Over the past 35 years, encapsulation technologies have played an important role in the development and diversification of cryopreservation techniques for cells and organized structures from numerous plant species. Encapsulation with calcium alginate was initially developed as a technological approach for the production of synthetic seeds. However, the coating of biological material with a hydrogel matrix subsequently promoted new applications that provided additional benefits to in vitro multiplication and preservation techniques. Alginate coatings can be complemented with growth regulators, antioxidants, and nanoparticles. These compounds have helped mitigate the toxic effects of cryoprotectants by regulating their penetration rate and have enabled the implementation of drastic desiccation treatments that would have otherwise been lethal. Furthermore, encapsulation facilitates the simultaneous handling of large quantities of samples, simplifying the ongoing handling required. Cryopreservation protocols based on encapsulation include Encapsulation-Dehydration, Encapsulation-Vitrification, V- and D-cryoplate methods. The objective of this review is to provide information on the impact of encapsulation of plant material on the advancement of cryogenic procedures and, consequently, on the understanding of tolerance of plant tissues to cryopreservation. In view of the advantages of encapsulation, it is considered that it will facilitate the long-term conservation of a greater number of plant species.

The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were briefly pre-cultured for 1 day at 23 °C in the dark on a medium containing 0.3 M sucrose and then embedded in calcium alginate gel within the wells of the aluminum cryo-plates. In the V cryo-plate protocol, dehydration was carried out at room temperature using the following vitrification solutions: original plant vitrification solution 2 (PVS2) and 90% PVS2 solution (for 20 and 40 min) and plant vitrification solution 3 (PVS3) (for 60 and 80 min). In the D cryo-plate protocol, desiccation was performed for 2, 2.5, or 3 h over silica gel at 23 °C. The effect of different treatments was evaluated by monitoring the regrowth of both non-frozen and cryo-preserved explants. After cryo-preservation, five genotypes achieved regrowth rates over 40% in at least one of the applied protocols, while two genotypes showed regrowth rates of around 10%. A significant improvement in regrowth success for all genotypes using both cryo-plate methods was achieved by pre-culturing shoot tips for 7 days on a medium containing 0.5 M sucrose in complete darkness at 4 °C. Shoots regenerated from cryo-preserved explants were further monitored in vitro. By the third subculture, they had not only regained but had even exceeded the multiplication capacity (index of multiplication, length of axial, and lateral shoots) of shoots regenerated from dissection controls. Following multiplication, the cryo-preserved shoots were successfully rooted and rooting ability was assessed by monitoring the percentage of rooting, number and length of roots, and height of rooted plantlets.

Although pineapple (Ananas comosus var. comosus) shoot tips have been cryopreserved but the possible effect of this process at the molecular level has not been studied. This communication describes the growth (plant fresh and dry weights; stem height; leaf length, width and area; and stem base diameter) and the Inter Simple Sequence Repeat (ISSR) analysis of pineapple plantlets of A. comosus MD-2; Red Spanish Florencia; and Hybrid 54 (Smooth Cayenne/Red Spanish) after 45 d of acclimatization. From each of these varieties, the acclimatized plants were obtained from: (1) conventional micropropagation (control 1); (2) from shoot tips submitted to pre-cryostorage conditioning treatments but not exposed to liquid nitrogen (LN) (treatment 2); and (3) from shoot tips exposed to cryostorage including use of LN (treatment 3). The ISSR-PCR method was used to study the genetic stability. There were no statistically significant differences between treatments for the phenotype indicators evaluated. On average, 45 day-old pineapple plants had 0.5 g fresh weight; 1.85 g dry weight; 12.2 cm stem height; 9.1 cm leaf length; 1.6 cm leaf width; 7.1 cm2 leaf area; and 1.4 cm stem base diameter. Also, the potential effects of cryopreservation at the DNA level were not revealed with the eight ISSR markers used, as no polymorphic bands were recorded, which represents 100% genetic stability. As far as we know, this is the first publication on ISSR analysis of pineapple plantlets after cryopreservation.

