R. Vollmer*, R. Villagaray, V. Egúsquiza, J. Espirilla, M. García, A. Torres, E. Rojas, A. Panta, N. A. Barkley and D. Ellis

Transcription

1 CryoLetters 37 (5), (2016) CryoLetters, THE POTATO CRYOBANK AT THE INTERNATIONAL POTATO CENTER (CIP): A MODEL FOR LONG TERM CONSERVATION OF CLONAL PLANT GENETIC RESOURCES COLLECTIONS OF THE FUTURE R. Vollmer*, R. Villagaray, V. Egúsquiza, J. Espirilla, M. García, A. Torres, E. Rojas, A. Panta, N. A. Barkley and D. Ellis International Potato Center, Av. La Molina 1895, La Molina, Lima, Peru *Corresponding author r.vollmer@cgiar.org Abstract BACKGROUND: Cryobanks are a secure, efficient and low cost method for the longterm conservation of plant genetic resources for theoretically centuries or millennia with minimal maintenance. OBJECTIVE: The present manuscript describes CIP s modified protocol for potato cryopreservation, its large-scale application, and the establishment of quality and operational standards, which included a viability reassessment of material entering the cryobank. MATERIALS AND METHODS: In 2013, CIP established stricter quality and operational standards under which 1,028 potato accessions were cryopreserved with an improved PVS2-droplet protocol. In 2014 the viability of 114 accessions cryopreserved in 2013 accessions were reassessed. RESULTS: The average recovery rate (full plant recovery after LN exposure) of 1028 cryopreserved Solanum species ranged from 34 to 59%, and 70% of the processed accessions showed a minimum recovery rate of 20% and were considered as successfully cryopreserved. CONCLUSION: CIP has established a new high quality management system for cryobanking. Periodic viability reassessment, strict and clear recovery criteria and the monitoring of the percent of successful accessions meeting the criteria as well as contamination rates are metrics that need to be considered in cryobanks. Keywords: cryopreservation, potato, PVS2-droplet, cryobank, Solanum INTRODUCTION Cryobanks are becoming increasingly important particularly for the secure longterm conservation of clonally propagated plant genetic resources. Low maintenance costs for cryopreserved accessions, optimized space usage, extended conservation periods (theoretically for hundreds of years), low risk of mixing or losing accessions, genetic stability, minimal selection pressure and a high probability of maintaining accessions pathogen-free longterm are some of the primary advantages of cryopreserving plant genetic resource collections. The International Potato Center (CIP) conserves the largest and most diverse potato collection worldwide, containing 4,723 cultivated, mostly landraces, accessions and 2,355 accessions of potato wild relatives (Solanum section Petota). Wild potato species are conserved as seed collections at -20 C, while cultivated landraces are maintained in vitro (6-8ºC) for medium-term storage as well as increasingly in a cryobank (-187ºC to -196ºC) for longterm conservation. All potato accessions in 318

2 the active collection at CIP are maintained in trust under Article 15 of the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA). CIP s cryobank currently contains 1,320 potato accessions. Other significant cryo-collections of potato are held at Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) in Germany (1,344 accessions), National Center for Genetic Resources Preservation (NCGRP) in USA (249 accessions), National Institute for Crop Science (NISC) in Korea (130 accessions), Czech Potato Cryobank (50 accessions) and National Genebank of China (24 accessions) (1, 4, 5, 7, 17). Since 2014 CIP successfully cryopreserves about 450 potato accessions per year using an improved methodology with an annual through-put of 56 accessions per person. CIP s modified protocol for potato cryopreservation and a detailed description of the establishment of quality and operational standards for CIP s cryobank which included a viability reassessment process of the newer material in the cryobank (year 2013) are highlighted. Historical overview of potato cryopreservation at CIP (from 1990 to 2015) Potato cryopreservation was initiated at CIP in the late 1990 s, when a set of 197 Andean potato landraces was