initiation would produce segregation
from which genotypes with improved callus inducibility
could be selected. The winter wheat cultivars 'Centurk
78', 'Norstar' and 'Norwin' were chosen as parents based on
their high callus initiation frequencies and diverse
genetic backgrounds. F2 populations from crosses between
each of these parents were evaluated for callus initiation
frequencies and callus vigor.
Heterosis was detected for callus initiation in the
Norwin/Norstar cross and for callus vigor in all crosses,
but is believed to be of minor importance. Heritability
estimates of .25 and .37 for callus initiation in the .
Centurk 78/Norstar and Norwin/Norstar crosses,
respectively, suggest substantial genetic variation is
available from these crosses for improving this trait.
Heritability estimates for callus vigor were less
promising.
I
INTRODUCTION
Anther culture provides plant breeders with a means of.
producing homozygous lines rapidly.
This could decrease
the time required to produce true breeding lines from at
least five years with conventional inbreeding, to one year
using anther culture, and result in an overall savings of
two to three years for variety development (Schnell et al.,
1980).
Somaclonal variation produced by anther culture has
also been cited as a possible source of novel genetic
variation for crop improvement (Evans et al., 1984).
The low frequency of success presently associated with
anther culture makes it cost and labor intensive to obtain
a representative array of the genetic variation available
from a cross, or to insure a high probability of isolating
favorable somaclonal variants.
This is limiting the
application of anther culture in crop breeding programs
(Fouroghi-Wehr et al., 1982).
Success frequency is influenced by environmental,
genetic, (Lazar et al., 1984a) physiological (Schaeffer et
al., 1979) and developmental factors (Ding-gang and Junwen, 1984).
Environment includes both the growing
conditions of the donor plant (Lazar et al., 1984a) and
culture conditions (Ouyang et al., 1983).
Culture media
2
have been studied extensively, with limited recent
improvements in success frequency (Kao, 1981; Chien and
Kao, 1983; Liang et al., 1982; Chuang et al., 1978).
The
optimum developmental stage for culturing is well
established at the mid to late uninucleate stage of
microspore development (Ding-gang and Jun-wen, 1984).
The
physiological factors in the donor plant which' promote
anther culture success are poorly understood, but
generally, more vigorous plants perform better (Schaeffer
et al., 1979).
Lazar et al. (1984a) found that field grown
wheat plants performed better than greenhouse or growth
chamber plants, but that donor plant environment had a less
signifigant effect than genotype.
Since little headway has
been achieved through recent investigations into anther
culture technique, researchers have begun exploring the
possibility of improving the capacity of cereals to undergo
the anther culture process.
It has been reported that normal microspores will not
respond to culture and only functionally impotent
microspores become embryogenic (Heberle-Bors and Reinert,
1980).
Heberle-Bors and Odenbach (1985) reported that
cytoplasmic male sterility, which blocks normal pollen
development, can produce large numbers of responsive
microspores and enhance callus yields.
3
Genetic factors include both nuclear and cytoplasmic
elements.
Nuclear genotypic differences for tissue culture
response have been reported extensively (Schaeffer et al.,
1979).
Heritability studies suggest that anther culture
responsiveness is a heritable trait which should be
amenable to breeding (Lazar et al, 1984b; Miah et al.,
1985; Charmet and Bernard, 1984).
Alien cytoplasms have
been reported to increase callus production from wheat and
triticale anther culture (Heberle-Bors and Odenbach, 1985;
Charmet and Bernard, 1984) and enhance in vitro
characteristics of wheat embryo cultures (Mathias et al.,
1986).
. ’
. ■
.
In Part I of this thesis, three nuclear genotypes of
wheat, each in three cytoplasms were studied.
The two
alien cytoplasms conferred cytoplasmic male sterility.
The
objective was to evaluate the effects of nucleus,
cytoplasm, nucleus X cytoplasm interaction and cytoplasmic
male sterility on callus initiation in anther culture of
wheat.
In Part II, three highly culturable winter wheats were
chosen as parents.
F2 populations from crosses between
these parents were evaluated for callus initiation
frequencies in anther culture to determine the feasibility
of improving callus initiation rates through selection.
4
PART I
EFFECTS OF NUCLEUS, CYTOPLASM AND CYTOPLASMIC MALE
STERILITY ON CALLUS INITIATION IN ANTHER CULTURE OF WHEAT
5
INTRODUCTION
Anther culture provides a rapid means of producing
homozygous lines in many crops.
Anther culture of
alloplasmic lines could expedite the breeding of restorer
lines for hybrid breeding programs (Ling et al.f 1978) or
of varieties in programs using cytoplasmic male sterile
(cms) facilitated crossing.
Picard and de Buyser (1973)
reported successful anther culture and haploid plant
regeneration from the cms wheat lC h a m p l e i n (T.
timopheevi). Meiosis and pollen development in cms wheat
with 2Ls_ timopheevi cytoplasm appear normal through the
first mitotic division (Joppa et al., 1966) which is beyond
the critical stage for anther culture (Ding-gang and Junwen, 1984).
Ling et al. (1978) reported increased callus
induction frequencies from anther cultures
of certain cms
lines of rice.
Normal tobacco flowers were found to produce dimorphic
pollen (Heberle-Bors and Reinert, 1980).
P-grains are
aberrant pollen grains which stain weakly with
acetocarmine, lack starch grains, and are functionally
sterile in situ, but potentially embryogenic in culture
(Heberle-Bors, 1982).
They reasoned that cytoplasmic male
sterility may block normal pollen formation such that high
6
levels of P-grains are produced.
