Cryopreservation of plant germplasm: The Cuban experience (original) (raw)
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Cryopreservation is currently the only safe and cost-effective method for long-term conservation of the germplasm of species that are vegetatively propagated. In the Bioplantas Centre of Ciego de Avila (Cuba) the research on cryopreservation begun in 1994 with the general objective to evaluate and develop feasible cryopreservation techniques for crops of great commercial importance of the province. In this sense, sugarcane and pineapple are considered examples. It has been possible by the scientific and financial support from the International Plant Genetic Resources Institute and the International Foundation for Science. In the case of sugarcane, a cryopreservation methodology for embryogenic calluses using a simplified procedure of slow cooling was established. The effect of cryopreservation on the structural and functional integrity of cell membranes and the field performance of plants both derived from cryopreserved sugarcane calluses has been studied. Recently, a droplet-vitrification...
Current Frontiers in Cryopreservation, 2012
23°C and-40°C was possible with little decrease in survival by using mixtures of glucose, dimethylsulfoxide and polyethylene glycol as cryoprotectants. However, no plants were recovered from the cryopreserved cell suspension. Later on, Gnanapragasam & Vasil (1990) reported that efficient plant regeneration was obtained from a cryopreserved embryogenic cell suspension of one commercial sugarcane hybrid established from leaf derived callus. They observed pregrowing the cells for three days in Murashige & Skoog (1962) basal medium supplemented with 0.33 M sorbitol was essential to the process. A regeneration efficiency of 92% was obtained and plants regenerated from cryopreserved cells, and grown to maturity in the greenhouse, were morphologically identical to regenerated control plants. Later, there were not detected differences at molecular level using RFLP technique comparing plants regenerated from cryopreserved and control cells for three sugarcane hybrids (Chowdhury & Vasil, 1993). 2.2 Embryogenic callus The first success for cryopreservation of sugarcane embryogenic callus was obtained by Ulrich et al., (1979) for the hybrid H50-7209. It was a pretreatment using a combination of 10% polyethylene glycol, 8% glucose and 10% DMSO, freezing rate of 2°C.min-1 until a first transfer temperature of-40°C and freezing rate of 5°C.min-1 until second transfer temperature of-80°C. However, the recovery of cryopreserved callus was achieved only with root regeneration. Ulrich et al., (1984) obtained after modifications of the same protocol a limited number of albino plantlets from cryopreserved calluses. Later on, high survival rates (ca. 90%) and recovery of whole plants were obtained by Jian et al., (1987), Eksomtramage et al., (1992) and Gnanapragasam & Vasil (1992). The conditions defined were different from that used by Ulrich et al., (1979, 1984). For cryoprotective treatment, a mixture of sorbitol and DMSO was used by Jian et al., (1987) and Gnanapragasam & Vasil (1992); Eksomtramage et al., (1992) employed a mixture of sucrose and DMSO. Freezing conditions were also different: 1°C.min-1 from 0°C to-10°C, and kept for 15min at the same freezing rate from-10°C down to-40°C and kept for 1-5 h, and finally immersed into liquid nitrogen (Jian et al., 1987); or 0.5°C.min-1 down to-40°C or-45°C with no plateau at the end of the controlled freezing sequence (Eksomtramage et al., 1992 and Gnanapragasam & Vasil, 1992). Moreover, the technique developed by Eksomtramage et al., (1992) was successfully applied to calluses of 10 varieties. These authors have followed the strategy known as dehydration by extracellular freezing, which uses a controlled freezing regime (Withers & King, 1980). However, this procedure requires expensive and sometimes complex programmable freezing devices, limiting its use to laboratories specializing in cryopreservation (Ashmore, 1997; Reed, 2001). Furthermore, their research has been focused on the cryopreservation of sugarcane calli obtained from segments of immature leaves belonging to in vitro cultured plants; however, such explants are known to have a limited morphogenetic capacity (Krishnaraj & Vasil, 1995) and it is widely acknowledged that immature embryos, as well as young inflorescences, are www.intechopen.com Current Frontiers in Cryopreservation 362 physiologically better explants for calli production because they retain their embryogenic capacities (Merkle et al., 1995). 2.2.1 Optimization of methodology for sugarcane callus Our research team (Martinez-Montero et al., 1998, 2006), using the cryo-research for sugarcane callus described above as starting point, published the results for establishing step by step a methodology for the cryopreservation of sugarcane calli with embryogenic structures obtained from immature inflorescence (Figure 1). We optimized the following aspects according to the in vitro survival and regeneration (plants per 500 mg of calli) percentages for: Selection of the cooling procedure, the effect of the cooling procedure and of the type of alcohol, the effect of the induction time of extracellular ice crystals, the effect of post-subculture time, the effect of sucrose and dimethylsulfoxide concentration in the cryoprotective medium, and the effect of the pre-freezing time.
