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Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification
Abstract
Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions
on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival
of vitrified sweet potato shoot tips cooled to approximately –208 °C was increased by preculturing with 0.3 M sucrose for 24 h at 22 °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod.
The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22 °C followed by dehydration with PVS2 for 16 min at 22 °C. Rapid cooling was used and achieved by
the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed
shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise
for cryopreserving sweet potato shoot tips.
... Dr-vi allows samples to obtain the ultra-fast cooling and warming rates, which are much faster compared to the methods using capped vials, due to the direct contact of explants with LN and the ULS [79,139,140]. Following the PVS2-based Dr-vi method, sweet potato shoot tips were firstly cryopreserved by Pennycooke & Towill [141] with 62% recovery reported. In this method, shoot tips (0.5-1 mm) were excised from 4-to 8-week-old in vitro stock cultures and then incubated in a liquid MS medium containing 2% sucrose at 25 • C for 1 day. ...
... Shoot tips were precultured in liquid MS medium supplemented with 0.3 M sucrose for one day at 25 • C, then osmoprotected with LS (2 M glycerol + 0.4 M sucrose in MS medium) for 1 h min at 22 • C. Osmoprotected shoot tips were exposed to PVS2 for 16 min at 22 • C and then transferred to PVS2 droplets of about 10 µL, placed on sterile aluminum foil strips, and directly immersed in partially solidified nitrogen (about −208 • C). For warming, the aluminum foils containing the shoot tips were immersed, for 20 min, in ULS (1.2 M sucrose in MS medium) at 22 • C, and transferred to recovery medium [141]. Shoot formation without intermediate callus was observed in all surviving shoot tips [141], showing a significant progress as compared to the traditional PVS2-Vi method, in which almost all the surviving sweet potato shoot tips formed callus after cryopreservation [122]. ...
... For warming, the aluminum foils containing the shoot tips were immersed, for 20 min, in ULS (1.2 M sucrose in MS medium) at 22 • C, and transferred to recovery medium [141]. Shoot formation without intermediate callus was observed in all surviving shoot tips [141], showing a significant progress as compared to the traditional PVS2-Vi method, in which almost all the surviving sweet potato shoot tips formed callus after cryopreservation [122]. Although considerable effort has been made to establish the Dr-vi method for the cryopreservation of sweet potato collections [128,142], accessions react very differently to the cryopreservation protocol, i.e., some of the cultivars achieved a regeneration rate of 66% while others barely reached 2% [142]. ...
Root and tuber crops (RTCs) are the second-most important carbohydrate commodity after cereals. Many species of the RTCs are vegetatively propagated, making their shoot tips the preferred material to be conserved for future uses. Shoot tip cryopreservation provides an important tool to support the long-term conservation of plant genetic resources. Over the past four decades, significant efforts have been undertaken to move shoot tip cryopreservation of RTCs from research projects to full-scale implementation in cryobanks. This comprehensive review focuses on the history of cryopreservation protocols developed in RTCs. The encapsulation and vitrification solution-based cryopreservation techniques followed by ultra-rapid freezing and thawing have been highly successful. Additionally, different strategies for improving the cryotolerance of shoot tips have been introduced to further increase post-cryopreservation recovery. Finally, the research conducted to explain the mechanism underlying cryoprotection and differential cryotolerance including the use of histological studies are highlighted.
... Para evitar este problema, algunos autores sugieren la aplicación de una fase de precultivo con sacarosa o polialcoholes como el sorbitol que pueden mejorar significativamente la supervivencia de los explantes crioconservados tras la deshidratación con la solución de vitrificación [20,[25][26][27][28]. ...
... Los explantes precultivados en sacarosa se dividieron en cinco grupos y la vitrificación se llevó a cabo en crioviales de polipropileno de 12,5 x 92 mm mediante la aplicación de distintas soluciones crioprotectoras y vitrificadoras, previas a la inmersión en NL, siguiendo tres procedimientos distintos de acuerdo con [29] para el Grupo 2, el trabajo de [30] para el Grupo 3 y [28] para el Grupo 4 (tabla 1). Los grupos restantes sirvieron como controles e incluían aquellos explantes que solo habían estado sometidos a un 2 % de sacarosa durante 24 horas, sin precultivo adicional. ...
... susbsp. leucantha basados en los trabajos previos de Langis [29], Benson [30] y Pennycooke [28]. En todos los casos, el tiempo de exposición a la solución de carga fue de 20 minutos. ...
Sideritis leucantha Cav. subsp. leucantha is a Valencian endemic medicinal plant that faces some conservation constraints. Therefore, this work aimed to test a preconditioning treatment for the further cryopreservation of shoot tip explants of this species. The results showed that pre-culture in sucrose 0.3 M for 24 hours resulted in the highest explant survival and regeneration after three vitrification approaches. These findings point out that preconditioning of explants may enhance cryopreservation performance in tissue cultured plantlets of S. leucantha subps. leucantha
... Matsumoto et al. (1994) reported that the absence of preculture in 0.3 M sucrose results in a reduced recovery rate in wasabi, which is similar to the results reported for cassava (Charoensub et al., 1999). Even in sweet potato, there was no survival observed without preculture (Pennycooke and Towill, 2000). Sucrose preculture provides some protection against dehydration from PVS2 exposure, even when no cryoprotectant preculture is used (Pennycooke and Towill, 2001 ...
... According to Pennycooke and Towill (2001) (Benson, 2008;Pennycooke and Towill, 2000;Towill and Jarret, 1992). Direct exposure without osmoprotectant preculture did not yield viable shoot tips in sweet potato (Towill and Jarret, 1992). ...
... Direct exposure without osmoprotectant preculture did not yield viable shoot tips in sweet potato (Towill and Jarret, 1992). Results from several studies have indicated that exposure of less tolerant shoot tips directly to the vitrification solution result in mortal damage, due to osmotic stress and/or chemical toxicity (Pennycooke and Towill, 2000;Volk et al., 2006;Wang et al., 2005). Adequate preconditioning by cryoprotective loading could be applied to mitigate these harmful effects (Langis and Steponkus, 1990). ...
Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulation-vitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at 25^{\circ}C. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.
... Secondly, the cryopreservation protocol was tested on more cultivars than most previous studies. Some previous studies only used one 20,22 , two 17 or three 18,45 cultivars, which might result in biases when interpreting the results and overgeneralizations, since this report shows that the cultivar type is a very important parameter, with 83.9 and 9.5% regeneration rate of the best and worst cultivar respectively. However, there is a trend in using multiple cultivars (≥ 5) when reporting on cryopreservation protocols 21,46 . ...
... However, there is a trend in using multiple cultivars (≥ 5) when reporting on cryopreservation protocols 21,46 . Thirdly, the research makes use of a well-defined post-cryopreservation reaction categorization, which is a valuable addition to the survival rate or "shoot elongation" often used in earlier reports 20,46,47 . This distinction is important since we show that survival does not equal plantlet regeneration, sometimes it is not even correlated. ...
Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3-9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.
... Among various methods for cryopreservation, droplet-vitrification offers relatively simple handling and generally produces higher survival since ultra-rapid cooling and warming are facilitated within this protocol (Kim et al., 2007a). To date, it has been successfully applied to cryopreservation of various plant species, including chrysanthemum (Dendranthema grandiflora) (Halmagyi et al., 2004), potato (Solanum tuberosum) (Kim et al., 2006b), sweet potato (Ipomoea batatas) (Pennycooke and Towill, 2000) and apple (Malus domestica) (Li et al., 2015). ...
... Plant vitrification solution (PVS) can protect cells from lethal intracellular freezing that occurs during cooling and thawing (Sakai et al., 1990). Among different vitrification solutions, PVS2 has been shown to be effective at improving tolerance of cryopreservation in many plant species (Halmagyi et al., 2004;Leunufna and Keller, 2003;Panis et al., 2005;Pennycooke and Towill, 2000;Li et al., 2015;Wang et al., 2017). Among Allium species, garlic has been successfully cryopreserved using PVS2 vitrification procedure (Volk et al., 2004); however, PVS3-based procedure produced better regrowth (Makowska et al., 1999;Kim et al., 2006aKim et al., , 2012. ...
