DanSeed Symposium 2014
10.-11.03.2014
Kobæk Strand
In vitro techniques
q
for propagation
p p g
and
breeding of horticultural crops
Traud Winkelmann
Institute of Horticultural Production Systems,
Leibniz Universitaet Hannover,
Hannover Herrenhaeuser Str.
Str 2
2,
D-30419 Hannover, Germany,
Outline
2

Commercial in vitro p
production in Germanyy

In vitro techniques for plant propagation

I vitro
In
it techniques
t h i
f plant
for
l t breeding
b di

Problems and limitations

Somatic embryogenesis in Cyclamen persicum

Endophytic bacteria in Prunus avium

Summary
Traud Winkelmann
11.03.2014
Commercial in vitro propagation in Germany
ADIVK (German Tissue Culture Association; www.adivk.de)
 About 70 members (½ institutes, ½ companies)

No. of plants
No
produced p.a.
No of commercial laboratories
No.
2002
2003
2004
2005
2006
2007
2008
2009
> 1.000.000
1 000 000
500.000 1.000.000
8
8
8
7
7
7
6
6
6
6
1
2
3
3
2
4
5
4
4
3
100.000 500.000
7
7
6
8
7
4
6
7
8
7
10.000 100.000
7
4
4
5
5
8
7
8
7
10
5
6
8
6
6
2
2
3
5
5
28
27
29
29
26
25
26
28
30
31
<10.000
Total
Courtesy: T. Geier, A. Meier-Dinkel ADIVK
3
2010 2011
Traud Winkelmann
11.03.2014
Commercial in vitro propagation in Germany
50
48,901
Small fruits
45
40
Woody plants
Perennials incl. aquatic plants
Production
n [million plantts]
Orchids
35
30
Other ornamental plants
33 543
33,543
Total
25
20
15
10
4,575
5
3,605
3,441
0
2,564
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Year
(Winkelmann et al. 2006, PCTOC 86: 319-327)
4
Traud Winkelmann
11.03.2014
Principle ways of plant propagation in vitro
90-95 %
5
George et al. (2008)
Traud Winkelmann
11.03.2014
Axillary shoot formation
Commercially most important
 Pros:

+ Easy
+ Applicable to many genotypes and species
+ Low risk for somaclonal variation

Cons:
- Labour intensive, therefore expensive
- Difficult to automate
- Needs several culture steps with different
requirements regarding media (rooting = extra step)
6
Traud Winkelmann
11.03.2014
Culture systems for in vitro propagation

Culture on solid media
+ High plant quality
- Solidifying agents: expensive
- Limited numbers per vessel/shelf…

Culture in liquid media
+ Easy handling
+ Fast growth and propagation
+ Scale-up and automation possible
- Physiological disorders
- Risk of contaminations

TIS = temporary immersion systems
+ High
g plant
p
qualityy
q
+ Fast growth and propagation
+ Scale-up and automation possible
- Risk of contaminations
- High prices for vessels
7
11.03.2014
Traud Winkelmann
http://perso.wanadoo.fr/vitropic/rita/en/ritanim.htm
Foto: S. Richartz
[email protected]
8
11.03.2014
Traud Winkelmann
8
In vitro techniques for plant propagation summary


9
Commercial micropropagation successful in plant
species that are

Difficult to propagate by conventional means

Efficiently propagated in vitro

High-priced
Different ways of propagation and culture systems
are described, but almost exclusively used for
production:

Axillary shoot formation

Culture on solid media in relatively small vessels
Traud Winkelmann
11.03.2014
1. Production of disease-free plants
Meristem/
meristem tip culture
 Thermotherapy
 Cryotherapy
Free of
pathogens


Important for all vegetatively
propagated ornamentals
(e.g. Pelargonium,
Chrysanthemum, petunias),
potato berry fruits
potato,
3 days
10
meristematic dome
meristem tip
Jansen et al. (1984)
Success in
establishing
4 weeks
Traud Winkelmann
6 weeks
Photos: Hydrangea
y
g
by A. Doil
11.03.2014
Meristem tip culture in Dahlia
1 mm
(MSc Thesis Sabine Oster)
6 weeks
6 weeks
0,5 cm
1 cm
1 cm
2 weeks
10 d
1 cm
11
Traud Winkelmann
11.03.2014
TSV (Tobacco Streak Virus) detection
Original plants
M pC D6 D10 D11 D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 nC M
17 out of
21
genotypes
infected
(81%)
TSV‐
amplificate
316 bp
After meristem tip culture
M pC D8.2B D2.1A D5.2A D5.2B D5.2D nC M
Elimination of TSV in 9
genotypes.
TSV‐
amplificate
316 bp
Oster et al. DGG p
proceedings
g
(2013)
12
Traud Winkelmann
11.03.2014
DMV (Dahlia Mosaic Virus) detection
After meristem tip culture
Original plants
L Dvf
Dvf
1
2
3
4
5
6
7
8
nC
L
L
1
2
3
4
5
6
7
8
nC
L
DMV
DMV690 bp
All tested dahlias were infected with DMV (100 %, n = 77)
Oster
O
t ett al.
l DGG proceedings
di
(2013)
13
Traud Winkelmann
11.03.2014
2. Interspecific hybridisation
1 mm
2





