Cryopreservation of Dormant Buds by PVS-Based Vitrification: A Highly Promising Strategy for Large-Scale Cryobanking of Plant Genetic Resources

Dormant bud cryopreservation

Dormant bud cryopreservation offers an ideal strategy to address these challenges, leveraging the natural cold hardiness and strong post-cryopreservation regrowth capacity of dormant buds. Sakai (1960) conducted the first cryopreservation study, demonstrating that winter bud sticks of poplar and willow could survive in LN. ¹⁵ Since then, dormant bud cryopreservation has gained increasing attention for conserving germplasm of woody plants, particularly fruit trees.^22-24 Dormant buds have proven to be efficient and cost-effective materials for cryopreservation, with simple protocols applicable to twigs of some temperate and tropical woody species. ²⁵ A study on cryopreservation of apple germplasm using dormant buds and in vitro shoot tips found that the time and labor for cryopreservation of dormant buds were 40% and 50% of those required for cryopreservation of in vitro shoot tips, respectively. ²⁶ Currently, the USDA-ARS facilities in Fort Collins and Corvallis preserve 2155 apple germplasm accessions using twigs with dormant buds. ⁴ Slow-freezing cryopreservation of apple twigs with dormant buds has achieved 90% survival after 10 years of storage. ²³ Traditional slow-freezing protocols for dormant buds involve dehydrating bud segments to low moisture content followed by gradual low-temperature acclimation.^4,26,27 However, this technique relies on expensive controlled-rate freezers to achieve freeze-induced dehydration and is primarily suited for cold-tolerant species and undifferentiated cultures and apices, which limits its widespread use.^8,20,28,29 In recent years, an increasing number of studies have reported successful dormant bud cryopreservation using PVS-based protocols. This method involves exposing explants to concentrated cryoprotectant solutions for dehydration and could induce a glassy state (vitrification) in cellular water to prevent ice crystallization and tissue damage during immersion in LN; thereby, ice crystallization is prevented and plant tissues survive.^8,10,12,30

Compared with slow freezing, PVS-based cryopreservation is simpler to perform, suitable for larger and complex organs, achieves higher post-cryopreservation survival rates, and requires no specialized equipment.^10,31 This protocol has been applied to over 100 plant species and is gaining prominence as a promising alternative to slow freezing for long-term plant genetic resource conservation.^1,10,30

PVS-based vitrification cryopreservation of dormant buds has achieved high regeneration rates in numerous species, including blackcurrant cultivars, ³² Malus, ³³ Salix, ³⁴ Vaccinium, ³⁵ cherry, ⁷ persimmon,^12,13,36 Betula pendula Roth, ³⁷ Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, ³⁸ and Gentiana straminea Maxim. ³⁹ Moreover, dormant bud cryopreservation has also exhibited distinguished superiority over the shoot tips of in vitro plants, such as Rosa canina, R. rubiginosa, ⁴⁰ R. pomifera, ¹⁶ persimmon,^41,42 and Actinidia arguta. ⁴³ Additionally, the possibility of genetic instability is minimized, and plant regeneration post-cryopreservation is easier than other propagules since the shoot tip of dormant bud is totipotent and could sprout directly or produce multiple shoots or bulblets in vitro without forming callus as an intermediate stage (Fig. 1). As a result, PVS-based vitrification cryopreservation holds great promise as an effective preservation strategy for perennial plants producing dormant buds whatever is woody and herbaceous plants. This review provides updated and comprehensive information on the development and recent progress of dormant bud cryopreservation with high regrowth levels across diverse species (Table 1).

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FIG. 1.

The anatomical structure of bulbs of Fritillaria przewalskii and the recovery process of dormant buds after cryopreservation. (A) Bulb morphology (1—Large scale petal; 2—Small scale petal; 3—Small scale leaf; 4—Dormant buds). (B) Dormant buds after cryopreservation. (C) Recovery of dormant buds cultured for 7 days (white arrows indicate viable dormant buds, black arrows indicate dead dormant buds). (D) Dormant buds produce small bulblets. (E) Subculture propagation of small bulblets. (F) Regenerated seedlings from bulblets.