Main conclusion This paper reviews the cryopreservation of the ornamental, carnation ( Dianthus caryophyllus L.), as an important method for the long-term preservation of this plant’s germplasm. Abstract Carnation ( Dianthus caryophyllus L.) is an important ornamental plant that is used as a potted plant as well as a cut flower. Important Dianthus germplasm would benefit from long-term strategies such as cryopreservation. Unlike the in vitro tissue culture literature of this ornamental, which has been studied in considerable detail, and with several genetic transformation protocols, surprisingly, the literature on its cryopreservation is still fairly scant, with barely two dozen or so studies, mostly having employed shoot tips. Early (< 2007) and more recent (2007–2020) cryopreservation techniques for carnation, including ultra-rapid cooling, encapsulation-vitrification, and encapsulation-dehydration, efficiently replaced programmed slow cooling processes used in early studies in the 1980s. Two large gaps (1997–2006, and 2016–2020) in which no carnation cryopreservation studies were published, requires future studies to cover new knowledge to fill gaps in information. Carnation cryopreservation research would benefit from testing a wide range of in vitro explants, new techniques such as the cryo-mesh, improved regeneration protocols for post-cryopreserved material, and the use of low-temperature storage as a mid- to long-term complementary germplasm storage strategy. This mini-review provides details of what has been achieved thus far and future objectives that could fortify cryopreservation research of this ornamental, as well as provide a robust long-term germplasm repository.

Adventitious root cultures of Tarenaya rosea were successfully cryopreserved using the encapsulation-vitrification technique. Histological analysis revealed useful information on the successive steps of cryopreservation. Coupled with complementary histochemical approaches, these studies provided cellular and tissue descriptions of T. rosea root cultures during cryopreservation and contributed to an understanding of cellular stress responses, as well as characterization of the anatomical pattern of root regeneration. The effects of exposure duration to PVS3 solution (0–120 min), unloading treatment (direct and gradual), and recovery medium (liquid and solid) on recovery of cryopreserved roots were investigated. The highest recovery (91%) after cooling in liquid nitrogen (LN) was reached with PVS3 treatment for 90 min, gradual rehydration in unloading solution, and recovery on solid MS medium. The cryopreserved roots showed high multiplication capacity, which was maintained for up to four subcultures. The effect of cryopreservation on root structure was investigated by histological and histochemical studies. Plasmolysis intensified during exposure to loading and PVS3 solutions, but decreased after unloading treatment. The proportion of intercellular spaces increased progressively throughout the cryopreservation protocol, culminating in root cortex disruption. Histochemical analyses revealed polysaccharides, proteins, and both lipidic and pectic substances in intercellular spaces. The vascular cylinder remained intact, ensuring the formation of new roots from the pericycle, showing that proliferative capacity of cryopreserved roots had not diminished.

Optimized cryopreservation protocols are essential for the safe, cost-effective and long-term conservation of germplasm of great economic interest, such as Guinea (Petiveria alliacea L.), a medicinal species that synthesizes a great diversity of bioactive substances, among them polysulfides with antitumor activity. Somatic embryos (SEs) produced from in vitro roots were cryopreserved using the V-cryoplate technique. Their viability was measured using the triphenyltetrazolium test and their recovery by counting the number of secondary SEs produced per cryopreserved SE 90 days after liquid nitrogen (LN) exposure. Structural alterations were evaluated qualitatively and quantitatively during the successive stages of the protocol. SEs were dehydrated in 0.5 M sucrose, attached to cryoplates using sodium alginate solution (3%) and treated with PVS2 for different periods. After immersion in LN, SEs were rewarmed in unloading solution (1.2 M sucrose) at room temperature (25 °C) for 20 min. Viability based on the triphenyltetrazolium test was 100% after 15 min of treatment with PVS2 and LN exposure, and recovery, based on multiplication rate, was 21 somatic embryos produced per cryopreserved somatic embryo after 90 days of culture. The histological analysis of cryopreserved somatic embryos showed plasmolysis in the different cell types after treatment with PVS2, with meristematic and parenchymatic cells presenting a higher plasmolysis level. However, after rewarming, the level of plasmolysis decreased over cultivation time, reaching only 2% in meristematic cells after 30 days, indicating the good ability of Petiveria alliacea SEs to develop cryotolerance under the experimental conditions tested.