( Allium cepa var. aggregatum ) shoot tips by droplet-vitrification
February 2019Acta horticulturae
DOI: 10.17660/ActaHortic.2019.1234.32
Min Rui WangMin Rui WangZhibo ZhangZhibo ZhangS. HaugslienShow all 7 authorsDag-Ragnar BlystadDag-Ragnar Blystad
Research Interest
0.3
Citations
... The cell dehydration process of C. figlalii calli was started with LS treatment before the chemical vitrification with PVS2. Sometimes, sucrose preculture alone may not be adequate to obtain higher viability, and direct PVS2 exposure of sensitive cells such as callus cells may produce damage to the tissues due to the chemical toxicity and high osmotic stress (44). Thus, the use of LS supports the precondition of the cell before treatment with PVS2 (45). ...
BACKGROUND: The natural population of Colchicum figlalii (Varol) Parolly & Eren grows in a narrow area of serpentine rock clearings at an altitude of 1900-2100 m in Southwestern Anatolia (Sandras Mountain, Mugla, Turkey). The species is regarded as endangered according to the IUCN Red List Categories. OBJECTIVE: To develop an optimum procedure for in vitro propagation and cryopreservation of germplasm of this rare endemic. MATERIALS AND METHODS: A total of 281 bulbs were used as in vitro culture starting material and after surface sterilization, clean material was obtained from 157 of them. Woody Plant Medium (WPM), Olive Medium (OM), and Murashige and Skoog medium (MS) were used for in vitro culture establishment. RESULTS: The maximum regeneration rate (~67.3%) was obtained after four weeks of incubation on OM. The calli were successfully induced by using OM supplemented with 10.7 µM NAA from leaves of in vitro grown C. figlalii bulbs. A PVS2-vitrification procedure was used for cryopreservation of C. figlalii callus tissue. After cryo-storage, the best result for regeneration (66.7%) was obtained from calli treated with PVS2 for 75 min before plunging into liquid nitrogen. All rooted seedlings derived from cryopreserved calli were successfully acclimatized to greenhouse conditions. CONCLUSION: This study is an effective reference for future long-term conservation of similar species that are difficult to cryopreserve.
... Initially, this technique was successfully used in potato (Schäfer-Menuhr et al. 1996) and asparagus (Mix-Wagner et al. 2000), where cryoprotectant solution comprising 10% DMSO was placed on aluminium foil strips, followed by placing the explants on them, and plunged in LN. Later on, Pennycooke and Towill (2000) used PVS2-based droplet-freezing for cryopreservation of sweet potato shoot tips and subsequently, this method was further modified and optimized by Panis et al. (2005) in Musa, which led to the development of one of the most efficient techniques of cryopreservation called Droplet-vitrification (DV). The major difference between vitrification and droplet-vitrification is freezing rate/cooling rate. ...
Plant genetic diversity (PGD) plays a significant role in attaining food, nutrition and economic security. Conservation of these valuable resources is essential in the current scenario of climate change and sixth mass extinction. Since the early twentieth century, conservation of PGD has been carried out by using in situ and ex situ methods. Advances in biotechnology have opened new avenues in the fields of PGD management, with several tools applied in varied activities viz., collecting, quarantine, multiplication, conservation, characterization and evaluation, exchange and utilization. Since the past three decades, significant progress has been made in the use of biotechnology for conserving ‘difficult-to-conserve’ species such as clonally propagated crops, rare and endangered species and crop wild relatives. Several protocols have been developed for in vitro collecting, micropropagation, normal growth, slow growth and cryopreservation in varied species. The present chapter provides the current status of application of various biotechnological tools for the conservation of PGD.
... Cryopreservation is the most reliable, cost and space-efficient long-term storage method (Benson and Reed 2008;Kaviani and Kulus 2022). Vitrification relies on exposure of explants with a highly concentrated cryoprotectant solutions to dehydrate materials, and thus all extraneous water that would otherwise freeze is removed from the cells, and both intra-and extra-cellular vitrify while avoiding the growth of intracellular ice crystals when directly immersion in LN (Benson and Reed 2008;Lambardi et al. 2000;Pawłowska and Szewczyk-Taranek 2014;Pennycooke and Towill 2000). High regeneration rates (52.9 to 90.0%) were yielded from the shoot tips of Colocasia esculenta (L.) Schott (Takagi et al. 1997), Mint (Hirai and Sakai 1999), Penthorum chinense (Zilani et al. 2022), Musa and Ensete , and Oil palm (Gantait et al. 2015). ...
Cryopreservation, an effective protocol for long-term conservation, requires minimal maintenance and space. Genetic resource collection of Pinellia ternata (Thunb.) Breit. is essential for breeding and genetic diversity. The purpose of this study is to develop a vitrification cryopreservation protocol and virus-free technology of P. ternata. Shoot tips of dormant buds recovered from cryopreservation yield a regeneration rate of 75.0% and the main steps of the optimized protocol are as following: 0.8 mm-long shoot tips excised from in vitro dormant tubers subjected to cold-acclimation for 14 days, and then treated with LS for 20 min and incubated in plant vitrification solution (PVS2) for 40 min followed by plunging directly into liquid nitrogen. After rapid thawing at 38°C for 2 min, the shoot tips were unloaded in a 1.2 M sucrose solution for 20 min and then inoculated into 1/2MS medium (half-strength salts) supplement with KT 0.5 mg·L−1, NAA 0.2 mg·L−1, 3% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. No obvious phenotypic variation was observed. Inter-simple sequence repeat (ISSR) detected only 0.063% variation bands in 44 cryo-derived plantlets of P. ternata. By means of shoot tips cryotherapy or thermotherapy in vitro plantlets at 35℃ for 4 weeks followed by shoot tips culture for regenerated plants, SMV and CMV could be efficiently eradicated. Thus, cryotherapy would hold great promise as an effective approach for validly eradication of P. ternata viruses, and can be simultaneously applied to long-term collect P. ternata germplasm and produce virus-free plants.
... ] was 0.5-1.0 mm long (Pennycooke and Towill 2000). The survival rate of 1.5-3.0 ...
Germplasm protection of an endangered plant Fritillaria przewalskii Maxim. is important to preserve genetic diversity, to store material for breeding. In the current study, A vitrifcation method was developed for cryopreserving dormant buds of F. przewalskii . Bulblets collected at July were stored at 4℃ for 3-4 months cold acclimation, 2-5 mm dormant buds dissected from the bulblets were pre-cultured on 0.5M sucrose 1/2 MS medium for 3 days. Then the dormant buds were treated with loading solution for 20 min at room temperature, dehydrated with vitrification solution 2 (PVS2) for 60 min, and finally directly plunged into liquid nitrogen. After rapid warming in water at 38°C, the dormant buds were directly plated on recovery medium without unloading. The recovery rate reached up to 93%. Successfully vitrified dormant buds developed bulblets within 5 weeks without intermediary callus formation. Freezing-thawing steps caused severe damage to the buds axis whereas cells in shoot apical meristem and leaf primordium were still intact and normal. Loading and appropriate PVS2 treatment resulted in hyperosmotic pressure leading to progressively cell plasmolysis which is beneficial to cell alive suffered ultra-low temperature. According to the morphology and the RAPD profiles of regenerated plants, no variation was found. As an superior cryopreserved material, dormant buds can assist in a faster and efficient development of new protocols or even in the creation of easy-to-use procedures.