cryopreserved using a vitrification-based protocol. Results showed that 75% of the accessions had at least one shoot tip surviving, with an average shoot survival (green elongated leaf tissue) of 46% and an average shoot recovery rate (stem elongation) of 20% (2, 15). During 2004 to 2006, CIP adapted a modified PVS2-droplet-vitrification protocol originally developed for Musa (8, 10, 13, 14). In this protocol, 2-3 mm shoot tips from 3-week old in vitro plants, were placed in loading solution (LS) for min, cryoprotected at 0-4 C with varying PVS2 exposure times (0-70 min) and rapidly frozen in liquid nitrogen (LN) on aluminium foil strips or within plastic straws (3 x 25 mm). Thawing was achieved by rewarming the shoot tips for 20 min in a 1.2 M sucrose rewarming solution (RS), followed by placing the shoot tips on a meristem culture medium MS salts (6), plus 0.04 mg l -1 kinetin, 0.1 mg l -1 gibberellic acid, and 0.3 M sucrose. For the following 2 d, shoot tips were transferred daily to medium containing sequentially reduced sucrose by 0.1 M until finally maintained on medium containing 0.1 M sucrose. Survival and recovery was evaluated 45 d after thawing with PVS2 exposure times of min showing the highest recovery rates of 8-47% depending on the genotype (11). The protocol was tested on 434 potato accessions assessing two different temperatures (6 and 22 C) for the culture of the shoot tip donor plants and 289 of 434 accessions (67%) showed a recovery rate (+LN) of 20% or higher (10). CIP s standard protocol for potato cryopreservation evolved with a series of improvements during (9). Plants were cold acclimated for three weeks at 6 C, followed by excision of smaller shoot tips ( mm) comprising the meristematic dome plus 4-5 primordial leaves. Prior to cryoprotection, shoot tips were incubated at room temperature for about 1 h on potato meristem medium (MMP) (MS salts supplemented with 0.04 mg l -1 kinetin, 0.1 mg l -1 gibberellic acid, 0.07 M sucrose and 2.8 g l -1 Phytagel TM ) followed by placement in LS (liquid MSA medium, 2 M glycerol, 0.4 M sucrose) for 15 min at room temperature and cryoprotected with PVS2 for 50 min at 0 C. Ten shoot tips were transferred to a PVS2 drop (10-15 µl) on an aluminium foil strip (0.5 x 2 cm). After 1 h of storage in LN, shoot tips were thawed for min in RS (liquid MSA medium, 1.2 M sucrose; ph: 5.8) and placed onto filter paper supported on potato meristem medium (MMP) with 0.3 M sucrose. Shoot tips were maintained in darkness and transferred to fresh MMP medium decreasing the sucrose concentration by 0.1 M daily for 2 d and then maintained on MMP with 0.07 M 319

3 sucrose. One week after warming, shoot tips were transferred from the filter paper directly onto the surface of fresh MMP and incubated at 22 C, with 45 µmol m -2 s -1 and a 16h/8h photoperiod. Survival and recovery was recorded 60 d after warming, based on 20 shoot tips. Explants showing green color were considered to have survived, and explants with a shoot size of at least 0.5 cm were counted as recovered. During January 2007 through March 2012, 755 potato accessions were cryopreserved by applying this protocol (50-80 shoot tips per run), with average survival and recovery rates of 75.0% and 51.3%, respectively (+LN). Solanum tuberosum subsp. andigena group (558 accessions), showed a recovery rate of 51.1% and had the largest number of accessions of any of the cultivated species in the cryobank (9). Since 2013, the cryopreservation protocol has undergone further changes, such as high density propagation of shoot tip donor plants (70 explants/deep Petri dish), a smaller shoot tip size of mm, modification of the composition of the recovery media with 20 ml l -1 of coconut water and a new recovery cycle with longer exposure to darkness and an elimination of the daily sequential decrease of sucrose. Strict assessment criteria for viability and standard protocols have been established. Recovery is now defined as the regeneration of a normal looking in vitro plantlet with a single elongated shoot with roots. Additional quality standards include the establishment of a protocol for a safety back-up of the cryobank, implementation of a protocol for long-term