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Cryopreservation of eucalyptus genetic resources
Abstract
The long-term preservation of forest genetic resources is a vital part of preserving our forest crops for future generations. Unfortunately, there are few genebanks dedicated to forest trees and very few methods for long-term preservation of forest genetic resources collections aside from field plantings of a limited number of seed-derived or elite clonal individuals. The use of cryopreservation for the long-term storage of elite germplasm is increasingly being used for the long-term preservation of clonal agronomic crops but for forest trees, such as Eucalyptus, the methodology for cryopreservation of diverse genetic resources collections has not been established. We report the successful cryopreservation of a germplasm collection of in vitro shoot cultures of thirteen Eucalyptus spp. lines consisting of two E. grandis x E. camaldulensis lines, seven E. urophylla x E. grandis lines, one E. grandis line, two E. grandis x E. urophylla lines, and one E. camaldulensis line. In a comparison of two cryopreservation methods, sucrose sensitivity limited the application of encapsulation-dehydration. However, with droplet-vitrification, all thirteen lines had good survival after cryopreservation in liquid nitrogen. A 30 min exposure to Plant Vitrification Solution 2 (PVS2) yielded post-liquid nitrogen survival between 38% and 85% depending on the line. One hundred shoot tips from all thirteen lines are currently in long-term storage as a germplasm collection.
... Thawing processes of direct and encapsulationvitrification techniques were the same. In this process, cryovials 24 storaged at least 24 hours in liquid nitrogen (Kaya et al., 2013) were directly plunged into the waterbath at 40 °C, after thawing, all explants were transferred to 1 M sucrose solution supplemented with standard liquid MS for 15 minutes, and then the samples were transferred to regeneration medium (MS medium including 4.44 µM benzyl adenine) at standard growthroom conditions [25 ± 2 °C, 16/8 hour photoperiod supplied via cool daylight fluorescent lamps (50 μmol −1 m −2 s −1 )]. Thawing process for droplet vitrification was achieved by direct transferring of frozen shoot tips to 1 M sucrose solution supplemented with standard liquid MS for 15 minutes at room temperature. ...
... and Saccharum spp. Kaya et al., 2013). Sugar accumulation in cells can cause slow removal of water content which could induce fatal ice-nucleation and improve cell membrane stability during dehydration and cryostorage (Salama et al., 2018;Lata et al., 2019). ...
... After cold hardening and sucrose preculture steps, explants of M. piperita were treated with PVS2 as cryoprotective/dehydration solution at different exposure times, long time exposure with PVS2 was toxic fo both of the control and the LN+ (Table 1-3). The ideal PVS2 treatment time can vary for different plant species, subspecies and, cultivars (Pennycooke and Towill, 2000; Kaya et al., 2013). In this study, droplet vitrification was the most effective cryopreservation technique for local M. piperita cultivars Candarli, Gomec and G-74 and the best PVS2 treatment time periods were 60, 75 and 30 min respectively (Table 1-3). ...
Peppermint is an important aromatic-medicinal plant species and it has valuable essential oil contents such as menthol, linalool and limonene. The main purpose of the current study was to optimize a protocol for cryopreservation of M. piperita local cultivars (G-74, Candarli and Gomec). The secondary aim was to investigate the genetic stability after cryopreservation using ISSR marker system. Three different single step freezing techniques were compared for long-term preservation of these local cultivars and the optimum regeneration percentages were acquired by using the droplet vitrification technique. The PVS2 treatments of this technique showed succesful long-term preservation of M. piperita cultivars with regeneration percentages of 72% to Candarli cultivar (60 min PVS2), 52% to Gomec cultivar (75 min PVS2) and 62.5% to G-74 cultivar (30 min PVS2) respectively. The ISSR PCR results showed that the genetic stability from cryopreserved M. piperita cultivars were high. While genetic stability percentage was ~99% for the Candarli cultivar, for the Gomec and G-74 cultivars the genetic fidelity was100%. The shoots that come from the cryopreserved shoot tips showed normal and health rooting, and all of them were also easily adapted to greenhouse conditions.
... After cold hardening and sucrose preculture, in cryoprotectant treatment, a chemical solution of a concentrated diffusing cryoprotectant solution is applied, followed by a vitrification solution. Treatment time, application temperature, and solution concentration may differ for plant species and cultivars [37][38][39][40][41]. ...
... The droplet vitrification method based on chemical vitrification and one-step freezing treatments was first reported by Sch€ afer-Menuhr et al. [55] using potato shoot tips. In this technique, 01-03 mm explants (Fig. 3a) are treated with the cryoprotectant solution (usually PVS2) put individually in 3-10 μL droplets of cryoprotectant solutions (depending on explant size) placed on a piece of [38] aluminum foil strip (Fig. 3b), which is then directly immersed in liquid nitrogen. For rewarming, the aluminum foils are directly plunged in liquid medium supplemented with 1-1.2 M sucrose, and after 20 min of unloading, shoot tips are transferred on regrowth medium (Fig. 3c). ...
... The main advantage of this method is the possibility of achieving very high cooling/warming rates due to the very small volume of cryoprotectant solution which the explants are placed. Although this is a newly developed technique, there are many reports obtained with a high regrowth percentage after immersion of liquid nitrogen [38,39,[56][57][58]. ...
Plant pathogens cause different diseases on crops and industrial plant species that result in economic losses. Pathogen-free plant material has usually been obtained by traditional procedures such as meristem culture, thermotherapy, and chemotherapy. However, there are many limitations of these procedures such as mechanical challenges of meristem excision and low regeneration rate, low resistance to high temperatures, phytotoxicity, and mutagenic effects of the chemicals used in the procedures. Cryotherapy is a newly developed biotechnological tool that has been very effective in virus elimination from economically important plant species. This tool has overcome the abovementioned limitations. This chapter aims to highlight the importance of the cryogenic procedures (vitrification, encapsulation-vitrification, droplet vitrification, two-step freezing, dehydration, encapsulation-dehydration) in order to generate virus-free germplasm.
... This LRR receptorlike protein interacts with CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (COL2) which has kinase domain but lakes the receptor domain. The third identified receptor in the CLV3p perception is RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOAD-STOOL2 (TOAD2) [38,39]. Triple mutant clv1 clv2 rpk2 Arabidopsis shows phenocopy of clv3 mutant which suggests that these three receptors are involved in main pathways of CLV3p perception. ...
... After cold hardening and sucrose Fig. 1 Schematic presentation of three different vitrification methods based on one-step freezing techniques: cryotherapy, vitrification, encapsulation-vitrification, and droplet vitrification [21] preculture, in cryoprotectant treatment, a chemical solution of a concentrated diffusing cryoprotectant solution is applied, followed by a vitrification solution. Treatment time, application temperature, and solution concentration may differ for plant species and cultivars [37][38][39][40][41]. ...
... The main advantage of this method is the possibility of achieving very high cooling/warming rates due to the very small volume of cryoprotectant solution which the explants are placed. Although this is a newly developed technique, there are many reports obtained with a high regrowth percentage after immersion of liquid nitrogen [38,39,[56][57][58]. ...
Plants are multicellular organism composed of different types of cells. These all kinds of cells are formed from pluripotent stem cells present at different positions in plant called stem cell niches. All these stem cell niches and their boundaries are maintained by complex regulatory mechanism at molecular level involving different genes, cofactors, and phytohormones. In this chapter, we discussed the regulatory mechanism and models of stem cell maintenance, specifying their boundaries at different stem cell niches.
... This LRR receptorlike protein interacts with CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (COL2) which has kinase domain but lakes the receptor domain. The third identified receptor in the CLV3p perception is RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOAD-STOOL2 (TOAD2) [38,39]. Triple mutant clv1 clv2 rpk2 Arabidopsis shows phenocopy of clv3 mutant which suggests that these three receptors are involved in main pathways of CLV3p perception. ...
