American Journal of Plant Sciences, 2013, 4, 2218-2226
Published Online November 2013 (
Open Access AJPS
Assessment of Polyamines and Trehalose in Wheat
Microspores Culture for Embryogenesis and Green
Regenerated Plants
Amina Redha, Patrice Suleman
Department of Biological Sciences, Faculty of Science, Kuwait University, Safat, Kuwait.
Received September 24th, 2013; revised October 20th, 2013; accepted October 25th, 2013
Copyright © 2013 Amina Redha, Patrice Suleman. 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.
Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treat-
ments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible
solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in
wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose
on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three
spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants
among polyamine treatments but trehalose had no effect (P 0.05). Polyamine treatments for 30 min generally pro-
duced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of
twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in
regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryo-
genesis and regeneration of green plants in this study. The study also showed that polyamines could be used to acceler-
ate cultivar development in wheat breeding.
Keywords: Androgenesis; Microspore Culture; Polyamines; Trehalose; Triticum Aestivum
1. Introduction
Microspores are haploid spores that develop into male
gametophytes in heterosporous plants. Immature micro-
spores can be stimulated in vitro to form embryos and
eventually develop to mature plants. Microspore culture
(androgenesis) is currently used to produce double hap-
loid plants for breeding and genetic research to obtain
homozygous lines [1-4]. Androgenesis has been apllied
to more than 250 plant species [5,6]. Wheat double hap-
loid lines obtained through maize pollination and anther
culture methods are being cultivated [7,8]. However, the
application of androgenesis in a number of crops includ-
ing wheat still requires improvement because many lines
are recalcitrant, have a very low response to the protocol
and/or often produce a high percent of albinos. Thus, in
wheat, microspore culture manipulation of conventional
protocols physically and/or chemically is one approach to
maximize the production of green plants [9]. Exogenous
application of polyamines (PAs) to wheat anthers im-
proved the green plant regeneration but overall effect
was dependent on duration of the pretreatment and on the
genotype [10].
The involvement of PAs in plant morphogenesis, root
growth, flower initiation and pollen is well documented
[11-13]. Polyamines are nitrogen containing compounds
of low molecular weight found in plants and animals.
The major forms: putrescine, spermidine and spermine
are present in all parts of the cell including the nucleus
[14] and appear to participate in all cell processes [12].
Polyamines in plants can serve as intracellular mediators
of hormonal activity [11]. The greening or antisenes-
cence role or effect of polyamines has been reported in
freshly isolated oat protoplasts [15] and spermine and
spermidine were involved in the retention of chlorophyll
and stabilization of thylakoid membranes [16,17]. Cur-
rent research on PAs is also devoted to the amelioration
of abiotic stresses such as osmotic stress, drought, heat,
chilling, light intensity, mineral nutrient deficiency and
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
UV irradiation [18]. Although PAs have been studied in
somatic embryogenesis in tissue culture [19,20] and in
androgenesis [10,21], the effects of these compounds in
microspore culture in wheat lines still require investiga-
tions to improve green plant regeneration.
PAs and trehalose accumulate and act as compatible
solutes in a number of stressed plants. Trehalose, a non-
reducing disaccharide, is found in some organisms in-
cluding a number of plants in which it serves as an os-
moprotectant. It stabilizes proteins and biological mem-
branes under a variety of stress conditions including hy-
drostatic pressure and osmotic stress [22]. Other pro-
posed roles of trehalose include regulation of carbohy-
drate utilization, plant growth and development [23],
especially in regulation of embryo maturation [24].
However, the effects of trehalose in wheat microspore
culture have not been studied. Currently, a number of
drought tolerant hexaploid wheat genotypes from Inter-
national Centre for Agricultural Research in the Dry Ar-
eas, Allepo, Syria (ICARDA) are being evaluated under
our local semi-arid conditions; hopefully using double
haploid plants. This study screened microspore culture of
three wheat genotypes for its application in our local
breeding and biotechnology programs. The objective of
this work was to investigate a protocol for efficient wheat
microspore culture by the application of polyamines and
trehalose, (two compatible solutes involved in plant
growth and development) to wheat microspores to im-
prove or increase the number of microspore-derived
double haploid green plants.