viability monitoring of the accessions in the cryobank, annual assessments of technicians working in cryopreservation, and the use of cryogenic bi-dimensional barcode labels and real-time data collection with pocket PCs in all stages of the cryo-process (since June 2015; Fig.1e). MATERIALS AND METHODS CIP s current protocol for cryobanking the in trust potato collection Propagation of plant material Potato accessions comprising mostly of native Andean landraces from seven different Solanum species are obtained from CIP s slow growth in vitro collection (2). In vitro plantlets are sub-cultured every three weeks on potato propagation medium (MSA) [4.33 g l -1 MS salts (Caisson), 2.0 mg l -1 glycine-hcl, 100 mg l -1 myo-inositol, 0.5 mg l -1 nicotinic acid, 0.5 mg l -1 pyridoxine-hcl, 0.1 mg l -1 thiamine-hcl, 25 g l -1 sucrose and 3.0 g l -1 Phytagel TM, ph adjusted to 5.6±0.02] in Magenta TM GA7 vessels with 25 plants per GA7. The in vitro plants are incubated at C with a 16/8h photoperiod and light intensity of μmol m -2 s -1. For the final multiplication cycle, terminal buds from in vitro plants are sub-cultured in deep Petri dishes with MSA medium (70 buds per Petri dish) and incubated for 2-3 weeks (genotypedependent) at 6-8 C with a photoperiod of 16h/8h provided by cool daylight fluorescent tubes with a light intensity of μmol m -2 s -1 measured at plant tip height with a solar electric quantum meter (Spectrum, Fieldscout). Excision of shoot tips Under a stereoscope (10x) shoot tips are excised from 2 to 3-week-old coldacclimated in vitro potato plantlets with forceps and scalpel containing a #11 scalpel blade on 2-3 sterile bond filter papers. Shoot tips contain 3-4 leaf primordia and have an approximate length of mm and a width of mm (length and width of shoot tip are genotype-dependent). The length of the stem segment of the shoot tip (that is the distance from the base of shoot tip to meristem zone), ranges from 0.3 to 0.5 mm (Fig.1a). The tip of the most external (longest) leaf primordia is slightly cut off ( mm) when necessary. A total of shoot tips are excised per accession and placed successively onto sterile filter papers (1x1 cm), until each filter paper contains 10 shoot tips (first one shoot tip is placed on each of the filter papers, then a second on each, and so on). The filter papers 320

4 are supported on meristem culture medium (MSA medium supplemented with 0.04 mg l -1 kinetin, 0.1 mg l -1 gibberellic acid and 2.8 g l -1 Phytagel TM ). Cryoprotection After shoot tip excision, the filter papers containing 10 apical shoot tips are placed into sterile 8 ml screw cap test tube with 2.0 ml filter-sterilized LS [liquid MSA medium, ml l -1 glycerol (= 2.0 M), g l -1 sucrose (=0.4 M); ph: 5.8]. Shoot tips are treated for 20 min with LS at room temperature (20-24 C) under ambient light in the laminar flow chamber (20-30 μmol m -2 s -1 ) (Fig.1b). Using a sterile glass Pasteur pipette with a 2 ml rubber bulb, the LS is carefully removed to avoid damage to the shoot tips and replaced with 2 ml of icecold sterile Plant Vitrification Solution 2 (PVS2) [liquid MSA medium, 240 ml l -1 glycerol (3.28 M), ml l -1 ethyleneglycol (2.42 M), ml l -1 dimethylsulfoxide (1.9 M), g l -1 sucrose (0.4 M), ph: 5.8]. Shoot tips are treated for 50 min with PVS2 on ice (0 C) at ambient light conditions in the flow chamber (20-30 μmol m -2 s -1 ). Freezing in liquid nitrogen (LN) At approximately 48 min, shoot tips are sucked up with a sterile glass Pasteur pipette ( of opening: mm). The pipette s tip is placed on the bottom of the test tube and the PVS2 is expelled from the pipette until the 10 shoot tips are in approximately 10 µl PVS2 in the tip of the pipette. Shoot tips are then placed along with the 10 µl droplet of PVS2 on a sterile aluminium foil strip (5x20 mm) which is slightly folded (2-3 mm) at one end to facilitate handling with forceps (Fig.1c). At exactly 50 min, after the initiation of PVS2 exposure, the aluminium foil strip is plunged into LN (-196 C) contained in a Cool Box 30 workstation (BioCision) (Fig.1d). The strip is moved under the surface of LN into a 1.8 ml cryovial (Nunc) that was previously labeled with a cryo-resistant