14
Many ornamentals = recent or ancient
interspecific hybrids
Potential for novelties not fully tapped
Identification of barriers by observing
in situ pollen tube growth
Pre-zygotic barriers  in vitro
fertilisation
Post-zygotic
Post
zygotic barriers  embryo rescue
Traud Winkelmann
pre-zygotic
postzygotic
11.03.2014
3
4
Overcoming post-zygotic crossing barriers
in Helleborus

Important factors:
Cross combination
 Cross direction
 Time of preparation
 Explant type

Meiners and Winkelmann, Plant Biology (2012)
15
Traud Winkelmann
11.03.2014
Embryo rescue and g
genetic distance
Number
Genetic
distance
♀x♂
0.069-0.264
all interspecific
crosses
crosses
carpels
prepared
ovules cultured
hybrids
obtained
661
3304
40885
217
0.5 %#
8
37
372
111
517
5287
Successful within sections
0 069
0.069
within
ithi Chenopus
Ch
0.081-0.141
within
Helleborastrum
97
26 %
106
2%
Successful between sections
0.241
H. foetidus x
H. argutifolius
21
51
307
0 255
0.255
H. x hybridus x
H. argutifolius
15
61
578
0.264
H. x hybridus x
H. niger
147
468
4640
#percentage
16
of all cultured ovules
2
0.7 %
1
0.2 %
11
0.2 %
Meiners and Winkelmann, Plant Biology (2012)
Traud Winkelmann
11.03.2014
3. In vitro mutagenesis
(i l di polyploidisation)
(including
l l idi ti )

In vitro mutagenesis:






Avoiding chimeras
High number of cells in a
small area
Closed environment
Mutagens better taken up
High propagation rate
Polyploidisation:
Colchicine,
 Oryzalin,...


Mutagenesis:
X- or gamma rays
X
 Chemicals (NMH, EMS)
 Transcription activator-like
effector nucleases
(TALENs)…

17
Traud Winkelmann
Pierik (1987)
after Haccius and Hausner (1975)
11.03.2014
3. In vitro mutagenesis
Flower colour and shape
 Growth habit
 Leaf variegation





18
Blütenfarbe (55 %)
Blüt f
Blütenform
und
d –größe
öß
Blattform und -farbe
Pfl
Pflanzenhabitus
h bi
Traud Winkelmann
Schum (2003)
11.03.2014
4. Genetic transformation
Transformation
19
George et al. (2008)
Traud Winkelmann
11.03.2014
5. Production of (double) haploid plants

Androgenesis



Anther culture
Mi
Microspore
culture
lt
Gynogenesis
Ovary culture
 Ovule
O l culture
lt

Regeneration of haploid plants
Polyploidisation to produce double
haploids
 Important for F1 hybrid breeding
programs: e.g. rape seed, Brassica,
begonias petunias,
begonias,
petunias cyclamen,
cyclamen lily
lily,
Helleborus, …


(Reynolds 1997)
(Eeckhaut
et al. 2001)
20
Traud Winkelmann
11.03.2014
5. Production of (double) haploid plants


Less successful compared
p
to agricultural
g
crops
p
Publications available for many ornamentals (Ferrie
and Caswell 2011)
Strongly genotype-dependent
 Could be ideally combined with mutation induction and
genetic transformation


Future approaches may focus on centromeremediated genome elimination (Ravi and Chan
2010)
Arabidopsis CENH3
 Mutated centromere-specific histone gene

21
Traud Winkelmann
11.03.2014
6. Protoplast
p
culture and fusion

Protoplast culture
direct transformation
somatic hybridisation (protoplast fusion)
 fundamental research (single cell level)



protoplast isolation




protoplast
l
culture
l



22
starting material
enzyme treatment
purification
culture system/immobilisation
osmotic potential
p
plant growth regulators
Traud Winkelmann
11.03.2014
Somatic hybridisation
Fusion