... After cold hardening and sucrose Fig. 1 Schematic presentation of three different vitrification methods based on one-step freezing techniques: cryotherapy, vitrification, encapsulation-vitrification, and droplet vitrification [21] preculture, in cryoprotectant treatment, a chemical solution of a concentrated diffusing cryoprotectant solution is applied, followed by a vitrification solution. Treatment time, application temperature, and solution concentration may differ for plant species and cultivars [37][38][39][40][41]. ...
... The main advantage of this method is the possibility of achieving very high cooling/warming rates due to the very small volume of cryoprotectant solution which the explants are placed. Although this is a newly developed technique, there are many reports obtained with a high regrowth percentage after immersion of liquid nitrogen [38,39,[56][57][58]. ...
In higher plants, the cells that form aboveground tissues and organs are derived from the shoot apical meristem (SAM). SAM is dynamic in nature and divided into central zone (CZ), peripheral zone (PZ), and rib meristem (RM). Stem cells reside in the CZ, and their progenitors differentiate to form lateral organs in PZ and stem tissue in RM. Besides zones, the SAM is also divided into distinct clonal cell layers that show patterned cell division. Here, we describe methods to tag and isolate cell types from both cell layers and zones of SAM in high purity using cell sorter. This method enable plant biologist in rapid isolation of desired cell types from SAM to record their transcriptome, epigenome, proteome, and metabolome. The information generated by this approach will elucidate the mechanism of stem cell self-renewal, differentiation, and organogenesis in SAM.
... After preculture, in loading phase, a dilute solution of a permeating cryoprotectant is applied, followed by a vitrification solution. Vitrification temperature and solution concentration may vary for different species [33,[37][38][39][40]. Several vitrification solutions have been improved by various resarchers worldwide [27] [41]. ...
... After loading, LS is removed from a bottle, and PVS is added newly for the dehydration of plant tissues. The same as with vitrification, the dehydration using PVS2 is performed at 0ºC in light of the toxicity to plant cells [36,38,51]. After dehydration of PVS2, encapsulated samples are moved to a cryotube containing fresh PVS2, and immersed in LN. ...
... After removal of the solution, unloading solution is added to a tube, and cryoprotectants are removed from plant tissues for 30 min at 25ºC. After unloading, samples are moved from the cryotube, and recultured [38,51]. ...
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.
... Under these conditions, infected vacuolated and differentiated cells are eliminated by the ultralow temperature effect, leaving only highly cytoplasmic meristematic cells VALKONEN, 2009 a, b;WANG et al., 2003b). Among the available cryopreservation techniques, encapsulation-dehydration, vitrification, encapsulationvitrification and droplet vitrification are the most commonly employed (WANG et al., 2018a;ROMADANOVA et al., 2016;WANG et al., 2016;LI et al., 2016;KAYA et al., 2013;WU et al., 1999;PAUL et al., 2000). ...
... Encapsulation dehydration is based on techniques proposed for synthetic seed production (BENELLI; DE CARLO;ENGELMANN, 2013). The protocol involves the following stages: excising of in vitro shoot tips; encapsulating shoot tips; incubation of encapsulated beads in cumulative sucrose concentrations; dehydrating beads; freezing at -196 °C; warming in a water bath at 40°C; and culturing on regeneration medium (WANG et al., 2018a;BETTONI et al., 2016;KAYA et al., 2013). In some cases, its application has resulted in the production of virus-free plants more quickly and economically than through the use of traditional methods . ...
Cryopreservation is the storage of biological materials at ultralow temperatures (-196 ° C), is currently the only safe method for the long-term storage of plant material, allows plant resources to be preserved in a small space and with minimal maintenance. Cryopreservation techniques are available for many plant species of economic importance and are increasingly being routinely applied in genebanks around the world. Recently, cryopreservation was observed beyond of the conservation, since in vitro plant tissues initially virus-infected when cryopreserved, generated virus-free plants. Thus, cryotherapy denomination was created, which provides a high frequency of virus-free plants among regenerants within a short time frame. Grapevine and apple represent the most economically important fruit crops in the world and diseases of viral etiology are major obstacles to the highly cost-effective development and production of these fruit species. This study aimed at establishing of practical and efficient grapevine species cryopreservation protocols using droplet-vitrification and V cryo- plate techniques and to evaluate the effectiveness of cryotherapy by encapsulation- dehydration and droplet-vitrification, based on cryopreservation protocols, in the virus elimination in apple tree accesses. Chapters I and II show droplet-vitrification and V cryo-plate methods for grapevine species cryopreservation, using apical shoot tips excised from in vitro cultures as the explants source and, also to droplet-vitrification method, in chapter III, a cryopreservation protocol for grapevine is presented using apical shoot tips excised from growth chamber source plants. Chapters IV and V demonstrate the effectiveness of encapsulation-dehydration and droplet-vitrification techniques to eradicate viral species in apple trees. The results found for grapevine species cryopreservation demonstrate a wide applicability of the droplet-vitrification technique with regrowth levels at least of 43 % in 12 Vitis species and 3 genotypes of grapevine using apical shoot tips derived from in vitro and ex vitro sources plants, respectively. In addition, the V cryo-plate cryopreservation technique described to two genotypes of grapevine was easily executed and results in high regrowth levels (≥70%). In the cryotherapy studies, axillary shoot tips from apple rootstock ‘Marubakaido’ and the cv. 'Monalisa' were exposed to liquid nitrogen, regrowth, and growth in the greenhouse for at least 6 months were submitted to the PCR analysis. All of the evaluated cryopreserved Marubakaido plants by encapsulation-dehydration were free of Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV), but 10% recovered plants were still infected with Apple stem grooving virus (ASGV). All of the evaluated cryopreserved Monalisa plants by droplet-vitrification that were tested were free of ASPV, 95% were free of ACLSV and 35% were free of ASGV. The technologies generated in this thesis can be used by institutions specialized in the maintenance and recovery of plant materials with agronomic potential, benefiting grapevine and apple production chain, by the conservation of plant genetic resources and to generate the high-quality plant material.
... Under these conditions, infected vacuolated and differentiated cells are eliminated by the ultra-low temperature effect, leaving only highly cytoplasmic meristematic cells (Bettoni et al., 2016;Wang et al., 2003). Among the available cryopreservation techniques, encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet vitrification are the most commonly employed Romadanova et al., 2016;Wang et al., 2016;Li et al., 2016;Kaya et al., 2013;Wu et al., 1999;Paul et al., 2000). ...
... Encapsulation dehydration is based on techniques proposed for synthetic seed production (Benelli et al., 2013). The protocol involves the following stages: excising of in vitro shoot tips; encapsulating shoot tips; incubation of encapsulated beads in cumulative sucrose concentrations; dehydrating beads; freezing at −196°C; warming in a water bath at 40°C; and culturing on regeneration medium Bettoni et al., 2016, Kaya et al., 2013. In some cases, its application has resulted in the production of virus-free plants more quickly and economically than through the use of traditional methods (Bettoni et al., 2016). ...
Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world’s apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from ‘Marubakaido’ apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered ‘Marubakaido’ plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material.
... Although cryoprotective solutions (especially those containing dimethylsulfoxide (DMSO) have protective effects during freezing, they may be toxic to the tissues under conditions of high concentration or temperature, or long exposure times. Kaya et al. (2013) reported superior results using droplet vitrification vs. encapsulation-vitrification for cryopreservation of Eucalyptus spp., reporting up to 84.8% post thaw recovery. Similarly, the droplet vitrification method proved to be the most effective in the present study. ...
... These bands were lost from NG 57-024 samples after treatment with PVS2 including DMSO, and these differences could be the result of DMSO toxicity. DMSO toxicity has been reported in several studies which indicated that DMSO increases benzene metabolism and the toxicity of other aromatic hydrocarbons (Kocsis et al. 1968;Ozudogru et al. 2011;Kaya et al. 2013 there were a few differences of ISSR band profiles, these profiles indicate that three lines of sugarcane had high rate of genetic stability after cryopreservation. Martin et al. (2014) used molecular markers (RAPD and AFLP) to compare two cryopreservation techniques for Mentha piperita L. (mint) and Chrysanthemum morifolium Ramat. ...
