Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat

doi:10.4236/ajps.2013.411278

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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.

Email: *[email protected]

Received September 29th, 2013; revised October 20th, 2013; accepted October 29th, 2013

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-

Open Access AJPS

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

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

Open Access AJPS

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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