Foto: Petunia, L. Meyer
Partner 2
Partner1
PEG
Selection of
Inhibitor 1
heterofusion products






23
Symmetric
Asymmetric
Cybrids
Combination of diploid
genomes
Combination of
extranuclear DNA
Inhibitor 2
inhibitors
markers
manual selection
cell sorting
l ki regeneration
lacking
ti ability
bilit
of one partner
Traud Winkelmann
11.03.2014
Somatic hybrids of C. persicum and C. coum
PEG mediated protoplast fusion
Prange et al. (2012)
Plant Cell Reports
31:723-735
24
Traud Winkelmann
11.03.2014
In vitro techniques for horticultural
l t breeding
b di - summary
plant
1.
Production of disease-free plants
Maintenance and propagation of breeding
material
25
2
2.
Embryo rescue for interspecific hybrids
3.
In vitro mutagenesis (incl. polyploidisation)
4.
G
Genetic
i transformation
f
i
5.
Production of (double) haploid plants
6.
Somatic hybridisation
Traud Winkelmann
11.03.2014
Problems and limitations
Labour costs; Automation
 Synchronisation
S
h i ti
 Quality evaluation
 Physiological disorders
 Endophytes
 Somaclonal variation
 Genotypic differences

26
Traud Winkelmann
11.03.2014
Somatic embryogenesis in Cyclamen persicum
Induction of embryogenic cells/cultures
½ MS-medium
2.0 mg/l 2,4-D; 0.8 mg/l 2iP
bar = 0.5 cm
½ MS-medium
2.0 mg/l 2,4-D;
0.8 mg/l 2iP
Cell growth
(solid or liquid culture)
embryogenic
Differentiation
(A) and
Diff
ti ti
d
germination (B) of
somatic embryos
Conversion
B
A
A
non embryogenic
Schwenkel and Winkelmann (1998)
Plant Tiss. Cult. Biotechnol. 4 (1): 28 - 34
27
hormone-free
½ MS-medium
Traud Winkelmann
11.03.2014
Somatic embryogenesis in
Cyclamen persicum


28
Applicable for many genotypes
Generally true to type
regenerants
Traud Winkelmann
11.03.2014
Applications
pp
in plant
p
propagation
p p g
1.
Propagation of parental lines of F1
h b id
hybrids
(numbers needed: ~1,000)
2
2.
Propagation
P
ti off sterile
t il iinterspecific
t
ifi
hybrids (‘Odorella‘)
((numbers needed: ~500,000)
,
)
3.
Mass propagation of single elite
genotypes, artificial seed
Foto: A. Ewald, IGZ
Winkelmann et al. (2004)
HortScience 39 (5):
1093-1097
29
Traud Winkelmann
11.03.2014
Limitations and drawbacks






Genotypic differences in
regeneration ability and
regeneration efficiency
Asynchronous formation and
development of somatic embryos
Malformations/ fused somatic
embryos
Precocious germination
Loss of embryogenic competence
Secondaryy somatic embryos
y
Insights in physiology of
embryogenesis
b
i
(transcriptomics/proteomics)
needed
Photos by S. Ratjens and from
 Model = zygotic embryo
Hoenemann et al. (2010)

30
Traud Winkelmann
11.03.2014
PhD project Christina Rode
Protein extraction – Plant material
Zygotic embryos
Somatic embryos
240 embryos in torpedo
stage (80 mg)
1cm
1cm
1mm
Traud Winkelmann
1mm
11.03.2014
PhD project Christina Rode
Protein separation by 2D SDS-PAGE
4 biological and technical replications
 about
b t 1,000
1 000 spots/gel
t / l

32
Traud Winkelmann
11.03.2014
Evaluation of gels
zygotic embryo
PhD project Christina Rode
somatic embryo
>1.5 in SE
1,013 spots
p
in total
>1.5 in ZE
137 spots higher abundant in
zygotic embryos
109 spots higher abundant in
somatic embryos
11.03.2014
Traud Winkelmann
33
Protein identification
(cooperation with D. Heintz and A. Van Dorsselaer, Strasbourg)
Zygotic embryos
Somatic embryos
Total
Spots
p
eluted
900
37
937
For mass spectrometry
263
37
300
Proteins identified
229
32
261
87 %
11.03.2014
Traud Winkelmann
34
Rode et al. (2011a)
Proteins of high abundance in somatic embryos
somatic embryo
11.03.2014
Traud Winkelmann
35
Proteins of high abundance
in zygotic embryos
zygotic embryo
Rode et al. (2011a)
36
Traud Winkelmann
11.03.2014
Enolases
37

Functional enzyme for glycolysis/gluconeogenesis and fatty acid
biosynthesis

„Small“ Enolases highly abundant in zygotic embryos
(MW 1/2-1/3 of functional protein)

Novel seed storage proteins?