2. Materials and Methods
2.1. Plant Material
Three spring wheat genotypes were used in the study.
The genotype DH83Z118.32 was selected for its agro-
nomic traits by the breeding department of the Swiss
Federal Research Station, Zurich-Reckenholz (FAL) and
for its high androgenetic response by ETH Zurich, Swit-
zerland [25]. Its androgenetic response was confirmed
for anther as well as isolated microspore culture. The
other two lines ICARDA17 and ICARDA39 were from
the International Center for Agricultural Research in the
Dry Areas (ICARDA), Aleppo, Syria. The anther culture
response for a number of ICARDA genotypes including
ICARDA17 and 39 was examined in an earlier study [10].
Seeds were germinated at 25˚C and after 10 days seed-
lings were planted in peat moss in 10 cm diameter pots,
then placed in growth chambers at a temperature cycle of
23/18˚C day/night, 16 h photoperiod and 290 - 310
µmol·m2·s1 light intensity. After two weeks plants were
transplanted to 15 cm diameter pots.
Spikes were collected when microspores were at mid-
to late-uninucleate stage [26]. The appropriate develop-
mental stage of microspores was determined by spike
and anther morphology as well as examination of iso-
lated microspores using acetocarmine stain [27]. Spikes
containing mid-to late-uninucleate microspores were
wrapped in a foil and kept at 3˚C - 4˚C for duration of 7
to 10 days for further studies.
2.2. Microspore Isolation and Treatment with
Spikes were surface sterilized with 70% ethanol and an-
thers were aseptically removed from the ensheathing
leaves into a glass vials containing 3 ml of AB medium
[28]. The microspores were isolated by stirring the an-
thers in AB medium with a magnetic bar for 2 - 3 min at
600 rpm and the suspension filtered through a 60 µm
sieve. Microspores were treated with putrecine (Put),
spermidine (Spd) and spermine (Spm) alone at 1.0 mM
or 0.5 mM combinations of the three PAs. The duration
of the treatments was 30 or 60 min and the microspores
were washed twice with 3 ml of AB medium, centrifuged
for 5 min and the pellet was re-suspended in A2 medium
[26]; and counted using a haemocytometer. Microspores
were cultured in Petri dishes at a density of 2 × 104
microspores/ml with six immature ovaries and incubated
at 27˚C in the dark for 4 days [28].
2.3. Microspore Culture Conditions
Microspores were kept at 27˚C ± 1˚C in the dark for a
period of 4 to 6 weeks. Developing embryos (2 mm)
were sub-cultured on embryo medium (EM) and the re-
generated plantlets were transferred to plant regeneration
medium (PM), [29]. Phytagel 3 g·l1 was added to EM
and PM media. The embryos were incubated at 27˚C
with a photoperiod of 16 h and a light intensity of 30 - 55
µmol·m2·s1. Similar conditions were used to regenerate
green plants for 3 weeks, which were then transferred to
peat moss for [30]. The green plants were maintained in
growth chambers till maturity.
2.4. Viability of PA Treated Microspores
Viability of microspores treated with different concentra-
tions of PAs or with trehalose was determined by stain-
ing with fluorescein diacetate (FDA) solution [31]. The
percentage of viable microspores was determined for at
least 500 microspores per treatment after 1 and 3 days
under fluorescent microscopy. Intense fluorescence and
swollen microspores were considered viable, while
non-stained and shriveled microspores were considered
Open Access AJPS
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
2.5. Treatment of Microspores with Trehalose
Experimental conditions and media for the evaluation of
trehalose treatments were the same as described above.
Isolated microspores were treated with 0.1, 0.2 and 0.4 M
of trehalose for 1, 2 and 3 days, and then transferred to
A2 medium. Developing embryos were sub-cultured on
embryo medium (EM) and regenerated plantlets trans-
ferred to plant regeneration medium (PM) under the cul-
ture conditions described above.
