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Summary of Work from DSM Project Period 1

Summary of Work from DSM Project Period 1. Basic In-Vitro and Cryopreservation Work. In-Vitro Culture Method Cryopreservation Method used Cryopreservation Method tested. Collection of Potato Cultivars used for CRYMCEPT. shoot regeneration after droplet freezing Unicopa (87 % )

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Summary of Work from DSM Project Period 1

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  1. Summary of Work from DSM Project Period 1

  2. Basic In-Vitro and Cryopreservation Work • In-Vitro Culture Method • Cryopreservation Method used • Cryopreservation Method tested

  3. Collection of Potato Cultivars used for CRYMCEPT • shoot regeneration • after droplet freezing • Unicopa (87 % ) • 2. Patawi (50 %) • Zeenuse (33 %) • Desiree (33 %) • (IPK = 30 %) • 5. Solena (25 %) • 6. Ijesseter (12,5 %) • 7. Tomensa (not tested) • 8. Virginia (not tested)

  4. Droplet method for cryopreservation 1. Propagation in Magenta boxes (MS medium + 20 g/l sucrose) 2. Excision of meristems at a plant hight of 6 – 8 nodes 3. Incubation over night on filter paper in MSTo medium 4. 2 hours incubation in MSTo medium + 10 % DMSO 5. Transfer of meristems on aluminum foil 7. Direct immersion in liquid nitrogen 8. Thawing in medium at room temperature 9. Transfer of meristems into agarose droplets

  5. Controlled rate method for cryopreservation (Towill et al. 1981) 1. Plant growth to ahight of 8 – 10 cm 2. Preparation of meristems (axillar with 3 – 4 leaf primordia) 3. 1 – 2 days incubation in thawing medium 4. 1 h incubation in thawing medium + 10 % DMSO 5. 10 min incubation in water bath at –5 °C 6. Seeding by touching vials with supercool forceps 7. 10 min more incubation at –5 °C 8. Cooling with –0,2 °C – -0,3 °C to –35 °C - -40 °C 9. Transfer to liquid nitrogen 10. Leave 10 Minuten at –196 °C 11. Thawing in water bath at 37 °C No positive results could yet be obtained

  6. Methods tested for Protein Isolation – The Phenol Method • 1. 20 mg plant tissue + 200 µl phenol + 200 µl Tris buffer (0,2 M, pH 8,0, 0,7 M sucrose, 5 % 2-mercaptoethanol) grinded with “Grinding Kit” or drill and pistil Eppendorf vial • 2. shake for 30 min • 3. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge • 4. separate phenolic phase and transfer in a new Eppendorf vial • 5. add same volume Tris buffer • 6, mix strongly (Vortex) and incubate 15 minutes at 4 °C • 7. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge • separate phenolic phase and transfer in new Eppendorf vial • add 4 fold volume of 0,1 M ammonium acetate in methanol • 10. incubate 1 h bei 4 °C • 11. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge • 12. discard upper phase and wash pellett 6 times with 200 µl methanol • 13. leave pellet over night in a desiccator or dry in SpeedVac for 30 minutes • 14. resuspend in 100 µl Amersham Lysis buffer and mix strongly (Vortex)

  7. Methods tested for Protein Isolation – The TCA 1 Method • 1. 20 mg plant tissue + 100 µl TCA protein isolation solution grinded with “Grinding Kit” or drill and pistil in 1,5 ml Eppendorf vial • incubate for 1 h at 4 °C • centrifuge for 10 minutes at 14 000 rpm • 4. wash pellet with 100 µl TCA protein isolation solution • 5. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge • 6. wash 6 x times with 0,1 M ammonium acetate in methanol • 7. leave pellet over night in a desiccator or dry in SpeedVac for 30 minutes • resuspend in 100 µl Amersham Lysis buffer and mix strongly(Vortex) • 9. centrifuge for 10 minutes and remove cell debris