Conservation of Saccharum spp. germplasm as ex situ collections of plants has a high cost, and in natural conditions, the plants remain exposed to pests, pathogens, and natural disasters. Long-term preservation of plant germplasm is important for agricultural biodiversity and food safety, so the aim of this study was to develop a cryogenic procedure for cryopreservation of sugarcane germplasm. The first study compared droplet vitrification and encapsulation-vitrification techniques for cryopreservation of in vitro shoot tips of Saccharum spp. variety Halaii. The best regeneration rate (70.9%) was obtained from 45-min PVS2 vitrification solution-treated shoot tips via the droplet vitrification technique. This technique was tested on two other Saccharum sp. varieties, and the best regeneration rates for varieties NG 57-024 and H 83-6179 were 63.3 and 76.3%, respectively. Shoots derived from cryopreserved shoot tip buds developed well-formed roots, and were easily acclimated to greenhouse conditions. The second study evaluated genetic stability of the cryopreserved varieties using ten inter-simple sequence repeat primers. A total of 211 (Halaii), 198 (H83-6179), and 201 (NG 57-024) reproducible bands, ranging from 125 to 5500 bp, were scored with this technique. One hundred genetic stability was detected from Halaii and H 83-6179 whereas 98.5% genetic stability was detected from varieties of NG 57-024. The PCR reactions showed that there was no crucial variation on genetic stability for all cryopreserved varieties.
... It is known that plant growth regulators are effective in the cell cycle and this causes somaclonal variation [9,110]. It is stated that the relative levels of cyto-kinin and auxin group growth regulators and the genome of the cell population are affected [2, 111 -113] ...
This reference is a timely compilation of studies of genome size and genetic stability of regenerated plants. It presents 13 book chapters that cover recent advancements in CRISPR/Cas-based genome editing, the use of molecular markers to analyze somaclonal variation in tissue culture, and genetic stability assessment in various plant species, including medicinally valuable plants like Valeriana and Coffea. The book also highlights the role of flow cytometry in investigating polyploidy and provides valuable insights into genetic fidelity assessment of micropropagated woody plants and orchids. The contributors have shed light on the intra-specific and inter-specific genome and chromosome number variation with reference to gene duplication and DNA sequence loss. Molecular techniques for detecting ploidy levels and genetic homogeneity in regenerated plantlets are also discussed. Additional highlights of the book include brief guidelines for experimental protocols for flow cytometry and molecular markers, coverage of a wide range of plants, and supporting references. This is an excellent reference for biologists, geneticists, and plant scientists exploring genetic homogeneity and genome size variation in diverse plant groups.
... In addition to taking an important place in the pharmaceutical industry with the active substances obtained from the secondary metabolites of medicinal plants, they are also used in cosmetics, spices, food, paints, insecticides, resins, gums, etc. It is also used in other areas, and its essential oils with aromatic properties are also used in the perfume industry [1,9]. ...
... It allows for the use of different parts of plants, such as the shoot tips, seeds, nodal and dormant buds, pollen grains, somatic and zygotic embryos, calli, and cell suspensions. The genetic stability also maintains for many years during cold storage [2,3]. However, during and after cryopreservation, the cells, tissues, and organs can suffer from freezing damage and even lose their viability, resulting in failed cryopreservation. ...
The different pre- and post-treatments are critical in cryopreservation procedures and affect
the shoot tip regrowth after freezing. In the present study, the long-term storage of four citrus cultivars
[Bodrum Mandarin (Citrus deliciosa Ten.); Klin Mandarin (Citrus nobilis Lauriro); White grapefruit and
Red grapefruit (Citrus paradisi L.)] were carried out by droplet vitrification methods, and the critical
points for effective cryopreservation of these species were determined. In this study, we investigated
the effect of explant size, cold hardening treatments, sucrose concentrations, and media combinations
on shoot regrowth after cryopreservation. The highest shoot tip regrowth, ranging from 13.3 to 33.3%,
was achieved when they were obtained from 0.3 to 0.7 mm explants excised from cold hardened
seedlings at 4 ◦C for three days that were then precultured in a medium containing 0.25 M of sucrose
and treated with PVS2 at 0 ◦C for 45 min. In addition, it has been determined that a regeneration
medium containing boric acid (H3BO3
) or ferric ethylenediaminetetraacetate (FeEDDHA) increased
the regeneration up to 33.3% after cryopreservation.
... The efficiencies of the cryopreservation of PVS2 and PVS4 had been demonstrated in other species (Kaya et al. 2013, Bustam et al. 2016, Kaya and Souza 2017, Tahtamouni et al. 2017, Volk et al. 2018, Uchendu et al. 2019, Bettoni et al. 2019, with survival rates similar to, or higher than, those seen with E. grandis. A study using Cannabis sativa likewise evaluated the efficiency of PVS2, PVS3, and PVS4 as cryoprotectants, and the explants exposed to PVS3 showed the lowest regeneration percentages (Uchendu et al. 2019). ...
... Most significantly, genetic stability of the cryopreserved regenerants was assessed using ISSR and EST-SSR markers. Since the first report of DV in potato shoot tips (Schafer-Menuhr et al. 1997), higher shoot recovery and regrowth in comparison to other cryopreservation techniques (such as V, ED, and encapsulation-vitrification) have been reported in a number of crops (Tanaka et al. 2011;Kaya et al. 2013;Le Bras et al. 2014;Kaya and Souza 2017;Wilms et al. 2020). However, in the present study, V technique was observed to be better (5 accessions), or equally suited (10 accessions), for cryopreservation of tested accessions of D. deltoidea, as compared to DV technique. ...
Dioscorea deltoidea Wall. ex Griseb. is a critically endangered, commercially important crop of high medicinal value. Several accessions of this valuable plant are conserved as slow growth cultures in the in vitro gene bank of Indian Council of Agricultural Research-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi, India. The purpose of this investigation was to develop a cryopreservation protocol that can be used for long-term conservation of 15 different D. deltoidea accessions. The vitrification technique of cryopreservation was compared to the droplet-vitrification technique. Shoot meristems excised from 4-wk-old in vitro plantlets were precultured on Murashige and Skoog (MS) medium containing 0.3 M sucrose for 16 h, treated with a loading solution for 20 min, dehydrated with plant vitrification solution (PVS2) for 90 min, and cryoconserved using both vitrification and droplet-vitrification protocols. Two-way ANOVA data subjected to post hoc Sidak’s multiple comparisons test (P ≤ 0.05) indicated that shoot regrowth after LN treatment, using vitrification (30 to 54%) and droplet-vitrification (21 to 51%) techniques, was statistically comparable in 66.7% accessions, while in 33.3% accessions vitrification gave significantly better results. Genetic stability of plants cryopreserved using both the protocols was confirmed using 39 Inter-Simple Sequence Repeat (ISSR) and 30 Expressed Sequence Tag-derived Simple Sequence Repeat (EST-SSR) markers. Results showed that there was no significant difference between mother plants and cryopreserved samples. The vitrification protocol has been implemented for long-term cryobanking of D. deltoidea germplasm (15 accessions), due to improved regrowth and ease of sample handling as compared to droplet-vitrification.
... With over 700 species and a number of hybrids and cultivars, Eucalyptus is one of the largest genera in the Myrtaceae [86]. Perhaps as a result of their typically orthodox seed, research into cryobiotechnology of this genus has been limited to conservation of valuable timber cultivars, hybrids, and elite clonal lines rather than 'wild' species [87]. In fact, 'unknown genetic diversity' in stored seed was listed as a problem for some authors aiming to conserve specific genotypes, limiting the applicability of these collections for conservation [88]. ...