2.6. Determination of Ploidy Level
Ploidy of regenerants was estimated using Partec CyFlow
space flow cytometer, (Partec GmbH, Münster, Germany)
and the data analysis was performed with FloMax soft-
ware, version 3, after transferring the plantlets to soil.
Samples were prepared using young emerging leaves of
10 - 12 day-old seedling. Young leaves were fixed in
ethanol-glacial acetic acid (3:1 v/v). Leaves were
chopped at 4˚C using a razor blade in 2 ml of Partec
DNA staining solution. Extracts with nuclei were filtered
through a 30 µm nylon gauze filter. More than 3000 nu-
clei were counted per sample. Leaf samples from seed-
lings of each genotype were used as reference. Ploidy
level of each sample was estimated by comparing the
peaks of histograms of the regenerated green plants to
that of its standard genotype (2n) as a control.
Green regenerated plantlets from the three wheat
genotypes were grown in the greenhouse and evaluated
for fertility and seed setting. Inflorescence of each plant
was protected from out-crossing using pollination bags
during flowering. Percentage of seed setting by plants of
each treatment was determined by checking for viable
2.7. Evaluation of Androgenic Response
Embryogenesis was recorded as the percentage of de-
veloped embryos (Embryos/100 microspores), green re-
generated plants (GRP/100 embryos), albino and total
regenerated plants per 100 microspores were also re-
corded. Data were statistically analyzed using ANOVA
and means were separated by Duncan’s multiple range
test (DMRT) with MSTAT-C, University of Michigan
Statistical package.
3. Results
3.1. Microspore Viability
Microspores harvested from the middle portion of inflo-
rescence of the three donor plants were mostly in mid- to
late-uninucleate stage (Figure 1). They were generally
spherical in shape, thin to dense cell walls with a large
nucleus that was off-centered and very close to the cell
Viable microspores treated with PAs or trahalose
showed intense fluorescence and were larger or swollen,
while nonviable ones were shriveled or misshaped (Fig-
ure 2). Regardless of the concentration of the PA treat-
ments, the viability of the ICARDA17 and 39 micro-
spores significantly declined (P 0.05) within 3 days
(Figures 3(a) and (b)). Similarly, viability declined with
increased concentration and duration of trehalose treat-
ments (data not shown).
Multicellular structures or pro-embryos emerged from
the exine wall of microspores within 7 - 10 days (Figure
4) which developed into mature embryos after 4 weeks
on EM medium.
3.2. Effect of PAs on Microspore Cultures
Generally, ICARDA39 produced the highest number of
Figure 1. Light microscopy of isolated wheat microspore at
mid-to late uninucleate stage (40×).
Figure 2. Fluorescent microcopy of viable microspores
stained with fluorescein diacetate. Non-viable microspores
ere shriveled, or misshaped and not-stained. w
Open Access AJPS
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
Open Access AJPS
Figure 3. Percent viability of microspores treated with PAs after 1 - 3 days (a) viable microspore in ICARDA17 and (b) in
ICARDA39. Abbreviations for treatments: T0 = control; T1 = 1 mM Put; T2 = 1 mM Spd; T3 = 1 mM Spm; T4 = 0.5 mM
(Put + Spd); T5 = 0.5 mM (Put + Spm); T6 = 0.5 mM (Spd + Spm); and for 60 min T7 = 1 mM Put; T = 8 mM Spd; T9 = 1
mM Spm; T10 = 0.5 mM (Put + Spd); T11 = 0.5 mM (Put + Spm); T12 = 0.5 mM (Spd + Spm); The bars represent the stan-
dard error of the mean.
GRPs per 100 embryoids compared to the other two
genotypes in the 30 min PA treatments (Figure 5(a)).
Two PA treatments, 1.0 mM Put and 1.0 mM Spm for 30
min produced the highest number of GRPs/100 embry-
oids in ICARDA39 whiles in DH83Z, the 0.5 mM com-
bination treatments of (Put + Spm) and (Spm + Spd)
yielded the highest GRPs/100 embryoids (Figure 5(a)).