  8. Methods tested for Protein Isolation – The TCA 2 Method 1. 20 mg plant tissue + 100 µl Amersham Lysis buffer grinded with Amersham “Grinding Kit” or drill and pistil in 1,5 ml Eppendorf vial 2. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge 3. separate liquid phase from cell debris and transfer to new Eppendorf vial 4. add the same volume of 30 % TCA 5. incubate 1 h at 4°C 6. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge 7. wash 6 times with 0,1 M ammonium acetate in methanol 8. leave pellet over night in a desiccator or dry in SpeedVac for 30 minutes 9. add 10 µl 0,2 M NaOH and incubate for 2 Minutes 10. add 90 µl Amersham Lysis Puffer 11. mix strongly for 1 - 2 Minuten (Vortex) and re-suspend

  9. Tools used for protein isolation After starting with the Amersham Grinding kit we are nowusing a drill with pistill and Eppendorf tubes for protein and DNA isolation

  10. Methods tested for protein quantification • Biorad Test • possible with Patterson buffer, no results with Amersham Lysis buffer • Pierce Test Kit • ? No results with Amersham Lysis buffer ? • Amersham Quant Kit • after initial difficulties good results with Amersham Lysis buffer Amersham protein test is performed in 96 multiwell plates

  11. Results of total protein measurements With unstandardized material total protein content varied among different experiments: extraction date µg/mg fresh weight Phenol method; leaf material: 26.3. 3,2 10.4. 2,39 15.4. 8,15 28.4. 3,81 TCA 1 method; leaf material 26.3. 2,68 15.4. 5,02 28.4. 4,06 TCA 2 method; leaf material 26.3. 2,68 15.4. 1,1 28.4. 9,9 10.6. 4,45 8.7. 7,5 11.8. 9,5

  12. Results of total protein measurements With unstandardized material total protein content varied among different experiments: extraction date µg/mg fresh weight Phenol method; stem material: 15.4. 0,11 28.4. 1,94 TCA 1 method; stem material 15.4. 1,28 28.4. 1,99 TCA 2 method; stem material 28.4. 3,4 10.6. 0,6 8.7. 4,0

  13. Results of total protein measurements Tests were performed with leaf material from plants of comparable age

  14. Results of total protein measurements Repeated protein isolation using the TCA 2 method (µg protein / mg fresh weight) Extraction date 2.9. 9.9. 10.9. Cultivar Desiree leaf 1,69 2,4 4,9 stem 1,47 1,8 2,1 Patawi leaf 1,4 8,5 7,2 stem 2,11 1,7 1,9 Results for stem material is relatively constant whereas the results for leaf material show a higher variation

  15. Elecrophoretic 2 D separation of proteins Equipment used so far: Amersham Multiphor Unit IPG strips 7 cm (IEP range 3 – 10) IPG strips 24 cm (IEP range 3-10) Homogeneous precast gel 12,5 % Gradient precast gel 12 – 14 % Rehydration of strips was carried out in spec. rehydration tray application of proteins was down by rehydration of the strips over night

  16. Elecrophoretic 2 D separation of proteins Test with different quantities of Biorad IEF standard using: 7 cm IPG strips Excel precast flatbed gel 12,5 % IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 120 IEF Standard 0,25 µl IEF Standard 0,5 ,µl IEF Standard 1 µl kD 10

  17. Elecrophoretic 2 D separation of proteins Analysis material: Leaf tissue, applied 50 µg protein Separated: 7 cm IPG strip (IEF 3 – 10) homogeneous Excel gel 12,5 % acrylamid Isolated by TCA 1 method Isolated by Phenol method IEP 3 - 10 IEP 3 - 10 120 kD 10

  18. Elecrophoretic 2 D separation of proteins Protein from leaf material isolated by the Phenol method separated by 7 cm IPG strips homogeneous Excel gel 12,5 % acrylamid applied 3 µg protein Isolated 17.4. Isolated 30.4 Isolated 28.5 IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 120 kD 10

  19. Elecrophoretic 2 D separation of proteins Protein from leaf material isolated by the TCA 1 method separated by 7 cm IPG strips homogeneous Excel gel 12,5 % acrylamid applied 3 µg protein Isolated 17.4 Isolated 30.4. Isolated 22.5. IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 120 kD 10