Genetic improvements in plant breeding are dependent upon having access to novel plant genetic resources that are available in plant genebanks. Many crops that are vegetatively-propagated are maintained as plants in the field or greenhouse, making them vulnerable to biotic and abiotic threats. Increasingly, plant genebanks are using cryopreservation technologies to secure vegetatively propagated collections at secondary locations. Droplet vitrification and cryo-plate cryopreservation methods have been used to successfully cryopreserve the shoot tips of many plant species. New propagule types, including small leaf square-bearing adventitious buds, stem disc-bearing adventitious buds, microtubers and rhizome buds are alternative explants for use in cryopreservation. This review describes new technologies for in-vitro based cryopreservation systems that have advanced the field of plant cryopreservation. Future advances will allow even more diverse germplasm to be successfully preserved in cryobanks. New technologies for in-vitro based cryopreservation systems have advanced the field of plant cryopreservation since the twenty first century. Further advances will certainly facilitate even more diverse germplasm to be successfully preserved in cryobanks.

BACKGROUND: Few cryopreservation studies have been reported with the genus Cleome. Due to the use of C. spinosa in traditional medicine and its valuable pharmacological potential, the long-term conservation of the species will allow the safe maintenance of its germplasm. OBJECTIVE: This study compares two vitrification-based techniques on the cryopreservation of shoot tips of C. spinosa. MATERIALS AND METHODS: The effect of sucrose preculture and different vitrification solutions was evaluated using vitrification and V Cryo-plate techniques. The supplementation of recovery medium with BAP was also assessed. RESULTS: The V Cryo-plate proved to be the most efficient technique. Treatment of shoot tips with PVS2 at 0 degrees C resulted in a higher regeneration response after cryopreservation when compared to treatment with PVS2 and PVS3 at 25 degrees C. The highest survival (83.3%) and recovery (76.6%) were achieved for shoot tips exposed to PVS2 for 90 min at 0 degrees C and recovered on MS medium supplemented with 0.5 mg L-1 BAP for 2 weeks. CONCLUSION: Plants regenerated from cryopreserved shoot tips maintained their in vitro multiplication capacity and showed a normal phenotypic aspect, demonstrating the efficiency of the cryopreservation protocol.

Conservation of pineapple (Ananas comosus L. Merr.) genetic resources - including cryopreservation in liquid N-2 at -196 degrees C - is essential for future breeding programmes to develop new varieties with improved agronomic performance. However, the potentially deleterious effects of cryopreservation on subsequent plant regrowth should be evaluated before large-scale development of cryobanks is implemented. This paper describes the histological analysis of pineapple plantlets regenerated from cryopreserved shoot tips. Two controls were included in the study: i) conventional micropropagation-derived plantlets, and ii) plants from shoot tips subjected to pre-cryostorage conditioning treatments but never exposed to liquid N-2. Histological studies of roots, leaves and stems were conducted after 45 days of hardening. No statistically significant differences with the controls were observed in any of the histological parameters evaluated, which supports the practical value of cryopreservation of pineapple germplasm.

Passiflora suberosa L. is a wild species of Passiflora, with great agronomic, ornamental and medicinal potential. In spite of this, there are few biotechnological studies aiming at its in vitro propagation and conservation. Thus, the development of cryopreservation protocols is considered of great relevance for this species. However, cryopreservation may be associated with oxidative damages, which cause injuries and may result in low recovery frequencies. The goal of this work was the establishment of a cryopreservation protocol for P. suberosa shoot tips with the V-Cryo-plate technique, evaluating the influence of the age of the explant and exposure to the vitrification solutions PVS2 and PVS3. In addition, the occurrence of oxidative stress at the different stages of the protocol was evaluated by monitoring lipid peroxidation and the activity of antioxidant enzymes. Plant recovery from cryopreserved shoot tips occurred at distinct frequencies, according to explant age and exposure to the vitrification solutions. Highest post-freezing recovery was observed in 40-day old shoot tips treated with PVS2 for 60 min (45%) or PVS3 for 45 to 90 min (50–60%). The occurrence of oxidative stress was evaluated by the quantification of lipid peroxidation through malondialdehyde detection, total protein content, and activity of the antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase. These assays revealed that oxidative stress mainly occurred at the osmoprotection and PVS3 dehydration stages, which were considered as the most critical of the V-Cryo-plate protocol for the cryopreservation of P. suberosa shoot tips.

