DanSeed Symposium 201410.-11.03.2014Kobæk Strand
In vitro techniques for propagation and q p p gbreeding of horticultural crops
Traud WinkelmannInstitute of Horticultural Production Systems,
Leibniz Universitaet Hannover Herrenhaeuser Str 2Leibniz Universitaet Hannover, Herrenhaeuser Str. 2, D-30419 Hannover, Germany,
OutlineOutline Commercial in vitro production in Germanyp y
In vitro techniques for plant propagation
I it t h i f l t b di In vitro techniques for plant breeding
Problems and limitations
Somatic embryogenesis in Cyclamen persicum
Endophytic bacteria in Prunus avium Endophytic bacteria in Prunus avium
Summary
Traud Winkelmann2 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 of commercial laboratoriesNo. of plantsproduced p.a.
No. of commercial laboratories
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
1 000 000 8 8 8 7 7 7 6 6 6 6> 1.000.000 8 8 8 7 7 7 6 6 6 6500.000 -1.000.000 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 -7 4 4 5 5 8 7 8 7 10100.000 7 4 4 5 5 8 7 8 7 10
<10.000 5 6 8 6 6 2 2 3 5 5Total 28 27 29 29 26 25 26 28 30 31
Traud Winkelmann3 11.03.2014
Total 28 27 29 29 26 25 26 28 30 31
Courtesy: T. Geier, A. Meier-Dinkel ADIVK
Commercial in vitro propagation in Germany
48,901
45
50Small fruits
Woody plants
33 54335
40
ts]
Woody plants
Perennials incl. aquatic plants
Orchids
Other ornamental plants33,543
25
30
n [m
illio
n pl
ant
Total
10
15
20
Prod
uctio
n
4,575
2,5643,4413,605
0
5
10
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004Year
Traud Winkelmann4
(Winkelmann et al. 2006, PCTOC 86: 319-327)
11.03.2014
Principle ways of plant propagation in vitro
90-95 %
George et al. (2008)11.03.20145 Traud Winkelmann
Axillary shoot formationAxillary shoot formation
Commercially most important Commercially most important Pros:
+ Easy+ Easy+ Applicable to many genotypes and species+ Low risk for somaclonal variation+ 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+ High plant quality- Solidifying agents: expensive- Limited numbers per vessel/shelf…
Culture in liquid media+ Easy handling+ Fast growth and propagation+ Fast growth and propagation+ Scale-up and automation possible- Physiological disorders- Risk of contaminations
TIS = temporary immersion systems+ High plant qualityg p q y+ Fast growth and propagation+ Scale-up and automation possible- Risk of contaminations- High prices for vessels
http://perso.wanadoo.fr/vitropic/rita/en/ritanim.htm
Foto: S. RichartzTraud Winkelmann11.03.20147
Traud Winkelmann11.03.20148 8
In vitro techniques for plant propagation -summary
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 Different ways of propagation and culture systemsare described, but almost exclusively used forproduction:
Axillary shoot formation
Culture on solid media in relatively small vessels
9
Culture on solid media in relatively small vessels
Traud Winkelmann 11.03.2014
1. Production of disease-free plants
Meristem/meristem tip culture
Free of pathogensmeristem tip culture
Thermotherapy Cryotherapy
meristematic dome
Important for all vegetativelypropagated ornamentals meristem tippropagated ornamentals(e.g. Pelargonium, Chrysanthemum, petunias), potato berry fruits
meristem tip
Jansen et al. (1984)potato, berry fruits Success in
establishing
Photos: Hydrangea
Traud Winkelmann10
y gby A. Doil
3 days 4 weeks 6 weeks
11.03.2014
Meristem tip culture in Dahlia (MSc Thesis Sabine Oster)
1 mm
6 weeks
6 weeks
0,5 cm1 cm
6 weeks
1 cm
2 weeks
1 cm
2 weeks
10 d
11 Traud Winkelmann 11.03.2014
TSV (Tobacco Streak Virus) detection
M pC D6 D10 D11 D13 D14 D15 D16 D17 D18 D19 D20 D21 D22 nC M
Original plants
17 out of17 out of21 genotypesinfected
After meristem tip culture
TSV‐amplificate
316 bp
infected(81%)
M pC D8.2B D2.1A D5.2A D5.2B D5.2D nC M
After meristem tip culture
Elimination of TSV in 9Elimination of TSV in 9 genotypes.