Embryos in 60 min treatments of DH83 Z and ICARDA17
developed less but significant number of green plants
(Figure 5(b)). A high percent (52% - 58 %) of these em-
bryos in these treatments however, developed into green-
ish calli with adventitious roots.
Green regenerated plants (GRPs) per 100 microspores
in the genotype ICARDA17 was not significantly differ-
ent among 30 min PA treatments (Figure 6(a)) but
ICARDA 39 and DH83Z produced significant numbers
of green plants among PA treatments. The highest num-
ber of GRPs/100 microspores was in DH83Z treated with
1mM Spm alone and 0.5 mM combinations of PAs. The
number of GRPs produced in the 60 min PA treatments
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
Figure 4. Multinucleate structure or a pro-embryo from a
microspore 7 - 10 days after treatment with Pas.
Figure 5. GRPs per 100 embryos for three wheat genotype
microspores treated with PAs for (a) 30 min and (b) 60 min.
Abbreviations for treatments: T0 = control; T1 = 1 mM Put;
T2 = 1 mM Spd; T3 = 1 mM Spm; T4 = 0.5 mM (Put + Spd);
T5 = 0.5 mM (Put + Spm); T6 = 0.5 mM (Spd + Spm). The
bars represent the standard error of the mean.
was also significantly different in the genotypes (P
0.05) but PA treatments for 30 min generally produce
more GRPs per 100 microspores than the 60 min treat-
Figure 6. GRPs per 100 microspores produced from PAs
treatments to microspores for (a) 30 min and (b) 60 min for
three wheat genotypes. Abbreviations for treatments: T0 =
control; T1 = 1 mM Put; T2 = 1 mM Spd; T3 = 1 mM Spm;
T4 = 0.5 mM (Put + Spd); T5 = 0.5 mM (Put + Spm); T6 =
0.5 mM (Spd + Spm). The bars represent the standard
error of the mean.
ments (Figure 6(b)). Microspores from ICARDA17
produced 1.5 times GRPs. Figures 7(a) and (b) show
representative green and albino plantlets obtained on PM
3.3. Effect of Trehalose
Treatment of microspores with trehalose resulted in total
inhibition of embryogenesis in DH83Z but in genotypes
ICARDA17 and 39 less than 10 embryoids developed
per treatment which were significantly lower compared
to the control treatments. None of these embryos devel-
oped into albino or green plants. Trehalose treatment at
0.4 M did not produce any embryos in all 3 genotypes.
3.4. Ploidy Analysis
Figures 8(a) and (b) show ploidy levels of GRPs deter-
Open Access AJPS
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
Figure 7. Plantlets obtained after PA treatments: (a) green
regenerated plantlets and (b) albino plants.
mined by flow cytometry based on the relative fluores-
cence intensity of stained nuclei in haploid and double
haploid plants. Double haploid plants occurred in all
three genotypes but not in all treatments. Seed setting in
DH83Z was 20.4% to 33.3%; in ICARDA17 it was
11.1% to 26.7% and 15.2% to 24.1% in ICARDA39. The
range of seed setting in controls was 12.43% to 16.85%.
Nine PA treatments in DH83Z were able to set seeds,
with three of them over 25% (Table 1). In ICARDA39
only two treatments failed to set seeds but seed setting
was less than 25% in all the treatments. ICARDA17
showed the lowest seed setting with only 4 treatments
setting seeds. Seed setting in control treatments of
DH83Z and ICARDA39 were lower compared to PA
treatments. Figure 9 shows representative potted green
fertile regenerants of the wheat genotypes.
4. Discussion
Polyamines play a positive role in plant growth and de-
velopmental processes including cell division, embryo-
Figure 8. Flow cytometry histograms depicting ploidy of
regenerated plants from PA treated microspores: (a) ploidy
of haploid plants and (b) double haploid regenerants.
