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Invitroandrogenesisoftriticaleinisolated
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Plant Cell, Tissue and Organ Culture 61: 221–229, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
221
In vitro androgenesis of triticale in isolated microspore culture
J
´
anos Pauk
1,∗
, Matti Puolimatka
2,4
, K. Lökös T
´
oth
3
&Tam
´
as Monostori
1
1
Cereal Research Non-profit Co., Department of Wheat Genetics and Breeding, Szeged, P.O.B. 391, H-6701,
Hungary;
2
Agricultural Research Centre of Finland, Plant Breeding Research Section, FIN-31600, Jokioinen,
Finland;
3
St. Stephen University, Department of Statistics, Gödöll˝o, P´ater K. u. 1., H-2103, Hungary;
4
present
address: PPIC, Seed Testing Dept., P.O.B. 111, FIN-32201, Loimaa, Finland; (
∗
requests for offprints; Tel: +36-
62-435-235, Fax: +36-62-434-163; E-mail: janos.pauk@gk-szeged.hu)
Received 17 February 1999; accepted in revised form 27 June 2000
Key words: haploid plant, regeneration, sporophytic development, tissue culture, X Triticosecale
Abstract
Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture.
Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale
genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2
mediumboth in the presence and absence of growth regulators.The highestinduction of microsporeembryogenesis
was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro devel-
opment of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More
than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in
soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid.
Abbreviations: ANOVA – analysis of variance; 2,4-
D – 2,4-dichlorophenoxyacetic acid; ELS – embryo-like-
structures; FDA – fluorescein diacetate; PAA – phenylacetic acid
Introduction
Triticale (X Triticosecale Wittmack) is a spontaneous
synthetic amphiploid cereal which has been consid-
erably improved through breeding (Rimpau, 1891;
Immonen, 1996) and is currently grown in about 2
million hectares world-wide (Rajaram et al., 1993).
Intensive research on triticale was started in the early
1950 (Kiss, 1966; Lelley, 1992). During the last
decade, genetic studies (Lelley and Gimbel, 1989),
somatic tissue culture (Stolarz and Lörz, 1986; Im-
monen, 1996), molecular genetics (Balatero et al.,
1995; Wang et al., 1996) and transgenic studies
(Zimny et al., 1995) have been reported.
Haploid tissue culture of several crops has been
studied (Heberle-Bors, 1989) and used (Bajaj, 1990;
Dunwell, 1996) and registered cultivars have been pro-
duced (Hu et al., 1986; De Buyser et al., 1987; Pauk
et al., 1995; Khush and Virmani, 1996). Two in vitro
cell and tissue culture methods are used to induce an-
drogenesis from microspores: anther- and microspore
culture. Since the induction of the first triticale an-
ther culture-derived haploid plantlets by Wang et al.
(1973), the method has been essentially refined (Bern-
ard, 1980; Charmat and Bernard, 1984; Lukjanjuk and
Ignatova, 1986; Martinez Garcia et al., 1992; Karsai
et al., 1994). The use of potato extract in induction
medium was suggested by Schumann and Hoffmann
(1989). The benefits of anther pretreatment and use of
conditioning anther culture media on triticale pollen
developmentwere reported by Keller (1991). Karsai et
al. (1994) optimised the pH and maltose concentration
in the induction medium. The beneficial effect of man-
nitol in the pretreatment solution and that of maltose in
the induction medium was reported by Gland-Zwerger
et al. (1994).
This report describes in vitro development of
blendor-isolated triticale microspores leading to pro-
duction of green and albino plantlets and the sub-
sequent doubled haploid lines from microspore-
222
derived embryo-like structures. The effects of
one growth regulator-free medium and two growth
regulator-supplementedmedia on microspore embryo-
genesis were studied.
Materials and methods
Plant material
Seeds of winter triticale donor genotypes - a cv
‘Presto’ and four F1’s (Tewo × Moniko, Presto × No-
visadi, Presto × Moniko, Novisadi × Moniko) - were
sown in the nursery in autumn. Each donor triticale
genotypewas a complete hexaploid(2n=6x=42, AAB-
BRR). The donor cultivars used in the crosses were
Polish ‘Tewo’, ‘Moniko’, ‘Presto’ and Yugoslavian
‘Novisadi’ and all are registered cultivars in Hungary.