PVS-based vitrification methods

Several vitrification-based cryopreservation technologies have been successfully developed for dormant buds, including vitrification, ⁵² droplet-vitrification (DV), ⁵³ and encapsulation-vitrification (EV). ⁵⁴ The standard vitrification protocol involves treating explants with a loading solution (LS; typically 2 mol/L glycerol + 0.4 mol/L sucrose, pH 5.7), ⁵⁵ followed by high-concentration vitrification solutions with the purpose of explant dehydration. Common types are PVS1 (22% glycerol + 13% ethylene glycol + 13% polyethylene glycol + 15% DMSO), ⁵⁶ PVS2 (30% glycerol + 15% ethylene glycol + 15% DMSO + 0.4 mol/L sucrose), ⁵² and PVS3 (50% glycerol + 50% sucrose). ⁵⁵ After PVS treatment, explants are plunged into LN for storage. For plant regeneration, explants are rapidly thawed in a water bath, exposed to an unloading solution (MS medium with 1.2 mol/L sucrose, pH 5.7) ⁵ to remove cryoprotectants, and finally recovered on hormone-supplemented medium. In DV, cryoprotectant solutions are applied as single droplets on aluminum foil to enable ultra-rapid cooling and thawing.^57,58 EV combines vitrification and encapsulation-dehydration, involving explant encapsulation in alginate beads, followed by loading and PVS dehydration.^5,59 Critical factors must be considered for applying dormant bud cryopreservation to germplasm conservation. This review summarizes current research on PVS-based cryopreservation of dormant buds and highlights factors enhancing survival and regeneration. Our goal is to stimulate further research and development in dormant bud cryopreservation, supporting the preservation of genetic diversity in perennial plants.

Plant species and genotypes

Genotype significantly affects the recovery rate in cryopreservation of most plant species, though its impact is less pronounced in PVS-based vitrification cryopreservation of dormant buds. Forsline applied slow-freezing cryopreservation to 64 apple accessions, with 60 of them achieving an average recovery growth rate exceeding 65%. ²² For Malus, 180 samples representing 32 species were cryopreserved using dormant buds over a 10-year period, yielding an average post-cryopreservation survival rate of 39%. ⁶⁰ In Pyrus, cryopreservation of dormant buds from 35 accessions across 21 species showed that the PVS2 method enabled more genotypes to meet the 40% survival baseline. ⁶⁰ Similarly, vitrification of dormant buds from 20 apple accessions resulted in shoot tip viability ranging from 58.3% to 74.2%. ⁶¹ Guyader et al. cryopreserved dormant buds from 15 Malus and Pyrus cultivars, recording average recovery rates of 31.8% and 26.9%, respectively. ⁴⁶ In DV of six persimmon accessions, survival rates exceeded 79%. ⁶²

The strong universality of PVS-based vitrification within genera is further demonstrated in blackcurrant, ³² Pyrus, ⁵⁰ almond, ⁶³ and grapevine. ⁶⁴ Even conspecific plants from diverse ecological environments achieved favorable results using dormant buds. For example, dormant buds from 24 apple varieties exhibited >70% survival, and protocols optimized for cold-hardy North American apple cultivars were adaptable to less cold-hardy varieties in Denmark’s temperate oceanic climate. ⁶⁵ Jenderek et al. confirmed no significant differences in post-cryopreservation viability among dormant buds from three apple cultivars grown across distinct U.S. locations. ⁶⁶ Additionally, a Shimane-developed persimmon dormant bud protocol was successfully applied to 16 temperate cultivars from Japan, China, and Korea, achieving an 89% average shoot formation rate. ¹³ Growing interest in dormant bud cryopreservation has driven protocol development for deciduous trees and shrubs, with varying success reported in peony, ⁶⁷ blueberry, ³⁵ aspen, ⁶⁸ currant, ⁶⁹ little walnut, English walnut, and black walnut. ⁴⁹ Subsequent studies extended vitrification to herbaceous species, including G. straminea Maxim.,^39,70 Codonopsis pilosula (Franch.) Nannf.,^70,71 and A. membranaceus. ³⁸ The above research results indicate that dormant buds enable cryopreservation of numerous accessions within a genus, with most achieving acceptable regrowth.