Genesis, and in tissue culture systems they play a posi-
tive role in morphogenesis [32,33]. PAs have also re-
sulted in inhibitory effects in somatic embryos [34] and
embryo abnormalities [35]. In this study PAs produced
positive results in two of the wheat lines (DH83Z and
ICARDA39). PAs applied to microspores for 30 min
improved the regeneration of green plants compared to
the 60 min pretreatments. Putrescine combined with Spd
or Spm significantly increased GRPs in DH83Z and
ICARDA17 but in ICARDA39; 1.0 mM Spd applied for
30 min significantly improved GRPs. The production of
GRPs in ICARDA17 improved in 0.5 mM (Put + Spd)
and 0.5 mM (Spd + Spm) in the 60 min treatments. It is
apparent from these observations that the need and role
or PAs may be limited in some genotypes of wheat. f
Open Access AJPS
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
Open Access AJPS
Table 1. Effect of PAs on percent seed setting of microspore derived plants in three wheat genotypes.
PA Treatments DH83Z ICARDA17 ICARDA39
T0 Control 16.85 16.06 12.43
T1 1.0 mM put for 30 min 23.33 26.67 17.81
T2 1.0 mM spd for 30 min 20.40 11.11 18.55
T3 1.0 mM spm for 30 min 0 20.00 0
T4 0.5 mM (put + spd) for 30 min 33.33 17.23 24.06
T5 0.5 mM (put + spm) for 30 min 20.83 0 16.67
T6 0.5 mM (spd + spm) for 30 min 21.97 0 0
T7 1.0 mM put for 60 mins 0 0 21.21
T8 1.0 mM spd for 60 mins 29.17 0 15.15
T9 1.0 mM spm for 60 mins 23.54 0 15.56
T10 0.5 mM (put + spd) for 60 mins 0 0 21.67
T11 0.5 mM (put + spm) for 60 mins 21.21 0 18.43
T12 0.5 mM (spd + spm) for 60 mins 29.82 0 20.32
Figure 9. Representative potted double haploid plants from
PA treated microspore culture of wheat genotypes.
Polyamines in this study also induced the formation of
embryos that developed into calli with adventitious roots
which is in agreement with the observations of other re-
searchers [28,36]. Some of these embryos developed into
greenish calli which has been observed in wheat anther
culture [10], in rice anther culture [37] and this could be
due to the presence of Spd and Spm which have been
shown to retain chlorophyll as well as stabilize thylakoid
membranes [38]. Undifferentiated greenish embryo cells
in wheat anther culture pretreated with PAs have been
shown to contain numerous agranal-like chloroplasts
Both trehalose and PAs have the capacity to alleviate
osmotic stress in plants. In this study embryogenesis and
regeneration of green plants were dependent on the type
as well as the concentration of osmoticum used because
trehalose and PA exhibited contradictory results. Similar
observations were observed with manitol in microspore
cultures in barley [39] and durum wheat [4]. In this study
the significant impact of PAs in wheat microspore cul-
ture was in embryogenesis which resulted in increased
green plant regenerants.
Also in this study lower numbers of double haploid
plants were produced in controls compared to PA treat-
ments and flow cytometry results using very young
leaves were very similar to the percent of seed setting
and to a large extent indicative of the fertility of the
green regenerated plants.
Finally, the results of this study showed that the appli-
cation of 1.0 mM Put, Spd and Spm alone or 0.5 mM
combinations of these PA treatments on isolated micro-
spores for 30 min improved the embryogenesis and pro-
duction of green plants in ICARDA39 and DH83Z
genotypes. Application of 0.1 - 0.4 M trehalose to
microspores however, had no impact on embryogenesis
and regeneration of plants. The effectiveness of PAs in
wheat microspore culture depended on the concentration
and duration of PA treatments as well as the genotype.
Treatment of microspores with polyamines not only im-
Assessment of Polyamines and Trehalose in Wheat Microspores Culture for
Embryogenesis and Green Regenerated Plants
proved plant regeneration but also significantly improved
seed setting and could be used to accelerate some cultivar
development in wheat breeding programs.
5. Acknowledgements
The Authors thank Research Administration of Kuwait
University for funding this project (# SL04/06). We also
thank Cynthia A. Menezes who was the Research Assis-
tant for the project.
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Embryogenesis and Green Regenerated Plants
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