The Myrtaceae is a very large and diverse family containing a number of economically and ecologically valuable species. In Australia, the family contains approximately 1700 species from 70 genera and is structurally and floristically dominant in many diverse ecosystems. In addition to threats from habitat fragmentation and increasing rates of natural disasters, infection by myrtle rust caused by Austropuccinia psidii is of significant concern to Australian Myrtaceae species. Repeated infections of new growth have caused host death and suppressed host populations by preventing seed set. Although most Myrtaceae species demonstrate orthodox seed storage behavior, exceptional species such as those with desiccation sensitive seed or from myrtle rust-suppressed populations require alternate conservation strategies such as those offered by cryobiotechnology. Targeting seven key Australian genera, we reviewed the available literature for examples of cryobiotechnology utilized for conservation of Myrtaceae. While there were only limited examples of successful cryopreservation for a few genera in this family, successful cryopreservation of both shoot tips and embryonic axes suggest that cryobiotechnology provides a viable alternative for the conservation of exceptional species and a potential safe storage method for the many Myrtaceae species under threat from A. psidii.
... The meristems were excised from in vitro grown C. avellana cv. "Palaz" shoots infected with ApMV (the shoots were cold hardened for 2 weeks in the dark at +4°C, Fig. 3A-B) and, for sucrose preculture, they were transferred to WPM medium supplemented with 4.44 μmol • l -1 BA and 0.4 M sucrose for 24 h (Kaya et al. 2013;Kaya and Souza 2017;Fig. 3C). ...
Numerous plant species around the world suffer from the presence of viruses, which especially
in economically important crops, cause irretrievable damage and/or extensive losses.
Many biotechnological approaches have been developed, such as meristem culture, chemotherapy,
thermotherapy or cryotherapy, to eliminate viruses from infected plants. These
have been used alone or in combination. In this work, meristem culture, thermotherapy and
cryotherapy were compared for Apple mosaic virus elimination from hazelnut local cultivar
“Palaz”. The virus-free plant was also confirmed by reverse transcriptase polymerase
chain
reaction (RT-PCR) after each treatment and, the best results were obtained by cryotherapy.
A one step freezing technique, droplet vitrification, was used for cryotherapy, and the best
regeneration percentage was 52%. After cryotherapy, virus-free seedlings of hazelnut local
cultivar “Palaz” were confirmed as being virus-free after three subcultured periods.
... A técnica de Droplet-vitrifcation foi realizada conforme Kaya et al. (2013) com adaptações. Os ápices caulinares (1-1,5 mm 2 ) foram excisados das plantas micropropagadas in vitro e pré-cultivados, por 24h em meio MS sólido suplementado com 0,2 M de sacarose, seguido por mais 24h em meio MS suplementado com 0,5M de sacarose, na ausência de luz. ...
... Dehydration time is a critical factor for tissue survival (Benson et al. 2007;Mathew et al. 2018). This technique has been successful with shoot tip cryopreservation of several plant species displaying varying degrees of tolerance to abiotic stresses; Eucalyptus spp. with regrowth rates of 38-85% (Kaya et al. 2013); Malus spp. with regrowth of 70% (Condello et al. 2011;Halmagyi et al. 2010); Prunus spp. with regrowth of 30% (Vujović et al. 2011); Rubus spp. with regrowth of 18% (Condello et al. 2011) and regrowth of 30% (Vujović et al. 2011) and Vitis spp. recording regrowth rates of 50% (Marković et al. 2013;Pathirana et al. 2016) and regrowth at least 43% for 12 Vitis species (Bettoni et al. 2019). ...
Cryopreservation combined with in vitro culture offers a safe and cost-effective method to conserve germplasm. Conservation of Persea spp. has been limited to heterozygous somatic embryos that are not true-to-type. A method for shoot-tip cryopreservation is vital to preserve the exact gene pool of interest. For the first time cryopreservation protocols for mature shoot tips of two avocado cultivars (cvs) ‘Velvick’ and ‘Reed’, were established. In vitro shoots were subjected to two different optimised pre-treatments; (1) cv ‘Velvick’—high sucrose (0.3 M) or (2) cv ‘Reed’—low temperature (10 °C) incubation, over a 2-week period prior shoot tip dissection. Two different plant vitrification solutions, plant vitrification solution 2 (PVS2) and vitrification solution L (VSL) were tested at 0 °C for 0, 10, 20, 30 and 40 min. Vitrified shoots were evaluated for survival and regrowth at 2 and 8 weeks after vitrification treatment and either with or without liquid nitrogen exposure. The study revealed that the optimal exposure time for each cultivar varied with the cryoprotectant used. After liquid nitrogen cv ‘Velvick’ highest regrowth levels were observed with 20 min exposure to either PVS2 or VSL, however, vigorous plants were produced only from VSL treated shoots. In the case of cv ‘Reed’ highest regrowth levels were observed with 10 min exposure to PVS2 however only morphologically normal plants were recovered from VSL treated shoots.
... As a result, conservation of genetic diversity and germplasm resources throughout the world has become an important subject of international concern (Krueger and Navarro, 2007;Kaya et al, 2017) Ex situ conservation strategies include maintenance and preservation of plant species as alive organisms outside their natural environments, in the form of in vitro cell, tissue and organ cultures, seeds, vegetative propagules, pollens and whole plants. Ex situ conservation procedures are usually used to complete in situ techniques, however, in sometimes are the just possible protocols to preserve spesific plant germplasms (Ramsay et al., 2000;Ozudogru et al., 2010;Kaya et al., 2013). ...
The fruit quality and other properties of Citrus spp. as germplasm resources are just only a small part of their usage. Such features as tolerance of environmet conditions and disease resistance, etc. are crucial properties of genotypes conserved in germplasm collections and utilized by scientist. Ecosystem loss can be caused by natural occurences and/or human activities. The damage of plant genetic resources has made indispensable the development of new conservation strategies. Developments in plant biotechnology provide new technological approaches for evaluation and preservation of plant germplasm resources. Biotechnological procedures such as in vitro propagation strategies, long-term conservation via cryopreservation techniques, and molecular marker systems offer a useful tools to plant diversity studies, management of genetic resources and ultimately conservation. This conference paper summarizes the current state of current information of citrus genetic resources and the status of current efforts in preservation of these resources via in vitro approaches and biotechnological applications.
... For obtaining calcium alginate beads, each shoot tips were then dropped 100 mM CaCl2 solution for polimerization. Finally, the calcium alginate beads washed with sterilled distile water to stop more hardening (Kaya et al, 2013). ...
Eucalyptus camaldulensis Dehn. having heavy, hard and durable wood is the one of most
widely planted trees over the World. Their woods are very important indusrial source of
timber, for pulp and paper production and also for fuel. Their oils are also used in medicine
for remedy of different helath problems such as migraine headaches, chronic bronchitis,
coughing, tuberculosis. Different tissue culture techniques have been developed for in vitro
propagation of Eucalyptus species. In this study was aimed to develop for efficient in vitro
propagation of E. camaldulensis by using synthetic seed technology. The results showed
that the somatic embryos encapsulated in the calcium alginate beads of E. camaldulensis
regenerated very easy and they produced healty shoots after incubation period at standard culture conditions.
... In vitro culture condition: The in vitro shoots of sugarcane were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 1% sucrose, 10 mgL -1 charcoal, 0,04 mg L -1 benzyladenine, 1,5 gL -1 gelrite and 3,5 gL -1 agar with a pH adjusted to 5,6. Cultures were maintained at 27±2°C temperature with a 16 hours light / 8 hours dark photoperiod provided by cool daylight fluorescent lamps (50 μmol −1 m −2 s −1 , Kaya et al., 2013). ...
Conservation of plant genetic resources has become highly important for food
security and crop improvement to reduction natural resources. The tissue culture protocols
providing aseptic and standard culture conditions is one of the most important tool for
developing of conservation strategies. Because sugarcane is significant industrial crop for
different countries over the World, their germplasm is needed to maintain. The sugarcane
grown in field genebanks are under the risk of their valuable germplasm loss due to hazards
of man and nature exist. Biotechnological approaches such as tissue culture techniques can
overcome all of these limitations for germplasm conservation of industrial crops. In the
current work aimed to develop an efficient protocol for mid-term conservation of sugarcane
germplasm using 3 and 6 six month culture periods at different temperatures. In this
incubation periods were analysed for determination best condition for mid-term
conservation. The maximum viability rate was 95%, and the regeneration and the rooting
rates were 100% after six month incubation at 15 °C.
