Final Report – Partner 6 / DSMZ

1. Final Report – Partner 6 / DSMZ

2. Final Report – Partner 6 / DSMZ Potato Cultivars used for CRYMCEPT • shoot regeneration • after droplet freezing • Unicopa (87 % ) • 2. Patawi (50 %) • Zeeuwse Blauwe (33 %) • Desiree (33 %) • (IPK = 30 %) • 5. Solene (25 %) • 6. Ijesselster (12,5 %)

3. Final Report – Partner 6 / DSMZ Small strips and gels did not yield a sufficiently high spot resolution Unicopa Ijsselster Desiree Scale down of methods for the measurement of protein pattern from isolated apices worked First analysis using 7 cm IPG strips and 10 x 10 cm SDS gels Large flatbed gels did not yield a sufficiently high sample throughput The initially used equipement turned out to be inadequate Self made spot analysis approaches turn out to be inadequate Finally new Amersham equipment was bought for second dimension runs, spot detection and spot analysis Desiree Ijsselster Unicopa

4. Final Report – Partner 6 / DSMZ Typical gel of the variety Ijsselster (after Incubation) (shows 12,5 % regeneration in freezing experiments) Typical gel of the variety Unicopa (after Incubation) (shows 87 % regeneration in freezing experiments) Increased in 2/3 resp 2/4 experiments Decreased in 2/3 resp 2/4 experiments

5. Final Report – Partner 6 / DSMZ Region 1 Typical gel Potato cultivar Unicopa

6. Final Report – Partner 6 / DSMZ Each experiment is represented by Independent pre-cultivation of potato plantlets Independent protein isolation 3 gels before and 3 gels after over-night incubation Region 1 Unicopa Ijsselster

7. Gel Region 1 Final Report – Partner 6 / DSMZ Unicopa unclear Exp 2 Exp 3 Ijsselster Exp. 4 down Exp. 2 Exp. 1 Exp. 3

8. Final Report – Partner 6 / DSMZ Region 2 Typical gel Potato cultivar Unicopa

9. R2/2 R2/3 R2/4 R2/1 Final Report – Partner 6 / DSMZ Each experiment is represented by Independent pre-cultivation of potato plantlets Independent protein isolation 3 gels before and 3 gels after over-night incubation Region 2 Unicopa Ijsselster

10. Gel Region 2 Final Report – Partner 6 / DSMZ Unicopa Spot R2/1 unclear/down Spot R2/2 unclear Ijsselster Exp. 4 Spot R2/1 down Spot R2/2 down Exp. 1 Exp. 3 Exp. 2

11. Final Report – Partner 6 / DSMZ Region 3 Typical gel Potato cultivar Unicopa

12. R3/1 R3/2 Final Report – Partner 6 / DSMZ Each experiment is represented by Independent pre-cultivation of potato plantlets Independent protein isolation 3 gels before and 3 gels after over-night incubation Region 3 Unicopa Ijsselster

13. Final Report – Partner 6 / DSMZ Gel Region 3 Unicopa Spot R3/1 Unclear/ down Spot R3/2 Unclear/ down Ijsselster Spot R3/1 Exp. 4 down Spot R3/2 down Exp. 3 Exp. 2 Exp. 1

14. Final Report – Partner 6 / DSMZ Region 4 Typical gel Potato cultivar Unicopa

15. Final Report – Partner 6 / DSMZ Each experiment is represented by Independent pre-cultivation of potato plantlets Independent protein isolation 3 gels before and 3 gels after over-night incubation Region 4 Unicopa Ijsselster

16. Gel Region 4 Final Report – Partner 6 / DSMZ Unicopa ? Exp 1 Ijsselster Exp. 4 ? / down Exp. 1 Exp. 2 Exp. 3

17. Final Report – Partner 6 / DSMZ Region 5 Typical gel Potato cultivar Unicopa

18. R5/2 R5/1 Final Report – Partner 6 / DSMZ Each experiment is represented by Independent pre-cultivation of potato plantlets Independent protein isolation 3 gels before and 3 gels after over-night incubation Region 5 Unicopa Ijsselster

19. Gel Region 5 Final Report – Partner 6 / DSMZ Unicopa Up ? Spot R5/1 Spot R5/2 Up ? Exp. 4 Ijsselster unchanged Spot R5/1 Spot R5/2 unchanged Exp. 1 Exp. 2 Exp. 3

20. Final Report – Partner 6 / DSMZ Use of Coomassie Stains – Pre-requisite for mass spectrometry 1. G. Candiano et al. Electrophoresis, 2004, 25, 1327 - 1333 2. classical 3. V. Neuhoff et al. Electrophoresis, 1988, 9, 255 - 265 Neuhoff et al. 1988 1. 23,5 ml/l Phosphoric acid (85%) 2 % 2. 1 g/l Coomassie G250 0,1 % 3. 100 g/l Ammoniumsulfate 10 % 4. 200 ml/l Methanol 20 % Candiano et al. 2004 1. 100 ml/l Phosphoric acid (85%) 8,5 % 2. 1,2 g/l Coomassie G250 0,12 % 3. 100 g/l Ammoniumsulfate 10 % 4. 200 ml/l Methanol 20 %