Current Frontiers in Cryopreservation, Prof. Igor Katkov (Ed.), ISBN: 978-953-51-0302-8, InTech, DOI: 10.5772/32047. , 2012
This Chapter comprises two main sections focusing on the establishment, optimization and application of cryopreservation techniques to different tissues of in vitro sugarcane and pineapple cultures. The first part presents the cryopreservation protocols developed for sugarcane apices isolated from in vitro grown plants, embryogenic calluses and somatic embryos, as well as some analytical techniques (electrolyte leakage, protein content and lipid peroxidation products), used to describe the impact of the successive steps of the protocol on the physiological state of the cultures, which are also useful to refine the cryopreservation protocol. The effect of cryopreservation on the phenotypical development, both in vitro and in the field, of sugarcane plants regenerated material will be also presented. The second section presents the studies performed to set up and refine a cryopreservation protocol for apices of pineapple in vitro plantlets. The protocol established following the vitrification approach was successfully applied for the first time to shoot tips of three pineapple varieties, and then extended to nine pineapple accessions belonging to the in vitro collection of Bioplantas Centre in Cuba. In addition, we present the preliminary assays developed using callus of two pineapple cultivars. In the conclusion, we discuss the possibilities and prospects of utilisation of cryopreservation techniques for the long-term storage of other vegetatively propagated tropical plant species.
Biotecnología Aplicada 23(4):344-359, 2006
Currently, cryopreservation is the only solution for avoiding the loss of embryogenic potential during the storage of sugarcane calli with embryogenic structures. However, implementing a methodology for cryopreservation implies overcoming technological drawbacks and validating the established procedure, wich are the main objectives of this work. We present our results on the development of a simple cryopreservation methodology for the storage of sugarcane calli with embryogenic structures, based on a slow freezing process. The proposed methodology successfully preserves the regenerative capacity of the calli, supported by ex vitro evaluation, for 27 months. We show the phenotypic characteristics of plants regenerated from cryopreserved calli, and compare them to non-cryopreserved controls. These studies are the first detailed evaluation of the effect of long-term cryogenic storage on the survival and regeneration of sugarcane plants from calli with embryogenic structures. Furthermore, we demonstrate the importance and implications of free radicals and oxidative stress as markers for cryopreservation-induced damaged, as evaluated by determinations of the products of lipid peroxidation during sugarcane culture. These results are fundamental for the efficient use and implementation of the established technologies, based on the use of calli for the large-scale production of vitroplants.
Cryopreservation of sugarcane embryogenic callus using a simplified freezing process
Cryo-letters, 19(3):171-176, 1998
A simplified freezing process was developed and successfully applied to embryogenic calluses of three sugarcane commercial hybrids (Saccharum sp. cv. CP 5243, C 91-301 and C 1051-73). To obtain optimal survival, the calluses were pretreated with a cryoprotective solution containing 10% DMSO and 0.3 to 0.75M sucrose. For freezing, the samples were immersed in an alcohol bath placed in a -40°C freezer, thus allowing a freezing rate comprised between 0.4 and 0.6"C/min. Samples were held at that temperature for 2 h before immersion in liquid nitrogen. The highest survival rates of cryopreserved calluses ranged between 20 and 94% depending on the variety, and fully developed plantlets could be obtained from regenerating calluses. Embryogenic calluses of one variety were stored for 14 months in liquid nitrogen without any effect on their survival rate and plantlet production.
Cryoletters, 2002
In this paper, we investigated if the differences consistently noted in survival and plantlet production between cryopreserved and non-cryopreserved, control sugarcane embryogenic calluses were related to modifications induced during cryopreservation in the structural and functional integrity of cell membranes. For this, the evolution of electrolyte leakage, lipid peroxidation products and cell membrane protein contents was measured during 5 d after cryopreservation. Differences between control and frozen calluses were observed only during the first 2 (electrolyte leakage) or 3 d (lipid peroxidation products and membrane protein content) after freezing. It was not possible to link these differences with the differences noted in survival and plant production between control and cryopreserved calluses. Additional studies are thus needed to elucidate which biochemical factors, linked to survival and plantlet regeneration, are affected by cryopreservation.
Cryo letters, 2006
Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allows the subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be highly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices.
Cryopreservation of sugarcane somatic embryos
Cryoletters 29(3):229-242, 2008
In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25°C, treatment with the PVS2 solution for 20-40 min at 0°C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 %.