... Long term conservation via cryopreservation is routinely used for wide range of plant species possible. The main advantages of this procedures are easy application, cheap and effective long-term storoge of plant materials such as herbaceous (figure 1a), woody (figure 1b,c; Kaya et al, 2013;), monocotyledones (figure 1d-f; Kaya, 2016;. These cryopreservation techniques are sufficiently improved to imagine their urgent usage for big-scale applications of vegetatively propagated species in genebanks. ...
Long-term conservation techniques via cryopreservation, the storage of
plant cells, tissues and organs at ultralow temperature, usually that of
liquid nitrogen (-196ºC), is the only available technique to provide the
safe and cheap conservation of plant germplasm. All cellular metabolic
activities, i.e., synthesis of biological material, cell division processes are
inhibited at this temperature, in this way, the biological material can be
preserved without change for long period of time. The aim of the current
study is to clarify of the various cryopreservation techniques available,
i.e., classical slowcooling and one step freezing methods, of the successes
made and limitations faced with cryopreservation of plant species, and of
the current usage of long-term storage methods for plant germplasm.
... So far, the droplet vitrification technique appears to be a promising method to overcome species-specific and genotypespecific responses to Vitis cryopreservation (Bi et al., 2018a;Volk et al., 2018). Droplet vitrification uses ultra-fast shoot tip cooling and warming conditions, which are an important requirement for successful cryopreservation protocols based on vitrification (Benson and Harding, 2012;Kaya et al., 2013;Souza et al., 2016). ...
The availability of and easy access to diverse Vitis species are prerequisites for advances in breeding programs. Plant genebanks usually maintain collections of Vitis taxa as field collections that are vulnerable to biotic and abiotic stresses. Cryopreservation has been considered an ideal method of preserving these collections as safety back-ups in a cost-effective manner. We report a droplet vitrification method used to cryopreserve 12 Vitis species (Vitis vinifera cvs. Chardonnay and ‘Riesling, V. actinifolia, V. aestivalis, V. jacquemontii, V. flexuosa, V. palmata, V. riparia, V. rupestris, V. sylvestris, V. ficifolia, V. treleasi, and V. ×novae angeliae) using shoot tips excised from plants grown in vitro. Our results demonstrated wide applicability of this technique, with regrowth levels at least 43% for 13 genotypes representing 12 Vitis species. We demonstrated that the droplet vitrification procedure can be successfully replicated by technical staff, thus suggesting that this method is ready for implementation.
... Attempts to store chrysanthemum plant material in liquid nitrogen (LN) have been made since 1990 (Fukai and Oe, 1990). In vitrogrown shoot tips are most suitable for cryopreservation (Lee et al., 2011;Kaya et al., 2013;Souza et al., 2016). Unfortunately, there are still some problems, such as low viability or regeneration potential of the cryopreserved chrysanthemum tissues (Osorio-Saenz et al., 2011), their hyperhydricity (Wang et al., 2014), disturbance of the chimeric structure (Fukai et al., 1994), or DNA sequence alternations (Martín and González-Benito, 2009;Kaya and Souza, 2017). ...
The aim of this research was to optimize the encapsulation-dehydration cryopreservation protocol of Lady Orange chrysanthemum explants on the preculture, pretreatment, and post-rewarming recovery steps. Shoot tips were precultured on MS medium with various abscisic acid concentrations (0–30 μM). Next, the encapsulated explants were osmotically dehydrated for 3 or 5 days and desiccated for 3–4 h and, after rewarming, they were subcultured on recovery media of various compositions (control and cytokinin/auxin-supplemented). A high explant survival rate, even up to 100%, was observed. The value of this parameter, however, changed depending on the post-rewarming culture duration. Moreover, not all the viable explants were capable of forming shoots. A
lower ABA concentration (15 μM) during preculture and the presence of 4.65 μM kinetin in the post-rewarming recovery medium enhanced cryopreservation efficiency with a high survival rate and typical microshoot formation. A higher ABA concentration and the presence of 6-benzylaminopurine in the recovery medium resulted in shoot multiplication, abundant callus formation, and root formation inhibition.
... In conifer clonal forestry, cryopreservation is used as be multiplied by thawing and propagating the cryopreserved embryogenic 2002; Sharma 2005). Cryopreservation (predominantly of shoot tips) is being increasingly applied for hardwood trees such as Populus, Robinia, Betula, Fraxinus, Morus (Häggman et al. 2008), Eucalyptus (Kaya et al. 2013), and Quercus (Barra-Jimenez et al. 2015). ...
The world’s total forest area is just over 4 billion hectares, or 31 per cent of the total land area. Forests provide wood, timber and a vast array of products and services. Forests also play a very significant role in carbon sequestration and host much of the world’s biodiversity. Forest trees have unique characteristics, including high heterozygosity, long generation intervals, vulnerability to inbreeding depression, narrow regional adaptation and the fact that the majority of the species are undomesticated, thus generating unique challenges and opportunities for biotechnology applications. Biotechnology has advanced considerably in the last decade and has contributed to improving characterisation, conservation and utilisation strategies for genetic resources, as well as advanced technologies for mass propagation, genetic improvement and biomass utilisation. Forest biotechnology is developing along a separate path from crop biotechnology. It is expanding very rapidly; covers an increasing number of taxa; and is no longer restricted to tree species used in plantation forestry. However, forest trees have received less attention than crop plants and domestic animals. The development and application of biotechnology are progressing at a much lower pace in developing countries than in industrialised countries. This paper briefly reviews the development of biotechnology and discusses its current and potential application in the forestry sector, with special attention to smallholders and to tropical areas.
... In recent years, many studies have been conducted on cryopreservation of citrus genetic resources. As a result, effective freezing protocols have been developed for different types of plant tissues and organs such as shoot tips (Wang and Deng, 2004;Kaya et al., 2013), embryonic axes (Cho et al., 2001(Cho et al., , 2002a(Cho et al., , 2002b, seeds (Cho et al., 2002c;Lambardi et al., 2004), somatic embryos (Marin and Duran-Vila, 1988), ovules (Gonzales-Arnao et al., 2003), nucellar cells (Sakai et al., 1991), and embryogenic calli (Olivares-Fuster et al., 2000). ...
An important method for plant germplasm conservation is offered by a biotechnology-based approach of cryopreservation.
Cryopreservation refers to the storage of plant material at ultralow temperatures in liquid nitrogen. A procedure for cryopreservation
of polyembryonic seeds was improved for selected citrus cultivars from Turkey. Seed dehydration was performed at different exposure
times, in sterile conditions of a laminar flow-hood. The tested cultivars showed the highest tolerance to low temperature storage when
the seeds were first dehydrated to a moisture content of 21.8% for Poncirus trifoliata Raf. × Citrus sinensis Osb. and to 17.6% for Citrus
limonia Osbeck. The postcryopreservation germinability ranged from 73.3% (Poncirus trifoliata Raf. × C. sinensis Osb. and Fortunella
margarita (Lour.) Swingle) to 93.3% (C. jambhiri Lush.). Dehydration was beneficial for germination of seeds from all of the tested citrus
species after the liquid nitrogen exposure. Seedlings derived from cryopreserved seeds had well-formed shoots and roots and were easily
acclimated to greenhouse conditions.
In this study, the effects of boron salt stress on in vitro cultivated Liquidambar orientalis (L. orientalis), a relict-endemic plant species, and the resulting changes in its phenolic appearance were investigated. Salt stress can cause negative impact on plant growth and production, especially in species with low salinity and drought tolerance, affecting metabolite expression and somaclonal execution. To evaluate the effects of different boron salts on meristem regeneration and progression, clonal in vitro L. orientalis meristems were exposed to boric acid, sodium perborate, sodium metaborate, and disodium octaborate salts. When compared with the control group examples where salt application was not performed, the highest regeneration percentage was determined to be 100% with the application of 1 mg/L disodium octaborate. In terms of the shoot formation capacity index, it was determined to be 5 mg/L. With a value of 4.94, the application of sodium perborate yielded the best result. In L. orientalis plants, the greatest change in phenolic compounds due to boron salt applications was observed in the concentration of Quercetin with the sodium perborate salt application at 1 mg/L concentration.