  20. Methods tested for Protein Isolation – The TCA 2 Method 1. 20 mg plant tissue + 100 µl Amersham Lysis buffer grinded with Amersham “Grinding Kit” or drill and pistil in 1,5 ml Eppendorf vial 2. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge 3. separate liquid phase from cell debris and transfer to new Eppendorf vial 4. add the same volume of 30 % TCA 5. incubate 1 h at 4°C 6. centrifuge for 10 minutes at 14 000 rpm in Eppendorf centrifuge 7. wash 6 times with 0,1 M ammonium acetate in methanol 8. leave pellet over night in a desiccator or dry in SpeedVac for 30 minutes 9. add 10 µl 0,2 M NaOH and incubate for 2 Minutes 10. add 90 µl Amersham Lysis Puffer 11. mix strongly for 1 - 2 Minuten (Vortex) and re-suspend

  21. Elecrophoretic 2 D separation of proteins Comparison of different protein samples from leaf material separated by 7 cm IPG strips homogeneous Excel gel 12,5 % acrylamid applied 3 µg protein Isolated by TCA 1 method Isolated by TCA 2 method IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 120 kD 10

  22. Elecrophoretic 2 D separation of proteins Different amounts of protein isolated from leaf material (Solena) isolated by TCA 2 method separated by 7 cm IPG strips (IEF 3-10) homogeneous gel 12,5 % acrylamid IEP 10 - 3 IEP 10 - 3 IEP 10 - 3 120 5 µg protein 10 µg protein 20 µg protein kD 10

  23. Elecrophoretic 2 D separation of proteins Different isolates of protein isolated from leaf material (Solena) isolated by TCA 2 method separated by 24 cm IPG strips (IEF 3-10) gradient gel 12 - 14 % acrylamid 50 µg protein applied 100 µg protein applied IEP 10 - 3 IEP 10 - 3 116 kD 12

  24. Technical problems still to be solved !!! Often sparkling occurs in IPG strips during the IEF step Set up of vertical electrophoresis for second dimension makes problems Decision has to be made whether scale down should be done using 7 cm or 24 cm strips Calculation: so far applied to 7 cm strips 3 – 20 µmg protein 24 cm strips 50 – 100 µg protein fresh weight of one apex (DSC) 0,2 – 0,5 mg protein content stem tissue: ca. 1,7 µg/ mg tissue leaf tissue ca. 8 µg/mg tissue needed for 3 µg protein from stems = equivalent of 4 - 8 apices from leafs = equivalent of 1-2 apices needed for 100 µg protein from stems = equivalent of 120 – 290 apices from leafs = equivalent of 25 – 65 apices

  25. Methods for „spot pattern“ anaylsis Automatic detection of spot is possible by the ROI (regions of interest) detection function of the Kodak 1D software Beside serial number 20 diffrenet parameter concerning intensity, geometry and position of each ROI are listed Automatic ROI detection Adjustment of criteria possible Adding ROIs possible Deleting ROIs possible Merging ROIs possible Separation of ROIs possible Problem: ROI serial numbers are not changed after manuell adjustments

  26. Methods for „spot pattern“ anaylsis Data listings from the Kodak 1D software can be transferred to Excel In Excel it is posssible to introduce a new serial number after manuell manipulation of ROIs Standardization of „spot patterns“ is possible by mathematical operations with X and yY values for the position of ROIs new old

  27. Methods for „spot pattern“ anaylsis Excel data listings can be re-transfromed into a diagramm Problems: The new diagramm does not show intensities In Excel points are not properly scaled Other software like „SigmaPlot“ will be tested to obtain properly scaled diagrams

  28. Discussion anf Future Work Discussion: Basic techniques for protein analysis have been set up using cell culture material, stem and leaf tissue of in-vitro plants Analysis of special patterns has not yet been performed Future work Scale down to use single apices has to be approached but seems to be possible at least with 7 cm IPG strips Measurement of leaf and stem protein pattern for different cultivars will be started Problems: Capacity problems may occur using 24 cm strips and large flatbed gels from a) electrophoresis b) apex production and excision

