American Journal of Plant Sciences, 2013, 4, 2259-2264
Published Online November 2013 (http://www.scirp.org/journal/ajps)
http://dx.doi.org/10.4236/ajps.2013.411278
Open Access AJPS
2259
Effect of Cold-Mediated Pretreatment on Microspore
Culture in Winter and Spring Wheat
Rituraj Khound1, Meenakshi Santra2, P. Stephen Baenziger3, Dipak K. Santra1*
1Panhandle Research and Extension Center, University of Nebraska-Lincoln, Scottsbluff, USA; 2Soil and Crop Sciences, Colorado
State University, Fort Collins, USA; 3Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, USA.
Received September 29th, 2013; revised October 20th, 2013; accepted October 29th, 2013
Copyright © 2013 Rituraj Khound et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Microspore culture of wheat generates completely homozygous (doubled haploid) plants in a single generation thereby
reducing the time required for wheat variety development. Success of microspore culture in spring wheat is relatively
higher than that in winter wheat. Cold mediated pretreatment was reported to improve response of microspore culture in
wheat. The objective of the study was to determine and compare the influence of cold pretreatment on microspore cul-
ture in spring and winter wheat. Three spring (“Chris”, “Express”, and “Macon”) and three winter (“Anton”, “Ante-
lope”, and “Camelot”) wheat cultivars were used. In cold pretreatment, excised anthers were incubated in solution B at
25˚C - 28˚C for 4 - 5 days followed by cold treatment at 4˚C for 5 days and were compared with the no-cold pretreat-
ment at 25˚C - 28˚C for 4 - 5 days. Isolated microspores were cultured in induction medium (MMS4) at 27˚C - 28˚C for
25 - 30 days in the dark. Embryos (1 - 2 mm size) were transferred to regeneration medium (MMS5). Numbers of mul-
ticellular structures, transferable embryos and green plants were counted and data were used for analysis of variance
using a generalized linear model. It was observed that cold pretreatment increased multicellular structures, transferable
embryos and green plants in both spring and winter wheat. However, the degree of improvement was higher in spring
wheat compared to winter wheat. The cultivars within spring and winter wheat responded differently. Development of
embryos from pro-embryos was 4 - 5 folds lower in winter wheat than that in spring wheat, indicating requirement of a
possibly different hormonal composition in induction medium for improving embryo induction in winter wheat. This
report may provide future direction of research to improve microspore culture response in winter wheat.
Keywords: Androgenesis; Doubled Haploid; Homozygosity; Biotechnology; Breeding
1. Introduction
Microspore culture is a tissue culture based on the me-
thod to produce completely homozygous (doubled haploid;
DH) plants from immature pollen grains. Single seed de-
scent (SSD) method is usually used for generating homo-
zygous lines in wheat (Triticum aestivum L.) breeding.
However, six generations of inbreeding are required to
obtain 98% homozygosity in SSD method. Microspore
culture generates 100% homozygous lines within one
generation which saves time, space and labor [1]. Since
winter wheat requires 6 to 8 weeks of vernalization to
induce flowering in every generation, microspore culture
can possibly increase the efficiency of breeding programs.
Besides breeding programs, DH plants have also been
used in plant transformation [2], gene mapping [3] and
mutation studies [4].
There are two basic methods of and cogenesis for pro-
duction of DH plants viz., 1) anther and 2) isolated mic-
rospore culture. In isolated microspore culture, micro-
spores are isolated from anthers prior to culture, whereas
anther culture involves culturing the whole anthers [5].
Microspore culture comprises three steps: pretreatment,
induction and regeneration. During pretreatment, micro-
spores are subjected to different stresses which switch
them from gametophytic to sporophytic development.
Carbohydrate starvation using cold shock [6] and heat
shock [7] are the most commonly used pretreatment me-
thods. During pretreatment, microspores are induced into
the embryogenic stage characterized by a star-like ap-
pearance, resulting from a fragmented vacuole and peri-
*Corresponding author.

Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat
2260
pheral cytoplasmic pocket containing the nucleus. For
pretreatment, both anthers and spikes are used as ex-
plants. Induction is the process of developing embryos
from embryogenic microspores. Regeneration involves
germination of microspore derived embryos, followed by
development of shoots and roots.
Success in microspore culture depends on several fac-
tors such as health and genotype of donor plants, condi-
tions during plant growth, pretreatment method, compo-
sition of induction and regeneration media, and density
of microspores in the induction medium [8,9]. Isolated
microspore culture derived wheat plant was first reported
by Mejza et al. (1993) [10] and Tuvesson and Ohlund
(1993) [11]. Since then, several methods to improve the
efficiency of embryo production from isolated micro-
spores have been published [5,6,12-14].
There are several reports of successful androgenesis
from pretreated anthers in wheat [14-18]. In these reports,
anther pretreatment temperature was in the range of 25˚C
- 33˚C. The success was somewhat limited due to set-
backs like low embryo formation rate [15] and high rate
of albinism [17]. Cold pretreatment at 4˚C for several
days was reported to have a beneficial effect on andro-
genic response and spontaneous chromosome doubling in
wheat [1,8,16]. Pretreatment at cold reportedly delayed
the mitotic division of the nucleus, thereby synchronizing
the stage of all the microspores during pretreatment re-
sulting in higher induction frequencies [16]. Moreover,
low temperature was speculated to increase the ratio of
green to albino plants [13].
Several spring wheat cultivars were found to be highly
responsive to microspore culture [14,19]. On the other
hand, winter wheat was observed to be less responsive
compared to spring wheat [17]. There are fewer reports
of cold temperature effect during pretreatment on micro-
spore culture in winter wheat compared to spring wheat.
Comparative analysis of microspore culture response to
cold and no cold pretreatments in spring and winter
wheat may provide some clue for future investigation to
improve response in winter wheat. However, no such
comparative analysis has been reported. Therefore, it is
imperative to learn if cold pretreatment can effectively
improve androgenesis in both spring and winter wheat.
The objectives of this study were to assess: 1) the effect
of cold pretreatment on microspore culture in spring and
winter wheat, and 2) the influence of genotype of wheat
on the success of microspore culture.
2. Materials and Methods
2.1. Plant Material and Growth Conditions
Three spring wheat cultivars viz. Macon [20], Chris [21]
and Express [22] and three winter wheat cultivars viz.
Antelope [23], Anton [24] and Camelot [25] were used
as donor plant material for this study. The plants were
grown in a greenhouse maintained at 20˚C - 23˚C
day/14˚C - 16˚C night temperatures with 18 h day/6 h
night photoperiod regime. The plants were watered every
al- ternate day and fertilized every week with 20:20:20
NPK water soluble fertilizer.
2.2. Pretreatment
The spikes were harvested at half-opened stage when
majority of the anthers contained mostly mid to late un-
inucleated stage microspores [6]. Each experiment con-
sisted of 1100 - 1200 anthers, which were dissected from
approximately 12 - 15 spikes and each experiment was
replicated three times. Two types of pretreatment meth-
ods were employed viz., no-cold and cold. For no-cold
pretreatment, the anthers were aseptically removed from
the surface sterilized spikes and placed in 4 ml solution B
[7] in 60 mm × 15 mm sterile petri dishes. Then the an-
thers were incubated at 25˚C - 28˚C for 4 - 5 days in dark
[26]. In case of cold treatment method, the anthers were
transferred to 4˚C for additional 5 days after 4 - 5 days at
25˚C - 28˚C.
2.3. Microspore Isolation and Induction
Isolation of microspores from pretreated anthers was
performed as described by Indrianto et al. (1999) [7]
with few modifications. The pretreated anthers were
transferred to 50 ml tubes and vortexed for 5 minutes.