Mentha x piperita L., one of the most important medicinal aromatic plants belonging to the
Lamiacea family, is a hybrid species formed by crossing watermint and spearmint. In addition
to being the active ingredient of many drugs with the secondary metabolites it contains, it is
used as a spice in a wide range from the food sector to the cosmetics industry thanks to its
valuable essential oils. Considering that M. piperita, which is known to be very important in
terms of medicine and economy, cannot be produced by seed and is easily affected by changing
environmental conditions, Mentha, a valuable plant, should be protected. Unlike short and
medium-term preservation methods, the cryopreservation technique, which provides long-term
preservation, can be performed in one or two stages. It has been determined as a result of
previous studies that the M. piperita plant successfully regenerates after the cryopreservation
application, which provides long-term protection by trying different parameters, and the genes
that are effective in the high success of survival after cryopreservation for the M. piperita plant
are not known. For this purpose, firstly, a study was conducted on the NCBI database of stress
genes in the M. piperita plant, which was successfully transferred to the in vitro environment.
Since there is no sequence data for the relevant stress in Mentha piperita plant, related genes
were detected in the model organism Arabidopsis thaliana, and then BLAST was performed
between the two species, again using the NCBI database. Sequences with the most percent
match to target genes were determined. Primers specific to these sequences were designed using
the Primer BLAST program. Total RNA isolation was performed from the leaf and stem
samples of the parent material grown in vitro, the material from sucrose preconditioning, and
the regenerated plant materiallays after cryopreservation application to see whether the relevant
genes were effective in regeneration after cryopreservation application in M. piperita plant. To
be used in real-time PCR, cDNA was synthesized from total RNAs with reverse transcriptase
enzyme. Real-Time PCR was performed to quantitatively detect the mRNA expression levels
126
of target genes. Real-Time PCR results were analyzed by the ΔΔCT method. mRNA expression
plots of six genes compared to the control group were generated by 18S rRNA normalization.
TTEST was performed for the significance of the results, and those with p ≤ 0.05 were
considered significant. According to the TTEST result, there was a significant increase in the
mac_4 (Hsp-80 like) gene.
on online. Papers were selected by the organising committee to be presented in oral and were subject to review by the invited reviewers. Abstract Conservation strategies can be divided into two parts, conventional and biotechnological. Traditional strategies that can provide relatively short-and medium-term protection have the disadvantages of skilled labor, high cost, and relatively short-term protection. In addition, conservation carried out in open environments such as field and garden collections can be under the pressure of both biotic and abiotic threats. However, cryopreservation guarantees safe and cheap long-term preservation of plant germplasm. From the first years it was developed to the present day, methods that ensure the protection of almost all kinds of plant species with biotechnological methods have been successfully applied. The basic principle in the method is the removal of the cellular water content of the explant of the plant species to be protected by physical and/or chemical methods and then its long-term preservation at ultra-low temperatures at liquid nitrogen (-196 °C). Thus, the water in the cell will be removed and the deadly ice crystal that may form during freezing will be prevented. In this context, many methods based on physical and chemical vitrification such as, dehydration, encapsulation dehydration, vitrification, encapsulation vitrification and droplet-vitrification have been developed and successfully applied on many plant species. The main purpose of this study is to describe the biotechnological methods aimed at long-term preservation of plant germplasm and to compare the advantages and disadvantages of these methods.
The management and protection of wild species and domesticated crops are essential for the maintenance of plant biodiversity. Plant tissue culture-based in vitro culture technique provides an alternative and valuable options for multiplication and conservation of a wide range of plant species, especially vegetatively propagated plants, plants having recalcitrant seeds, and plant species at risk of extinction. The plant tissue culture technique allows for propagating such plant species with high multiplication rates and conservation for short to medium-term or long-term. The slow growth culture technique is used to achieve short- to medium-term preservation of plant germplasm under growth-limiting conditions, which allow the storage of germplasm from few months to 1-2 years by extending subculture intervals. The strategies adopted for the storage of plant germplasm under slow growth storage include the incubation of cultures at low temperature, minimal growth medium by minimizing nutrient components in the medium or applying growth retardants or osmoticum for limiting the growth of cultures. Cryopreservation is an ideal and most viable technique for the long-term conservation of plant genetic resources. Refinement in technology mainly, newly developed droplet-vitrification and cryo-plate methods increased the applicability of cryopreservation for long-term preservation of plant genetic resources. Instead of several limitations, both slow growth storage and cryopreservation methods are now routinely used to conserve a wide range of plant species. This chapter briefly presents the recent progress on short- to medium and long-term conservation of plant germplasms by slow growth storage and cryopreservation. The role of in vitro culture technique and their applications and limitations in the conservation of plant biodiversity is also discussed.
EFFECT OF SOLAR THERMAL RADIATION AND MAGNETIC FIELD ON THE FLOW OF CASSON FLUID
Abstract
In this paper, the behavior of the non-Newtonian fluid and heat transfer is considered in an
exponentially stretching surface in the occurrence of porous magnetic field, as it has vast
applications in the field of several industries. An explicit Finite difference method is applied
for the solution of governing equations. By considering governing equations of the
mathematical model as a platform non-linier differential equation have been reduced to
ordinary differential equations by using similarity transformations. BVP4C software
technique is used for finding the solutions and results are presented in the form of tables and
graphs for several equations. After simulation, it is found that the heat transfer rate decreases
with higher values of the magnetic field as well as the radiation. The temperature profile and
velocity profile of the Casson fluid flow is directly related to several parameters. Finally, it is
observed that several parameters such as Casson fluid parameter, radiation parameter, Prandtl
number, and Eckert number are stable at the point of convergence.
Eucalyptus has traditionally been used for the treatment of asthma, shortness of breath, rheumatism and hemorrhoids, as well as toothache relief, expectorant, antiparasitic. The responses of plants to salt stress vary greatly according to environmental factors and especially genotype. As with many other stress factors, salt stress causes an increase in the amount of some enzymes, polyamines and amino acids. The responses of plants to salt stress vary greatly according to environmental factors and especially genotype. As with many other stress factors, salt stress causes an increase in the amount of some enzymes, polyamines and amino acids. The role of proline amino acid is especially important in terms of salt resistance, and it is known that many salt-tolerant plant species have a higher proline amount when compared to the sensitive ones. Proline is an amino acid with protective properties, the amount of which increases under stress conditions, participates in the detoxification of free oxygen radicals and plays an important role in resistance to stress conditions. The current work aimed to investigate the proline effect on in vitro regeneration of Eucalyptus camaldulensis against salt stress. When compared to the control group (no NaCl), the best proliferation rates were obtained from WPM medium supplemented 100 mgL-1 proline after 25, 50, 100 mM salt application.
Vriesea bahiana, Hohenbergia castellanosii and Encholirium spectabile are endemic Brazilian species that are considered endemic or endangered. Development of strategies to conserve these species is important to prevent irreversible genetic erosion. The objective of this study was to evaluate the post-seminal development and seed cryopreservation of three endemic or in danger of extinction bromeliad species in Brazil, to obtain a protocol that can safeguard the genetic variability of these species. In the seed cryopreservation assay, we evaluated five desiccation periods. The seeds in the cryotubes were taken from the desiccator and immediately plunged into liquid nitrogen. For the analysis of post-seminal development, seeds in different germination stages were collected and evaluated by light and scanning electron microscopy. Vriesea bahiana seeds frozen in liquid nitrogen presented almost 100% germination, indicating dormancy break of this species. Vriesea bahiana can be cryopreserved with 5.9% water content after being dried for 24 hours. Hohenbergia castellanosii and E. spectabile seeds did not need to be desiccated before being cryopreserved. The most relevant morphological traits for differentiation of genera and subfamilies of Bromeliaceae are the shape and type of seed appendages. In this study, all three species presented well-differentiated size and shape of their structures.