... It can be considered the first "generic" cryopreservation method for hydrated tissues as it combines droplet-freezing and vitrification procedures [81]. The first report related to the PVS2-based droplet-vitrification method was presented by Pennycooke and Towill [82] on cryopreservation of sweet potato shoot tips. Panis et al. [36] optimized the droplet-vitrification procedure in a study with shoot tips of banana (Musa spp.). ...
Horticultural crops comprise various economic species extending from fruits, nuts, vegetables, spices and condiments, ornamentals, aromatic, and medicinal plants. Ornamental and fruit plants are produced mainly for their nutritional and aesthetic values, respectively. Unfortunately, many tropical and subtropical species are in danger of extinction because of climate change and (a)biotic stresses. It is imperative to preserve the germplasms of these species for the present and future genetic improvement programs. Cryopreservation, i.e., maintenance of tissues at the ultralow temperature of liquid nitrogen, is a promising long-term preservation technique, alternative to seed or in vitro banks, which can be applied for both vegetatively and generatively (through seeds) propagated crops, including those with recalcitrant seeds. It is a technology of choice not only for the preservation of plant biodiversity but also for virus elimination in the proficient administration of large-scale micropropagation. The main advantages of cryopreservation are the lowering of in vitro culture expenditures, needed space, contamination risk, and operator errors. However, tropical species are temperature delicate and one of the foremost challenging issues is preconditioning treatments that stimulate physiological reactions to sufficiently enhance tolerance to dehydration and cryogenic procedures. In recent years, several cryopreservation methods based on encapsulation-vitrification, droplet-vitrification, the use of aluminum cryo-plates, and cryo-mesh have been established. Combined cryo-techniques, gene/DNA conservation, as well as studies on perceiving bio-molecular events and exploring the multistage process from the beginning to end of cryopreservation are receiving more emphasis. The development of cryobiomics delivers a conceptual framework to assess the significance of cell signaling mechanisms on cellular functions, the influence of cryoinjury factors on sample viability, and the implications for genetic stability following cryo-storage. The aim of this mini-review article is to provide a succinct synthesis of the developed cryogenic procedures and their use for the storage and exchange of genetic resources of tropical and subtropical horticultural crops, particularly fruit crops and ornamental plants under the threat of extinction.
... The potential of SA to induce regeneration of meristems via plant growth and cell division was confirmed, it has been associated with signaling and regulation of other hormones such as auxins, cytokinins, gibberellins, and abscisic acid (Zarghami Moghaddam et al., 2014). Cytokines have also been linked in the regeneration of shoots following a cryogenic process (Lambardi et al., 2000;Pennycooke & Towill, 2000;Wang et al., 2003). ...
Elicitation is the term used to describe the utilization of molecules that activate plant defense responses, generating increased resistance to biotic and abiotic stresses. Elicitors are defined as natural or synthetic substances that, when applied to plants in small concentrations, initiate or increase the synthesis of secondary compounds related to plant defense. Additionally, elicitors may play an important role in stimulating the biosynthetic pathways for the production of commercially important secondary compounds. In this sense, elicitation has been considered as a technological tool widely tested in order to maximize the production of bioactive compounds, both in medicinal plant species and species categorized as functional foods, bringing significant economic benefits to the pharmaceutical and therapeutic industry (including nutraceuticals). The elicitation can be applied in the culture of cells and tissues and in intact plants. Salicylic acid (SA) is classified as an abiotic elicitor that alters the expression of genes encoding key enzymes of secondary plant metabolism, significantly increasing the production of bioactive compounds such as essential oils, phenolic acids, flavonoids, tannins, alkaloids, tannins, among others. In this chapter we will address the theme “Elicitation of Plants with Salicylic Acid” and adopt the following sequence of topics: (a) definitions of elicitation and elicitors, (b) elicitation as a tool to increase the production of bioactive compounds in plants, (c) SA mechanisms of action as an eliciting molecule and (d) examples of studies about SA elicitation in medicinal plants.
... The potential of SA to induce regeneration of meristems via plant growth and cell division was confirmed, it has been associated with signaling and regulation of other hormones such as auxins, cytokinins, gibberellins, and abscisic acid (Zarghami Moghaddam et al., 2014). Cytokines have also been linked in the regeneration of shoots following a cryogenic process (Lambardi et al., 2000;Pennycooke & Towill, 2000;Wang et al., 2003). ...
Salicylic acid (SA) is an essential phytohormone that regulates plant growth, development, and defense during stress conditions. SA, in low amounts, participates in the coordination of physiological processes such as closure of stomata, uptake of nutrients, synthesis of chlorophyll pigments, protein synthesis, homeostasis of phytohormones, transpiration and photosynthesis in plants. It also plays an important signaling molecule in local and systemic disease resistance response of plants after pathogen attack. Similarly, SA-dependent signaling pathways regulate plant responses to abiotic stress factors altering antioxidant enzyme activities. Besides the vegetative development, SA induces flowering by increasing cell metabolic rate, increasing flower life, and retarding senescence. Although there are several studies on the effects of SA during vegetative development, few studies on the relation in reproductive organ development is available. Some of them concerns pollen viability and pollen tube growth. In a case study, different concentrations of SA (0.005, 0.025, 0.05 and 0.25 mM) improved the pollen germination in kiwifruit (Actinidia deliciosa). In recent years SA has been the focus of intensive research due to its physiological functions during growth-development and stress defense. The discovery of its targets and the understanding of its molecular mechanisms in developmental and physiological processes could help to clear the complex SA signaling network, confirming its critical role in plant growth, development, and defense. In the present chapter, we aimed to focus the role of SA during plant reproductive development under regular and/or stress conditions.
... The droplet vitrification (DV) technique is one of the modifications of the vitrification method. The aforementioned technique used a PVS2 solution and was developed for sweet potato shoot tips [19] and Musa germplasm conservation [20]. During DV, explants were frozen in a single droplet of vitrification solution, placed on small pieces of aluminium foils, which were immersed in LN. ...
Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.
... mm) are placed into a droplet of cryoprotectant and then quickly plunged into liquid nitrogen. Initially DMSO was used as a cryoprotectant, but later it was broadly replaced by PVS2 and its application led to a breakthrough of successful cryopreservation of many crops [2,9,10]. ...
Cryopreservation is currently the only method which allows long-term conservation of living clonal plant material in the vapor or liquid phase of nitrogen (at −140 to −196 °C) allowing tissue to be viable for decades or perhaps centuries. Specifically, for species with recalcitrant seeds or requiring constant vegetative propagation, it is the method of choice for the long-term conservation of its genetic resources. The protocol described here is a modification of a previously developed plant vitrification solution 2 (PVS2)—droplet vitrification method of potato shoot tips, adapted from Musa species. Utilizing this protocol, the International Potato Center (CIP) has successfully stored in the cryobank more than 3000 cultivated potato accessions, belonging to seven species and nine different taxa [16], originating principally from ten countries in South and Central America. As part of CIP’s quality management system, all vegetative material placed in cryo is routinely subsampled, thawed, and assessed to confirm that whole plantlets can be produced after storage in liquid nitrogen. Complete plant recovery rates of thawed shoot tips range from 20% to 100% (average rate: 60%). This chapter describes the complete set of steps from the routine procedure of cryopreserving potato shoot tips for long-term conservation.
... In these treatments, the explants were immersed in polypropylene vials containing 3 mL of a mixture of 1.29 M EG or 1.28 M DMSO for 60 min and then subjected to vitrification in a solution composed of 8.05 M EG, 0.82 M sorbitol, and 0.9 mM bovine serum albumin (BSA) for 15 min at room temperature (approximately 22°C). Treatments 8 to 11 were based on the study conducted by Pennycooke and Towill (2000) with sweet potato (Ipomoea batatas (L.) Lam.) and consisted of two exposure times of 30 and 60 min to 3 mL of LS (0.4 M sucrose plus 2 M GLY in MS liquid medium) in polypropylene vials. LS was then replaced by PVS2, and two exposures of 15 and 30 min were studied at room temperature (approximately 22°C). ...