The suspended solution of microspores was sieved
through 90 μ mesh and microspores were collected as a
pellet after centrifugation at 800 g for 5 minutes. Isolated
embryogenic microspores were collected from the sus-
pended pellet by a density gradient centrifugation tech-
nique using 21% maltose [6]. The isolated microspores
were co-cultured in 2 - 4 ml MMS4 induction medium
with 5 - 7 wheat ovaries in a 60 mm × 15 mm petri plate,
followed by incubation at 27˚C - 28˚C for 25 - 30 days in
dark. Plates were inspected weekly for loss of media
owing to evaporation, and 0.2 - 0.5 ml fresh medium was
added as needed. Brown ovaries were replaced with fresh
ovaries whenever required.
2.4. Regeneration
After 3 - 4 weeks in induction medium, microspore de-
rived embryos were transferred aseptically to MMS5
regeneration medium [6]. Embryos (1 - 2 mm in size)
were transferred with a sterile forceps from induction
medium into solid regeneration medium in 90 mm plates
and incubated in dark for 3 - 4 days. Then the embryos
were transferred to full light growth chamber at 24˚C (16
h day and 8 h dark). After two weeks, regenerated green
plants were transferred to magenta boxes containing
Murashige and Skoog solid media without plant hor-
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Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat 2261
mones and incubated at 24˚C with 16 h day/8 h night
photoperiod until they reached the 4 - 5 leaves stage.
2.5. Data Recording and Statistical Analysis
After two weeks of culturing the embryogenic cells in
induction medium, number of multicellular structures
(MCS), also known as pro-embryos, was counted using
an inverted microscope and an average was taken from
three replications. Numbers of transferrable embryos (TE)
and green plants (GP) were also counted visually after 3 -
4 and 6 weeks, respectively. The data were transformed
to stabilize the variance effectively [27] where embryos
without plant production were assigned a value of zero
and data were transformed by

X1. Data were ana-
lyzed using a generalized linear model analysis of vari-
ance [28]. Least significant difference (LSD) was used to
separate significantly different means.
3. Results
3.1. Differential Effect in Spring and Winter
Wheat
Spring and winter wheat responded differently to micro-
spore culture when cold and no cold pretreatments were
used (Table 1). When MCS was compared between cold
pretreated spring and winter wheat, it was observed that
cold-mediated pretreatment produced higher number of
MCS in winter wheat (44.26) than that in spring (34.43)
but the difference was non-significant. Similarly, MCS
was higher in winter wheat after no-cold pretreatment
compared to spring wheat and the difference was sig-
nificant. However, the TE and GP were significantly
higher in spring wheat (13.76 and 7.64, respectively)
than that in winter wheat (2.85 and 1.39, respectively)
after cold pretreatment. Significantly higher TE and GP
were also observed in spring wheat compared to winter
wheat in case of no cold pretreatment. When compared
with no cold pretreatment, cold pretreatment increased
MCS, TE, and GP from 22.75 to 34.43 (51% increase),
6.90 to 13.76 (99%), and 3.80 to 7.64 (101%), respec-
tively in spring wheat. Similar results were also obtained
in winter wheat but the degree of multiplication was less.
Cold pretreatment increased MCS, TE, and GP from
32.56 to 44.26 (36%), 2.14 to 2.85 (3%) and 1.09 to 1.39
(3%), respectively in winter wheat.
Green plant recovery was significantly lower in winter
wheat than in spring wheat although MCS yield was
higher in former than later irrespective of the pretreat-
ment methods. To understand this, developmental pro-
gress was compared in both spring and winter wheat un-
der both pretreatment conditions (Table 2). Under cold
pretreatment, 39% of MCS developed into TE in spring
wheat, whereas it was 7% in winter wheat. Under no cold
pretreatment, 31% of MCS developed into TE in spring
Table 1. Microspore culture response of spring and winter
wheat on multicellular structure (MCS), transferable em-
bryos (TE) and green plants (GP) under cold and no cold
pretreatments.