bi-dimensional barcode label and chilled with LN. Thirteen to 15 cryovials ( shoot tips) are initially cryopreserved per accession. After a minimum of 24 h in LN three cryovials are thawed for an initial viability check to ensure the cryo-run is successful and as a baseline for viability assessments at later dates. The remaining vials are transferred to transitory cryotanks. If the viability assessment (baseline) shows a minimum recovery rate of 30% (single run) or 20-30% (in two successive duplicate runs) the cryovials are transferred to the cryobank and safety-backup tanks. Shoot tips for accessions not meeting this minimum standard are discarded and are not placed into the cryobank. Thawing Aluminium foil strips containing 10 frozen shoot tips are quickly removed from the cryovials and immediately plunged into sterile 8 ml screw cap test tubes containing 4 ml filter-sterilized RS [liquid MSA medium, g l -1 sucrose (=1.2 M); ph: 5.8]. Shoot tips are rewarmed with RS for 20 min at room temperature (20-24 C) under ambient light conditions (20-30 μmol m -2 s -1 ). Shoot tips are removed from the test tubes with a Pasteur pipette and placed for s on a sterile filter paper (1 x 1 cm) supported on sterile bond papers to wick away excess RS. 321

5 shoot tips are supported on filter papers when transferred. a) b) c) g) Figure 1. Steps of CIP s potato cryopreservation protocol. (a) Dimensions and example of potato shoot tip used for cryopreservation; (b) Introduction of 10 shoot tips to LS (at room temperature); (c) Shoot tips placed on an aluminium foil strip, within a droplet of PVS2 (approx. 10 µl); (d) Freezing d) of samples in liquid nitrogen (LN); (e) Transfer e) f) of cryovials to cryoboxes (freezing, thawing, evaluation, transfer and viability reassessment data is recorded by using of bidimensional barcode and pocket PC); (f) Assessment of recovery 30 d after thawing (in Petri dishes); (g) Final recovery assessment 60 d after thawing (individually in 13x100 mm test tubes). Recovery The shoot tips are then placed on a filter paper (2.5 x 2 cm) in a Petri dish containing Potato Recovery Medium I (PRM-I) [semisolid MSA, 0.4 mg l -1 kinetin, 0.1 mg l -1 gibberellic acid, 20 ml l -1 coconut water (Sigma C5915), g l -1 sucrose (=0.3 M), 2.8 g l -1 Phytagel TM, ph: 5.6]. Three filter papers each containing 10 shoot tips are placed in each Petri dish. Shoot tips are incubated on PRM-I for 3 d in darkness at C, followed by culturing on Potato Recovery Medium II (PRM-II) and III (PRM-III), for 3 d on each media (in darkness at C). PRM-II and PRM-III contain the same composition as PRM-I, but with lower sucrose levels of 68.4 g l -1 (0.2 M) and 34.2 g l -1 (0.1 M), respectively. During culturing on PRM-I to PRM-III, After 9 d (3 d each on PRM-I, PRM-II and PRM-III), shoot tips are removed from the filter paper and placed directly onto Potato Recovery Medium IV (PRM-IV) [same composition as PRM-I, but with 25 g l -1 sucrose (0.07 M)] under diffuse light at C with a 16h/8h photoperiod, followed by normal light conditions ( μmol m -2 s -1 ). After 30 d, surviving shoot tips are transferred individually onto PRM- IV in 13x100 mm test tubes. Recovered samples without root formation are cut slightly at the base before transferring to remove senescent tissue. Viability assessment Viability is assessed 30 and 60 d after thawing (Fig.1f-g). Survival rate assessment 322

6 at 30 d is based on whether a shoot tip shows green tissue and some organized development. Recovery rate at 60 d is based on whether a shoot tip has elongated into a normal looking shoot. A shoot is classified as recovered if it has developed into a complete and normal looking in vitro plant (elongated stem, functional apex, leaves and roots) (Fig. 2a). Samples that show only shoot formation are sub-cultured onto fresh MSA medium to confirm if a complete in vitro plant can be obtained. If samples do not develop into complete in vitro plants they are recorded as survived but not recovered (Fig. 2b-c). Shoot tips that develop into deformed plants (Fig. 2d-e), have only leaf formation (Fig. 2f) or signs of vitrification (Fig. 2g), are classified as survived but not recovered. Shoot tips that do not