Droplet-vitrification methods for apical bud cryopreservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Rob.]

Abstract

This study aimed to develop a cryopreservation protocol for the long-term preservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.)], an Andean crop with high fructooligosaccharide content in its tuberous roots. Initially, the cryopreservation protocol was developed using a yacon clone originated from Ecuador classi ed as ECU 41. Osmotic dehydration of apical buds (2-3 mm long) was carried out by assessing two plant vitri cation solutions, PVS2 (15, 30, and 60 min) at 0°C and PVS3 (30, 45, 60, and 75 min) at 22°C. After cryopreservation, the apical buds were thawed and placed on MS medium ± 0.1 mg l − 1 N 6 -benzyladenine (BA). The survival rates ranged from 37 to 90% within all treatments, with those subjected to PVS2 and PVS3 for 60 min showing the highest survival rates on MS medium without BA (87 and 90%, respectively). At 12 weeks post cryopreservation, these treatments also provided the highest regrowth rates, both reaching 73% of normally growing (shooting, rooting) plantlets. Survival rates on MS + 0.1 mg l − 1 BA regrowth medium reached up to 90%; however, regrowth into normally rooted plantlets did not exceed 67% post cryopreservation. The optimized protocols were then applied to 4 additional yacon clones originated from Bolivia and Peru, classi ed as BOL 22, BOL 23, PER 12, and PER 14. This resulted in survival and regeneration rates ranging between 79.7-94.1% and 66.3-75.4% respectively. Our study shows that optimal cryopreservation protocols for the long-term conservation of yacon can be based on both PVS2 and PVS3 vitri cation solutions. Key MessageAn e cient PVS2 and PVS3 based cryopreservation protocol for yacon was developed, ensuring shoot tip survival of up to 94.1% and subsequent regrowth up to 75.4% after cryopreservation.

Overcoming Challenges for Shoot Tip Cryopreservation of Root and Tuber Crops

“…H.Rob.] [149]. Dr-vi is currently the most widely applied cryoprotocol for cryopreserving plant germplasm within genebanks, including those for potato [66,69,150,151], cassava [69], sweet potato [151] and yam [70].…”

“…They found that PVS2 or PVS4 incubations at 23 • C for 15-30 min achieved satisfactory shoot regrowth, while PVS3 was impractical for Dr-vi due to its weak adhesion to the aluminum foil [117]. In contrast, the PVS3 dehydration was later optimized and applied successfully in Dr-vi for shoot tip cryopreservation of potato [131], yacon [149], and ulluco [155]. Additionally, PVS3 has been routinely applied for cryopreserving potato shoot tips at the gene bank of Gatersleben, Germany [131].…”

“…Shoot tip thawing procedures that achieve fast warming rates are suggested to ensure the speedy transition from glass to liquid without ice recrystallization within plant cells [79,132,212]. In ultra-rapid freezing methods such as Dr-vi and V/D cryo-plate, cryopreserved shoot tips were rapidly warmed in ULS with high level of sucrose (0.8-1.2 M) [73,108,124,148,149] at room temperature [104,109] or pre-warmed to 35-40 • C for 30 s, then transferred to room temperature and held for 15-20 min prior to plating onto appropriate recovery media [133,144]. The use of 1.2 M sucrose in ULS is used as a standard concentration for many species, including RCTs.…”

Vitrification Solutions for Plant Cryopreservation: Modification and Properties

“…The droplet-vitrification method is derived from the DMSO droplet methods proposed by Kartha et al [146], and Kaczmarczyk et al [149] and Schaefer-Menhur et al [150]. The procedure is similar to the droplet method but with highly concentrated cryoprotective solution PVS2 [104,147,[150][151][152] or PVS3 either in original or in its modification PVS3-M4 [122] or both PVS2 and PVS3 [97,113,126] before ultra-fast cooling. Rewarming of the samples is usually done in unloading solution temperated in a 40 • C sterile water bath for 1-2 min.…”

“…Therefore, there is no generic time for PVS2. For example, in apple the droplet-vitrification method had the highest regrowth percentage after 30-50 min PVS2 exposure at room temperature [109]; in potato droplet-vitrification had the highest regrowth percentage after 50 min PVS2 exposure at 0 • C [110]; in shallot droplet-vitrification had the highest regrowth percentage after 40-60 min PVS2 exposure at 0 • C [111]; in grapevine droplet-vitrification had the highest regrowth percentage after 90 min PVS2 exposure at 0 • C [112], and in yacon droplet-vitrification had the highest regrowth percentage after 60 min PVS2 exposure at 0 • C [22,113]. DMSO and Gly penetrate the cell wall membrane and increase cellular osmolality avoiding ice formation [7,38,114].…”