Sideritis leucantha Cav. subsp. leucantha is a Spanish endemic medicinal plant that faces conservation problems. Therefore, this work is aimed to develop a protocol for the in vitro propagation and cryopreservation of shoot tip explants of this species. The morphogenic responses were recorded using Murashige and Skoog medium containing 6-benzyladenine, 2-isopentenyladenine, kinetin, or thidiazuron for multiplication and 1-naphthaleneacetic acid or indole-3-butyric acid for rooting. Maximal shoot proliferation was observed with medium containing 0.088 M sucrose plus 0.22 or 0.44 μM 6-benzyladenine. The longest shoots and greatest number of nodes per shoot were obtained from cultures incubated on medium with 0.44 μM 2-isopentenyladenine. Optimal rooting was achieved with 1-naphthaleneacetic acid at concentrations of 0.05 μM (highest percentage of rooted shoots) and 0.26 μM (highest number of roots). The morphological traits of the regenerated plantlets did not differ from those of wild-type plants, and these plantlets were successfully adapted to ex vitro conditions. After cryopreservation by vitrification, the highest percentage of regenerated shoot tips (70 ± 6%) was obtained with the treatment consisting of 60 min of loading solution (0.4 M sucrose plus 2 M glycerol in liquid medium) followed by exposure to plant vitrification solution 2 for 30 min at room temperature and then immersion in liquid nitrogen for 60 min. This study constitutes the first work on the micropropagation of S. leucantha subsp. leucantha and the first work on the cryopreservation of Sideritis species. The results obtained could be applied commercially and also in the conservation for other species of Lamiaceae.
... Since first reported for sweet potato shoot tips (Pennycooke and Towill 2000), the DV technique has been successfully applied to a large number of plant species owing to high post-thaw recovery and being adopted for long-term conservation of many crops in the cryobanks across the world (Panis 2019; Wang et al. 2020). However, there are no publications regarding its implementation in Gentiana spp. ...
Gentiana kurroo Royle, a critically endangered medicinal plant species of India is conserved as in vitro slow growing cultures in the In Vitro Genebank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Shoot tips (about 1 mm in length) excised from 8-weeks-old stock cultures subjected to cold hardening for four weeks at 8 °C (dark) were precultured on Murashige and Skoog (MS) medium supplemented with 5% Dimethylsulfoxide (DMSO) at 8 °C (dark) for two days. Thereafter, shoot tips were dehydrated with PVS2 solution (30 min) at 0 °C and cryopreserved using conventional vitrification (V) and droplet-vitrification (DV) techniques. Average post-thaw regeneration after 4 weeks was ~ 35% by V technique and ~ 60% by DV in all the three accessions tested. Genetic stability assessment on the basis of 40 Inter Simple Sequence Repeats and 30 Expressed Sequence Tagged-Simple Sequence Repeats markers revealed no variation between in vitro-cryopreserved plants and controls, in any of the genotypes. Thus, standardized DV method has the potential for long-term conservation of germplasm of G. kurroo and the three accessions are now safely cryobanked at ICAR-NBPGR.
... Droplet-vitrification methods combine the advantages of the rapid cooling rates obtained with droplet freezing along with vitrification solution properties that result in high levels of successfully cryopreserved shoot tips (Panis et al. 2005;Panis 2019;Sakai and Engelmann 2007;Sakai et al. 2008). Pennycooke and Towill (2000) were the first to report PVS2-based droplet-vitrification methods for sweet potato shoot tip cryopreservation and Panis et al. (2005) further optimized the droplet-vitrification procedure. In the study of Panis et al. (2005), shoot tips were excised from 4 week old in vitro grown banana stock shoots and treated at room temperature for 20 min with a loading solution composed of 0.4 M sucrose and 2 M glycerol in Murashige and Skoog (1962) medium (MS). ...
A droplet-vitrification protocol was described for cryopreservation of shoot tips of kiwifruit ‘Yuxiang’ (Actinidia chinensis var. deliciosa). No significant differences were found in root formation and shoot growth between the in vitro-derived shoots (the control) and cryo-derived ones when cultured in vitro. No significant differences were detected in survival and vegetative growth between the in vitro-derived plants (the control) and cryo-derived ones after re-establishment in greenhouse conditions. Inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) did not detect any polymorphic bands in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-establishment in greenhouse conditions. These data indicate rooting ability, vegetative growth and genetic stability are maintained in the cryo-derived kiwifruit plants recovered from the droplet-vitrification cryopreservation. Methylation sensitive amplification polymorphism (MSAP) detected 12.8% and 1.6% DNA methylation in the cryo-derived shoots when cultured in vitro and the cryo-derived plants after re-established in greenhouse conditions, respectively. This droplet-vitrification was applied to five cultivars and three rootstocks belonging to A. chinensis var. deliciosa, A. chinensis var. chinensis, A. macrosperma, A. polygama and A. valvata. The highest (68.3%) and lowest (22.5%) shoot regrowth were obtained in A. macrosperma and A. chinensis var. chinensis ‘Jinmi’, respectively, with an average of 46.4% shoot regrowth obtained across the eight genotypes. The droplet-vitrification protocol described here can be considered the most applicable cryopreservation method so far reported for the genus Actinidia. Results reported here provide theoretical and technical supports for setting up cryo-banks of genetic resources of Actinidia spp.
... Droplet-vitrification methods combine the advantages of the rapid cooling rates obtained with droplet freezing along with vitrification solution properties that result in high levels of successfully cryopreserved shoot tips (Panis et al. 2005;Panis 2019;Sakai and Engelmann 2007;Sakai et al. 2008). Pennycooke and Towill (2000) were the first to report PVS2-based droplet-vitrification methods for sweet potato shoot tip cryopreservation and Panis et al. (2005) further optimized the droplet-vitrification procedure. In the study of Panis et al. (2005), shoot tips were excised from 4 week old in vitro grown banana stock shoots and treated at room temperature for 20 min with a loading solution composed of 0.4 M sucrose and 2 M glycerol in Murashige and Skoog (1962) medium (MS). ...
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.
... To our best knowledge, these results represent the first time that 2-butanol and 2,3-butanediol have been found in mangrove plants. Alcohols have been proved to lower freezing point [39], the possible mechanisms of which include: (i) a viscosity increase, retarding the diffusion of water during freezing and creating smaller crystals; (ii) "binding" of water, reducing the total amount of ice formed; and (iii) reduction in crystal growth rate, permitting the nucleation of additional crystals with an ultimate smaller size [40]. So, the results suggest that 2-butanol and 2,3-butanediol are specific substances produced by mangrove under low-temperature stress. ...
In this study, mangrove seedlings Kandelia obovata were firstly introduced to Zhoushan in Eastern China at 29° 93′ N from Xuwen in South China at 20° 34′ N in April 2016. In order to analyze ecophysiological differences of K. obovata seedlings domesticated in Zhoushan, the growth status and antioxidant system of K. obovata exposed to natural low temperature were studied through situ measurements. The results showed that K. obovata seedlings introduced artificially to Zhoushan grew slowly when subjected to natural cold stress. The chlorophyll contents exhibited a decreased tendency. In addition, 2-butanol and 2,3-butanediol were firstly found in K. obovata after being moved to Zhoushan, which are specific substances produced by K. obovata under low-temperature stress. Moreover, there was a synergistic competition mechanism in the antioxidant enzyme system in K. obovata, in which superoxide dismutase (SOD) would convert oxygen radicals to hydrogen peroxide, and then catalase (CAT) and peroxidase (POD) could work together to remove hydrogen peroxide. This study provides a foundation for better understanding of the response of mangroves to natural low temperature at high latitudes.