Physiological state of dormant bud

Developing highly efficient cryopreservation protocols for germplasm banks requires optimizing not only the operation technology but also the physiological status of the shoot tips,^5,44,72 which is mainly determined by the extent of cold acclimation.^25,73-75 The reason is that adequate natural cold acclimation induces cell dehydration^22,24,76,77 and promotes synthesis and accumulation of freezing tolerance solutes, such as soluble sugars, sugar alcohols, organic acids, amino acids, and polyamines in plants. ⁷⁸ Most dormant bud studies collect branches or scions in December–February, as early fall samples often lack cryotolerance and are susceptible to suffer freeze damage.^75,77 However, optimal timing varies by species and region. For peony, buds collected in late November—late February yielded 66%–74% regrowth, but dropped to <1% in late March. ⁶⁷ In Finland, the regeneration of hybrid aspen buds post-cryopreservation increased from 9.8% (September) to 75% (October–February). ⁶⁸ Dracocephalum austriacum buds collected in late December achieved 100% regeneration, declining to 76% in February and 30% in March. ⁷⁹ Moreover, late December collection is critical for high survival in Pyrus, ⁵⁰ blueberry,^35,79 American elm, ⁷² and A. arguta. ⁴³

It is often desirable to collect materials in their cold-hardiest state from the field. However, different accessions or species exhibit distinct acclimation kinetics, and the degree of cold hardiness may vary annually due to temperature fluctuations, so this state is not always easy to discern or test. In temperate species such as apple and pear, it has been documented that buds should only be harvested after the plants have been exposed to subzero temperatures for a minimum of 72 hours. ⁷⁷ Ravish (2023) ²⁶ reported on dormant buds harvested from subtropical zones, where temperatures do not drop below 12°C in any season. Moisture levels in buds during three winter months—when leaf abscission occurred—were used as a criterion. During natural acclimation, buds are reported to lose water content to varying extents in December, January, and February. The results showed that buds harvested in February with the lowest moisture level were optimal for preservation. In addition, cold acclimation can be artificially induced and significantly improves the post-cryopreservation survival rate of shoot tips. Jenderek et al. found that blueberry buds harvested under conditions where the average maximum air temperature was below 11.2°C showed higher post-cryopreservation viability, ranging from 52% to 80%, in Fort Collins, CO. ³⁵

For in vitro plants, cold acclimation is a highly effective method, typically achieved by exposing them to low temperatures (0°C–4°C) or alternating between daytime exposure at 20°C and nighttime exposure at 3°C for more than 2–3 weeks. ⁸⁰ For field-grown plants, woody branches or organs bearing dormant buds can be stored under low-temperature conditions for further acclimation, which enables the dormant buds to tolerate the stresses incurred during cryopreservation. The most common approach involves enclosing the material in polyethylene bags and storing it in a refrigerator at −5°C to 3°C for 1 week to 3 months prior to use, which generally enhances cold hardiness while maintaining plant viability.^75,77 However, the optimal storage duration varies across species. Towill and Ellis observed that apple branches stored for 6–8 months could be successfully cryopreserved, whereas the regenerative capacity of sweet cherry branches stored for more than 4–6 months declined significantly. ²⁵ For herbaceous perennials, subterranean organs with dormant buds must be collected pre-soil freezing, though cold acclimation is often insufficient. Our studies showed that excavating C. pilosula ⁷¹ and F. przewalskii subterranean organs in October—early November and storing them in moist soil within polyethylene bags at 2°C–4°C for 1–3 months could gradually improve dormant bud survival rate post-cryopreservation with prolonged the preservation time (unpublished, Fig. 1). Notably, A. membranaceus dormant buds collected in March achieved 100% survival after 2 weeks of artificial cold acclimation. ³⁸