develop, and/or never turn green, are recorded as dead (Fig. 2h-i). Samples with signs of fungal or bacterial contamination are replaced by thawing one or more additional vials. If the contamination recheck also shows signs of contamination, the cryo-run for that accession is discarded from the temporary storage tank and the accession is cryoprocessed again. Transfer to the cryobank and safety-backup tank Accessions with a minimum recovery rate of 20% (based on 30 shoot tips) are transferred to the cryobank. If the 60-d viability assessment of the first run has a recovery rate of 30% or higher, this single run is conserved and 8-10 vials are transferred to the cryobank (Chart MVE 819P-190AF-GB) while the remaining two vials are transferred to the safety back-up tank. If the first run has a recovery rate between 20% and 30%, a second cryo-run is performed for that accession. For runs with a recovery rate of 20-30%, 6-8 vials of each run are stored in the cryobank and 4 vials are placed in the safety back-up tank. Accessions with less than 20% viability are discarded from the temporary tank, the viability data is recorded in the corporate database and the accession is slated for cryopreservation at a later date when more parameters can be experimentally imposed. Data storage The recovery data is manually entered into the cryobank database (January 2013 to June 2015). Since July 2015 all data is recorded in real-time on pocket PCs (Motorola MC55A0) (Fig.1f) with labels automatically produced on a barcode printer (Zebra Z400M) with cryogenic ribbon (5095 Performance) and hand-held barcode printers (Zebra RW 220). The bidimensional barcodes of the cryovials contain information on the identification number of the accession, ID of cryo-run, date of cryopreservation, and the ID of the used protocol. Data analysis Survival and recovery data are expressed as a percentage of the total assayed shoot tips. In normally distributed data, means are assessed by analysis of variance (ANOVA) and t-tests (with confidence levels of 95%). Homogeneity of variance and normality of distribution are assessed with the Levene and Anderson- Darling tests, respectively (p-value 0.05). In cases of non-normally distributed data, medians are assessed with the Kruskal- Wallis multiple comparison test (with a simultaneous confidence level of 95%). Statistical analysis and graphical presentation is done with MINITAB and Microsoft Excel 2010 software (Version ). Viability reassessment of CIP s current cryobank (year 2013) In order to assess the robustness of the current protocols and quality standards, a single vial (holding 10 shoot tips each) from 114 accessions cryopreserved in 2013 was thawed in 2014 and reevaluated. Since 2013, potato accessions have been cryobanked with 1 or 2 runs depending on the rate of recovery (see Method section). The 323

7 historical viability data (+LN) is based on a sample size of 30 shoot tips RESULTS CIP s cryobank ( ) As of November 2015, CIP s potato cryobank contained 1,320 cultivated potato accessions, the majority of which (1028 accessions) were cryopreserved from with a new quality management system imposing strict viability and quality criteria. The cryobank represents a genetically diverse group of potato landraces successfully cryopreserved with the described cryopreservation method. Based on the taxonomy of Hawkes (3) the accessions cryopreserved include seven different Solanum species: S. tuberosum (761 accessions), S. stenotomum (131 accessions), S. phureja (41 accessions), S. chaucha (32 accessions), S. juzepczukii (8 accessions), S. ajanhuiri (7 accessions), S. curtilobum (1 accession) and non-classified Solanum species (47 accessions). Within S. tuberosum, the accessions belong principally to the subspecies andigenum (717 accessions) but also to the subspecies tuberosum (44 accessions). The S. stenotomum collection consists of subspecies stenotomum (100 accessions) and subspecies goniocalyx (31 accessions). Average survival was 68.8% and average recovery 55.4% (Table 1). Over three quarters (805 of 1028 or 78.3%) of the cryobanked accessions showed a minimum recovery rate of 40.0% or higher. No significant differences were observed