Mean value of trait after
cold pretreatment Mean value of trait after no cold pretreatment
Treatments
(growth
habit type)MCSaTEb GPc MCSa TEb GPc
Spring 34.43 A#13.76 A7.64 A 22.75 B 6.90 A3.80 A
Winter 44.26 A2.85 B1.39 B 32.56 A 2.14 B1.09 B
LSD (0.05)11.233.35 2.06 11.11 0.81 0.39
a = Multicellular structure counted after 2 weeks. b = Transferable embryos
counted after 3 - 4 weeks. c = Green plants counted after 6 weeks. # = Num-
bers followed by the same letter are not significantly different at 0.05 level.
Table 2. Microspore culture response as measured by rate
of conversion of multicellular structure (MCS) into trans-
ferable embryos (TE), transferable embryos (TE) into green
plants and multicellular structure (MCS) into green plants
(GP) in spring and winter wheat under cold and no cold
pretreatments.
% conversion after cold
pretreatment % conversion after no cold pretreatment
Treatments
(growth
habit type)MCS to
TE MCS to GP TE to GP MCS to TE MCS to GP TE to GP
Spring 39 A#22 A 56 A 31 A 18 A62 A
Winter 7 B 4 B 57 A 8 B 4 B 56 A
LSD (0.05)7 3 17 3 4 25
wheat, while it was 8% in winter wheat. Therefore, con-
version of MCS into TE was significantly (4 - 5 folds)
lower in winter wheat than that in spring wheat under
both cold and no cold pretreatments. However, that was
not true in case of TE into GP. Conversion of TE into GP
was similar in both spring (56%) and winter (57%) wheat
under cold pretreatment. It was also similar under no
cold pretreatment in both spring (62%) and winter (56%)
wheat.
3.2. Genotype Effect
Differential responses to microspore culture were ob-
served among all the spring and winter wheat cultivars
(Table 3). After both cold and no cold pretreatments,
MCS in all the three spring cultivars were not signifi-
cantly different. Macon had the lowest MCS (25.58)
among the three spring wheat cultivars after cold pre-
treatment. Under cold pretreatment, the difference in TE
in Macon was significantly lower than Chris and Express,
which had similar TE. However, TE was non-significant
among the three spring cultivars in case of no cold pre-
treatment. Significant difference was observed among the
cultivars in GP. In both cold and no cold pretreatments,
Chris generated highest GP and was significantly higher
than Macon and Express. In case of winter wheat,
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Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat
2262
Table 3. Microspore culture response of three spring and
three winter wheat cultivars on multicellular structure
(MCS), transferable em bryos (TE) and green plants (GP) in
spring and winter whe a t under two pretreatment methods.
Mean value of trait after
cold pretreatment Mean value of trait (NC)
Treatments
(Variety) MCSa TEb GPc MCSa TEbGPc
Macon (spring) 25.58B 7.870B 4.90C 19.30C 7.50A3.60B
Chris (spring) 42.45AB# 17.83A 10.75A 25.50BC 6.70A4.35A
Express
(spring) 35.25AB 15.59A 7.28B 23.46BC 6.48A3.44B
Antelope
(winter) 26.33B 1.71C 1.26D 16.64C 2.06B1.14C
Anton (winter) 53.33A 2.63C 1.79D 33.14B 1.66B1.14C
Camelot
(winter) 53.12A 4.20C 1.14D 48.19A 2.70B1.00C
a = Multicellular structure counted after 2 weeks. b = Transferable embryos
counted after 3 - 4 weeks. c = Green plants counted after 6 weeks. # = Num-
bers followed by the same letter are not significantly different at 0.05 level.
MCS in Antelope were significantly lower than Anton
and Camelot for cold and no cold pretreatments. More-
over, MCS in Camelot was significantly higher than that
of Anton after no cold pretreatment. Surprisingly, no sig-
nificant difference was observed among the three winter
wheat cultivars in terms of TE and GP after both pre-
treatments.
4. Discussion
Effect of cold on microspore culture response was dif-
ferent in various reports. Cold pretreatment was observed
to have negative effects on microspore induction in hex-
aploid wheat but positive effect on plant regeneration in
durum wheat (T. durum L.) [29]. In other reports, cold
pretreatment was found to have neither positive nor ad-
verse effects on microspore embryogenesis [8]. In cur-
rent report, cold was found to increase MCS, TE and GP.