Pollen from 88 cms wheat
lines was studied, and 13 , including lines in Ti timopheevi
'
'
-"X
and A e . speltoides cytoplasms, were identified as high
producers of P-grains.
Two of these lines in Ti timopheevi
cytoplasm were anther cultured and formed embryogenic
structures, while corresponding fertile lines did not
(Heberle-Bors and Odenbachf 1985).
Ti timopheevi cytoplasm
has also been reported to increase embryogenesis in anther
cultures of triticale (Charmet and Bernard, 1984), although
no mention was made of male sterility.
The effect of nuclear genotype on tissue culture
response in wheat has been well documented (Schaeffer et
al.f 1979).
Alien cytoplasms of TriticUm and Aeailops
species produce many phenotypic effects in wheat (Mukai et
al.f 1978).
Recently, several investigators studied
effects of alien cytoplasms on tissue culture of wheat.
Kinoshita and Mikami (1984) measured the growth of callus
derived from seeds of a euplasmic and 17 alloplasmic lines
of wheat with the nuclear genotype of cv. 'Chinese Spring1.
They reported that euplasmic Chinese Spring produced the
most prolific callus while several alloplasmic lines,
including one in Aeailops speltoides cytoplasm, had reduced
growth rates.
Mathias et al. (1986) compared euplasmic and seven
alloplasmic Chinese Spring lines for response to immature
7
embryo culture.
The euplasmic line was near average for
growth rate and all but one alloplasmic line had higher
plant regeneration frequencies.
The line in Triticum
timopheevi cytoplasm exceeded the euplasmic line in all
parameters.
Although effects of nuclear and cytoplasmic factors on
the tissue culture of wheat have been demonstrated, nucleocytoplasmic interactions are known to occur in the
expression of several phenotypic traits (Maan, 1979).
This
has not been studied in regard to tissue culture.
Environment can also affect the expression of cytoplasmic
factors (Maan, 1979) as well as the anther culture response
of wheat (Lazar et al., 1984a).
The objective of this
study was to evaluate nuclear, cytoplasmic, nucleus X
cytoplasm and cms effects on callus initiation frequencies
in anther cultures of wheat grown in different
environments.
8
MATERIALS AND METHODS
Three euplasmic and six alloplasmic wheat lines were
evaluated for callus initiation ability.in anther culture.
Euplasmic lines included the spring wheat (Triticum
aestivum L .) cultivars 'Chris', 'Butte', and 'Coteau'.
Alloplasmic lines consisted of the same nuclear genotypes,
each in the cytoplasms of Triticum timopheevi Zhuk. and
Aeailops speltoides Tausch.
Seed was kindly provided by
Drs. S. S . Maan and K . A. Lucken of North Dakota State
University, Fargo.
The alloplasmic Chris lines were
derived from over twenty generations of backcrossing. The
alloplasmic Butte and Coteau lines resulted from a minimum
of four backcrosses to alloplasmic Chris (Lucken, 1987).
Plants were grown in three environments.
The first
set was grown spaced on benches in the glasshouse during
early spring 1985.
Lighting was natural and temperature
was unregulated but not allowed to drop below I C .
The
maximum temperature reached was 23 C . The second set of
material was space planted in the field under dryland
conditions during the summer of 1985.
The third set was
started in 5 X 25 cm conetainers in a growth chamber with
20 C day, 10 C night temperatures, a 12 hr daylength and
cool white fluorescent light source. After six weeks, the
9
plants were transferred to 8 inch pots in the glasshouse
and grown at 20 +/- 5 C constant temperature with 14 hour
daylength.
Natural light was supplemented about two hours
in the evening with sodium halide lamps.
Spikes for cytological study were fixed in a 3:1
solution of 95% ethanol:glacial acetic acid for 24 hours
and then stored in 70% ethanol. Anther squashes were
stained with acetocarmine to study pollen development
(Smith, 1947).
Some pollen preparations were stained for
starch with IKI.
Spikes were harvested for culturing at the mid to late
uninucleate microspore stage, wrapped in foil and
prechilled at 4 C for 7-10 days.
Anthers were cultured on
60 X 15 mm petri dishes containing 12.5 ml potato medium
(Schaeffer et al., 1979) made with cv. 'Kennebec1 potatoes
and supplemented with Img/I thiamine HCl.
Each plate
consisted of the anthers cultured from one spike.
At least
15 anthers, with a mean of 22, were cultured from each
spike.
Cultures were kept in the dark at 26 C for eight
weeks at which time macroscopic calli were counted.
Frequencies of callus induction were determined for
each spike as the number of callusing anthers/anthers
cultured.
Frequencies were weighted according to the .
1/2
number of anthers cultured and transformed by arcsin X
prior to statistical analysis.
10
RESULTS
Cytological examination confirmed that pollen
development in cms-(3% timopheevi) wheat appeared normal
through the first pollen mitosis (Joppa et al., 1966).
Pollen development in cms-(A e ..speltoides) wheat was very
similar to cms-(T» timophOevi).
Microsporogenesis appeared
normal through meiosis to the late uninucleate stage,
producing microspores with sparse cytoplasms.
In some
grains, the cytoplasm degenerated at this stage, while
others continued development beyond the first pollen
mitosis.
No pollen was observed to undergo the second
mitotic division.
IKI produced no stain reaction
indicating a lack of starch deposits in cms-(A e .
speltoides) pollen.
Mean callus induction frequencies for environments,
nuclear genotypes, and cytoplasms are shown in Table I.I.
Nucleus and cytoplasm both had significant effects on
callus initiation.
The nuclear genotype of Chris had a
significantly higher callus initiation frequency than Butte
or Coteau.