between median survival rates of species and/or subspecies. However, the recovery rates were significantly higher for the tetraploid species S. tuberosum (subsp. andigenum and subsp tuberosum., 56.0% and 59.5% respectively) than for the diploid species S. stenotomum (subsp. stenotomum and subsp. goniocalyx, 40.0% and 43.0 % respectively). The differential between survival and recovery rates was higher for the S. stenotomum and S. ajanhuiri species (18.4 to 19.2%) than for the other species ( %). Viability reassessment of CIP s current cryobank Confirmation of viability after a year in cryopreservation is a component of CIP s new cryobanking standard. One hundred and nine of the 114 tested accessions (cryopreserved in 2013) showed a minimum recovery rate of 20% or higher. The remaining five of the reassessed accessions showed low recovery or bacterial contamination and were removed from the cryobank and re-processed (data not shown). The two sample t-test of the 109 accessions with successful reassessment showed significant differences between the original and reassessment data for survival (p=0.002) but not for recovery rates (p=0.076) (Fig. 3). However, the average rates for survival (66.7%) and recovery (50.3%) of the original assessment were lower than the survival (74.9%) and recovery rates (55.1%) of the reassessment. Nevertheless, historical data in the database is based on 30 shoot tips, while the reassessment data has a smaller sample size of 10 shoot tips and the low sample size of the reassessment could account for these differences. 324

8 Species/ Subspecies Ploidy level Number of cryobanked accessions Total number of accessions (in vitro bank) Accessions cryobanked (%) Table 1. Survival and recovery rates (+/- SE) of 1,028 potato accessions cryobanked at CIP from The accessions belong to nine taxa (seven species), based on the taxonomy of Hawkes (3) with four different ploidy levels (2x, 3x, 4x and 5x). All accessions were cryopreserved with the PVS2-droplet cryopreservation method (LS, 20 min; PVS2, 50 min on ice; RS, 20 min). Per accession shoot tips were cryopreserved of which 30 were thawed after a minimum of 24 h in liquid nitrogen (+LN). Survival and complete plant recovery was assessed 60 d after thawing. Means Medians (1) Post +LN survival (% ± SE) Post +LN recovery (% SE) Post +LN survival (%) Post +LN recovery (%) Solanum tuberosum subsp. tuberosum Solanum tuberosum subsp. andigenum 4x ± ± A x ± ± A 56.0 a Solanum chaucha 3x ± ± A 49.3 a b c Solanum juzepczukii 3x ± A 52.5 a b c Solanum phureja 2x ± A 46.0 a b c Solanum stenotomum subsp. stenotomum Solanum stenotomum subsp. goniocalyx 2x ± A 40.0 c 2x ± A 43.0 b c Solanum ajanhuiri 2x ± A 46.0 a b c Solanum curtilobum 5x ± (2) - (2) - (2) Solanum sp.? ± A 56.0 a b Total Mean 68.8± ± 0.6 (1) Different letters indicate significant differences (p<0.05) for the Kruskal-Wallis [KW] Multiple Comparison Test (p<0.05). Significant differences between survival rates are indicated with capital letters and differences between recovery rates were indicated with lower case letters. (2) Solanum curtilobum was not included in the statistical analysis because only 1 accession was cryopreserved. 325

9 Figure 2. Description of categories for survival/recovery assessment (+LN). (a) Complete plant recovery (after 60 d); (b) and (c) Only shoot formation, samples were sub-cultured on MSA medium to assess if complete plant recovery can be achieved (if samples did not develop into complete in vitro plants they were recorded as survived but not recovered); (d) Deformed apex (survival only); (e) Deformed plant (survival only); (f) Only leaf formation (survival only); (g) Vitrified plant (survival only); (h) and (i) Production of phenolic compounds and no growth of green tissue (dead) Success rate: low viability and contamination (as of Nov/2015) The success rate for cryopreservation of potato (percentage of accessions reaching 30% viability with the first cryo-run or 20% with two cryo-runs) increased substantially from 60.3% in the years (567 of 940 accessions) to 86.2% in 2015 (461 of 535 accessions; Fig. 4). The general success rate for potato cryopreservation in the period was 69.7% (1028 of 