... In particular, SA exhibited the potential to induce regeneration of meristems via its ability to increase plant growth and cell division, as well as via its ability to signal and regulate other hormones such as auxins, cytokinins, gibberellins, and abscisic acid (Zarghami Moghaddam et al. 2014). Cytokines have also been hypothesized to play an important role in the regeneration of shoots following a cryogenic process (Lambardi and Caccavale 2000;Pennycooke and Towill 2000;Wang et al. 2003a, b). It has been demonstrated that the exposure of meristems to ultra-low temperatures (e.g., −196°C) facilitates the elimination of viruses, since many viruses are not able to infect meristematic cells (Martin and Postman 1999). ...
Potato virus S (PVS) is one of the most difficult viruses to eliminate by thermotherapy due to its thermal deactivation point. Pre-treatment methods involving salicylic acid (SA) have been successfully carried in conjugation with cryogenic methods to reduce oxidative damage and eliminate viruses. In the present investigation, the effect of SA to protect Solanum tuberosum plants infected with PVS from oxidative damage from subsequent cryotherapy was studied. Vulnerable genotypes to cryogenic protocol were selected, with two SA treatments examined. Potato clones were pretreated with SA (0, 10⁻⁵, and 10⁻⁶ M), plant development was evaluated and then exposed to cryotherapy. This was followed up by a plant development evaluation and virus testing. After the initial treatment with SA, the plants exhibited an increase in the variables evaluated before cryotherapy. After cryotherapy, between 66.6% and 100% of the treated plants were found to be virus-free compared to control plants which exhibited 0% survival. Thus, the SA-cryotherapy treatment combination described appears to enhance plant survival and eliminate PVS from potato plants.
... Glycerol is considered a very efficient cryoprotectant as it imparts better membrane stabilization due to its small size and stereochemically oriented OH groups along one side of the molecule that resulted in better hydrogen bonding qualities with membrane phospholipids (Turner et al., 2001). Preculture treatment may not be sufficient to produce higher survival and direct exposure of less tolerant cells and shoot tips to PVS2 could be harmful to the cells due to osmotic stress and chemical toxicity (Pennycooke & Towill, 2000). Thus, loading is a step where the cells are preconditioned with cryoprotective solution before exposure to PVS2 (Langis & Steponkus, 1990;Sakai & Engelmann, 2007). ...
A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 h gave significantly higher survival (75%) compared to 24 h preculture (50%) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83%) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50% PVS2 followed by 10 min with 100% PVS2 solution improved survival of the shoot tips after cryopreservation (41.67%). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67%) than MS with 1.2 M sucrose (25%) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g⁻¹ dw for fresh shoot tips down to 2.93 ± 0.28 g g⁻¹ dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana.
... The survival rate for the cryopreservation method of vegetatively propagated plants is high. Some species such as Allium (Pandey et al., 1992), yam (Mandal et al., 1996), and potato (Pennycooke and Towill, 2000) have a 100% survival rate in this method. The meristematic zone of apices, from which the growth derives, constitutes a homogenous population of small and actively dividing cells, with a high nucleocytoplasmic ratio and little vacuoles. ...
Never before in history has the issue of preserving biodiversity been of such significance than today, to the extent that it has become an important global issue. Nowadays, humans have a massive impact on biodiversity whether in rural, urban, or wilderness settings. Besides, biodiversity today is also being significantly constrained by climate change and the introduction of new varieties of plants. Plant tissue culture is a method that can be useful for collecting, storing, and multiplicating of plant germplasms. Nowadays, many valuable germplasms are in danger of extinction, especially in Iran. Thus, there is a necessity to preserve valuable Iranian ornamental geophytes such as Oxalis articulata, Eminium jaegeri, Muscari kurdicum, Leopoldia tijtijensis, Gagea calcicola, Tulipa faribae, Gagea alexii, and Allium. In this study, plant tissue culture as well as in vitro conservation techniques as means for medium- and long-term conservation of Iranian ornamental geophytes is reviewed. There are only few studies on plant tissue culture of Iranian ornamental geophytes despite their great importance in Iran. To our knowledge, there is no report on cryopreservation of the abovementioned plants. In conclusion, the methods presented in this review can be utilized for conserving these valuable germplasms for future generations. © 2018, Institute of Bioorganic Chemistry. All rights reserved.
... (Schäfer-Menuhr et al. 1997;Panta et al. 2014), sweet potato(Pennycooke and Towill 2000), mint(Towill and Bonnart 2003), Musa spp.(Panis et al. 2005), Malus(Halmagyi et al. 2010), and Limonium serotinum(Barraco et al. 2011). ...
Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L⁻¹ 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L⁻¹ TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.
... In the droplet method, in order to make a plant sample cool quickly, Wesley-Smith et al. [54] used not liquid nitrogen but a slush nitrogen (-210ºC) and an isopentane (-160ºC). In addition, the droplet method can reportedly obtain a high regrowth percentage after cryopreservation in tropical plants difficult to cryopreserve [38,[55][56][57][58]. In the droplet vitrification protocol, shoot tips are loaded, treated with the PVS2 vitrification solution, put individually in 5-10 µl droplets of PVS2 placed on a piece of aluminium foil, which is then immersed in LN. ...
Plant diseases are caused by microscopic pathogens including viruses, viroids, phytoplasmas, bacteria, and fungi. Pathogenic microorganisms are difficult to diagnose and treat as a consequence of their small size and thus rely on technologically advanced methodologies for recognition of chemicals, proteins or molecular fragments specific to different organisms. Cryotherapy is an coming out method stemming from the use of cryopreservation, a cold treatment used for the long-term storage and preservation of all plant materials. It has recently been accepted that cryotherapy is an effective method for elimination viruses from plant shoot tips. This review covers different one step-freezing methods based on vitrification of cryotheraphy for virus eliminations from plants. In addition, the advantages and disadvantages as well as future prospects of each methods have also been discussed.
... However, limitations of ex situ conservation include large space and specialized facilities needed, considerable labor, and limited number of species. Field gene banks are also subjected to environmental stresses, climate change, and various plant diseases (Pennycooke and Towill 2000;Matsumoto et al. 2001). ...
The Orchidaceae is one of the largest and most diversified families of flowering plants including over 880 genera and 25,000 species distributed worldwide, with the largest diversity occurring in the tropics. The Royal Horticultural Society reports that more than 110,000 hybrids have been generated over the last 150 years. Their ornamental appeal and medicinal value place them among the most popular plants worldwide. Orchid biotechnology is a complex subject that encompasses a wide array of technologies and topics, including genomics, gene technology, gene sequencing, genetic transformation, phylogeny, germplasm conservation, cryopreservation, disease control, and postharvest technology, among other basic aspects. This chapter provides an overview of the current biotechnology methods as applied to orchid production and conservation, including advances and the latest technologies available. It focuses on in vitro propagation, bioreactor, synthetic seed, and cryopreservation technologies.
... This technique generally produces higher survival than droplet-freezing or vitrification alone, since ultrarapid cooling and warming are facilitated within this protocol. Droplet-vitrification has been successfully applied to in vitro grown shoot tips of various plant species (1,4,10,13,16,22,23) and to field grown garlic bulbil primordia (12). ...
The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest regrowth of cryopreserved primordia (82.7-85.0 %) was achieved after preculture for 2-4 days at 10°C on solid medium with 0.1-0.3 M sucrose, loading for 50 min in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 solution for 90-150 min, cooling primordia in 5 μl droplets of PVS3 placed on aluminum foil strips, warming them by plunging the foil strips into 40°C, 0.8M sucrose solution. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regrowth percentages ranging between 77.4-95.4 %. This protocol was further applied to the Korean garlic collection for safety back up of germplasm conservation. A total of 351 accessions were tested (with regrowth of 75.6-84.7%) and, among them, 260 accessions of three clonal Allium species were stored in liquid nitrogen.
Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.
