A preliminary review of the literature on cryopreservation of different plant species using the droplet-vitrification method showed that most experiments traditionally used a RS containing a 1.2 M sucrose concentration for unloading (~ 70% of the reviewed publications used a 1.2 M RS sucrose concentration). Specifically, for potato, 11 publications reported four different sucrose concentrations for unloading in RS: 0.09 M, 0.8 M, 1.0 M and 1.2 M. As all steps of a protocol can influence directly or indirectly the efflux of CPAs (unloading efficiency) and influx of water (osmotic stress), a detailed comparison of the methodologies reported was warranted (Table 1).
The age of the in vitro plantlets used for shoot tips excision varied from 2 to 7 weeks and the shoot tip size from 1 to 4 mm. The intracellular water and ion content of shoot tips coming from older and younger plants may vary, consequently the intrinsic osmotic potential could be different, affecting the influx of water during unloading. In larger shoot tips (2–4 mm) unloaded CPAs and incoming water may need to move longer distances than in shorter shoot tips (1–2 mm). Further, cold hardening and pre-culture steps can significantly influence the intracellular water content of potato shoot tips as shown by Folgado et al. (2015). Pre-culture and cold-hardening times, as well as pre-culture sucrose concentrations (0.09–0.7 M), varied considerably between the reviewed protocols. As the potato genotypes were treated with different cryoprotectant agents, it makes sense that RS sucrose concentrations may also need to be varied. For example, unloading of DMSO may, or may not, require different RS sucrose concentrations and unloading times than PVS2 (Kryszczu et al. 2006; Schäfer-Menuhr et al. 1997; Kaczmarczyk et al. 2008). Additionally, the conditions of recovery (light vs. darkness) and the composition and sucrose concentration of the recovery media may cause different post-unloading stresses as the differential of sucrose concentrations between RS and recovery media may affect water influx during recovery. Finally, different response variables were used for the assessment in the reviewed publications. Some authors reported complete plant recovery (Schäfer-Menuhr et al. 1997; Vollmer et al. 2017), while others reported only survival (swollen shoot tips of > 3 mm) or shoot formation (Kim et al. 2006; Panta et al. 2015).
On a smaller scale, the effect of RS sucrose concentration and unloading times was previously assessed by Kim et al. (2006) where an assessment of a combination of unloading times (10, 30, 60 min) and sucrose concentrations (0.3 M, 0.8 M, and 1.2 M) was done using two potato varieties, var. ‘Dejima’ (S. tuberosum) and ‘STN13’ (S. stenotomum subspecies stenotomum). The best unloading time and sucrose concentration based on these two varieties (30 min exposure to 0.8 M sucrose RS) was tested on a set of 12 genotypes (belonging to 4 taxa). The reported survival rates ranged from 64 to 94% (+ LN). Consequently, it may be possible that the unloading time in the present study of 20 min is not long enough to permit complete unloading of the cryoprotection solution (PVS2) or that transferring to a new RS solution (washing) is required.
Towill and Bonnart (2003) reported that cracking of external glass during warming did not result in a decrease of viability. Based on their results, it seems that the critical steps for successful and fast rewarming are not related to devitrification or thawing, but maybe influenced by a subsequent efflux of CPAs and influx of water into the shoot tips.
In the present study, the initial experiment with 16 potato accessions showed for the non-frozen samples (− LN) a significantly higher median of RR with a RS sucrose concentration of 0.6M (means: 92.9% [− LN], 86.3%[+ LN]), compared to the 0.0 M RS-sucrose concentration (means: 83.0% [− LN], 66.5% [+ LN]). This small set of accessions (16) showed no significant differences between RR medians for RS in the remaining sucrose concentrations 0.3 M, 0.6 M, 0.9 M, and the routinely utilized 1.2 M (means: 81.1–86.8%) (Table 2).
The data was evaluated to determine if genotype played a role in recovery rates. CIP 705458 (S. stenotomum subsp. goniocalyx) (mean RR: 93.8%) showed a significantly higher median RR (+ LN) than CIP 702353, CIP 704047, and CIP 706825 (S. stenotomum subsp. goniocalyx, S. × chaucha, and S. phureja, respectively) (means of RR: 64.9–69.8%). Interestingly, when the data was pooled across all genotypes, cryopreserved shoot tips showed a low differential of 4.5% between the average SR (85.5%) and RR (81.0%) (Table 2). Three different patterns were observed among the genotypes which included: (I) six accessions (CIP 701611, CIP 702152, CIP 702439, CIP 704061, CIP 704087 and CIP 704043) had stable RRs (+ LN), independent of the RS sucrose concentration (0.0 M–1.2 M), (II) five accessions (CIP 702190, CIP 704052, CIP 705569, CIP 705458 and CIP 706250) showed lower RRs when sucrose was removed completely from RS (0.0 M), (III) five accessions (CIP 701561, CIP 702353, CIP 704541, CIP 704047 and CIP 706825) showed a higher RR with one of the intermediate RS sucrose concentrations (0.3 M, 0.6 M, or 0.9 M) (Fig. 1).
Somewhat surprising, complete removal of sucrose from RS (0.0 M) resulted in an acceptable RR range of 46.7–86.7% (Fig. 1). This suggests genotype is an important factor that should be considered in response rates and further species and ploidy level do not seem to play a role in RR.