TSV‐amplificate
316 bp
Oster et al. DGG proceedings
12 Traud Winkelmann 11.03.2014
p g(2013)
DMV (Dahlia Mosaic Virus) detection
Original plantsL Dvf Dvf 1 2 3 4 5 6 7 8 nC L L 1 2 3 4 5 6 7 8 nC L
After meristem tip cultureL Dvf Dvf 1 2 3 4 5 6 7 8 nC L L 1 2 3 4 5 6 7 8 nC L
DMVDMV-690 bp
All tested dahlias were infected with DMV (100 %, n = 77)
O t t l DGG di
13 Traud Winkelmann 11.03.2014
Oster et al. DGG proceedings(2013)
2. Interspecific hybridisation
2
1 mm
Many ornamentals = recent or ancient interspecific hybrids
2
interspecific hybrids Potential for novelties not fully tapped Identification of barriers by observing
3pre-zygotic Identification of barriers by observing
in situ pollen tube growth Pre-zygotic barriers in vitro Pre-zygotic barriers in vitro
fertilisation Post-zygotic barriers embryo rescue
4
post-zygotic
Post zygotic barriers embryo rescue
Traud Winkelmann14 11.03.2014
Overcoming post-zygotic crossing barriersin Helleborusin Helleborus
Important factors: Cross combination Cross direction Time of preparation Time of preparation Explant type
Traud Winkelmann15
Meiners and Winkelmann, Plant Biology (2012)
11.03.2014
Embryo rescue and genetic distanceg
NumberNumber
Genetic distance ♀ x ♂ crosses carpels
prepared ovules cultured hybrids obtained
all interspecific 2170.069-0.264 all interspecificcrosses 661 3304 40885 217
0.5 %#
Successful within sections
0 069 ithi Ch 8 37 372 970.069 within Chenopus 8 37 372 26 %
0.081-0.141 withinHelleborastrum 111 517 5287 106
2 %Successful between sectionsSuccessful between sections
0.241 H. foetidus xH. argutifolius 21 51 307 2
0.7 %
0 255 H. x hybridus x 15 61 578 10.255 H. argutifolius 15 61 578 0.2 %
0.264 H. x hybridus xH. niger 147 468 4640 11
0.2 %
11.03.2014Traud Winkelmann16
#percentage of all cultured ovules Meiners and Winkelmann, Plant Biology (2012)
3. In vitro mutagenesis(i l di l l idi ti )(including polyploidisation)
In vitro mutagenesis: Avoiding chimeras High number of cells in a
small area Closed environment Mutagens better taken up High propagation rate
Polyploidisation: Colchicine, Colchicine, Oryzalin,...