Plant pathogens such as viruses, bacteria, fungi affecting field crops, culture and wild plant species cause great economic losses. The crucial step toward disease anagement under natural field conditions is to appropriately detect the pathogen and then immediately treated the infected plant. There are many different traditional ways to prevent for virus contaminations such as biological and chemical control of the vector being often an insect transmitting viruses, growing virus resistant crop varieties being made via genetic transformations, using virus-free planting material and the protection of disease placement in fields where viruses do not yet occur. In biotechnological approaches, pathogen-free plant material has usually been obtained by traditional procedures such as meristem culture, thermotherapy, and chemotherapy and newly developed procedures such as cryogenic techniques. For successful viral elimination via meristem culture, it is usually necessary to excise shoot tips that are lower than 1 mm in size. Thermotherapy method connected with meristem culture is also a difficult process requiring specific conditions such as virus-specific treatments and specific equipments. An alternative method is chemotherapy based on the usage of antiviral chemicals associated with thermotherapy or meristem culture for plant virus elimination and it was successfully used for virus elimination of some infected plants such as apple. On the other hand cryotherapy being newly developed biotechnological tool has been very effective for virus elimination for many important plant species such as strawberry, potato, sweet potato, grape, nut and apple. This study aims to highlight the importance of biotechnological approaches in order to generate virus-free germplasm and to compare the efficiency of these traditional and newly developed proecedures.
Dünya nüfusundaki artış nedeniyle giderek büyüyen gıda ihtiyacı, verim ve kalite açısından üstün çeşitlerin varlığı ve eldesi ile karşılanabilir. Bu amaçla yeni çeşitlerin geliştirilmesi için yapılacak ıslah çalışmalarında genetik kaynakların koruma altına alınması çok önemlidir. Genetik kaynakların önemini anlayan gelişmiş ülkeler dondurarak muhafaza konusunda yoğun çalışmalar sürdürmekte en son tekniklerle genetik kaynakların korunmasına ve muhafaza altına alınmasına çalışmaktadır. Sahip olduğumuz bitki genetik kaynakları çeşitli nedenlerle genetik erozyona uğramakta ve yok olma tehlikesi ile karşı karşıya kalmaktadır. Özellikle kültür formlarına ait bitki gen kaynaklarındaki çeşitliliğin korunması bitkisel üretimin sürdürülebilirliği bakımından son derece önemlidir. Ayrıca çeşitli hastalık ve zararlılar ile insan tahribatı gibi birçok nedenden dolayı yabancı döllenen türler, tohum miktarı az olan ya da tohum yoluyla çoğaltılamayan türler yok olmaktadır. Bu sebeple öncelikli olarak kültüre alınmış bitki gen kaynaklarını ve ıslah çalışmalarında kullanılabilecek yabani formları kriyoprezervasyon yöntemiyle muhafaza etmek zorunlu hale gelmiştir. Kriyoprezervasyon tekniği bitkiden alınan sürgün uçları, meristemler, kültüre alınmış hücreler, somatik embriyolar, protoplastlar, embriyo, endosperm, polen, anter, ovul, tohum gibi bitki organ ve organellerinin sıvı azot içerisinde çok düşük derecelerdeki sıcaklıkta (-160 o C) uzun süre muhafaza edilebilmesine olanak sağlamaktadır. Bu çalışmada kriyoprezervasyon tekniği ile ilgili yöntem ve uygulamalara ilişkin bilgiler sunulmuştur. Anahtar Kelimeler: dehidrasyon, yavaş büyütme, vitrifikasyon, damlacık dondurma, sıvı azot, kriyobanka. ABSTRACT The growing need for food due to the increase in the world population can be met by the existence and production of superior varieties in terms of yield and quality. For this purpose, it is very important to protect the genetic resources in breeding studies to develop new varieties. Developed countries, who understand the importance of genetic resources, are working hard on freeze conservation and trying to protect and conserve genetic resources with the latest techniques. Our plant genetic resources are subject to genetic erosion due to various reasons and are in danger of extinction. In particular, preservation of diversity in plant genetic resources of culture forms is extremely important in terms of sustainability of crop production. In addition, for many reasons such as various diseases and pests and human destruction of allogamous species, the amount of seed or species that can not be reproduced by seed is destroyed. For this reason, it has become necessary to preserve the cultivated plant gene resources and wild forms that can be used in breeding studies by cryopreservation method. The cryopreservation technique allows plant organs and organelles such as shoot tips, meristems, cultured cells, somatic embryos, protoplasts, embryos, endosperms, pollen, anther, ovules, seeds from plants to be kept in liquid nitrogen for a long time at very low temperatures (-160 o C). In this study, methods and applications related to cryopreservation technique are presented.
Long-term storage of synthetic seeds can be accomplished using cryopreservation
techniques. Cryopreservation allows the viability of encapsulated plant
material to be conserved and maintained over a long period without modifications or
genetic changes because the material is exposed to ultralow temperatures in liquid
nitrogen (�196 �C), which decreases or even halts cellular metabolism. Cryopreservation
has been found to be practical and efficient for the conservation of many
species due to the small volume of material needed for storage, the simplification of
transportation procedures and the minimal maintenance required compared to conventional
storage methods. The main cryopreservation techniques applied to synthetic
seeds are encapsulation-dehydration and encapsulation-vitrification. These
techniques have been shown to be highly applicable for small explants that are
sensitive to the conventional cryopreservation process, such as meristems and
somatic embryos. However, the success of cryopreservation techniques for synthetic
seeds depends on the type of encapsulated explant, on the capsule constitution and
consistency and on research on the different cryopreservation stages in order to
optimize the survival and regeneration of the plant material. Therefore, the present
chapter is based on studies of the different stages of cryopreservation related to
encapsulation techniques developed over time and on the major advances and
innovations in cryopreservation.
Cryotherapy newly developed biotechnological tool has been very
effective for pathogen elimination from ecomical important plant species
and this tool has overcome limitations such as mechanical challenges of
meristem excision and low regeneration rate, low resistance to high
temperatures, phytotoxicity and mutagenic effects of chemicals. Current
work aims to determine the cryogenic procedure, droplet vitrification,
based on chemical vitrification for pathogen elimination, and also
compare with traditional methods such as meristem culture,
thermotherapy, and chemotherapy.
Globally, forests are of great economic importance and play a vital role in maintaining friendly ecological environments, sustainability of eco-systems, and biodiversity. Harsh environments, human activities and climate warming have long threatened the diversity of forest genetic resources. Among all conservation strategies, cryopreservation is at present time considered an ideal means for long-term conservation of plant genetic resources. To date, studies on cryopreservation of forest trees have been far behind agricultural and horticultural crops. The present review provides a comprehensive and update information on recent advances in cryopreservation of shoot tips, somatic embryogenic callus and seeds of forest trees. Assessments of genetic stability in the regenerants following cryopreservation were also analyzed and addressed. Further studies on cryopreservation of forest trees are proposed and needed. By doing so, we expect to re-evoke research interests and promote further developments in forest tree cryobiotechnology, thus assisting to ensure maintenance of biodiversity of genetic resources of forest trees.
Conservation of forest genetic resources is essential for meeting the demand for future wood products. Genotypes with improved characteristics are nowadays widely produced through conventional breeding programmes, by selection of mature superior trees, by genetic transformation procedures, etc., which are important for increasing the productivity of forestry clonal plantations. Strategies for forest biodiversity conservation today are well defined, among which cryopreservation is viewed as a complementary storage method, important for plant tissues with specific characteristics (vegetatively propagated species). In addition, many hardwood forest trees produce recalcitrant seeds (seeds that cannot be stored for long periods under conventional conditions) that only would be stored on a long-term basis through cryopreservation. The availability of simple, reliable and cost-effective strategies for conservation of hardwood forest species (with special attention to recalcitrant species of the Fagaceae family) will be highlighted in this review. Specifically, emphasis will be addressed to the following topics: (i) medium-term conservation through slow growth storage; (ii) cryopreservation techniques; (iii) selection of explants for cryopreservation: in vivo collection of embryonic axes and dormant buds and in vitro collection of shoot tips and embryogenic cultures; and (iv) genetic stability of cryopreserved material. The limited application of cryopreservation to the development of large cryobanks of hardwood forest species also will be mentioned.