Euplasmic and T.timopheevi cytoplasms produced
the highest induction frequencies t and Ae. speltoides was
significantly lower. Although main environmental effects
were nonsignificant, there was a significant environment X
11
nucleus interaction.
The large number of factors known to
affect anther culture (Vasil, 1980) make it impossible to
determine a specific cause of the interaction.
There was
no detectable environment X cytoplasm interaction.
Table I.I. Mean callus induction percentages for
environments, nuclear genotypes, and cytoplasms.
Environment
Nucleus
Cytoplasm
I
0.97
A*
Chris
3.5
T . aestivum
2.0
A
2
1.3
A
Butte
0.46
B
T . timopheevi
2.1
A
3
2.1
A
Coteau
0.75
B
A e . speltoides
0.75
*
Means followed by different letters are significantly
different at the .05 level by Tukey's Studentized Range.
A
. To facilitate the analysis of nuclear and cytoplasmic
effects, data were pooled across environments.
The mean
squares from analysis of variance (Table 1.2) indicate the
nuclear genotype has a greater effect than cytoplasm.
A
nucleus X cytoplasm interaction was also significant.
Means of nucleus-cytoplasm combinations are presented
in Table 1.3.
Chris had the highest initiation frequency
followed closely by Chris-(Ti timopheevi).
Callus
initiation was low for all lines in A e . speltoides
cytoplasm.
Although Butte and Coteau had low callus
initiations in all cytoplasms, Butte performed best in its
normal cytoplasm while Coteau performed best in Ti
B
12
timopheevi.
The relative performances are apparent in
Figure I.I.
Table 1.2. Analysis of variance for nuclear and cytoplasmic
effects with data pooled across environments.
Source
DF
Mean Square
F-value
Nucleus
2
21.01
39.63
**
Cytoplasm
2
4.72
8.90
**
Nuc.X Cyt.
4
2.67
5.04
**
663
0.53
Residual
** significant at .01.
Table 1.3.
Results of nucleus-cytoplasm combinations.
Line
Number
Spikes
Number
Anthers
Number
Calli
Percent
Initiation
Chris
89
1969
83
4.2
Chris-(T. timooheevi)
86
1936
79
4.1
Chris-(Ae. speltoides)
66
1449 .
22
1.5
Butte
67
1353
12
0.89
Butte-fT. timooheevi)
55
1180
3
0.25 F
Butte-(Ae. speltoides)
76
1554
5
0.32 F
Coteau
80
1752
10
0.57
E
Coteau-(T. timopheevi)
83
1937
20
1.0
CD
Coteau-(Ae. speltoides)
70
1545
8
-
0.52
A
B
C
D
E
I. Means followed by different letters are significantly
different at the .05 level by Tukey's Studentized Range.
13
5-1
-BH*.
1-
Ae. speltoides
Figure I.I
—a
•-©
Butte
I . timopheevl
Cytoplasm
T. aestlvum
Performance of nuclear genotypes across
cytoplasms for callus initiation.
14
DISCUSSION
The development of pollen in cms-(Ae. speltoides)
wheat closely resembles that reported for cms-(T.
timopheevi) (Joppa et al., 1966).
No anther sectioning was
done but it is likely that a failure of tapetal breakdown
and/or reduced vasculature of the anthers, as reported for
cms-(T\ timopheevi) wheat, also occurs in cms-(A e .
speltoides).
The absence of environmental main effects is somewhat
surprising considering the diverse growing conditions.
Plants were generally vigorous in all environments which
may be the major consideration.
A genotype X environment
interaction has previously been reported for callusing of
Wheat anthers (Lazar et al., 1984a).
Many factors differed
between environments, making it impossible to assign any
specific cause to the interaction.
Qian et al. (1986)
reported a temperature X genotype interaction on pollen
development in wheat.
Temperature may be the most
important variable in the three environments since the
first greenhouse environment was generally quite cool,
while temperatures in the field often exceeded 32 C .
Photoperiod and light intensity can also affect
androgenetic response (Vasil, 1980) and may have
15
contributed to the interaction.
Although nuclear genotype produced the greatest
overall effect on callusing, A e . speltoides cytoplasm
caused a large reduction in the initiation frequency, of
Chris.
This indicates responsive genotypes will not
perform well in unfavorable cytoplasmic combinations.
The
Chris-(Ae5. speltoides) plants were the least vigorous of
any line, which lends credence to the suggestion that
cytoplasms may affect tissue culture indirectly via the
physiology of the donor plant (Mathias et al., 1986).
The
results from Butte and Coteau show that different genotypes
respond better to culture in different cytoplasms.
The
Coteau nuclear genotype performed significantly better in
T. timopheevi cytoplasm than in normal Ti aestivum
cytoplasm.
Overall, wheats in T . timopheevi cytoplasm performed
as well as euplasmic wheats.
Since Ti timpheevi cytoplasm
is the most common source of cms in wheat (Lucken., 1973) ,
it should be possible to implement anther culture in cms
facilitated breeding programs.
It.would be important to
evaluate the anther culture performance of specific
nucleus-cytoplasm combinations in the breeding lines to
avoid the wasted effort of culturing nonresponsive types.
Cytoplasmic male sterility did not enhance callus
initiation frequencies.
The low frequency of callus
16
induction from cms-(Ae . speltoides) lines shows that there
are factors influencing androgenetic competence which can
not be observed cytologically as described by Heberle-Bors
(1982) . It is possible that the increased anther culture
response reported in two cms wheat lines by Heberle-Bors
and Odenbach (1985) was due to a favorable nucleus X
cytoplasm interaction and was not a direct result of
cytoplasmic male sterility.