Mutagenesis: X or gamma rays X- or gamma rays Chemicals (NMH, EMS) Transcription activator-like
effector nucleases
Pierik (1987) after Haccius and Hausner (1975)
Traud Winkelmann17
effector nucleases(TALENs)…
11.03.2014
3. In vitro mutagenesis Flower colour and shape Growth habit
Blütenfarbe (55 %)Blüt f d öß
Schum (2003)
Leaf variegation
Blütenform und –größe Blattform und -farbe
Pfl h bi Pflanzenhabitus
18 Traud Winkelmann 11.03.2014
4. Genetic transformation
Transformation
19George et al. (2008)
11.03.2014Traud Winkelmann
5. Production of (double) haploid plants
Androgenesis Anther culture
Mi lt Microspore culture Gynogenesis
Ovary cultureO l lt Ovule culture
Regeneration of haploid plants Polyploidisation to produce double
haploidshaploids Important for F1 hybrid breeding
programs: e.g. rape seed, Brassica,begonias petunias cyclamen lilybegonias, petunias, cyclamen, lily, Helleborus, …
(Eeckhaut et al. 2001)
(Reynolds 1997)
20 Traud Winkelmann 11.03.2014
5. Production of (double) haploid plants
Less successful compared to agricultural cropsp g p Publications available for many ornamentals (Ferrie
and Caswell 2011) Strongly genotype-dependent Could be ideally combined with mutation induction and
genetic transformationgenetic transformation
Future approaches may focus on centromere-mediated genome elimination (Ravi and Chanmediated genome elimination (Ravi and Chan 2010) Arabidopsis CENH3 Mutated centromere-specific histone gene
Traud Winkelmann21 11.03.2014
6. Protoplast culture and fusionp
Protoplast culture direct transformation direct transformation somatic hybridisation (protoplast fusion) fundamental research (single cell level)
protoplast isolation starting material enzyme treatment enzyme treatment purification
l l protoplast culture culture system/immobilisation osmotic potentialp plant growth regulators
22 Traud Winkelmann 11.03.2014
Somatic hybridisation Fusion Fusion
Symmetric Asymmetric Asymmetric Cybrids Combination of diploid
Foto: Petunia, L. Meyer
Partner1 Partner 2genomes
Combination ofextranuclear DNA PEGextranuclear DNA
Selection ofheterofusion products
Inhibitor 1 Inhibitor 2heterofusion products
inhibitors markers manual selection cell sorting
l ki ti bilit
23
lacking regeneration abilityof 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
Traud Winkelmann24
Plant Cell Reports 31:723-735
11.03.2014
In vitro techniques for horticulturall t b diplant breeding - summary
1. Production of disease-free plantsMaintenance and propagation of breedingMaintenance and propagation of breedingmaterial
2 Embryo rescue for interspecific hybrids2. Embryo rescue for interspecific hybrids3. In vitro mutagenesis (incl. polyploidisation)
G i f i4. Genetic transformation5. Production of (double) haploid plants6. Somatic hybridisation
Traud Winkelmann25 11.03.2014
Problems and limitations
Labour costs; AutomationS h i ti Synchronisation
Quality evaluation Physiological disorders Endophytes Somaclonal variation Genotypic differences Genotypic differences
Traud Winkelmann26 11.03.2014
Somatic embryogenesis in Cyclamen persicumInduction of embryogenic cells/cultures
½ MS-medium 2.0 mg/l 2,4-D; 0.8 mg/l 2iP
bar = 0.5 cm
Diff ti ti dConversion
Differentiation (A) and germination (B) of somatic embryos
B
Cell growth(solid or liquid culture)
embryogenic
½ MS-medium 2.0 mg/l 2,4-D; 0.8 mg/l 2iP
A B
A
non -embryogenic
11.03.2014Traud Winkelmann27
Schwenkel and Winkelmann (1998) Plant Tiss. Cult. Biotechnol. 4 (1): 28 - 34
hormone-free½ MS-medium
Somatic embryogenesis in Cyclamen persicum
Applicable for many genotypes
Cyclamen persicum
Applicable for many genotypes Generally true to type
regenerants
Traud Winkelmann28 11.03.2014
Applications in plant propagationpp p p p g
1. Propagation of parental lines of F1 h b idhybrids (numbers needed: ~1,000)