Plant genetic resources are the basic requirement for crop improvement program. Many species are lost due to climate change and biotic and abiotic stresses. Horticultural crops comprise diverse economic species ranging from the fruits/nuts, vegetables, spices and condiments, ornamentals, aromatic and medicinal plants. It is utmost important to preserve the germplasms for a long period for genetic improvement program. Cryopreservation is an alternative technique for the long-term preservation of the germplasm either vegetatively propagated or seeds. In recent years, some cryopreservation methods based on encapsulation vitrification, droplet vitrification, the use of aluminum cryo-plates (D and V types), and cryo-mesh have been developed. In recent time, combined techniques, gene/DNA conservation, genetic stability of cryopreserved species are given more emphasized. This chapter is to highlight different cryopreservation techniques and their use for the storage and exchange of genetic sources of tropical and subtropical horticulture crops especially fruits and ornamental plants under the threat of extinction.
Orchid micropropagation is an indispensable tool for seed and clonal propagation, especially for commercial purposes. It has been interestingly researched and used for enormous species and hybrids. In contrary, many wild orchid species are in endangered and extinction because of deforestation and natural disaster. Only outstanding horticultural characteristic orchids are cultivated. Therefore, orchid conservation is urgently needed by various means, such as living collection, seed and pollen storage, in vitro conservation, and cryopreservation for small aseptic materials, such as protocorms, protocorm-like bodies (PLBs), buds, root tips, meristem, callus, and cell suspension. In this review, orchid cryopreservation which is a long-term storage with genetic stability is focused for the development and successful use.
Potato seed production requires virus-free plants. Potato virus X (PVX) is normally cleaned by thermotherapy, however, potato virus S (PVS) is one of the most difficult viruses to clean by thermotherapy. Cryotherapy is an optional technique to eliminate difficult viruses by traditional techniques. Both therapies induce oxidative damage leading to low survival. In the present investigation, the effect of SA to protect Solanum tuberosum plants infected with PVX or PVS from damage due to thermotherapy or cryotherapy was studied. Previously to therapies, nodal explants from plants PVX or PVS positive were sub-cultured for 30 d on MS medium containing SA 10⁻⁵ or 10⁻⁶ M. Thermotherapy: Plants PVX positive were subcultured for a supplementary 30 d period on MS SA-free, followed by thermotherapy (32–42 °C) for 35 d. Results showed SA induced thermotolerance during thermotherapy. PVX-free plants obtained in SA were significantly higher than in the control. SA increased CAT activity and reduced the H2O2 content. Cryotherapy: Plants PVS positive, following cryotherapy, plant development and virus testing were carried out. Between 66.6% and 100% of the treated plants were virus-free compared to control plants which exhibited 0% survival. Thus, SA treatment in combination with thermotherapy or cryotherapy described, enhanced plant survival and virus cleaning in potato plants.
Today, in vitro culture is a strategic tool to support medium and long-term conservation of plant genetic resources by using the slow growth storage of shoot cultures and the cryopreservation of organs and tissues. Over the last 30 years, considerable progresses were made in the development of both techniques that are nowadays considered as ex situ conservation strategies complementary to traditional seed banks and in-field clonal collections. Efficient protocols were developed for the conservation of a large number of crops, including important vegetables of the temperate environment (garlic, artichoke, asparagus, mint, potato, sweet potato, tomato, red chicory, thyme). Conservation in slow growth storage consists in modifying the medium and/or culture conditions to reduce the growth of plant material without affecting the viability and regrowth potential of shoots when moved back to standard culture conditions. The technique allows medium-term crops conservation, with a storage time of vegetables ranging from a few months to two years and more, without recurring subculturing typical of micropropagation. Cryopreservation preserves plant organs and tissues at ultra-low temperature, as liquid nitrogen temperature (-196°C). Currently, various techniques are available, based on cold tolerance induction by cell cytoplasm vitrification during the fast ultra-freezing through in liquid nitrogen immersion of explants. The term “vitrification” refers to the solidification of a liquid without crystallization. If induced in plant cells, it avoids the formation of lethal intra-cellular ice crystals during the ultra-freezing process, keeping tissues at stopped metabolism condition but vital. Working with vegetables, the techniques based on direct immersion of specimens in liquid nitrogen (“one-step freezing”, such as the “droplet-method”, the “PVS2 vitrification”, the “encapsulation-based” and the “cryo plate-based” procedures) are the most used. Further, the “two-step freezing”, i.e. the slow explants cooling before immersion in liquid nitrogen, still finds some applications. The experimental activity mainly focused on three economically important species, i.e., Allium sp., potato and sweet potato. Currently, almost 38,000 accessions of garlic, cassava, mint, potato, sweet potato, taro, yam are maintained in 17 genetic resources conservation centers, located in 12 Countries and 5 Continents (Europe, Asia, Africa, North and South America). Approximately 4/5 of these accessions are maintained in vitro by means of slow growth storage of shoot cultures, but more recent cryopreservation is constantly growing, and over 7,500 accessions from vegetables are stored at -196 °C. The germplasm of potato (Solanum sp.) is by far the most collected in the world, with almost 17,700 accessions presently maintained in in vitro banks and cryobanks.
The Food and Agriculture Organization predicts that the global population
will reach 9.2 billion by 2050, and there will be high demand for a large
quantity of foods. With limited farming land and freshwater resources,
traditional systems cannot cater to the increasing demand. Adaptation of
modern technology is one of the alternatives that can be used by the
farmers to increase their production in a sustainable manner. However, the
poor knowledge on the modern technology is one of the limitations for
technology adoption. Therefore, the main purpose of this chapter is to
introduce modern technologies that can be used in the farming to improve
the productivity and sustainability of the agricultural production.
Technologies developed in the last century have created to huge
improvement in land preparation, crop management, harvesting and
breeding. Modern technologies like nano technology, sensor technology,
molecular markers, remote sensing, wireless sensor networks, geographic
information system (GIS), Global Positioning System (GPS), drones,
photovoltaic technology, and information and communication technology
etc are discussed in this chapter.
Today, application of in vitro culture by means of slow growth storage of shoot cultures
and cryopreservation of organs, tissues and cells in liquid nitrogen presents a remarkable
strategic tool to support medium- and long-term conservation of plant genetic resources.
Over the last 30 years, considerable progresses have been made in the development of both
methods that are currently considered as ex situ conservation strategies, complementary to
traditional seed banks and in-field clonal collections. Efficient protocols were developed
for the conservation of a large number of crops, including strategically-important vegetables,
such as garlic, artichoke, asparagus, cassava, Jerusalem artichoke, mint, potato,
sweet potato, chicory, taro, thyme and yam. As a consequence, more than 45,000 accessions
of vegetable crops are maintained in 22 genetic resources conservation centers
(biobanks), located in 16 countries and 6 continents (Europe, Asia, Africa, Oceania, North
and South America). Approximately 4/5 of these accessions are maintained in vitro by
means of slow growth storage of shoot cultures, but cryopreservation is also constantly
growing, with almost 8300 vegetable accessions being stored in liquid nitrogen at
- 196 �C.
In vitro conservation of Ipomoea batatas (L.) Lam allows the exchange of germplasm and its availability for breeding programs. The objective of this work was to determine the effect of mannitol and abscisic acid in the conservation through minimum growth of I. batatas cultivars from INIVIT Germplasm Bank. Treatments included abscisic acid (ABA) (5 and 10 mg l-1) and mannitol (1.0, 1.5 and 2.0%) in a basal MS culture medium, the combination of ABA (10 mg l-1) and mannitol (1.0, 1.5 and 2.0%). As controls were used the MS basal culture medium and MS with sorbitol (1.0%) and glucose (1.0%). In the cultivars 'Cautillo' and 'INIVIT BS 16-2006' the best results of survival, growth decrease and green but small leaves were obtained in the treatments that contained the basal culture medium with mannitol (10, 15 and 20%) . Growth of the explants was not achieved in culture media with abscisic acid. The MS culture medium with 2 mg l-1 of thiamine, 100 mg l-1 of myo-inositol and 1.0% of mannitol, allows the in vitro conservation of sweet potato cultivars between six and eight months.