Mathias et al. (1986) reported that related cytoplasms
react similarly in culture but the results of Kinoshita and
Mikami (1984) do not support a relationship between
cytoplasmic phylogeny and in vitro reaction.
There is
strong indication that the cytoplasmic donor of T_s_ aestivum
is Ae ^ longissima (Tsunewaki and Ogihara > 1983).
As a
diploid, A e . speltoides is considered to be its own
cytoplasm donor (Ogihara and Tsunewaki, 1982).
The origin
of the Ti timopheevi cytoplasm is more confusing.
On the
basis of restriction endonuclease fragment length
polymorphisms in chloroplast DNA, Tsunewaki and Ogihara
(1983) believe Ti aucheri is the donor and that the three
cytoplasms utilized in the current study of separate plasma
types.
Mitochondrial genome analysis also indicates that
all the current cytoplasms are distinct (Vedel et al.,
1978).
Based on morphological characteristics of eu- and
alloplasmic lines of different wheats, Maan and Lucken
17
(1971) propose that A e . speltoides donated the T«
timopheevi cytoplasm.
This is further complicated by the
fact that the alloplasmic (A e . speltoides) lines developed
by Tsunewaki are morphologically indistinguishable from
common wheat, while those of Maan cause complete male
sterility (Mukai et al ., 1978).
It appears these
researchers may be reporting on different cytoplasms.
Vedel et al. (1978) made no mention of whether the Ae.
speltoides cytoplasm they studied confers cms to wheat.
The lines used in this study were developed by Drs.
Maan and Lucken, so it is perhaps better to assume their
classificatioh (Maan and Lucken , 1971).
case , A e . speltoides and
That being the
timopheevi would be closely
related but behave differently in culture, while Tjl
aestivum and 3L*. timopheevi would be less closely related,
but behave more similarly.
Whichever classification is
correct, it is difficult to draw conclusions about how .
cytoplasmic relationships affect wheat anther culture from
this small sample of cytoplasms.
The nucleus X cytoplasm
interaction further complicates any generalizations.
_
The results of this study demonstrate that cytoplasmic
male sterility per se does not increase callus initiation
from anther culture of wheat, although one cms line did
perform better than its respective euplasmic line.
Nuclear
genotype had the greatest effect on callus initiation
18
frequency.
Significant effects due to cytoplasm and
nucleus X cytoplasm interaction were also observed. This
emphasizes the interrelationship between cellular
components in the response of plant tissues to culture.
There was no clear relationship between cytoplasmic
phylogeny and callus initiation in these lines.
These
results suggest that efforts for improving the anther
culture capacity of wheat would best be directed toward the
nuclear genotype.
19
PART II
GENETIC VARIATION FOR ANTHER CALLUS INDUCIBILITY
IN CROSSES OF HIGHLY CULTURABLE WINTER WHEATS
20
INTRODUCTION
Anther culture technique has been studied extensively
with limited recent progress.
Investigators have begun
exploring the possibility of improving the genetic capacity
of cereals to undergo the anther culture procedure.
Successful anther culture depends on callus induction, and
subsequent green plant formation (Charmet and Bernard,
1984; Lazar et al ., 1984b),
although some authors report
the involvement of up to five separate steps (Henry and
DeBuyser, 1985; Keyes et al., 1980).
Success rates for
each step are genotype dependent (Schaeffer et al., 1979;
Foroughi-Wehr et al., 1982), and independently inherited
(Foroughi-Wehr et al., 1982; Lazar et al., 1984b; Charmet
and Bernard, 1984) .
Control of these traits appears to be due mainly to
additive gene action, but significant dominance effects
have also been reported (Charmet and Bernard, 1984; Lazar
et al., 1984b; Miah et al., 1985).
In wheat, narrow sense
heritability estimates range from 0.62 to 0.71 for callus
initiation and 0.60 to 0.72 for plant regeneration
frequencies (Lazar et al., 1984b).
Broad sense
heritabilities in triticale have been estimated at 0.66 for
embryogenesis and 0.54 for green plant regeneration
21
(Charmet and Bernard # 1984).
Narrow sense heritability for
callus induction in rice was estimated at 0.92 and broad
sense at 0.97 (Miah et al., 1985).
These reports suggest
that cereals should respond to selection for better
culturability. Alfalfa with increased ability to
-
regenerate plants from callus has been successfully bred
using recurrent selection (Bingham et al., 1975).
Transgressive segregation for androgenesis has been
demonstrated in crosses of wheats with low embryogenic
rates (Henry and DeBuyser, 1985), but there have been no
reports in which highly culturable parents were used.
Since subsequent steps in anther culture are dependent
on successful callus initiation, it was decided to study
the possibility of breeding for improved callus
inducibility.
This study's objective was to determine if
crosses between highly callus inducible parents would
produce segregation for callus inducibility in F
2
populations from which superior genotypes could be
selected.
22
MATERIALS AND METHODS
Seven field grown winter wheat (Triticum aestivum L .)
lines were evaluated for ability to form callus in anther
culture.
lCenturk 1, lNorstar1 and lNorwin1 were selected
as highly culturable cultivars for use as parents.
lCenturk 781 replaced Centurk because of heterogeneity in
Centurk.
These cultivars are of diverse genetic
backgrounds.
Centurk 78 is a five head selection from
Centurk (Schmidt et al.f 1981) which has the following
pedigree:
'Kenya 58'/2/lNewhatch'/3/'Hope'/2*'Turkey'/4/
'Cheyenne'/5/'Parker' (Schmidt et al., 1973).
Norstar
originated from a 'Winalta'/'Alabaskaya' cross (Grant,.
1980).
Norwin is derived from a 'Froid'/'Minoka'//MT6928/
'Trader' cross (Taylor et al., 1986).