2 P ti f t il i t ifi2. Propagation of sterile interspecific hybrids (‘Odorella‘)(numbers needed: ~500,000)( , )
3. Mass propagation of single elite genotypes, artificial seed
Foto: A. Ewald, IGZ
Winkelmann et al. (2004)
Traud Winkelmann29
HortScience 39 (5): 1093-1097
11.03.2014
Limitations and drawbacks Genotypic differences in
regeneration ability andregeneration efficiencyregeneration efficiency
Asynchronous formation anddevelopment of somatic embryos
Malformations/ fused somaticembryos
Precocious germination Precocious germination Loss of embryogenic competence Secondary somatic embryosy y
Insights in physiology ofb iembryogenesis
(transcriptomics/proteomics) needed
11.03.201430
Model = zygotic embryo Photos by S. Ratjens and fromHoenemann et al. (2010)
Traud Winkelmann
Protein extraction – Plant materialPhD project Christina Rode
Zygotic embryos Somatic embryos240 embryos in torpedostage (80 mg)stage (80 mg)
1cm
1cm
1mm1mm 1mm1mm
11.03.2014Traud Winkelmann
Protein separation by 2D SDS-PAGEPhD project Christina Rode
4 biological and technical replicationsb t 1 000 t / l about 1,000 spots/gel
11.03.2014Traud Winkelmann32
zygotic embryo somatic embryo
Evaluation of gelsPhD project Christina Rode
>1.5 in SE
1,013 spots in total>1.5 in ZE
p
137 spots higher abundant in zygotic embryos
109 spots higher abundant in109 spots higher abundant in somatic embryos
3311.03.2014 Traud Winkelmann
Protein identification (cooperation with D. Heintz and A. Van Dorsselaer, Strasbourg)
Zygotic embryos Somatic embryos Total
Spots eluted 900 37 937p
For mass spectrometry 263 37 300
Proteins identified 229 32 261
3411.03.2014 Traud Winkelmann
87 %
Proteins of high abundance in somatic embryosRode et al. (2011a)
somatic embryo
3511.03.2014 Traud Winkelmann
Proteins of high abundancein zygotic embryos
zygotic embryo
in zygotic embryos
11.03.2014Traud Winkelmann36
Rode et al. (2011a)
Enolases
Functional enzyme for glycolysis/gluconeogenesis and fatty acid Functional enzyme for glycolysis/gluconeogenesis and fatty acidbiosynthesis
„Small“ Enolases highly abundant in zygotic embryos(MW 1/2-1/3 of functional protein)
Novel seed storage proteins?Rode et al. 2011a
Recycling of amino acids?
11.03.2014Traud Winkelmann37
Rode et al. 2011aPlant Mol Biol 75: 305-319
Digitial proteome reference map (www.gelmap.de)
3811.03.2014 Traud Winkelmann
Rode et al. (2011b) Journal of Proteomics 74: 2214-2219
Proteomic analysis of somatic b diff ti tiembryo differentiation
Experiment A: Embryogenic suspension
cultures Transferred to PGR free
medium Protein extraction after Protein extraction after
1, 3, 7, 21 and 28 days
Experiment B:
Rode et al. (2012) Planta 235: 995-1101
Experiment B: 28 d old somatic embryos
treated or not with 10 mg/L b i i idabscisic acid
Traud Winkelmann39 11.03.2014
Traud Winkelmann40
Rode et al. (2012)
11.03.2014
Alterations in protein abundances during differentiationduring differentiation
Callus: enzymes related to energy supply protein metabolism Callus: enzymes related to energy supply, protein metabolism
Somatic embryos: controlled proteolysis (1 d and 21d), auxinmetabolism, storage proteins, isoelectric point switch in catalase
Traud Winkelmann41
Rode et al. (2012)
11.03.2014
Alterations in protein abundances in t ABAresponse to ABA
ABA: proteins of primary metabolism and stress response higher- ABA: proteins of primary metabolism and stress response higherabundant
+ ABA: storage proteins, HSP 70g p ,
Traud Winkelmann42
Rode et al. (2012) Planta 235: 995-1011
11.03.2014
Endosperm developmentMwangi et al. (2013) Plant Science 201–202: 52–65
p pWeeks After Pollination (WAP)
0 21 3 54 111098764 WAP 5 WAP 9 WAP7 WAP 11 WAP
11.03.2014Traud Winkelmann43
MS-based protein identificationZeaxanthin
Mwangi et al. (2013)