Recycling of amino acids?
Traud Winkelmann
Rode et al. 2011a
Plant Mol Biol 75: 305-319
11.03.2014
Digitial proteome reference map (www.gelmap.de)
Rode et al. (2011b) Journal of Proteomics 74: 2214-2219
11.03.2014
Traud Winkelmann
38
Proteomic analysis of somatic
embryo
b
differentiation
diff
ti ti
Experiment A:
Embryogenic suspension
cultures
 Transferred to PGR free
medium
 Protein extraction after
1, 3, 7, 21 and 28 days

Experiment B:

39
28 d old somatic embryos
treated or not with 10 mg/L
abscisic
b i i acid
id
Traud Winkelmann
Rode et al. (2012)
Planta 235: 995-1101
11.03.2014
Rode et al. (2012)
40
Traud Winkelmann
11.03.2014
Alterations in protein abundances
during differentiation

Callus: enzymes related to energy supply,
supply protein metabolism

Somatic embryos: controlled proteolysis (1 d and 21d), auxin
metabolism, storage proteins, isoelectric point switch in catalase
Rode et al. (2012)
41
Traud Winkelmann
11.03.2014
Alterations in protein abundances in
response to
t ABA
- ABA: proteins of primary metabolism and stress response higher
abundant
g p
proteins,, HSP 70
+ ABA: storage
Rode et al. (2012) Planta 235: 995-1011
42
Traud Winkelmann
11.03.2014
Mwangi et al. (2013) Plant Science 201–202: 52–65
Endosperm
p
development
p
Weeks After Pollination (WAP)
0
4 WAP
43
1
2
3
5 WAP
4
5
6
7 WAP
Traud Winkelmann
7
8
9
9 WAP
10
11 WAP
11.03.2014
11
Mwangi et al. (2013)
MS-based protein identification
Zeaxanthin
epoxidase (ZEP)
Xyloglucan
endotransglucosylase
F-box protein
Ethylene recptor 1
416
291
F-box protein
Bet V I allergen
family protein
Major latex protein
Seed maturation
protein
326
312
314
179
313 346
176
196
Leucine-rich repeat
receptor protein kinase
246
11WAP
F-box protein
15
Zeaxanthin
epoxidase (ZEP)
349
287
59
Wax synthase
y
427
331
Vacuolar processing
enzyme (VPE)
Annexin
CHO metabolism (13)
protein processing (12)
defence response (6)
ABA signalling pathway (4)
stress response (3)
lipid pathway (2)
transport (4)
48 spot => 62 proteins
9 spots
p
not identified
7WAP
44
Sugar carrier protein C 313,
314 326,
314,
326 346
Traud Winkelmann
11.03.2014
Role of endosperm in embryogenesis
Nutrition of the developing embryo
 Insulates embryo from mechanical pressure imposed by the
seed coat
 Endosperm-embryo signaling influencing



45
Developing embryo
Maturation/growth arrest (ABA)

Regulates
g
germination
g
timing
g

Developing an artificial endosperm for somatic embryos?
Traud Winkelmann
11.03.2014
Endophytes
What are Endophytes?
Microorganisms internally colonizing plants
and establishing neutral or beneficial
interactions with their host
(Anand et al. 2006)
 Most endophyte/plant relationships are
not well understood.
 In vitro culture:

Often regarded as negative

Causing losses during culture, especially
during rooting and acclimatisation

First reports of beneficial endophytes:
Paenibacillus (Ulrich et al. 2008, PCTOC 93:
347-351)
347
351)
46
Traud Winkelmann
11.03.2014
Prunus avium for timber production
 Fast growing hardwood for the production of
high quality furniture.
B
Breeding
di goals:
l straight
t i ht stem,
t
good
d wood
d
quality, fast growth.
 To achieve these characteristics single trees
with a good habitus are selected and
propagated as in-vitro clones.
 A mixture of several Prunus avium clones in
each batch to ensure plant diversity in the field.
47
Traud Winkelmann
11.03.2014
(Mona Quambusch, PhD project)
In vitro culture of Prunus avium
48
Traud Winkelmann
11.03.2014
(Mona Quambusch, PhD project)
Endophytes in Prunus avium
Identification of endophytic bacteria
 Quantification of endophytes (qPCR)

 in
different culture phases
 under stress conditions
Understanding the balance of endophytic bacteria
 Isolation of beneficial bacteria
 Inoculation
Inoc lation of cultures
c lt es with
ith beneficial bacteria
bacte ia