Shoot tip size

Shoot tip size significantly affects post-cryopreservation recovery. ²⁷ Optimal sizes range from 1 to 3 mm ²⁷ with most protocols using about 1 mm.^27,59,81-83 Cryopreservation of 0.5–1 mm shoot tips dissected from dormant buds of American elm, ⁷² Diospyros kaki Thunb.,^13,84 and almond ⁶³ all achieved 80%–100% regeneration. Smaller apices are susceptible to vitrification solution toxicity, reducing vitality and survival, and larger apices, conversely, exhibit poor cryoprotectant permeability, leading to incomplete dehydration, and high water content reduces cold tolerance of shoot tips and ultimately results in lower survival. ⁷¹

However, dissecting 1 mm-long shoot tips from growing plantlets is time-consuming and often reduces explant vigor. In contrast, a simple method can enable efficient isolation of small shoot tips from dormant buds. First, dormant buds can be slittered at an appropriate angle, exposing inner shoot tips, and then excised with a scalpel to yield abundant, vigorous tips rapidly.^38,39,70,71 Moreover, our research found 2–4 mm shoot tips from F. przewalskii dormant buds achieved optimal survival by vitrification (unpublished), and even whole dormant buds of C. pilosula in big volume also exhibited strong survival and regeneration ability, ⁷¹ which suggested that vitrification was a viable long-term preservation method for larger explants and could greatly simplify the steps of shoot tip dissection.

Shoot tip preculture

Preculture is a treatment in which shoot tips are cultured in a medium supplemented with high concentrations of sugar, DMSO, sorbitol, or mannitol. ⁸⁰ These substances could increase medium osmotic pressure leading to cell dehydration or permeate cells and reduce free water, which could reduce the cell freezing point and then further decrease the formation of ice crystals. ⁸⁰ Sucrose is the most widely used sugar in preculture, with proven efficacy for dormant bud cryopreservation in B. pendula Roth., ³⁷ G. straminea Maxim., ³⁹ Tamarix taklamakanensis, ⁴⁵ Prunus dulcis, ³ and almond. ⁶³ Typically, shoot tips are cultured in medium adding 0.3–1.0 mol/L sucrose for 1–7 days,^5,27,51 but optimal preculture duration must be determined experimentally as species-specific variation exists. Preculture is unnecessary for dormant buds collected at optimal times, such as persimmon, ⁸⁴ C. pilosula (Franch.) Nannf., ⁷¹ and F. przewalskii (unpublished).

Loading

Loading involves immersing materials in cryoprotectant solutions at room temperature for a specified period to reduce cellular water content and prevent osmotic shock caused by PVS solution. ⁶ Commonly used loading solutions include 0.2 mol/L glycerol + 0.4 mol/L sucrose or 60% PVS2, with a 20-minute exposure—a routine parameter across most species and explant types.^5,9 Loading solutions have been repeatedly validated for in vitro-grown and dormant bud shoot tip cryopreservation.^2,13,85 Rasl et al. found that while loading did not affect survival, it significantly inhibited regeneration of Lamiaceae dormant buds cryopreserved via DV. ⁷⁹ Notably, satisfactory survival of D. kaki Thunb. dormant buds was achieved via DV without loading. ⁸⁴