ذخيره ژنتيكي گياهان دارويي با توجه به كاربرد هاي روز افزون آنها در دارو سازي و مواد آرايشي و بهداشتي يكي از سرمايه
هاي هر كشور محسوب مي شود. بيوتكنولوژي با توسعه روش هاي آزمايشگاهي براي حفظ ژرم پلاسم كمك شاياني به حفظ
براي نگهداري مواد در شرايط انجماد را بوجود (Cryopreservation) ذخاير ژنتيكي نموده و رشته اي به نام حفاظت انجمادي
مي باشد . در Lamiaceae گياهي دارويي متعلق به تيره نعناع و خانواده (thymus officinalis L.) آورده است. گياه آويشن
اين آزمايش براي اولين بار حفاظت انجمادي بر روي اين گونه انجام مي شود. در اين آزمايش گياهچه هاي بذري با عمر 3 روز
و يك محلول تغيير يافته PVS و 3 PVS از جوانه زني با دو محلول شناخته شده در حفاظت انجمادي گياهان به نام هاي 2
120 ،90 ،60 دقيقه ، مورد تيمار قرار گرفتند. زمان قرار گرفتن گياهچه ها در محلول هاي حفاظتي با چهار سطح زماني 30
مورد بررسي قرار گرفتند. نمونه ها پس از تيمار در نيتروژن مايع به مدت 18 ساعت باقي ماندند و سپس گياهچه ها بازيابي و
كشت شدند. اين آزمايش در قالب طرح كاملا تصادفي و با سه تكرار بصورت (MS) در محيط كشت موراشيك و اسگوك
فاكتوريل انجام شد. نتايج حاصل از تجزيه واريانس داده هاي آزمايش نشان داد نوع محلول هاي حفاظتي و زمان تيمار، از
نظر ميزان زنده ماندن گياهچه ها داراي تاثير معني داري در سطح 0.01 بوده است. اثرات متقابل در اين آزمايش معني دار
داراي بيشترين ميانگين و داراي تفاوت معني PVS نبود. مقايسه ميانگين ها به روش چند دامنه اي دانكن نشان داد محلول 2
داري با ساير محلول ها در سطح 0,05 بوده است. در مقايسه ميانگين زمان تيمار با محلول ها نيز تيمار 120 دقيقه داراي
بيشترين ميانگين و داراي تفاوت معني دار با ساير زمان ها در سطح 0,05 بود .
This issue of In Vitro Cellular and Developmental Biology—Plant is dedicated to current developments in liquid-nitrogen cryopreservation methods and their use in plant biology and germplasm preservation. The development of cryopreservation for storage of plant cells, tissues, and organs began in the 1960s and continues to this day. Long-term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains an important requirement for many scientists, organizations, and companies. The continued development of cryopreservation techniques and their application to new plants is the subject of this issue.
Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.
In an attempt to evaluate the current research status of genetically modified (GM) plants, the scientific research publications in Korea as well as in international SCI journals were screened. About 190 research articles related to the development of GM plants were searched from 10 different domestic journals in the last 12 years (Jan. 1990 to Sept. 2002), The researches in 65 articles were carried out with tobacco plant, 20 with rice, 19 with potatoes, and less then 9 articles from each other plant species, respectively, In total, 38 different plant species were being subjected for the development of GM plants. In particular, there was only one article for each major staple grains such as wheat, barley, soybean, and maize. In more than 47% of total published articles, scientists mainly focused on the basic research such as developing transformation system (46 articles), gene expression study in transgenic plants (34), and vector constructions (10). In addition, 28 articles which main authors are Korean scientists were searched from 11 different international SCI journals. Again, major plants for GM research were tobacco (10) and rice (7). More than 50% of published articles were focused on the basic research, gene expression study with transgenic plants (16). The publications on the research of disease-resistant plants were 7 articles, 3 for the development of stress-resistant and 2 for the herbicide-resistant plants, respectively. It is believed that the last 10 year`s investment through government organizations has just strengthen the capacity for the next big stride on agricultural biotechnology in Korea.
Cryopreservation of embryogenic callus derived from apical meristem culture was attempted by slow prefreezing method (two-step method) with various cryoprotectants in sweetpotato cv. `Yulmi` Precultured embryogenic calli on medium containing 10 mg/L ABA prior to slow prefreezing in liquid nitrogen indicated higher survival rate than 1.0 mg/L ABA preteatment. The cryoprotectant comprising 1.28 M DMSO in 0.4 M sucrose solution gave the best survival (over 46%) of sweetpotato cells exposed to liquid nitrogen as determined by TTC reduction and FDA staining method. Cryopreserved calli cultured on MS medium with 1.0 mg/L 2,4-D were grown for 4 weeks in the dark and induced embryos after another 4 weeks. They were subcultured on MS medium supplemented with 0.1 mg/L 2,4-D?0.1 mg/L kinetin for 2 weeks and regenerated into normal plantlets in MS basal medium.
Plant tissue culture is a significant contribution in micropropagation of ornamental and forest trees, production of pharmaceutically interesting compounds and plant breeding for improved nutritional value of staple crop plants as well as in the improvement of tree species. Plant tissue culture can provide high-quality planting material for the fruits, vegetables and ornamental plants and forest tree species throughout the year, irrespective of season and weather, thus opening new opportunities to producers, farmers and nursery owners. The biotechnological approaches like haploid induction, somaclonal variation, etc. to improve traits are also its important applications. Plant tissue culture is a noble approach to be used in bioproduction, bioconversion or biotransformation of the valuable secondary products for large-scale production and biosynthetic studies.
Plant tissue culture has been routinely done for conservation of germplasm to be used for improving secondary metabolite production and agronomic traits of crops to increase yields. Production of artificial seeds has unravelled new vistas in in vitro plant biotechnology, such as large-scale clonal propagation, delivery of clonal plantlets, germplasm conservation and breeding of plants in which propagation through normal seeds is not possible.
Background:
The increasing demand for hypericins and hyperforins, the unique pharmaceuticals found in the Hypericum genus, requires the development of effective tools for long-term storage of cells and tissues with unique biochemical profiles.
Objective:
To determine the temperature of crystallization (TC) and of ice formation of 14 cryoprotectant mixtures (CMs) for their use in cryoprotection of H. perforatum L. cell suspensions and to evaluate the impact of the lowest Tc on post-cryogenic recovery.
Materials and methods:
TC was determined by real-time microscopy of ice formation during slow cooling to -196 degree C and heating to 20 degree C.
Results:
Exposure of cells to CMs CM2 (PVS3) containing sucrose and glycerol or CM12 and CM13 containing sucrose, glycerol, dimethylsulfoxide and ethylene glycol decreased TC below -60 degree C, prevented intracellular crystallization and considerably reduced both the size of crystals and the rate of extracellular ice propagation.
Conclusion:
The selected CMs proved suitable for cryopreservation of H. perforatum cell suspensions with the maximum of 58 % post-thaw recovery.
Tropical apical meristems excised from in vitro-grown plants which were sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2, 7.8 M) survived subsequent plunging into liquid nitrogen (LN) and regenerated plants (recovery growth 80%). Excised meristems of cassava (Manihot esculenta Grantz) were precultured with 0.3 M sucrose for 16 hr and then enhanced for tolerance to PVS2 with a mixture of 2 M glycerol and 0.4 M sucrose (LS) for 20 min at 25 °C. These osmoprotected apices were then sufficiently dehydrated with PVS2, so that the cytosolic concentration required for vitrification was attained upon rapid cooling into LN. Vitrification refers to a phase transition from a liquid into amorphous glass, while avoiding crystallization. In the vitrification protocol, enhancing tolerance to PVS2 and the mitigation of injurious effects during dehydration were crucial for ensuring the survivals.