Donor tillers were cut between the 2nd and 3rd node
with scissors when the primary anthers of the most
mature florets contained mid- to late uninucleate mi-
crospores. Except for the flag leaf, the leaves were cut
and the tillers were put into Erlenmeyerflasks contain-
ing fresh common tap water. Tillers were covered by a
PVC bag to maintain high humidity. Microsporedonor
tillers were cold pre-treated under a dim fluorescent
light at 4
◦
C for about two weeks.
The developmentalstage of the microporeswas de-
termined microscopically after squashing the anthers
in a drop of water. The number of isolated microspores
was estimated with a haemocytometer. The differ-
ent structures in microspore cultures were counted in
representative fields using an inverted microscope.
Isolation of microspores
After cold treatment, about ten spikes containing the
late uninucleate to early binucleate microspores were
removed from the leaf sheath and surface-sterilised
in 2% sodium hypochlorite for 20 min, and then
rinsed three times with sterile water. The sterilised
heads were cut into 1-cm sections and put into 100
ml Waring Micro Blendor container (Eberbach Cor-
poration. Ann Arbor, Michigan, USA) and 60 ml of
0.3M autoclaved mannitol solution (5.47% mannitol
in bidestillated water) was added. Microspores were
isolated by blending twice for 5 sec at low speed, each
time the quality of maceration was visually monitored
through the plastic cap of the vessel. The crude mi-
crospore suspension was filtered through 160 and 80
µm sterile nylon sieves to remove raw spike debris.
The filtrate was divided between four centrifuge tubes
(each 10 ml volume) and centrifuged at 80 g for 5 min.
The pellet was resuspendedin 2 ml 0.3M mannitol and
the microspore suspension was carefully layered over
a 21% autoclaved maltose solution using a pipette.
The solutions were centrifuged at 60 g for 10 minutes.
The viable microspores were collected in a band at the
maltose/mannitol gradient interphase using a Pasteur
pipette. They were resuspended, washed in 0.3M man-
nitol (8 ml/tube) and centrifuged again at 60 g for 5
minutes.
Microspore culture
Following the final centrifuge, the pelleted mi-
crospores were resuspended in 1 ml of culture me-
dium. The quantity of microspores was estimated
using a Burker chamber. The viable microspores were
identified by staining with fluorescein diacetate (Wid-
holm, 1972). Subsequently, the culture density was
adjusted to 0.9-1 × 10
5
microspores ml
−1
by adding
culture medium. Two ml of micropore suspension
was put into a Greiner 35 mm plastic Petri dish. In
the different experiments, four to seven replicates per
genotype were made.
Parafilm-closed cultures were kept in darkness at
80% humidity at 28
◦
C. In the fifth week of subcul-
ture, the ELS (embryo-like-structures) were plated on
Gelrite-solidified microspore culture medium and ex-
posed to a 16-h photoperiod provided by cool white
fluorescent tubes with 20 µmol m
−2
s
−1
irradiance, in
a tissue culture chamber at 28
◦
C.
Regeneration and transfer of green plantlets
When the ELS reached the bipolar stage, the in-
dividual structures were transferred to 190-2 regen-
eration medium (Pauk et al.,1991) in glass culture
tubes. The cultures were kept under a natural pho-
toperiod. The well-tillered and rooted plantlets (about
four weeks after subculture) were transplanted into
non-sterilized 1:1 ratio peat/sandy soil. During the
following two weeks, the plantlets were acclimatized
in a Conviron growth cabinet at 80% humidity. The
irradiance was 200 µmol m
−2
s
−1
with a 16-h pho-
toperiod. Subsequently, the plants were grown under
greenhouse conditions. Haploid plantlets were treated
with colchicine before vernalization, as published for
wheat anther culture-derived plantlets (Pauk et al.,
1995). Vernalization was carried out in a cold chamber
for six weeks at 2–4
◦
C under continuous fluorescent
light at 40 µmol m
−2
s
−1
irradiance.
223
Culture media
Formicrosporeculture inductionmedia, thebasic 190-
2 medium (Zhuang and Jia, 1983) was supplemented
with 3 mM
L-glutamine and the following growth reg-
ulator combinations:190-D/K = 1.5 mg 1
−1
2,4-D and
0.5mgl
−1
Kinetin; 190-PAA = 10 mg l
−1
PAA; 190-0
= without growth regulators.
Each culture medium included 175 mM maltose
(Scott and Lyne, 1994) and the pH was adjusted to
5.8 with 1M KOH. Osmotic pressure was determined
for each preparation using an osmometer. The media
were filter sterilised and stored at room temperature,
but medium older than four weeks was not used.