Parent and F
populations from crosses between each of
2
these three cultivars were grown in four environments. The
first environment was an early spring planting in dryland
field conditions.
Plants in environments 2, 3 and 4 were
started in 2.5 X 15 cm conetainers and vernalized for 9, 10
arid 11 weeks respectively in a controlled environment
chamber under 15 C day temperature, 2 C night temperature,
and 13 hour daylength with a sodium halide light source.
Plants were transplanted into 15 cm pots and placed in a
23
glasshouse with 20 +/- 5 C constant temperature with a 14
hour daylength and natural light supplemented with sodium
halide lamps.
Population 2 of each cross inadvertently
received a severe moisture stress during tillering. .
Individual plants were sampled with two spikes per
plant as subsamples.
Spikes were harvested at the mid to
late uninucleate stage of microspore development, wrapped
in foil and prechilled at 4 C for 7-12 days.
Anthers from
the middle of each spike were aseptically plated on a 60 X
15 mm petri dish containing 12.5 ml potato medium
(Schaeffer et al ., 1979) supplemented with I mg/1 thiamine
HCl. At least 18 anthers were cultured from each spike
with a mean of 28.4.
Plates were incubated in the dark at
26 C for 8 weeks at which time macroscopic call! were
counted and scored.
Callus initiation frequency was
calculated for each spike as the number of callusing
anthers/anthers cultured.
Spike callus initiation
frequencies were weighted according to the number of
1/2
anthers cultured and transformed by arcsin X
prior to
.
statistical analyses. " Calli were visually scored for
vigor.
Calli up to approximately I mm in diameter were
given a score of I, from I to 4 mm scored 2 and over 4 mm
scored 3.
Watery or necrotic calli were dropped a score
and very small watery or necrotic calli were not counted.
Heterosis was calculated from population means using
24
the following formula:
1982).
(F -mp)/mp (Mather and Jinks,
2
Between plant variances were petitioned out for
each population and used to calculate broad sense
heritabilities using the following equation:
(P1+P2)/2)/F
(Mather and Jinks, 1982).
2
(F 2
25
RESULTS
The lines surveyed for ability to initiate callus in
anther culture showed a wide range of mean callus induction
frequencies.
These results are shown in Table II.I.
Norstar, Centurk and Norwin had the three highest means and
were chosen as parents for crosses designed to improve
callus initiation ability, with Centurk 78 replacing
Centurk.
Table II.I.. Mean callus initiation frequencies of winter
wheat cultivars tested for use as parents.
Redwin
0.004
A*
MT8003
0.005
A
MT7811
1.1
A
Winalta
1.6
A B
Norstar
3.0
B
Centurk
CO
*
% Callus Initiation
B
Norwin
CO
Line
B
Frequencies followed by the same letter are not
significantly different at the .05 level by Tukey's
Studentized Range Test
26
In the parent and F
populations, analysis of variance
2
showed significant effects due to pedigree and pedigree X
environment interaction on both callus initiation frequency
and callus vigor rating.
There was no significant effect
of donor plant environment on either trait.
Means for the
parents and F
populations over all environments are
2
presented in Table II.2. The Norwin/Norstar and Centurk
78/Norstar F
populations had the highest mean callus
2
initiation frequencies. The Centurk 78/Norstar F
2
population also had the highest mean callus vigor rating.
Table II.2.
Mean callus initiation frequencies and callus
vigor ratings of the parent and F2
populations over all environments.
% Callus
Initiation
Callus
Vigor
Pedigree
N
Norwin
98
3.6 A*
1.7 A
Centurk 78
82
5.5 A B
1.8 A B
Norstar
86
6.2
1.9
B
B
: 2.0 C B
Centurk 78/Norwin
219
5.4 A B
Norwin/Norstar
193
7.0
B
2.0 C B
Centurk 78/Norstar
186
7.0
B
2.1 C
*
Means followed by the same letter are not significantly
different at the .05 level by Tukey's Studentized Range
Test.
Comparison of the F
population means to midparent
2
means produced the estimates of heterosis presented in
27
Table II.3.
Only the Norwin/Norstar cross showed
significant heterosis for callus initiation in the F
2
population. All three crosses showed significant heterosis
for callus vigor.
Table II.3.
Estimates of heterosis for callus initiation
and callus vigor in the F2 populations.
% Callus Initiation
Cross
Callus Vigor
Centurk 78/Norwin
.10
.11 **
Norwin/Norstar
.20 **
.10 **
Centurk 78/Norstar
.10
.15 **
**
Significant at .01 according to F2 vs. midparent
contrasts.
Between plant variances for callus initiation
frequency and callus vigor were petitioned out for each
parent and F
population. These were used to calculate
2
broad sense heritability estimates (Table II.4). for each
cross.
The Norwin/Norstar cross produced the highest
heritabilities for both callus initiation and callus vigor.
The heritability estimate of -0.15 for callus initiation
ability in the Centurk 78/Norwin cross was considered 0.
Figures II.I - II.3 show the percentage distributions
of callus initiation frequency in each F
compared to its parents.
population
2
There is a substantial decrease
in the proportion of zeros in all the F
populations
2
28
compared to their parents.
However, the Centurk 78/Norwin
F
population distribution shows little shift toward higher
2
callus initiation frequencies, while in the Centurk 78/
Norstar and Norwin/Norstar F
populations there are shifts
2
toward higher callus initiation frequency as compared to
the parents.
Table II.4.
Broad sense heritability estimates for callus
initiation frequency and callus vigor.
