Cultivation and Conservation of Competent Endophytes May In-Vitro Culture help?

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1 COST Action FA1103 Core Group 3 Meeting New concepts and strategies for longterm cultivation and conservation of competent endophytes for plant growth and plant protection Cultivation and Conservation of Competent Endophytes May In-Vitro Culture help? Nicole Brinkmann, Joachim Keller, Jutta Schulze, Elke Heine-Dobbernack and Heinz Martin Schumacher Leibniz Institute DSMZ Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, Braunschweig, Germany, mas@dsmz.de

2 The Leibniz-Institute DSMZ German Collection of Microorganisms and Cell Cultures GmbH runs a collection of app. 720 undifferentiated plant cell cultures since 1988

3 Undifferentiated plant cell Cultures are supposed to be axenic Routine sterility testing of cell cultures by cultivation tests 14 cell lines raised suspicion of latent infection Material was plated on 3 different media and cultivated at 2 different temperatures: DSMZ M92 Trypticase Soy Broth Yeast extracts DSMZ M215 Infusion of potato Glucose DSMZ M129 Brain Heart Infusion Cysteine Na 2 S

4 Undifferentiated plant cell Cultures are supposed to be axenic 1. They may be contaminated during sub-culturing 2. Endophytic microorganisms may persist during initiation Surface sterilization Appilcation of plant hormones

5 Results for routine sterility check caused a project for sytematic identification of contaminations with improved methods: Model systems investigated: 1. In-vitro plants from different species provided by IPK Gatersleben 2. Undifferentiated callus cultures provided by DSMZ 3. Embryogenic cultures of wheat and barley provided by Jutta Schulze, TU Braunschweig 4. Embryogenic Pelargonium cultures provided by E. Klocke JKI Quedlinburg

6 Investigation of in-vitro plants (by culture dependent approach) Isolated from Solanum Isolated from Allium Isolated from Mentha Isolated from Sechium Bacillus cereus Curtobacterium flaccumfaciens Pseudomonas cedrina Candida parapsilosis Pseudomonas koreensis Pseudomonas cedrina Methylobacterium radiotolerans Rhizobium pusense Stenotrophomonas rhizophila Wickerhamomyces onychis Pseudomonas graminis

7 Observed after cyropreservation of potato shoot tips: After thawing regrowing shoot tips often show microbial contamination Often only some shoot tips of one batch show contamination during regrowth after cryopreservation Potato shoot tips regrowing after cryopreservation (IPK Gatersleben)

8 Investigation of cell cultures (by culture independent approach) Catharanthus roseus Solanum tuberosum Mycobacterium interjectum Mycobacterium interjectum Tabernanthe iboga Methyloferula stellata + Paenibacillus sp. 1 Polygonum tinctorium Mycobacterium sp. + Paenibacillus sp. 1 Ludwigia octavalvis Paenibacillus sp. 1 Eschscholtzia californica Paenibacillus sp. 1 Melanoselinum decipiens Paenibacillus sp. 1 Stephania wightii Paenibacillus sp. 2 Dicrocaulon prostatum Pseudomonas sp.

9 Paenibacillus spec. Clone 1 1. Isolates show less than 98% (97%) sequence similarity to their next relatives Clone 2 2. None of the isolates forms spores 3. Isolate 1 is nifh negative

10 Colonization is different depending on on host and endophyte Colonization is different in different parts of a callus Further investigation of localization and growth conditions is on the way Paenibacillus sp.1 on Melanoselinum decipiens Paenibacillus sp.2 on Stephania whightii

11 Investigation of embryogenic barley and wheat cultures Isolated from wheat and barley cultures: Uncultured alpha proteobacterium clone GC0AA3ZA09PP1 16S ribosomal RNA gene Next cultured relative for all isolated strains: NCBI blast: AY Afipia felis (cat scratch disease bacillus) [99] Embryogenic cell line of Hordeum vulgare Lomerit Photos J. Schulze, Inst. Plant. Biol.

12 Investigation of embryogenic cultures of Pelargonium hortorum 1a 1b Isolated by culture dependent and /or culture independent approach: Mycobacterium ssp Micrococcus luteus Enhydrobacter sp. Moxarellea sp. Staphylococcus hominis Contaminants were detected in a specific phase during the regeneration of an embryogenic culture

13 Summary: A diverse range of bacteria could be isolated from different in vitro culture systems Which species occur seems to be determined by laboratory and în-vitro culture type Open questions: Are the isolated bacteria really endophytes or contaminations? How specific is the relationship between cell cultures and endophyte/latent contaminants?

14 May in-vitro culture help to cultivate and conserve endophytes? 1. In-vitro cultures may be used to produce substrates for endophyte cultivation 2. In-vitro cultures may serve as substrates for endophyte cultivation 3. In-vitro cultures may help for cryopreservation of endophytes

15 Is the production of substrates for endophyte cultivation possible by in vitro culture? 1. The large scale production of substrates for endophyte cultivation is probably too expensive 2. Cell cultures may be used for the investigation of specific nutritional requirements of endophytes 3. Cell cultures may be used for the isolation and production of compounds triggering or supporting the growth of endophytres

16 May in-vitro cultures serve as substrates for endophyte the cultivation of endophytes? 1. Endophytes can survive in in-vitro cultures and cultures may be used for maintenance and propagation of endophytes at the laboratory scale 2. Whether axenic cultures can be inoculated with defined endophytes for maintenance and propagation has to be demonstrated 3. The possibility of maintenance and cultivation of endophytes in in-vitro sytems may depend on the specificity of cell culture/ endophyte interaction

17 Preliminary results How specific is the interaction between cell cultures and endophytes 16 different cell lines of Catharanthus roseus were infected with the same amount of different Paenibacilli They showed very different growth behaviour after infection Ctr Inf. Ctr Ctr Ctr Inf. Inf. Inf.

18 May in-vitro cultures help for the cryopreservation of endophytes? At IPK various bacteria (probably) survived cyropreservation in their in-vitro cultured host plants At DSMZ at least Mycobacterium interjectum survived in a cryopreserved cell culture Is there a need for cryopreservation together with hosts plants? Future question: Can an intact consortium of host plant and endophytes be cyropreserved without loss or selection of endophytes?

19 Thank you for your attention

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