For subculture of induced ELS, Phytagel (2.5%)
solidified microspore culture medium was used, and
the 175 mM maltose content was reduced to 80 mM.
For the regeneration of ELS collected from the plated
cultures, 190-2 medium was used (Zhuang and Jia,
1983; Pauk et al., 1991).
Ploidy level determination
Chromosome numbers were determined from root
meristem preparations. Donor plant root tips were pre-
treated at 4
◦
C for 24 hours in a cool chamber. The
pre-treated tips were collected and treated in a satur-
ated oxiquinolin suspension for five hours and fixed
in 3:1 ethanol and glacial acetic acid. Root tips were
hydrolysed in 1N HCl at 60
◦
C for 10 minutes be-
fore staining in aceto carmine. Squash preparations
were made in 45% acetic acid and chromosomes were
counted in three wellspread cells from each root tip.
The length of stomatal guard cells was determ-
ined using an ocular micrometer from a 10-mm distal
leaf segment taken from microspore-derived plants.
Chlorophyll was extracted in 70% alcohol and the leaf
segments were mounted in a drop of water on a glass
slide with a coverslip. The length of stomatal guard
cells was measured using a micrometer ocular. The
length of stomatal guard cell of haploids was found
to be 40–50% shorter than that of control hexaploid
plants.
Analysis of data
The evaluation of data (Table 3 and 4) started with
the descriptive statistical analysis (mean, standard
deviation, coefficient of variation) of the three an-
drogenetic parameters sorted by treatments (media).
Standard deviation of data proved to be very high;
therefore, logarithmic transformation for data of ELS
was performed prior to further analysis. Data of albino
and green plants given as percentage were transformed
using arcsin transformation. After transformation, the
data show approximately normal distribution making
further analysis possible. ANOVA was performed for
analysing the effects of the three media on the three
androgenic traits. In the case of the regeneration of
albino and green plants, oneway multiple comparison
of the means of different media was based on the LSD.
The mean values in ELS production were compared in
case of similar deviations by a two sample t-test, and
in the case of different deviations by the Welch-probe.
ANOVA and other statistical tests (Fowler and Cohen
1990) were computed using appropriate programes
from the MiniTab statistical package.
Results and discussion
Characteristic stages of triticale androgenesis in
microspore culture
Viable, mid- and late-uninucleate microspores (Fig-
ure 1a) could be recovered from the blendor after
the crude macerate had been sieved twice and cent-
rifuged with a maltose/mannitol gradient (Figure 1b).
The characteristic stages of triticale androgenesiswere
studied and the data were recorded (Table 1) for the
F
1
cultures of Tewo-Moniko. FDA staining showed
that 64% of the freshly isolated microspores were vi-
able. After two days of culture, 25% of the viable
micropores began cell division. The remaining mi-
crospores ruptured. Nine percent of the dividing cells
produced multicellular colonies. These structures be-
came visible under an inverted microscope within one
week of isolation (Figure 1c). Thirty-five percent of
the multicellular colonies were able to burst through
the exine and develop into globular structures (Figure
1d). Within the next two-three weeks, they quickly
developed into ELS, which were easily detectable on
the surface of the culture medium (Figure 1e). Dur-
ing the fifth week of culture the structures were plated
on solidified induction medium (Figure 1f) and after
one-two weeks, the ELS were transfered to a regen-
eration medium, where they continued to grow. The
well-developed ELS were separated and transferred
into individual regeneration vials, where they germin-
ated, producing green or albino plantlets. Nineteen
percent of the proembryos developed into plantlets.
After the eigth to nine week of culture, well tillered
and rooted regenerants were transplanted into soil and
224
Figure 1. In vitro androgenesis of isolated triticale microspores: (a) freshly isolated late-stage uninucleated microspores in 0.3M mannitol, (b)
separation of viable (star) and dead (arrow) microspores from blendor macerated crude spike extract on a maltose/mannitol cushion (c) dividing
multicellular and non-responsive (dead) microspores one-week after isolation; (d) rapidly-growing microspore-derived aggregates two weeks
after isolation, (e) ELS in the fourth week of culturing, non-divided microspores are visible in background; (f) ELS on a microspore culture
medium before plating on the solidified medium, at five weeks after isolation; (g) microspore-derived colchicine-treated androgenetic triticale
spike in isolation bag with fertile sector (arrow).