21. Final Report – Partner 6 / DSMZ Identification of salt stress proteins with cell cultures 5 spot succesfully measured 3 identified IPG strip 3 – 10 NL

22. Final Report – Partner 6 / DSMZ Conclusions: Standardization of protein patterns measured with isolated apices is more difficult than expected Possible reasons: 1. Inadequate electrophoretic technique ? 2. Different physiological state of plant material ? 3. Differences in the preparation of plant material ? (different amounts of leaf and meristem tissue) 4. Time difference in “over-night” incubation ? (16 h incubation +/- 4 h time for isolation) Evidence for different up and down regulation of similar spots in different varieties is therefore weak Spot identification is necessary before any conclusion can be made There is so far no evidence that “induced” or “up-regulated” “protective” proteins are present in the shoot tips at the time when “droplet freezing” takes place. More evident is a loss of proteins in the bad performing cultivar

23. Final Report – Partner 6 / DSMZ “Résumé” on protein work at DSMZ Achievements: Establishment of techniques for proteomic studies with plant cells and tissues Establishment of scale down methods to achieve protein patterns from shoot apices Definition of some spots which different cultivars have in common and show slightly different reactions after excision

24. Final Report – Partner 6 / DSMZ “Résumé” on protein work at DSMZ Problems and Failures: Unexpected staff problems Initial under-estimation of the technical challenges (use of in-adequate equipment for too long time) Unexpected problems with standardization of protein pattern of isolated shoot tips Lack of flexibility for using an adequate model system ?

25. Final Report – Partner 6 / DSMZ DSC Measurements / Improvement of Methods shoot regeneration after droplet freezing 1. Unicopa (87 % ) 2. Patawi (50 %) 3. Zeeuwse Blauwe (33 %) 4. Desiree (33 %) 5. Solene (25 %) 6. Ijsselster (12,5 %) Potato apex thermogram type P1 : freshly excised or left overnight Directly after excision ice formation differs for different cultivars After over-night incubation and DMSO treatment no differences between cultivars could be observed Potato apex thermogram type P2 : 2h DMSO pre-treatment Experiments for DMSO treatment without over-night incubation have not been carried out

26. Final Report – Partner 6 / DSMZ DSC Measurements / Improvement of Methods Directly = without over-night incubation + with 2 h DMSO treatment Over-night = with over-night incubation + with 2 h DMSO treatment After 3 days = with 3 days incubation + with 2 h DMSO treatment For each treatment ca. 40 apices have been used

27. Final Report – Partner 6 / DSMZ DSC Measurements / Improvement of Methods Directly = without over-night incubation + with 2 h DMSO treatment Over-night = with over-night incubation + with 2 h DMSO treatment After 3 days = with 3 days incubation + with 2 h DMSO treatment For each treatment ca. 40 apices have been used

28. Final Report – Partner 6 / DSMZ Cryopreservation in different MSTo media Salt and sugar concentration differed Hormone concentrations remained unchanged Ijsselster Unicopa

29. Final Report – Partner 6 / DSMZ “Résumé” on DSC work at DSMZ DSC in the standard configuration is not adequate for measuring the true behaviour of potato shoot apices during droplet freezing Nevertheless the measurements performed gave an extremely interesting new view on early stage s of droplet freezing These new view on early stages of droplet freezing led to slight modifications which may improve the results for cultivars showing insufficient regeneration rates

30. Final Report – Partner 6 / DSMZ Oxidative Stress Previous results using vitamin C as additive in cryopreservation of undifferentiated potato cell cultures could not be obtained with Potato shoot tips during droplet freezing Application of vitamin C before freezing with two culticars ( Unicopa and Early Rose) resulted in Decreased survival rates Experiments with more cultivars may be beneficial Future experiments with undifferentiated cell cultures are more promising

31. Final Report – Partner 6 / DSMZ “Résumé” on oxidative stress work at DSMZ “Droplet freezing” seems to be not much affected by treatments with antioxidants before freezing More work on oxidative stress would have been desirable for the needs of freezing at DSMZ The compiled manual on oxidative stress is extremely useful for work at DSMZ Continued co-operative contacts would be even more desirable

32. Final Report – Partner 6 / DSMZ Strawberry Experiments 8 Experiments have been carried out using the conventional method 2 Experiments use aluminium foils and droplets instead of cryovials Experiments have been carried out with ca. 50 meristems: 10 for control 10 after PVS2 30 after freezing technical failure

33. Final Report – Partner 6 / DSMZ “Résumé” of CRYMCEPT for DSMZ Personal contacts which should be maintained Knowledge about new techniques important for cryopreservation Establishment of new techniques which are important even beyhond cryopreservation (proteomics) Not much benefit for cryopreservation work with DSMZ resources (undifferentiated cell cultures) Not much progress at DSMZ in techniques important for undifferentiated cell cultures (oxidative stress) Patent application at WIPO for differentiated plant material cultured in-vitro