416291F-box protein
Ethylene recptor 1
Zeaxanthin epoxidase (ZEP)
Xyloglucan endotransglucosylase
326
179 176
312314
196346313
F-box proteinF-box protein
Sugar carrier protein C 313, 314 326 346
Bet V I allergen family protein
196
246 Leucine-rich repeat receptor protein kinase
314, 326, 346Major latex protein
Seed maturation protein
59
15
349
427
11WAP
Wax synthase
F-box proteinZeaxanthin epoxidase (ZEP)
protein
287331
427
Annexin
y
Vacuolar processing enzyme (VPE) CHO metabolism (13)
protein processing (12)
48 spot => 62 proteins9 spots not identified
defence response (6)ABA signalling pathway (4)stress response (3)lipid pathway (2)
11.03.2014Traud Winkelmann44
7WAP
p lipid pathway (2)transport (4)
Role of endosperm in embryogenesis
Nutrition of the developing embryo Insulates embryo from mechanical pressure imposed by the Insulates embryo from mechanical pressure imposed by the
seed coat Endosperm-embryo signaling influencing
Developing embryo Maturation/growth arrest (ABA)
Regulates germination timingg g g
Developing an artificial endosperm for somatic embryos?
11.03.2014Traud Winkelmann45
EndophytesWhat 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)
Traud Winkelmann46
347 351)
11.03.2014
Prunus avium for timber production Fast growing hardwood for the production of
high quality furniture.
B di l t i ht t d d Breeding goals: straight stem, good wood quality, fast growth.
To achieve these characteristics single trees 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.
11.03.2014Traud Winkelmann47
(Mona Quambusch, PhD project)
In vitro culture of Prunus avium
11.03.2014Traud Winkelmann48
Endophytes in Prunus avium(Mona Quambusch, PhD project)
Endophytes in Prunus avium
Identification of endophytic bacteria Identification of endophytic bacteria Quantification of endophytes (qPCR)
in different culture phases in different culture phases under stress conditions
Understanding the balance of endophytic bacteria Understanding the balance of endophytic bacteria Isolation of beneficial bacteria Inoc lation of c lt es ith beneficial bacte ia Inoculation of cultures with beneficial bacteria
(cooperation with Institut für Pflanzenkultur, Schnega)
COST action FA1103: www endophytes eu
Traud Winkelmann49
COST action FA1103: www.endophytes.eu
11.03.2014
Identification of bacteria(Mona Quambusch, PhD project)
Culture-dependent Culture-papproach independent
approach
DNA extracted from plant materialDNA from bacterial isolates
11.03.201450 Traud Winkelmann
Identification of bacteriaIsolation of bacterial endophytes
Plant material Pure culture
Nep
tun
+N
ria ±
es
Aste
r+G
enot
yp no bacteria
Dem
eter
Growth media: nutrient agar and medium #523 (Viss et al 1991) Growth media: nutrient agar and medium #523 (Viss et al. 1991) Cultivation at RT for 5 weeks
11.03.201451 Traud Winkelmann
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.phylum Proteobacteria.
Demeter (+) Two bacterial isolates sequenced Two bacterial isolates sequenced. D-I1:
100% identical to Microbacterium( ) h l b(genus), phylum Actinobacteria.
D-I3: 100% identical to Bacillus (genus), (g ),
phylum Firmicutes.
11.03.201452 Traud Winkelmann
Quambusch et al. (2014) Tree Physiology: accepted
Identification of bacteriaPCR on 16S rDNA
±± + +--
bacterial amplicon mitochondrial ampliconbacterial amplicon, mitochondrial amplicon* Indicates amplicons used for cloning and ARDRA
DNA extracted from in vitro plant material of propagation phase
d f d Primers used: 799f and 1492r. (Chelius and Triplett, 2001)
11.03.201453 Traud Winkelmann
Identification of bacteriaAmplified rDNA Restriction Analysis (ARDRA)
Restriction patterns of 95 bacterial 16S rDNA
11.03.201454 Traud Winkelmann
Restriction patterns of 95 bacterial 16S rDNAfragments of a plant sample of Neptun (+).