% Callus Initiation
Callus Vigor
Centurk 78/Norwin
O
.13
Centurk 78/Norstar
.25
.11
Norwin/Norstar
.37
.24
Cross
Linear regression showed a weak correlation between
2
callus initiation frequency and callus vigor with an r
value of .26.
29
Population
E53 CENTURK 7 8
CZI NORWIN
Percent Callus Initiation (upper bound)
Figure II.I
Percentage distribution of callus initiation
frequencies in the parent and F2 populations
of the Centurk 78/Norwin cross.
30
Population
ESI NORSTAR
□
CENTURK 7 8
Percent Callus Initiation (upper bound)
Figure II.2
Percentage distribution of callus initiation
frequencies in the parent and F2 populations
of the Centurk 78/Norstar cross.
31
Population
ESI NORSTAR
C D NORWIN
Percent Callus Initiation (upper bound)
Figure II.3
Percentage distribution of callus initiation
frequencies in the parent and F2 populations
of the Norwin/Norstar cross.
32
DISCUSSION
The genetic control of in vitro traits and
heritability for these traits have been well documented in
many small grain crops (Foroughi-Wehr et al ., 1982; Lazar
et al., 1984b; Charmet and Bernard, 1984; Miah et al.,
1985) .
This suggests that in vitro traits should respond
to selection.
The heritability studies, however, have all
been limited to diallel analyses, and involved crosses
between poorly and highly cultutable genotypes.
Breeding
strategy generally involves selecting parents of superior
genotypes and preferably of diverse genetic backgrounds.
In this study, segregation of callus inducibility and
callus vigor were examined in F
populations from crosses
2
designed for improving callus initiation.
The broad sense heritability estimates of .25 and .37
for callus inducibility in the current study would
generally be considered low, but since the parents were
phenotypically similar, and superior performers for this
trait, these values represent substantial genetic variation
for selecting improved genotypes.
These are broad sense
heritability estimates of which only the additive genetic
variance is fixable.
The heterosis effects imply dominance
genetic variance is also involved, but Figures II.I - II.3
33
suggest that the distributions of these populations for
callus initiation frequency approximate a Poisson
distribution.
Since the mean equals the variance in a
Poisson distribution, it is impossible to determine if the
increase in population mean of the F
over the midparent is
2
due to an increased genetic variance produced by
segregation, or if the increased variance is an artifact of
heterosis.
Data from F
or more advanced generations would
I
be required to definitely determine the importance of each
factor. However, heterotic effects are generally limited in
wheat (Hayes and Foster, 1976), and would be less in F
2
generations than F .
Since previous reports indicate a
1
predominance of additive gene action (Lazar et al., 1984b),
the shift in the F
population distributions relative to
2
their parents is likely due to segregation for callus
inducibility.
Callus vigor produced low estimates of heritability
and heterosis in each cross.
The heritability estimate
of .24 for the Norwin/Norstar cross suggests some potential
for improvement, but the other estimates are not
encouraging, especially since the parents were not chosen
on the basis of callus vigor.
Heterosis has previously
been reported for callus growth (Keyes and Bingham, 1979),
but in this case it is surprising because the callus is
presumably haploid or doubled haploid and thus of a
34
completely inbred nature.
This effect could be explained
by gametic selection as suggested by Wenzel and Uhrig
(1981).
In the virtually homozygous parent cultivars,
microspores would have little selective advantage over one
another in culture.
Genetic segregation within the
plants , creates a
2
heterogeneous population of microspores. Those genetically
sporogenous tissues of the F
more suited to the tissue culture environment could
outcompete less suited ones and produce more vigorous
calli.
Gametic selection probably does not explain the
heterotic effect for callus initiation which is thought to
be under sporophytic control (Raquin , 1982; Pei-Iun et al .,
1978) .
The correlation between callus initiation frequency
and callus vigor suggests the possibility of some linkage
or pleiotropy between these traits.
Selection for high
callus initiation may produce a slight trend toward higher
callus vigor.
The lack of significant environmental main effects was
unexpected.
Effects of donor plant environment are well
documented (Lazar et al., 1984a; Sunderland, 1978) and the
growing conditions of the different sets of plant material
were quite diverse.
There was a significant difference in
the parent means between the preliminary survey and the
other four environments.
That, and the significant
35
pedigree X environment interaction emphasize the influence
of environment on the expression of quantitative
characters.
These results indicate that breeding efforts could
increase the callus initiation rates of wheat above those
currently present in the best lines.
It is likely that
plant regeneration ability would respond similarly and that
breeding for both traits could produce lines with
outstanding in vitro characteristics.
Since these traits
have never been selected for , it is probable that initial
advances would be rapid.
Highly culturable lines would
enhance the efficiency of anther culture research and
breeding.
While placing a major emphasis on improving
culturability would not be practical for most breeding
projects, it should be feasible to develop some elite lines
with excellent in vitro characteristics and good agronomic
traits.
These lines could be used to confer culturability
to nonresponsive lines and eventually high culturability
could be incorporated into most breeding lines, making it
feasible to routinely use anther culture in wheat breeding.
36
SUMMARY
In Part I , callus initiation frequency was evaluated
in three euplasmic and six alloplasmic wheat lines.
Nuclear genotypes of the spring wheat varieties 'Chris',
'Butte' and 'Coteau' were each present in normal Triticum
aestivum cytoplasm and the alien cytoplasms of Triticum
timopheevi and Aeailops speltoides.
The alien cytoplasms
both conferred cytoplasmic male sterility.
Breakdown of pollen development occurred after the
critical stage for anther culture in all cms lines.
Cytoplasmic male sterility per se did not enhance callus
initiation, although one cms line outperformed its
respective euplasmic line.