Plant vitrification solutions

Application of concentrated vitrification solutions is critical for cryopreservation: they accelerate dehydration, lower freezing points, and induce vitrification (amorphous solidification) of cellular fluids, which avoid the formation of large amount of ice crystals and thereby reduce ice-induced injury. ⁶⁰ PVS2 is the most widely used vitrification solution.^6,37,52,86 DMSO as a key component exhibits lower toxicity at 0°C–5°C than at 20°C–25°C. ²⁷ Explants can tolerate longer PVS2 exposure at lower temperatures; thus, PVS2 treatment is typically performed at 0°C. ²⁷ For example, axillary buds of greenhouse-grown Rosa exposed to PVS2 at 0°C via DV retained 100% survival even after 90-minute exposure, whereas room-temperature PVS2 treatment significantly reduced survival. ⁸⁷ Similar findings were reported for shoot tips of Nephelium ramboutan-ake, ⁸⁸ mint, ⁸⁹ and Populus alba L. ² Optimal PVS2 exposure varies by species, with 20–120 minutes most common. For instance, DV of shoot tips from test-tube plantlets of Rosa, ⁴⁰ potato, ⁹⁰ Colocasia esculenta var. esculenta, ⁹¹ shallot, ⁹² kiwifruit, ⁹³ and yacon ⁹⁴ used 20–60 minutes PVS2 exposure at 0°C. In contrast to dormant buds, they exhibited broad PVS2 tolerance due to high dehydration and cold stress resistance. A. arguta dormant buds treated with PVS2 for 120–300 minutes at 0°C achieved >60% survival ⁴³ ; G. straminea Maxim. buds exposed for 20–60 minutes yielded >65% survival ³⁹ ; R. pomifera buds treated for 10–30 minutes achieved >58% survival ¹⁶ ; and R. canina and R. rubiginosa dormant buds treated for 0–30 minutes via DV achieved >90% regeneration. ⁴⁰ When dormant buds of C. pilosula were incubated for 20, 40, 60, and 80 minutes at room temperature, all resulted in a high survival rate of more than 65% using vitrification. ⁷¹ For A. membranaceus dormant buds, 20, 50, and 80 minutes incubation resulted in a survival rate of more than 80%. ³⁸ In cryopreservation of persimmon dormant buds, the survival rate increased with the longer exposure duration, and the best exposure time was 90 minutes by vitrification. ¹² In another report, persimmon dormant buds exposed to PVS2 for 10, 20, and 30 minutes obtained a survival rate higher than 60%. ¹³ 10, 20, and 30 minutes incubation in PVS2 attained a survival rate of higher than 58% in cryopreservation of dormant buds of R. pomifera.

PVS3 is also a kind of widely used plant vitrification solution and is recommended for samples sensitive to chemical toxicity and tolerant of osmotic stress, while its penetrative property generally necessitates a longer exposure time compared with PVS2.^27,95 Kami et al. demonstrated that a balanced composition of PVS A3 solution can be more beneficial for the recovery of cryopreserved samples compared with the original PVS2 solution, while PVS3 solution is recommended to use for samples sensitive to chemical toxicity and tolerant to osmotic stress. ⁹⁶ In mint shoot tips, PVS3 has shown lower toxicity to sensitive shoot tips than PVS2.^27,89 Bae et al. reported an average regrowth rate of 61.5% in 26 strawberry accessions cryopreserved using the PVS3-based DV protocol. ⁹⁷ In the cryopreservation of dormant pear buds, results indicated that PVS3 yielded the highest viability after cryopreservation compared with other cryoprotectants, including honey, PVS2, and PVS4. ⁹⁸

Thawing and unloading

When the germplasm is needed, thawing is first carried out, which is essential for all cryopreservation protocols.^27,90,99 Severe cellular damage often occurred during thawing, such as shrinkage and cytoplasmic melting, which were observed in peripheral cells of G. straminea Maxim. ⁷⁰ and C. pilosula (Franch.) Nannf. (unpublished) after thawing. Slow-cooled dormant twigs are typically thawed slowly at 0°C–4°C for 24 hours^36,42,100 or at 37°C for 3–4 minutes. ⁶⁸ In contrast, PVS-based vitrification of dormant buds uses rapid thawing in 37°C–40°C water baths to prevent recrystallization,^5,27,54,101 while DV often uses 20–30 minutes of room-temperature thawing.^5,27,102

In order to avoid the PVS toxicity to shoot tips post-thawing and to prevent uncontrolled rehydration of shoot tips, unloading is necessary.^5,27,103 Unloading is typically washing the explants in MS medium with 1.2 mol/L sucrose for 20 minutes. ⁵⁴ However, A. membranaceus ³⁸ and F. przewalskii (unpublished) dormant buds achieved high survival without unloading, which suggested dormant bud was a wonderful preservation material.