Identification of bacteria(Mona Quambusch, PhD project)
Results of culture-independent analysis
Endophytic population of four Prunus avium genotypes
80%
100%
clon
es
40%
60%
orti
on o
f
Rhodopseudomonas spp.
other α-Proteobacteria
Microbacterium spp.
0%
20%
tive
pro
po
oba u spp
Mycobacterium spp.
uncultured bacterial clone
Fama - Achilleus - Neptun + Demeter +
Rel
at
Prunus genotype
11.03.201455 Traud Winkelmann
Identification of bacteria(Mona Quambusch, PhD project)
Summary of results from culture independent and -dependent method
Phylogenetic tree based on 16S rDNA showing the relationship of clones andPhylogenetic tree based on 16S rDNA showing the relationship of clones and isolates from different Prunus avium genotypes to reference sequences.
11.03.201456 Traud Winkelmann
Inoculation with EndophytesQuambusch et al. (2014) Tree Physiology: accepted
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 5 min treatment with bacterial suspension in 10mM MgSO4 Evaluation of rooting after three weeks, n = 8 vessels with 5 shoots
***** *
**
11.03.201457 Traud Winkelmann
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).
Summary
In vitro culture techniques
Diverse applications in propagation and breeding
Potential not fully tappedy pp
Deeper understanding of regeneration processes aspired
Use of molecular tools reasonable Use of molecular tools reasonable
New view on endophytes?
New plant growth regulators
Traud Winkelmann58 11.03.2014
ReferencesMEINERS, 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: 38-47MEINERS, 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: 576-585MWANGI, J.W., RODE, C., COLDITZ, F., HAASE, C., BRAUN, H.P. AND T. WINKELMANN (2013): Proteomic and histological analyses
of endosperm development in Cyclamen persicum as a basis for optimization of somatic embryogenesis. Plant p p y p p y gScience 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-03-2013/dgg-pr-03-03-so-2013.pdf
QUAMBUSCH M PIRTTILÄ A M MYOSORE V T WINKELMANN T AND M BARTSCH (2014): Endophytic bacteria in plant tissueQUAMBUSCH, M., PIRTTILÄ. A.M., MYOSORE, V. T., WINKELMANN, T. AND 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 i d C t ll i tibl i Pl t C ll R 31 723 735Cyclamen persicum and C. coum, two sexually incompatible species. Plant Cell Rep. 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 persicumMill. Plant Mol Biol 75: 305-319
RODE, C., SENKLER, M., KLODMANN, J., WINKELMANN, T. AND H.-P. BRAUN (2011B): GelMap – A novel software tool for building ( ) p gand 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 Plant Tiss Cult Biotechnol 4 (1): 28 – 34persicum Mill.. Plant Tiss. Cult. 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
S S 2006 ff l f l l d f bWINKELMANN, T., SPECHT, J. AND M. SEREK 2006: Efficient plant regeneration from protoplasts isolated from embryogenicsuspension cultures of Cyclamen persicum Mill. Plant Cell Tiss. Org. Cult. 86: 337-347
11.03.2014Traud Winkelmann59
Acknowlegdements:Melanie BartschSvenja RatjensSvenja RatjensJenniffer MwangiChristina RodeSamuel BreselgeSamuel BreselgeCathleen NeitschAnnika PrangeMona QuambuschMona QuambuschJulia MeinersBarbara RaffeinerMaike WarwasMaike WarwasViola MussmannEwa 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)
60 Traud Winkelmann 11.03.2014
Anna Maria Pirttilä (University of Oulu)