However, degree of improvement was better in spring
wheat than winter wheat. Although cold pretreatment
increased GP production compared to no cold pretreat-
ment, the improvement was not significant. In general,
stress during pretreatment induces recombination of nu-
clear genes or chloroplast genome, which results in low
regeneration, albino plants and somaclonal variation [18].
However, cold pretreatment was found to improve the
green plants to albino ratio in our study (data not shown)
as suggested by Liu et al. (2002) [13].
Spring wheat in general showed significantly better
response to microspore culture than winter wheat similar
to earlier report by Tuvesson and Ohlund (1993) [11].
Although they observed good induction in the cultivars
of both spring and winter wheat, the multicellular struc-
tures in winter wheat cultivars did not develop further.
On the other hand, they successfully regenerated green
plants from the spring wheat cultivars. We also found
similar results in winter wheat. There are more reports
where spring cultivars showed better response in green
plant regeneration than those of winter wheat [17,18]. All
these findings suggest that spring wheat is superior than
winter wheat in terms of response to microspore culture.
The results from comparison between the cultivars in
both spring and winter wheat clearly indicate that re-
sponses to isolated microspore culture vary among dif-
ferent genotypes. Effect of genotype on microspore em-
bryogenesis was shown in many reports [8,9,17,18,30].
In fact, there are certain genotypes which are generally
quite recalcitrant in their in vitro response [9]. Tuvesson
and Ohlund (1993) [11] reported significantly different
green plant regeneration efficiency between two spring
cultivars. It is hence apparent that genotypes belonging to
even the same growth habit (spring or winter) of wheat
show variable responsiveness to microspore culture.
Among the three winter wheat cultivars Camelot pro-
duced the highest number of multicellular structures. Yet,
Camelot’s GP was lower than Antelope and Anton. Tu-
vesson and Ohlund (1993) [11] reported similar results
where one of two spring cultivars produced no green
plant at all. Non-viable structures were also included
during counting of multicellular structures since it was
not possible to distinguish non-viable structures from
viable ones. These non-viable structures did not proceed
in further development to form embryos and conse-
quently regenerated plants. It seems that such non-viable
structures were significantly higher in Camelot than in
Antelope and Anton.
It was indicated in the results that green plant recovery
was significantly lower (~4-folds) in winter wheat than in
spring wheat although yield of multicellular structures
(pro-embryos) was higher in former than later. It was
also observed that conversion of multicellular structures
(pro-embryos) into embryos was significantly (4 - 5 folds)
lower in winter wheat than that in spring wheat. There-
fore, it seems that in winter wheat, development of em-
bryos from multicellular structures (pro-embryos) was
not as efficient as in spring wheat. This may be the rea-
son of low green plant recovery in winter wheat. In mic-
rospore culture, hormones play a crucial part in induction
and regeneration [31]. The level of endogenous hor-
mones may vary between spring and winter wheat with
different growth habits. A common induction medium
may not be equally effective for both spring and winter
wheat. Levels of endogenous phytohormones in micro-
spore-derived embryos of spring and winter wheat may
be different. This may contribute to the significant dif-
ference in development of embryos from pro-embryos
(multicellular structures) between spring and winter
wheat. Similar factors may be the part of the reason for
genotypic differences. This indicates that the hormones
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Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat 2263
in induction medium may have to be manipulated to im-
prove microspore culture response in winter wheat. Fur-
ther study is necessary to address this issue.
5. Conclusion
Green plant regeneration was improved by use of cold
during pretreatment especially in spring wheat. Success
of microspore culture was dependent upon growth habit
(spring and winter) and genotype of wheat. Changes in
hormones in induction medium may be necessary to im-
prove microspore culture response in winter wheat. This
is the first report on comparative assessment of micro-
spore culture response in spring and winter wheat.
6. Acknowledgements
The project was funded by Nebraska Wheat Board. Con-
tribution from Allison Hazen is acknowledged.
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