We recorded the crypreservation effects (direct immersion) on various parameters of early germination stages of maize seeds (0, 7 and 14 days). Percentages of germination; fresh mass of different seedling parts; levels of chlorophyll pigments (a, b); carotenoids; malondialdehyde; other aldehydes; phenolics (cell wall-linked, free) and proteins were determined. Various statistically significant effects of seed exposure to liquid nitrogen (LN) were recorded. Maize seeds did not seem to be affected by LN exposure either visually or regarding fresh weight or germination rate. However, delayed growth was observed in seedlings recovered from cryopreserved seeds. This trend indicated an increase in the effect of seed cryopreservation on growing plants. The most significant effects of LN exposure were recorded in the combined fresh weight of stems and leaves at day 7 of germination and in fresh weights of roots, stems and leaves at day 14. At the biochemical level, numerous indicators varied following LN exposure, but the most significant effects were recorded in carotenoids, malondialdehyde and other aldehyde contents. LN exposure modified 50.0% of indicators in cotyledons, 48.1% in stems and leaves, 38.8% in roots and 11.1% in seeds. LN storage modified 11.1% of the variables measured at day 0 of germination, 37.0% at day 7, and 52.7% at day 14. Field performance of cryostored seed-derived plants should be evaluated to measure the durability of the changes observed.

Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L−1 α-naphthalene acetic acid and 0.5 mg L−1 thidiazuron. Small leaf squares (SLSs, 3 × 4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.

BACKGROUND: A cryopreservation protocol has been established for oil palm somatic embryos (SEs), the efficiency of which must be evaluated, both in terms of regeneration and of long-term storage capacity, before its large-scale routine use.OBJECTIVE:To test the survival and recovery of 29 clones of oil palm somatic embryos cryostored for 20 years.MATERIALS AND METHODS:Clumps of SEs were pregrown for 7 days on medium containing 0.75 M sucrose, dehydrated in air-tight containers containing silica gel to moisture contents between 19-35% fresh weight, and then immersed directly in liquid nitrogen and stored in cryotanks for 20 years.RESULTS:Survival of SEs cryopreserved and rewarmed immediately displayed an average value of 19.1% for the 29 clones tested while survival of SEs rewarmed after 20 years of cryostorage was significantly higher, with an average of 33.2% for the 28 surviving clones. Out of these 28 surviving clones, three were lost due to contamination or regrowth decline, six produced only shoots and the rest proliferated.CONCLUSION:It is possible to cryostore oil palm SEs for extended periods and to regenerate proliferating cultures and plantlets from the cryopreserved material. The cryopreservation protocol established can thus be efficiently used to store oil palm germplasm and to manage large-scale production in industrial laboratories.

In this chapter, we describe a cryopreservation (liquid nitrogen, -196 degrees C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5 mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171 g/L sucrose for 3 days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20 min with a loading solution containing 184 g/L glycerol and 136.8 g/L sucrose. They were then dehydrated for 90-120 min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15 min in an unloading solution containing 410.4 g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3 days in the dark on posttreatment medium containing 102.6 g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.

Oil palm (Elaeis guineensis Jacq.), a tropical plant, is the leading source of edible oil. This review deals with the cryopreservation of oil palm as a way to preserve this important tropical germplasm. Somatic embryos have been the most popular source of material for cryopreservation as they are propagules that are effectively produced during micropropagation. In contrast, fewer studies exist on the cryopreservation of pollen, zygotic embryos, seeds, kernels and embryogenic cell suspensions. This review highlights the ideal protocols, in detail, in a bid to offer guidance for further advances in oil palm cryopreservation.

Many publications describe cryopreservation techniques but only a few studies have focused on the biochemical and physiological changes occurring in plants regenerated from seeds exposed to liquid nitrogen. This paper aims at describing the effect of common bean seed cryostorage on mineral nutrition of young plantlets. The following elements were measured on leaves of 10-day-old plantlets from non-cryopreserved and cryopreserved seeds: Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Se, Sr and Zn. At 10 days after sowing, both treatments (control and cryopreserved seeds) showed 100% seed germination without any visual phenotypic difference. However, contents of several elements in the leaves were different. Exposure of seeds to liquid nitrogen decreased Cu, Cd and Na uptake and increased absorption of B and Al. Further studies are required to understand the mechanisms underlying the relationship between seed exposure to liquid nitrogen and mineral nutrition during the early stages of plantlet growth.