Cryopreservation of Rosa hybrida cv. Helmut Schmidt by PVS2 vitrification method using in vitro fragmented explants (IFEs)

This is the first report on cryopreservation via PVS2 vitrification method on roses using in vitro fragmented explants (IFEs) as the starting material. The aim of this study is to optimize the efficient plant recovery and regeneration system for cryopreservation of Rosa hybrida cv. Helmut Schmidt using IFEs. Some important parameters have been optimized in this study are the effect of ascorbic acid (0.3 mM) examined separately and in combination at all steps in cryopreservation procedure (preculture, loading, unloading and growth recovery), loading type, loading duration, and PVS2 duration. The highest growth recovery of 43.33% was obtained when 3-4 mm size IFEs precultured on 0.25 M sucrose media supplemented with full-strength MS for one (1) day, followed by loading treatment supplemented with 1.5 M glycerol + 0.4 M sucrose + 5% DMSO + 0.3 mM ascorbic acid for 20 minutes, dehydration with PVS2 solution for 30 minutes and then treated with unloading solution supplemented with 1.2 M sucrose + 0.3 mM ascorbic acid for 20 minutes. This finding implies that long-term storage of Rosa Hybrida cv. Helmut Schmidt by PVS2 vitrification method was successful with essential biomolecules.


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Growth was observed in loading solutions which were supplemented with a higher 263 concentration of DMSO. These results may also be due to the antioxidant impact of DMSO 264 which is a free radical scavenger and may be the reason for the reduced oxidative damage.  295 Figure 2A shows that the greatest survival was achieved with an optimum loading 296 duration at 20 minutes (A490nm:1.36) followed by 10 minutes of treatment (A490nm: 297 0.224). Therefore, the survival of IFEs was influenced by the loading duration. There was no 298 regrowth on IFEs that were exposed to loading duration for 5 and 40 minutes. Less exposure 299 and extreme exposure to loading solution caused a detrimental effect on IFEs. Thus, the 300 loading duration of 20 minutes was chosen and used for the following experiments.

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Cryopreserved IFEs (Fig 3A)  , the proline accumulation reduced to 6.52 nmol/mg with no significant difference to the 362 control IFEs (Fig 4). Loading, PVS2, and growth recovery 1 stage had no significant 363 difference with one another (Fig 4).

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For cryopreserved IFEs, the highest amount of carbohydrate content (1.757 mg/g) was 370 observed at the unloading stage (Fig 5). However, after 1 day in growth recovery medium 371 (recovery 1) (0.528 mg/g), there was a drastic decrease in carbohydrate content ( Figure 5).

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SOD activity increased during the cryopreserved stages (Fig 6) from the preculture to 378 growth recovery stage. There was an increase in SOD activity at the preculture stage 379 (1516.015 U.g -1 ) with no significant difference to growth recovery stage 1 (1432.950 U.g -1 ).
380 The highest SOD activity was observed at the thawing stage followed by PVS2 stage. The 381 least SOD activity was observed at unloading and growth recovery stage 2 (1168.736 U.g -1 ) 382 (Fig 6).

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The water content of IFEs (Fig 9) was examined to discover the percentage of water 407 lost at each stage of cryopreservation. The water content for cryopreserved IFEs was lowest 408 at the PVS2 dehydration stage (39.21%). Water content which was reduced after control 409 treatment from 93.17% to PVS2 treatment (39.21%) amounted to a total water loss of about 410 53.96%. After dehydration with PVS2 solution, there was a progressive increase in water 411 content from thawing, unloading, recovery stage 1 and 2 for cryopreserved IFEs (Fig 9).