Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

doi:10.4236/ajps.2013.411275

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American Journal of Plant Sciences, 2013, 4, 2218-2226

Published Online November 2013 (http://www.scirp.org/journal/ajps)

http://dx.doi.org/10.4236/ajps.2013.411275

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.

Email: aminaredha@gmail.com, [email protected]

Received September 24th, 2013; revised October 20th, 2013; accepted October 25th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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

2219

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·m−2·s−1 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

PAs

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·l−1 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·m−2·s−1. 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

nonviable.

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Assessment of Polyamines and Trehalose in Wheat Microspores Culture for

Embryogenesis and Green Regenerated Plants

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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

seeds.

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

wall.

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

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Assessment of Polyamines and Trehalose in Wheat Microspores Culture for

Embryogenesis and Green Regenerated Plants

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(a)

(b)

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

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Figure 4. Multinucleate structure or a pro-embryo from a

microspore 7 - 10 days after treatment with Pas.

(a)

(b)

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-

(a)

(b)

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

medium.

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-

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Assessment of Polyamines and Trehalose in Wheat Microspores Culture for

Embryogenesis and Green Regenerated Plants

2223

(a)

(b)

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-

(a)

(b)

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

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Assessment of Polyamines and Trehalose in Wheat Microspores Culture for

Embryogenesis and Green Regenerated Plants

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Table 1. Effect of PAs on percent seed setting of microspore derived plants in three wheat genotypes.

Genotype

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

[10].

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

2225

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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