The results from this initial experiment, were expanded to a set of 85 diverse potato accessions, to determine if the RS sucrose concentration can be reduced to 0.3 M, 0.6 M, or 0.9 M. RS sucrose concentrations of 0.3–0.9 M resulted in significantly higher medians of the RR (means: 55.2–61.1%) compared to the 0.0 M and 1.2 M RS-sucrose concentrations (RRs: 36.4–43.9%) (Table 3), suggesting that RR is affected by the RS concentration employed.
No statistical differences were observed between the analyzed species/subspecies for the analysis of the decrease of the average recovery rate under osmotic stress conditions (0.0 M and 1.2 M sucrose in RS) in relation to the best bet RS sucrose concentration of 0.6 M. Although statistically non-significant, the S. phureja accessions showed a relative decrease of 16.6% [absolute RR: 36.7%] in its average RR with a RS sucrose concentration of 1.2 M, compared to a relative decrease of 59.1% [absolute RR: 18%] when rewarmed in RS without sucrose (0.0 M). S. xchaucha showed a nearly 3-time higher relative decrease of 46.2% in RR for the same comparison (Table 3, right column). In general, no clear correlation between species/subspecies and its response to increased osmotically stressful RS sucrose concentrations (0.0 M or 1.2 M) was observed.
Potato genotypes belonging to the same species/subspecies, show a variable response to the assessed RS sucrose concentrations suggesting that it is likely genotype specific response. As an example, the accessions from S. tuberosum subsp. andigenum and S. xchaucha include both accessions that were sensitive (IDs 1, 2, 7, and 19) and tolerant to 0.0 M (IDs 33, 34, 35, and 47) (Fig. 2). Similar results can be observed for other taxa. This led to the notion, that within the different potato taxa specific accessions are more tolerant to stresses during unloading with RS than others. It is assumed that water content of the cells before cryoprotection, influx and efflux capacity of cryoprotectant agents (CPAs) and water, and sensitivity to CPAs’ toxicity and osmotic stress, vary greatly between genotypes of the same species/subspecies. Further, it might be possible that homologous groups of genes, that are present in specific accessions of different taxa, are strongly involved in the loading/unloading process of CPAs and/or control of influx/efflux of water (osmotic stress).
An analysis of the 85 screened accessions showed that 66 of the 85 accessions had the highest RR with a RS sucrose concentration of 0.6 M (42 genotypes) or 0.9 M (24 genotypes). In contrast, removal of sucrose from RS still resulted in a recovery rate of 30% or higher in 44 of 85 accessions. The minimum acceptable recovery standard used at CIP is a RR of 30% (Vollmer et al. 2015). Six genotypes (CIP 703288, CIP 703314, CIP 703762, CIP 703952, CIP 706845, and CIP 707336) showed RRs of 80–100% when sucrose was completely removed from RS (0.0 M); five of them belong to S. stenotomum subsp. stenotomum. Nine of the 85 genotypes showed a RR of 80% or higher for a RS sucrose concentration of 1.2 M (CIP 701531, CIP 703520, CIP 703709, CIP 704501, CIP 704767, CIP 705352, CIP 705575, CIP 706213, and CIP 706764). These nine accessions belong to six taxa: S. tuberosum subsp. andigenum (4), S. xchaucha (1), S. stenotomum subsp. stenotomum (1), S. stenotomum subsp. goniocalyx (1), S. xajanhuiri (1), and S. phureja (1) (Fig. 2), again suggesting little correlation of taxa with RR in their response to sucrose levels in the RS.
Seventy-seven of 85 accessions were classified based on their response to the maximum and minimum RS sucrose concentrations (0.0 and 1.2 M), as well as, to their tolerance along the complete range of tested concentrations. The accessions were ordered and classified in four groups: (a) accessions sensitive to RS sucrose concentration of 0.0 M (IDs 1–24), (b) accessions sensitive to a RS sucrose concentration of 1.2 M (IDs 25–33), (c) accessions tolerant along the whole range of tested RS sucrose concentrations (IDs 34–70), (d) accessions sensitive to RS sucrose concentrations of 0.0 M and 1.2 M (IDs 71–75) (Fig. 2). The remaining eight of 85 accessions could not clearly be assigned to one of these four groups.
The best bet RS sucrose concentration of 0.6 M showed a significantly higher RR (72.9–75.9%) in S. tuberosum subsp. andigenum and S. xchaucha accessions than the S. phureja accessions (44.0%). It was decided to use the term “best bet” instead of “optimum”, as the real optimum RS sucrose concentration will not exactly coincide with the here tested RS sucrose concentrations (e.g. no results are available for 0.5 M or 0.7 M of RS sucrose concentration). For the experiment with the 85 accessions the highest average RR (61%, Table 3) was observed with a RS sucrose concentration of 0.6 M (“best bet”).
Nevertheless, based on the knowledge of the authors, the present study reports for the first time the assessment of a diverse set of 101 potato genotypes (16 acc. + 85 acc.) over a wide range of RS sucrose concentrations (0.0 M, 0.3 M, 0.6 M, 0.9 M, 1.2 M). The results of this study showed, that complete removal of sucrose (0.0 M), as well as, high RS sucrose concentration (1.2 M) significantly reduces the RR of cryopreserved potato accessions. It was noted that the use of 1.2 M sucrose in RS is used as a standard concentration for many species and genotypes.
We postulate that cryopreserved potato clones show a highly genotype specific response to RS sucrose concentration during rewarming. Experimental results obtained with a limited number of accessions (16) should not be generalized for larger population sizes or collections (as is frequently done) because it does not always reflect the whole story as seen here. It would be interesting to perform similar experiments with species other than potato, to study the extent the most frequently reported RS sucrose concentration of 1.2 M can be optimized.