(cooperation with Institut für Pflanzenkultur, Schnega)

49
COST action FA1103: www
www.endophytes.eu
endophytes eu
Traud Winkelmann
11.03.2014
(Mona Quambusch, PhD project)
Identification of bacteria
Culture-dependent
p
approach
DNA from bacterial isolates
50
Cultureindependent
approach
DNA extracted from plant material
Traud Winkelmann
11.03.2014
Identification of bacteria
Isolation of bacterial endophytes
Pure culture


51
Asterria ±
no
bacteria
Demeter +
Genotypes
Neptun +
N
Plant material
Growth media: nutrient agar and medium #523 (Viss et al
al. 1991)
Cultivation at RT for 5 weeks
Traud Winkelmann
11.03.2014
Identification of bacteria
Neptun (+)
 Three bacterial isolates tested (N-I2, N-I3, N-I4).
The sequences are identical.
 99% identical to Rhodopseudomonas (genus),
phylum Proteobacteria.
Demeter (+)
 Two bacterial isolates sequenced
sequenced.
 D-I1:
 100% identical to Microbacterium
(
(genus),
) phylum
h l
Actinobacteria.
b
 D-I3:
(g
),
 100% identical to Bacillus (genus),
phylum Firmicutes.
Quambusch et al. (2014) Tree Physiology: accepted
52
Traud Winkelmann
11.03.2014
Identification of bacteria
PCR on 16S rDNA
-
-
±
±
+
+
bacterial amplicon,
amplicon mitochondrial amplicon
* Indicates amplicons used for cloning and ARDRA
 DNA extracted from in vitro plant
material of propagation phase
 Primers used:
d 799ff and
d 1492r. (Chelius and
Triplett, 2001)
53
Traud Winkelmann
11.03.2014
Identification of bacteria
Amplified rDNA Restriction Analysis (ARDRA)
Restriction patterns of 95 bacterial 16S rDNA
fragments of a plant sample of Neptun (+).
54
Traud Winkelmann
11.03.2014
(Mona Quambusch, PhD project)
Identification of bacteria
Results of culture-independent analysis
Relattive propo
ortion of clones
Endophytic population of four Prunus avium genotypes
55
100%
80%
Rhodopseudomonas spp.
60%
other α-Proteobacteria
40%
Microbacterium
oba
u spp
spp.
20%
Mycobacterium spp.
uncultured bacterial clone
0%
Fama -
Achilleus - Neptun + Demeter +
Prunus genotype
Traud Winkelmann
11.03.2014
(Mona Quambusch, PhD project)
Identification of bacteria
Summary of results from culture independent and -dependent method
Phylogenetic tree based on 16S rDNA showing the relationship of clones and
isolates from different Prunus avium genotypes to reference sequences.
56
Traud Winkelmann
11.03.2014
Quambusch et al. (2014) Tree Physiology: accepted
Inoculation with Endophytes
Experimental design
 Two genotypes: Fama(–) and Achilleus (–)
 Inoculation with two isolates: N-I-2 (Rhodopseudomonas) and D-I-1
(Microbacterium)
 5 min treatment with bacterial suspension in 10mM MgSO4
 Evaluation of rooting after three weeks, n = 8 vessels with 5 shoots
** *
***
**
Asterisks indicate significant differences between the treatments and corresponding control by
Dunnett’s test (*, ** and *** indicate p ≤ 0.05, 0.01 and 0.001 respectively).
57
Traud Winkelmann
11.03.2014
Summary

58
In vitro culture techniques

Diverse applications in propagation and breeding

Potential not fullyy tapped
pp

Deeper understanding of regeneration processes aspired

Use of molecular tools reasonable

New view on endophytes?