1475 accessions). The overall contamination rate (fungal and bacterial) decreased from % (years 2013/14) to 6.3% (year 2015). Improvement was also made in lowering the percentage of cryopreserved accessions with a subpar viability rate (0-20%) which decreased from 31.8% (67 of 211 accessions) to 27.7% (202 of 729 accessions) in 2013 and 2014 respectively, to 7.5% (40 of 535 accessions) in DISCUSSION The newly established potato cryocollection ( ; 1,028 accessions) shows a higher average recovery rate (55.4%) compared to the previous years (51.3%) ( ; 755 accessions) as should be expected with protocol improvements (9). Particularly, in the large Solanum tuberosum subsp. andigenum collection (717 accessions) a positive increase of 6.1% in the recovery rate was noted, i.e., 57.2% compared to the average recovery rate of 51.1% reported for (9). The smaller and more uniform shoot tip size ( mm) used at CIP for the newly established cryobank ( ) is approximately half of size of previously reported shoot tip dimensions of mm (9) and might be one of the major explanations for the improved recovery rate. The other major potato cryobank, held by IPK, reported mean regeneration of 49.2% for its 1,344 accession potato cryobank using the DMSO-droplet cryopreservation method (5). The National Genebank of China reported regeneration 326

10 rates of 47-76% on a set of 24 potato accessions using the droplet vitrification cryopreservation method (17), while the Czech Potato Cryobank obtained an average recovery rate of 29% on a set of 50 potato accessions with a dehydration-vitrification cryopreservation method (1). Recently, a new cryopreservation method using V-plate and D-plates was tested on 16 potato accessions and showed regrowth rates of %. Using the V-plate method, high regrowth rates of 100% were reported with small shoot tips of 0.5 and 1.0 mm, and lower regrowth rates of 96.7% and 56.7% with larger shoot tips of 1.5 and mm, respectively (16). Other components, such as recovery media, transfer times, and assessment criteria for recovery, have undergone valuable improvements affecting the rate of success. Additionally, the monitoring and improvements of the physical components of the cryobank system have also advanced. Improved monitoring of LN level, automatic LN level controllers, regular temperature monitoring with individual tank alarms and the self-generation of LN have all helped consistently in running a cryobank. The 2014 reassessment of accessions cryopreserved in 2013 confirmed that 96% of the reassessed accessions (109 out of 114) did accomplish the reassessment test based on reaching a minimum viability per vial of 20% after one year in LN. Nevertheless, it was also noted that 4% of the reassessed accessions did not accomplish the minimum standards for viability and quality. The reassessment of CIP s new cryobank helps to close the quality cycle and identify outlier events that happened during the transfer of the accessions as a consequence of human error during routine cryopreservation or data recording (i.e., contamination, high standard deviation between vials, bad data points, etc.). The discarding of cryopreserved accessions, as a consequence of contamination or low viability, was reduced substantially from 2013/14 to 2015 in the CIP cyrobank. Large-scale ramping up of the cryopreservation capacity started in October This necessitated the hiring and training of new staff with no prior cryopreservation experience, and in some cases no prior tissue culture experience. A program to train these staff, by a senior technician with more than 15-years of experience in cryopreservation, impacted on the success in cryopreservation by reducing the contamination rate. Overall, cryopreservation success increased by 26% from 2013/14 to 2015 and contamination rates decreased by 4.3% in the same period. b A Figure 3. Comparison of original (assessed in 2013) and reassessed (in 2014) recovery and survival rates of 109 potato accessions cryobanked in 2013 with the PVS2-droplet method. Original and reassessed data are based on n=30 and n=10 shoot tips per accession, respectively. Different letters indicate significant differences (p<0.05) for two sample t-test. For recovery (capital letters), no significant differences between original and reassessed recovery rates were observed. a A 327