  29. Work Package 1 – DSC Measurements Basic Problem: Transportation of plant material Measurements performed: Different suspension cell lines representing different performance in cryopreservation procedures Catharanthus roseus, Cytisus canariense, Lycopersicon esculentum Atropa bella-donna, Berberis wilsoniae (2 different strains Apices of different potato cultivars afetr different treatments Cultivars: Unicopa (87%), Patawi (50%), Zeenuse (33%), Deisree (33%), Solena (25%),Ijesseter (12,5), Steps: * directly after excision * after incubation over night in MSTo medium * after incubation over night and 2 hours DMSO treatment

  30. DSC Measurement with Medium Comoponents MSTo Medium MSTo Medium + 10% DMSO 451 J/g 311 J/g

  31. DSC Measurements with Single Potato Apices Potato apex thermogram type P1 : freshly excised or left overnight Potato apex thermogram type P2 : 2h DMSO pre-treatment 211 J/g 134 J/g

  32. DSC Measurement – Major Results - All suspensions showed the same freezing characteristic - Measurements with single apices are possible - Although only two apices were measured results were similar - Cultivars showed differences in freezing energy directly after excision - Cultivars showed almost the same freezing energy after incubation over night in MSTo medium - DMSO treatment for two hours reduced freezing energy

  33. DSC Measurements - Discussion Working hypothesis: A correlation exists between plant regeneration and content of freezable water in the meristems Results of Measurements: There may be a correlation between plant regeneration and water content but for more complex reasons Problems and further action Cooling rates of droplet freezing cannot be achieved with the DSC machine Further experiments should use preincubation in media with varying osmotic strength (so far not performed) subsequently new DSC measurement may be performed

  34. Work Package 7 – Oxidative Stress Working hypothesis from results of previous work Refinement of vitamin C effect in droplet freezing Screening for suitable material by testing for MDA formation During droplet freezing

  35. Results from previous unpublished work Application of Vitamin C in Droplet freezing Unicopa Survival rate: without Vitamin C 100 % Vitamin C added 11% Early Rose Survival rate: without Vitamin C 40 % Vitamin C added 0 %

  36. Screening for high MDA formation in potato cultivars Method from: Benson, Lynch, Jones: The detection of lipid peroxidase products in cryoprotected and frozen rice cells,: consequences of post thaw survival, Plant Science, 85, 1992, 107 – 114. The method was scaled down to work with 125 mg plant tissue Instead of apical meristems (250 apices resemble 125 mg) either leaf or stem material. 1. suspend 125 mg tissue material in 250 µl dest. water in Eppendorf vial 2. prick a hole in the lid of the Eppendorf vial 3. add 375 µl 0,5 % TBA in 20 % TCA 4. incubate for 30 minutes in an incubator at 100 °C 5. transfer quickly into an ice bath 6. remove cell debris by centrifugation 7. measure at 532 nm, correct for absorption at 600 nm

  37. Measurement of MDA formation in untreated leaf material and after ultra rapid exposure to liquid nitrogen

  38. Measurement of MDA formation in untreated stem material and after ultra rapid exposure to liquid nitrogen

  39. Work Package 7 – Further Action Discussion Is this approach useful to find free radical sensitiv cultivars ? Does ultra rapid exposure to liquid nitrogen temperatures cause any free radical damage ? Possible further action Repeat the method with more cultivars ? Further scale down and perform the method with apices ? Perform the method also under slow freezing conditions ? Screen more cultivars (10 more aquired from IPK) ?

  40. Problems to be faced in the near future Financial: Purchase of further equipment from own resources is doubtful (Ettan Dalt vertical electrophoresis, spec. software) Location: Move of the laboratory has to organized early next year working conditions will improve afterwards Staff: EU Technician will be on maternity leave for 14 weeks We may have to work with 2 half time employees afterwards

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