New plant growth regulators
Traud Winkelmann
11.03.2014
References
MEINERS, J., DEBENER, T., SCHWEIZER, G. AND T. WINKELMANN (2011): Analysis of the taxonomic subdivision within the genus
Helleborus by nuclear DNA content and genome-wide DNA markers. Scientia Horticulturae 128: 38-47
MEINERS, J. AND T. WINKELMANN (2012): Evaluation of reproductive barriers and realisation of interspecific hybridisations
depending on the genetic distances between species in the genus Helleborus. Plant Biology, 14: 576-585
MWANGI, J.W., RODE, C., COLDITZ, F., HAASE, C., BRAUN, H.P. AND T. WINKELMANN (2013): Proteomic and histological analyses
p
development
p
in Cyclamen
y
persicum as a basis for optimization
p
p
of somatic embryogenesis.
y g
Plant
of endosperm
Science 201–202: 52–65
OSTER, S., MAISS, E. AND T. WINKELMANN (2013): Detection and elimination of plant viruses in Dahlia. DGG-Proceedings Vol.
3, May 2013, No. 3: 1-5, DOI: 10.5288/dgg-pr-03-03-so-2013 http://www.dgg-online.org/proceedings/vol-032013/dgg-pr-03-03-so-2013.pdf
QUAMBUSCH, M.,
M PIRTTILÄ. A.M.,
A M MYOSORE, V.
V T.,
T WINKELMANN, T.
T AND M.
M BARTSCH (2014): Endophytic bacteria in plant tissue
culture: differences between easy- and difficult-to-propagate Prunus avium genotypes. Tree Physiology (accepted)
PRANGE, A.N.S., SEREK, M., BARTSCH, M. AND T. WINKELMANN 2010: Efficient and stable regeneration from protoplasts of
Cyclamen coum Miller via somatic embryogenesis. Plant Cell Tiss. Org. Cult. 101:171–182
PRANGE, A.N.S., BARTSCH, M., MEINERS, J., SEREK, M. AND T. WINKELMANN (2012): Interspecific somatic hybrids between
C l
Cyclamen
persicum
i
and
d C.
C coum, two
t
sexually
ll incompatible
i
tibl species.
i
Plant
Pl t Cell
C ll Rep.
R
31:723-735
31 723 735
RODE, C., GALLIEN, S., HEINTZ, D., VAN DORSSELAER, A., BRAUN, H.-P. AND T. WINKELMANN (2011A): Enolases: Storage
compounds in seeds? Evidence from a proteomic comparison of zygotic and somatic embryos of Cyclamen persicum
Mill. Plant Mol Biol 75: 305-319
RODE, C., SENKLER, M., KLODMANN, J., WINKELMANN, T. AND H.-P. BRAUN ((2011B)): GelMap
p – A novel software tool for building
g
and presenting proteome reference maps. Journal of Proteomics 74: 2214-2219
RODE, C., LINDHORST, K., BRAUN, H.-P. AND T. WINKELMANN (2012): From callus to embryo - a proteomic view on the
development and maturation of somatic embryos in Cyclamen persicum. Planta 235: 995-1011
SCHWENKEL, H.-G. UND T. WINKELMANN (1998): Plant regeneration via somatic embryogenesis from ovules of Cyclamen
persicum Mill..
Mill Plant Tiss.
Tiss Cult.
Cult Biotechnol.
Biotechnol 4 (1): 28 – 34
WINKELMANN, T., MEYER, L. UND M. SEREK (2004): Germination of Encapsulated Somatic Embryos of Cyclamen persicum.
HortScience 39 (5): 1093-1097
WINKELMANN, T., MUßMANN, V. UND M. SEREK (2004): Cryopreservation of embryogenic suspension cultures of Cyclamen
persicum Mill. Plant Cell Rep. 23 (1-2): 1-8
WINKELMANN, T., SPECHT, J. AND M. SEREK 2006:
2006 Efficient
ff
plant
l
regeneration ffrom protoplasts
l
isolated
l d from
f
embryogenic
b
suspension cultures of Cyclamen persicum Mill. Plant Cell Tiss. Org. Cult. 86: 337-347
59
Traud Winkelmann
11.03.2014
Acknowlegdements:
Melanie Bartsch
Svenja Ratjens
Jenniffer Mwangi
Christina Rode
Samuel Breselge
Cathleen Neitsch
Annika Prange
Mona Quambusch
Julia Meiners
Barbara Raffeiner
Maike Warwas
Viola Mussmann
Ewa Schneider, Bärbel Ernst, Friederike Schröder
Hardy Rolletschek, Henning Tschiersch
(IPK Gatersleben)
Hans-Peter Braun (Leibniz Universität Hannover)
Dimitri Heintz, Alain van Dorsselaer
(University Strasbourg)
Karsten Niehaus (Universität Bielefeld)
Anna Maria Pirttilä (University of Oulu)
60
Traud Winkelmann
11.03.2014