The osmotic dehydration by PVS2 which is enable ceHs and tissues to survive at 196°C by vitrification is significantly advantaged over the freeze-induced dehydration in dehydrating cystosol more effectively, uniformly, speedy and less injuriously at non-freezing temperature, even in tropical plants .
Somatic embryogenesis is as an excellent technology for potential use in plant mass production, germplasm conservation, or genetic engineering. We examined the effect of cold storage using 3 embryogenic callus lines with different levels of embryogenesis competence derived from immature zygotic embryo cultures of Kalopanax setemlobus. Somatic embryo induction, germination and plant conversion were evaluated after 1, 3 and 6 months storage at 4°C in the dark. Most cold-stored embryogenic calli formed somatic embryos normally even after 6 months; however, the induction rate was gradually decreased by increasing the storage period. The most competent line tended to show a slight decline in somatic embryo induction rate, as compared with other lines after cold storage. In general, cold storage resulted in reduced somatic embryo germination and plant regeneration, although 93% somatic embryo germination and 91% plant conversion were achieved regardless of the storage period. Cold storage led to cell browning and degradation. Additionally, the cell structures were confirmed by the aceto-carmine and evans blue dye evaluation. Collectively, our results showed that embryogenic callus of K. Septemlobus could be preserved at 4°C without subculture for 6 months, and suggested the need for storage of relatively more competent embryogenic calli lines to support somatic embryo induction.
Cryopreservation of tissue-cultured plant, cells, meristems and organ is an important method for longterm storage of plant genetic resources using a minimum of space and maintenance. In this work, shoot tips from two Egyptian cultivars (Black Matrouh and Bez El-Anza) obtained from greenhouse were successfully cryopreserved by two step vitrification protocol. Shoot tips were excised cultured on 1/2 MS +0.16 mM glutathione +0.14 mM ascorbic acid in addition to different concentration 0.1M, 0.2M and 0.3M of sucrose and then treated with a mixture of glycerol and sucrose for 20 min at 25°C. They were then dehydrated with mixture of 1/2X PVS2 and PVS2 for 150-20 min. then plunged into LN for 1 hr. Samples were then warmed rapidly in water at 40°C. Precultured shoot tips with high sucrose concentrations to form Osmo-protected shoot tips were showed using 0.3M sucrose that gave the highest recovery before plunging into LN. After pluning into liquid nitrogen the recovery percentage were ranged 12-10 for Black Matrouh and Bez El-Anza, respectively. The recovery of the shoot tips amounted to approximately 40-46% after dehydration with PVS2 and plunging in liquid nitrogen. This method incorporates a simple and reliable approach for providing high levels of growth recovery. It also appears promising for the cryopreservation of Vitis germplasm.
The effect of shoot tip pre-culture on media with varying sucrose levels (0.0-0.6 M) followed by treatment with Plant Vitrification Solution 2 [PVS2] (15, 30, 45, 60 min) was evaluated with six sweet potato accessions [Ipomoea batatas (L.) Lam.], using the "PVS2-droplet vitrification" cryopreservation method. No significant differences in the percent of shoot regeneration were observed when shoot tips were pre-cultured on sucrose concentrations ranging from 0.1 to 0.4 M. The highest mean shoot regeneration rate of 57.7 ± 5.5% after rewarming from cryopreservation (+LN) was obtained with a sucrose concentration of 0.35 M. Intermediate exposure times in PVS2 of 30 and 45 min showed statistically better results than shorter or longer exposure, with a maximum mean shoot regeneration (+LN) of 68.5 ± 4.5% with a 30 min exposure. Screening a separate larger group of 24 sweet potato accessions with a 0.35 M sucrose pre-culture and 30 min PVS2 treatment (+LN) resulted in shoot formation rates ranging from 1.7 to 66%.
The ability of shoot tips from carnation (Dianthus caryophyllus L., var. Eolo) cultured in vitro to develop resistance to freezing in liquid nitrogen depends on the physiological state of the cell material and the pretreatment conditions. Regrowth rates close to 100% have been obtained with apical shoot tips isolated from 2 month-old stems, precultured on medium supplemented with sucrose (0.75M) and treated with dimethylsulfoxide (5% or more). Resistance of axillary shoot tips decreased progressively as a funtion of their distance from the apical shoot tip. During the development of the stem from axillary buds (obtained by cutting), progressive increases in the regrowth rate of frozen apices were noted, from 30% before cutting (axillary buds) to 98% after 3 weeks of culture.
In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.
Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25C for 20 min or at 0C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.
Evidence is reviewed that freezing and dehydration are fundamentally different stress vectors: (a) Proteins, membranes, phospholipids, and living cells and organisms all contain about 0.25 g nonfreezable H2O/g dry weight. By definition, this H2O is not removed by freezing. (b) Dehydration, by contrast with freezing, can remove the nonfreezable H2O. Removing this H2O results in profound changes in the physical properties of biomolecules, particularly phospholipids and proteins, (c) The mechanisms of preservation of proteins during freezing and drying are completely different. The specificity for solute requirements for stabilization of proteins during freezing is low; any solute that is preferentially excluded from the hydration shell of a protein is also a cryoprotectant. (d) By contrast, stabilization of proteins during drying requires direct interaction between the stabilizing molecule and the protein, probably involving hydrogen bonding between the stabilizer and polar residues in the protein. The specificity is very high in this case; only carbohydrates are effective, and of those that have been tested trehalose is the most effective, (e) Less is understood about the mechanism of stabilization of phospholipid bilayers during freezing, but it is clear that while many solutes will preserve liposomes during freezing, only a few (of which trehalose is the most effective) will preserve them during drying. Stabilization of bilayers during drying requires direct interaction between the sugar and polar head groups of the phospholipids.
Embryogenic tissue of six sweet potato (Ipomoea batatas (L) LAM) genotypes of differing geographic origin was maintained on Murashige and Skoog (1962) medium (MS) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates (9 - 12 mg fresh mass) were precultured on MS medium supplemented with up to 0.7M sucrose prior to rapid freezing in liquid nitrogen. Survival of embryogenic tissue of all the genotypes was obtained, with four of these ranging from 37% to 87%. Minor adjustments in the sucrose levels before and after freezing had differing effects on the survival of embryogenic tissue of different genotypes. Embryogenic tissue recovered after cryopreservation appeared to develop normally.
The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.
Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (-196°C) exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.
Embryogenic tissue of two sweet potato (Ipomoea batatas (L) LAM) genotypes, TIB 10 and Nemanete (Nem), was established from in vitro axillary meristems on Murashige and Skoog (1962) media supplemented with 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid respectively. Embryogenic aggregates of approximately 1.5-2.0 mm in diameter were subjected to a rapid or a two-step freezing protocol in liquid nitrogen following alginate encapsulation, sucrose preculture and varying degrees of dehydration. Up to 28% of encapsulated embryogenic aggregates of TIB 10 survived rapid freezing without dehydration. This was not enhanced by dehydration prior to freezing. However, survival after dehydration was enhanced up to 74% by incorporating an initial slow cooling step prior to plunging the tissue into liquid nitrogen. Following freezing, embryogenic tissue appeared to develop normally and retained its competence to produce mature embryos and plantlets. Similar results were obtained with Nem, although the survival percentages were much lower.
In vitro-grown apical meristems of hybrid statice (Limonium cv. Blue Symphonet) were cryopreserved by three cryogenic procedures; (1) vitrification with encapsulation, (2) vitrification without encapsulation, and (3) a revised encapsulation/dehydration technique. When dehydration tolerance was well developed by preconditioning and cryogenic procedures were well optimized, these three procedures produced nearly the same levels of growth recovery (70–75%). These results support our theory that the acquisition of dehydration tolerance is sufficient for specimens to survive to cryopreservation.
Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.
A simplified protocol for cryopreservation of embryogenic tissue of sweet potato [Ipomoea batatas (L) Lam.] utilizing sucrose preculture only Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules
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A revised medium for rapid growth and bioassays with tobacco tissue cultures
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