11 Figure 4. Bar chart for successfully cryobanked and discarded potato accessions at CIP from (data from only the first 10 months of 2015). Unsuccessfully processed potato accessions were discarded from the cryobank if they showed low viability (0%, 1-10%, 11-20%), or signs of contamination (fungal, bacterial). The numbers of successfully cryopreserved accessions are indicated (black bar, white dots), as are the numbers of processed but discarded accessions (all other bars). The PVS2-droplet protocol is a multistep process and even the most experienced and trained staffs are challenged at times with a contaminated accession. Thus it is important to have multiple control points to avoid transferring contaminated accessions or ones with low viability to the cryobank. Outliers can be identified by periodical viability assessment of the cryobanked accessions. Although, a minimum recovery standard of 40% has been suggested as baseline for storage of cryopreserved material (12), for cryopreservation of potato landraces at CIP we determined a lower recovery rate was acceptable as a minimum for our quality standards. We also determined that our strict criteria of recovery of a whole elongated plantlet permitted security with a lower viability rate. Finally, our standard of reassessment gives added security for these viability rates. The established cryobank (years ) includes 15.4% more accessions (805 of 1028 accessions) with a minimum recovery rate of 40.0% than previously held in the CIP cryobank (9). Although a large achievement is the cryopreservation rate of 450+ potato accessions per year, a successful cryopreservation of 56 diverse accessions per worker in our potato cryopreservation group is the largest accomplishment. CONCLUSION CIP has established a new high quality management system for potato cryobanking. The development of reliable protocols applicable on a wide range of diverse genotypes has been successful but remains an evolving process. Periodical viability reassessment, strict and clear recovery criteria and the monitoring of success and contamination rates are aspects that need to be considered when implementing a cryobank. Cryobank managers have to ensure that future 328

12 generations will inherit reliable cryobanks that include the predictable recovery of complete and normal looking plants. Acknowledgements: We express our sincere gratitude and thanks to the Global Crop Diversity Trust and the Genebank CGIAR Research Program for funding this work. REFERENCES 1. Faltus M, Bilavcik A, Zamecnik J, Svoboda P & Domkarova J (2008) In Cryopreservation of Crop Species in Europe (Agrifood Research Working papers 153) (eds) J Laamanen, M Uosukainen, H Häggman, A Nukari & S Rantala. Abstract, p Golmirzaie A & Panta A (2000) In Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Application (eds) F Engelmann and H Takagi, JIRCAS, Tsukuba/IPGRI, Rome, pp Hawkes JG (1990) The Potato: Evolution, Biodiversity, and Genetic Resources. The Smithsonian Institution Press 259 pp 4. Hirai D (2011) CryoLetters 32, Keller ERJ, Senula A, Grübe M, Diekmann K & Dehmer HJ (2014) Acta Horticulturae 1039, Murashige T & Skoog F (1962) Physiologia Plantarum 15, Niino T & Valle AM (2015) Breeding Science 65, Panis B, Piette B & Swennen R (2005) Plant Science 168, Panta A, Panis B, Ynouye C, Swennen R, Roca W, Tay D & Ellis D (2015) Plant Cell Tissue Organ Culture 120, Panta A, Tay D, Ynouye C & Roca W (2011) Acta Horticulturae 908, Panta A, Panis B, Ynouye C, Swennen B & Roca W (2006) Cryobiology 53, Reed BM (2001) CryoLetters 22, Sakai A, Kobayashi S & Oiyama I (1990) Plant Cell Reports 9, Schäfer-Menuhr A, Schumacher HM & Mix-Wagner G (1994) Landbauforschung Völkenrode 44, Steponkus PL, Langis R & Fujikawa S (1992) In Advances in Low Temperature Biology (vol. 2) (Steponkus PL ed.). JAI Press. London. pp Yamamoto S, Wunna, Rafique T, Valle Arizaga M, Fukui K, Cruz Gutierrez EJ, Castillo Martinez CR, Watanabe K & Niino T (2015) American Journal of Potato Res. 92, Zhang J, Xin X, Yin G, Lu X & Chen X (2014) Acta Horticulturae 1039,