The „hidden“ endopytes in protoplast cultures – a critical thinking

The „hidden“ endopytes in protoplastThe „hidden“ endopytes in protoplastcultures – a critical thinking about

Evelyn Klocke, Simone Abel, Karina Weinzierl

JKI, Institute for Breeding Research on Horticultural and Fruit Crops, Quedlinburg, Germany

[email protected]

www.jki.bund.deEuropean Endophyte Research, COST Action FA1103Workshop 28-30 March 2012, Reims, France

Endophytes and plant tissue culture

• Endophytes live within a plant.

• They do not cause apparent disease.

www.jki.bund.de

evelyn.klocke@jki,bund.deEuropean Endophyte Research, COST Action FA1103Workshop 28-30 March 2012, Reims, France

MutualismTwo organisms of different species

interact

There is a fitness

benefit for both.

CommensalismTwo organisms of different species

interact

There is a fitness

benefit for one, the other is neutral.

WhatWhat wewe knowknow aboutabout thesethese hiddenhidden endophytesendophytes??

Protoplast culture + fusion

Tissue digestionProtoplast isolation

www.jki.bund.de


Protoplast isolation

Protoplast mixing

Fusion

Similarities:

Endophytes in protoplast cultures of Hydrangea and Pelargonium

-

Similarities and differences

Protocols for isolation of protoplasts and fusion are well established.

Regeneration of cell wall and first divisions were observed.

Development of microcolonies / calluses in liquid culture medium.

Visible endophytes on solid regeneration medium.


Hydrangea Pelargonium

Visible endophytes on solid regeneration medium.

Differences:

Endophytes in protoplast cultures of Hydrangea and Pelargonium

-

Similarities and differences

Hydrangea Pelargonium

Development of green

compact callus on solid

Development of green

compact callus and white


compact callus on solid

medium

Root development – yes

Shoot development – no

No plant regeneration

compact callus and white

soft callus

Root development – yes

Shoot development – yes

Successfull plant regeneration

Endophytes in protoplast cultures of Hydrangea -

Observations and attempts to overcome them

Addition of Timentin 1 day after fusion

to the protoplast cultivation medium

Use of antibiotic Timentin in Hydrangea protoplast culture

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Plants in vitro with antibiotic Timentin

Calluses

Endophytes visible on solid medium

after ca. 4 weeks, especially on lower

surface to the medium

Endophytes in protoplast cultures of Hydrangea -


Media composition – Influence on callus formation and time of browning

Control protoplasts

Regeneration after fusion of H. macrophylla (x) H. paniculata protoplasts

www.jki.bund.deEuropean Endophyte Research, COST Action FA1103

Workshop 28-30 March 2012, Reims, France

PPM3 PRM12

Green

microcalli

Brown

microcalli

evelyn.klocke@jki,bund.de

PPM3

PRM 12

Endophytes in protoplast cultures of Pelargonium -


Observation of endopytes in all fusion experiments with Pelargonium ssp.independent from:

• source of protoplasts (hypocotyl, leaf, suspension)• species used: e. g. P. x hortorum, P. quinquelobatum, P. graveolens, P.

caylae

• method of fusion: PEG or electrofusion

In liquid protoplast culture medium no endophytes visible.

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In liquid protoplast culture medium no endophytes visible.Although

developing structures could be differentiated in:a) compact structures regeneration later possibleb) soft, lucid aggregates no regeneration, plenty endophytes



For regeneration it is important :• the at the right time

• the right structure

• on the right medium

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PPM 3 PRM 12



For regeneration it is important :• Separation of compact green structures from soft callus with endophytes under the stereomicroscope





Successful regeneration – no more endophytes visible!



Attempts for endophyte identification

No growth on several bacterial media:

Glycerine Bouillon Agar

Tryptone Soy Agar (solid + liquid)



LB – medium liquid


Results by Raster / Electron microscopy:

Results very scarce.

Difficult to find structures which could be identified as bacterias. This is

surprising because the callus material investigated was overgrown with

endophytes.



Pictures by REM


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Bacteria visualised in ultrathin sections of Hydrangea callus showingsymptoms of browning /endophytes.

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Putative bacterias have been found (about 1 µm, typical cell membrane), but surprisingly rare, always near the plant cell wall.In leaves of Hydrangea in vitro no endophytes observed.


Molecular characterisation

Amplification of 5‘ end of bacterial16S-rDNA with primer pairs for the

highly conserved sequences

Sequencing of the PCR-products with hypervariable regions of 16S-rDNA

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Source:http://www.bioinformatics-toolkit.org/Help/Topics/hypervariableRegions.html


Multialignment of 9 samplesafter sequencing

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Molecular characterisation

BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

• uncultured prokaryote clone, 16 ribosomal RNA gene clone

• Cronobacter sakazakii

• Escherichia coli (various strains)

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Sequences are never specified as plant-

derived!!!

Control reactions with plant DNA : sequences

revealed the cp DNA of the plant.

Methods should be adapted.

Difficult to enrich the endophytes as

template for PCR.

•Discussion – A thinking about

The „hidden“ endophytes

• Our knowlegde about the interactions among plants and

microorganisms is limited up to now.

• The most we know about parasites or very useful

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• The most we know about parasites or very useful

microorganisms.

• The coexistence plant / microorganisms is very complex; there

are endophytes without or with positive influence on the plant.

The coexistence is in balance. In stressful situations this balance

could be disturbed.

• With the right management the balance can be restored.

Thanks to my coworkers:

Karina Weinzierl

Simone Abel

Thanks for support:

www.jki.bund.de


Elke Zimmermann: for preparations and pictures from TEM

Katja Richert-Pöggeler: for pictures from REM

Katja Thiele: for providing tips for molecular characterisation

European Endophyte Research, COST Action FA1103Workshop 28-30 March 2012, Reims, France

I thank COST Action FA1103 for reimbursement of my

travelling costs.

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The „hidden“ endopytes in protoplast cultures – a critical thinking