In vitro techniqqppgues for propagation and breeding of ...agro.au.dk/fileadmin/danseed/danseed-traud.pdf · Embryo rescue and genetic distance Number Genetic distance ♀x ♂ crosses

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


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

[email protected]

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


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


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


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


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


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)


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


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


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


Problems and limitations

Labour costs; AutomationS h i ti Synchronisation

Quality evaluation Physiological disorders Endophytes Somaclonal variation Genotypic differences Genotypic differences


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


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


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


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


87 %

Proteins of high abundance in somatic embryosRode et al. (2011a)

somatic embryo


Proteins of high abundancein zygotic embryos

zygotic embryo

in zygotic embryos


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?


Rode et al. 2011aPlant Mol Biol 75: 305-319

Digitial proteome reference map (www.gelmap.de)


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 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


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)


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?


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.


(Mona Quambusch, PhD project)

In vitro culture of Prunus avium


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


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


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.


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)


Identification of bacteriaAmplified rDNA Restriction Analysis (ARDRA)

Restriction patterns of 95 bacterial 16S rDNA


Restriction patterns of 95 bacterial 16S rDNAfragments of a plant sample of Neptun (+).


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



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.


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

***** *

**


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


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


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)


Anna Maria Pirttilä (University of Oulu)

In vitro techniqqppgues for propagation and breeding of ...agro.au.dk/fileadmin/danseed/danseed-traud.pdf · Embryo rescue and genetic distance Number Genetic distance ♀x ♂ crosses

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