Nuclear genotype had the
greatest influence on callus initiation frequency, but
cytoplasm and nucleus X cytoplasm interaction effects were
also significant. . Chris was the superior nuclear genotype
in all cytoplasms, and overall, the euplasmic and cms-(-T.
timopheevi) lines outperformed the cms-(A e . speltoides).
These results suggest that efforts to improve the capacity
of wheat to initiate callus in anther culture would best be
directed toward the nuclear genotype.
In Part II, crosses were designed for improving callus
initiation frequency in anther culture.
The three winter
37
wheat cultivars, lCenturk 78', lNorstar1 and .'Norwin1, were
selected as parents on the basis of high callus initiation
frequencies.
F
populations from crosses between each of
2
these parents were evaluated for callus initiation
frequency and callus vigor.
Significant heterosis was detected for callus
initiation in the Norwin/Norstar cross, and for callus
vigor in all crosses.
Heterosis for callus vigor was
attributed to possible in vitro microspore selection since
heterosis is theoretically
impossible in haploid or
doubled haploid tissue.
Heritability estimates of 0, .25 and .37 were obtained
for callus initiation in the Centurk 78/Norwin, Centurk
78/Norstar and Norwin/Norstar crosses, respectively.
These
estimates would normally be considered low, but since both
parents were superior performers, substantial genetic
variation may be available in the Centurk 78/Norstar and
Norwin/Norstar crosses for improving callus initiation
rates.
F
Due to the callus initiation distributions in these
populations it is impossible to distinguish between
2
effects of heterosis and segregation. However, based on
previous knowlege of gene action in callus induction in
anther culture of wheat, it is believed that segregation is
the predominant contributor.
These results suggest that
transgressive segregation can occur in crosses of highly
38
culturable wheats and that, through breeding, callus
initiation frequencies could be improved oyer those
currently present in the best lines.
39
REFERENCES
40
REFERENCES
Bingham, E . T., L. V. Hurley, D. M. Kaatz and J. W.
Saunders. 1975. Breeding alfalfa which regenerates
from callus tissue in culture. Crop Sci. 15: 719-721
Charmet, G. and S . Bernard. 1984. Diallel analysis of
androgenetic plant production in hexaploid Triticale
(X . tritosecale, Wittmack). Theor. Appl. Genet. 69:
55-61
Chien, Y. C . and K . N. Kao. 1983. Effects of osmolality,
cytokinin and organic acids on pollen callus formation
in Triticale anthers. Can. J . Bot. 61: 639-641
Chuang, C. C., J. W. Ouyang, H. Chia, S . M . Chou and C. K .
Ching. 1978. A set of potato media for wheat anther
culture. In: Proc. Symp. Plant Tissue Culture,
Science Press, Peking. p. 51-56
Ding-gang, H. and 0. Jun-wen. 1984. Callus and plantlet
formation from cultured wheat anthers at different
developmental stages.
Plant Sci. Lett. 33: 71Evans, D . A., W. R. Sharp and H . P . Medina-Filho. 1984.
Somaclonal and gametoclonal variation. Amer. J. Bot.
71: 759-774
Foroughi-Wehr, B., W. Freidt and G. Wenzel. 1982. On the
genetic improvement of androgenetic haploid formation
in Hordeum vulaare L. Theor. Appl. Genet. 62: 233-239
Grant, M. N. 1980.
Sci. 20: 552
Registration of lNorstar1 wheat.
Crop
Hayes, J. D. and C . A. Foster. 1976. Heterosis in selfpollinating crops, with particular reference to
barley. In: A. Janossy and F . G. H. Lupton (eds.)
Heterosis in Plant Breeding. Elsevier Scientific Pub.
Co., NY. p. 239-256
Heberle-Bors, E . and J. Reinert. 1980. Isolated pollen
cultures and pollen dimorphism. Naturwissenshaften
67: 311
41
Heberle-Bors, E . 1982. In vitro pollen embryqgenesis in
Nicotiana tabacum L . and its relation to pollen
sterility, sex balance, and floral induction of the
pollen donor plants. Planta 156: 396-401
Heberle-Bors, E . and W. Odenbach. 1985. %n vitro pollen
embryogenesis and cytoplasmic male sterility in
Triticum aestivum. Z. Pflanzenzuchtg. 95: 14-22
Henry, Y . and J. DeBuyser. 1985. Effect of the 1B/1R
translocation on anther culture ability in wheat
(Triticum aestivum L.). Plant Cell Reports 4: 307-310
Joppa, L. R., F . H. McNeal and J. R. Welsh. 1966. Pollen
and anther development in cytoplasmic male sterile
wheat. Crop Sci. 6: 296-297
Kao, K. N . 1981. Plant formation from barley anther
cultures with Ficoll media. Z. Pflanzenphysiol. 103:
437-443
Keyes, G. J., G. B. Collins and N . L. Taylor. 1980.
Genetic variation in tissue cultures of red clover.
Theor. Appl. Genet. 58: 265-271
Keyes, G. J. and E . T. Bingham. 1979. Heterosis and
ploidy effects on the growth of alfalfa callus. Crop
Sci. 19: 473-476
Kinoshita, T . and T . Mikami. 1984. Alloplasmic effects on
callus proliferation and streptomycin resistance in
common wheat. Seiken Ziho 32: 31-38
Lazar, M. D., G. W. Schaeffer and P . S . Baenziger. 1984a.
Cultivar and cultivar X environment effects on the
development of callus and polyhaploid plants from
anther cultures of wheat. Theor. Appl. Genet. 67:
273-277
Lazar, M . D., P. S. Baenziger and G. W. Schaeffer. 1984b.
Combining abilities and heritability of callus
formation and plantlet regeneration in wheat (Triticum
aestivum L .) anther cultures. Theor. Appl. Genet. 68:
131-134
Liang, G. H., N. Sangduen, E . G. Heyne and R. G. Sears.
1982. Polyhaploid production through anther culture
in common wheat. J. Heredity. 73: 360-364
42
Ling, T., H . Huang, C . Liang and P . Chun. 1978. The study
of anther culture in "three lines" breeding and
utilization of heterosis in Oryza sativa subsp. Hsein.
In: Proc. Symp. of Plant Tissue Culture, Science
Press, Peking p. 213-222.
Lucken, K . A. 1973. Comparative use of cytoplasmic male
sterility-fertility restoration systems in hybridwheat breeding. In: Proc. 4th Intl. Wheat Genetics
Symp. p . 361-366
Lucken, K . A. 1987. Agronomy Dept., North Dakota State
University, Fargo. Personal communication.
Maan, S . S . and K . A. Lucken. 1971. Nucleo-cytoplasmic
interactions involving Aeailops cytoplasms and
Triticum genomes. J. Hered. 62: 149-152
Maan, S . S . 1979. Specificity of nucleo-cytoplasmic
interactions in Triticum and Aegilops species (a
review).
Wheat Inf. Serv. 50: 71-79
Mather, K . and J. L. Jinks. 1982. Biometrical Genetics
(3rd ed.). Chapman and Hall, New York.
Mathias, R. J-., K . Fukui and C . N. Law. 1986. Cytoplasmic
effects on the tissue culture response of wheat
(Triticum aestivum) callus. Theor. Appl. Genet. 72:
70-75
'
Miah, M. A. A . ,.E . D . Earle and G. S . Khush. 1985.
Inheritance of callus formation ability in anther
cultures of rice, Oryza sativa L. Theor. Appl. Genet.
70: 113-116
Mukai,'Y., S . S . Maan, I . Panayotov and K . Tsunewaki.
1978. Comparative studies of the nucleus-cytoplasm
hybrids of wheat produced by three research groups.
Proc. 5th Int. Wheat Genet. Symp. I: 282-292
Ogihara, Y. and K . Tsunewaki. 1982. Molecular basis of
the genetic diversity of the cytoplasm in Triticum and
Aeailops♦ I. Diversity of the chloroplast genome and
its lineage revealed by the restriction pattern of ctDNAs. Jpn. J . Genet. 57: 371-396
Ouyang, J. W., S. M. Zhou and S . E . Jia. 1983 The
response of anther culture to culture temperature in
Triticum aestivum. Theor. Appl. Genet. 66: 101-109
43
Pei-Iunf S ., C. Chi-Iiang and P. Wen-chung. 1978. The
induction effect of somatic tissues in the anther
cultured in vitro on androgenesis and its control.
In: Proc. Symp. Plant Tissue Culturef Science Press,
Peking p . 143-150
Picardf E . and J. de Buyser. 1973. Obtention de plantules
haploides de Triticum aestivum L . a partir de culture
d'antheres la vitro. C . R. Acad. Sc. Paris 277-D:
1463-1466
Qian/ C.M., A. Xu and G. H. Liang. 1986. Effects of low
temperatures and genotypes on pollen development in
wheat. Crop. Sci. 26: 43-46
Raquinf C . 1982. Genetic control of embryo production and
embryo quality in anther culture of petunia. Theor.
Appl. Genet. 63: 151-154
Schaeffer, G. W.f P. S. Baenziger and J. Worley. 1979.
Haploid plant development from anthers and in vitro
embryo culture of wheat. Crop Sci. 19: 697-702
Schmidtf J. W . f V. A. Johnsonf P . J. Mattern and A. F .
Dreier. 1973. Registration of 'Centurk' wheat. Crop
Sci. 13: 776
Schmidtr J . W . , V. A. Johnsonf P . J. Matternf A. F . Dreier
and D . V. McVey. 1981. Registration of 'Centurk .78'
wheat. Crop Sci. 21: 989
Schnellf R. J.f IIf E . A. Wernsman and L. G. Burk. 1980.
Efficiency of single-seed-descent vs. anther-derived
dihaploid breeding methods in Tobacco. Crop Sci. 20:
619-622
Smith, L . 1947. The acetocarmihe smear technique.
Tech. 22: 17-31
Stain
Sunderlandf N. 1978. Strategies in the improvement of
yields in anther culture. In: Proc. Symp. of Plant
Tissue Culture, Science Pressf Peking p. 65-86
Taylorf G. A . f G. H . Spitlerf C. F . McGuire, J. W. Bergman,
A. L. Dubbsf G. Carlson, G. F . Stallknecht and V. R,
Stewart. 1986. Registration of 'Norwin' wheat. Crop
Sci.'26: 1086-1087
44
Tsunewaki, K . and Y . Ogihara. 1983. The molecular basis of
genetic diversity among cytoplasms of Triticum and
Aeailops species. II. on the origin of polyploid wheat
cytoplasms as suggested by chloroplast DNA restriction
fragment patterns. Genetics 104: 155-171
Vasil, I . K . 1980. Androgenetic haploids. In: I . K .
Vasil (ed.) Perspectives in Plant Cell and Tissue
Culture. Intl. Rev. Cytol. Suppl. IlA. Academic
Press, NY
Vedel, F., F . Quetier, F . Dosba and G. Doussinault. 1978.
Study of wheat phylogeny by EcoRI analysis of
chlbroplastic and mitocondrial DNAs. Plant Sci. Lett.
13: 97-102
Wenzel, G. and H. Uhrig. 1981. Breeding for nematode and
virus resistance in potato via anther culture. Theor.
Appl. Genet. 59: 333-340