Türkiye, bitki biyoçeşitiliği ve endemizm açısından oldukça zengin bir ülkedir. Ancak son yıllarda dünyada olduğu gibi Türkiye'de de biyoçeşitlilik ve bitki germplazmı ciddi tehdit altındadır. Hem beslenme hem de tıbbi amaçlı kullanılan kültür bitkileri ve yabani ırklara ait genetik çeşitliliğin in situ veya ex situ germplazm koleksiyonları olarak korunması büyük önem arz etmektedir. Bitki biyoteknolojisinin kullanıldığı ve temelleri in vitro doku kültürü tekniklerine dayanan muhafaza stratejileri tükenme riski taşıyan türlerde başarı ile uygulanmaktadır. Bu derleme çalışmasında bitki germplazmının kısa, orta ve özellikle kriyoprezervasyon tekniği ile uzun süreli muhafazası, kriyoprezervasyonun klasik ve yeni teknikleri ile kriyoterapi gibi farklı kullanım alanları belirtilmiş ve yeni tekniklere ait çalışmalar özetlenmiştir. Abstract Turkey is a very rich country in terms of plant biodiversity and endemism. In the recent years, However, as in the World, biodiversity and plant germplasm have been under serious threatening in Turkey. As in situ or ex situ protection of genetic diversity of culture plants and their wild types used both food and medicinal is great important. Protection strategies based on in vitro tissue culture techniques and use plant biotechnology are successfully implemented to species at risk of extinction. In this review were pointed out short, mid-and especially long-term conservation via cryopreservation techniques of plant germplasm, new and classic techniques and different usage of cryopreservation such as cryotherapy, and were summarized works on new techniques.
Arpanın (Hordeum vulgare) gen merkezlerinden birisi olan Türkiye ve yakın çevresinde 2-
sıralı yabani arpa (Hordeum spontaneum) çok yaygın olarak bulunmaktadır Yabani arpa
(Hordeum spontaneum) hastalıklara dayanıklılık yönünden çok değerli bir kaynaktır. Hordeum
spontaneum ile yapılan dayanıklılık çalışmalarında ümitvar sonuçlar elde edilmiştir. Bu
derlemede Hordeum spontaneum ve hastalıklara dayanıklılık çalışmaları özetlenmiştir.
Micropropagation protocols for multiplication of highly important Eucalyptus hybrids have been achieved. Two Eucalyptus hybrids namely as FRI-5 (Eucalyptus camaldulensis Dehn x Eucalytpus tereticornis Sm) and FRI-14 (Eucalyptus torelliana F. V. Muell x Eucalyptus citriodora Hook) selected for micropropagation using nodal segments as explants from the mature trees (30 - 32 years old) for the two hybrids. These Eucalyptus hybrids were produced by Forest Research Institute, Dehradun, in the early 1960 - 70 through controlled crossing. These are available in limited number and need multiplication true to type for field - testing to ascertain its superiority under varied agro - climatic conditions. These hybrids showed 3 - 5 superior in terms of total biomass and wood volume when compared to their parents. Clonal cultures were established and multiplied on MS medium supplemented different concentration of BAP either alone or in with combination of auxins i.e. IBA/NAA. MS medium was found to be effective and suitable for all the experiments during plant production. 85 - 92% rooting was achieved on half strength MS medium supplemented with IBA. 90 - 98% of the plantlets survived hardening and acclimatization prior to field transfer. Parameters like height, DBH, clear bole length and self pruning capability of both the hybrids were recorded up to three years to identify the suitability of these hybrids in different varied climatic zones of Uttarakhand.
Cryopreservation of apices sampled on in vitro plantlets of #Poncirus trifoliata$ (L.) Raf. was achieved using the encapsulation-dehydration technique. The highest survival rates after cryopreservation (up to 50%) were obtained when encapsulated apices were pregrown for 3 d either directly in liquid medium containing 0.5 M sucrose or in media with progressively increasing sucrose concentration from 0.3 to 0.75 M, desiccated to 20-25% moisture content and frozen slowly (0.5°C/min) from +20°C to -40°C before immersion in liquid nitrogen. Recovery of whole plantlets from cryopreserved apices took place directly, without transitory callus formation. (Résumé d'auteur)
The status of genomics and genetics research in the Myrtaceae, a large family of dicotyledonous woody plants, is reviewed with Eucalyptus as the focal genus. The family contains over 5,650 species in 130 to 150 genera, predominantly of neo-tropical and Southern Hemisphere distribution. Several genera are well known for their economic importance worldwide. Myrtaceae are typically diploids with small to intermediate genome size. Microsatellites have been developed for several genera while higher throughput marker systems such as diversity arrays technology and single nucleotide polymorphism are available for Eucalyptus. Molecular data have been fundamental to current perspectives on the phylogeny, phylogeography and taxonomy of the Myrtaceae, while numerous studies of genetic diversity have been carried out particularly as it relates to endangered, rare, fragmented, overharvested or economically important species. Large expressed sequence tag collections for species of Eucalyptus have recently become public to support the annotation of the Eucalyptus grandis genome. Transcriptomics in Eucalyptus has advanced by microarrays and next-generation sequencing focusing on wood development. Linkage maps for Eucalyptus display high synteny across species and have been extensively used to map quantitative trait loci for a number of traits including growth, wood quality, disease and insect resistance. Candidate gene-based association genetics have successfully found marker–trait associations for wood and fiber traits. Genomic selection experiments have demonstrated clear potential to improve the efficiency of breeding programs while freeze-tolerant transgenic Eucalyptus trials have recently been initiated. The recently released E. grandis genome, sequenced to an average coverage of 8×, will open up exceptional opportunities to advance Myrtaceae genetics and genomics research.
A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N6-benzyladenine (BA, 2 mg l–1) and -naphthaleneacetic acid (NAA, 1 mg l–1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l–1 and NAA at 0.5 mg l–1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.
Preservation of clonal genotypes of most fruit crops requires vegetative propagation, so cryopreservation is an ideal method
for long-term germplasm storage (Engelmann 2004). Some of the first and most successful experiments with the cryopreservation
of apical meristems began with a temperate berry crop, strawberry (Kartha et al. 1980; Sakai et al. 1978). These initial studies
showed that slow cooling of <1 mm shoot tips in cryoprotectant solutions was a feasible option for long-term storage of clonally
propagated plants. During the last two decades, cryopreservation methods were developed for many species of fruit crops (Reed
2001).
Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri V. rupestris), 41B (V. vinifera V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.
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.
This paper discusses the importance of the successive steps of the vitrification technique and reviews the current development and use of vitrification and of the two derived protocols, encapsulation-vitrification and droplet-vitrification. Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Samples are thus cryopreserved without detrimental intracellular ice formation. In a standard vitrification protocol, excised explants are precultured on medium enriched with sucrose, treated (loaded) with a loading solution composed of 2 M glycerol + 0.4 M sucrose, dehydrated with a highly concentrated vitrification solution [e.g. the PVS2 vitrification solution, which contains 30 percent (w/v) glycerol, 15 percent (w/v) ethylene glycol and 15 percent (w/v) DMSO and 0.4 M sucrose], frozen and rewarmed rapidly, unloaded with basal culture medium supplemented with 1.2 M sucrose, and then transferred to standard culture conditions. In the encapsulation-vitrification technique, the explants are encapsulated in alginate beads, loaded and dehydrated with a vitrification solution before rapid immersion in liquid nitrogen. In the droplet-freezing technique, excised explants are loaded, treated with the vitrification solution and frozen in individual microdroplets of vitrification solution placed on aluminium foils, which are immersed rapidly in liquid nitrogen. These three techniques have been applied to different tissues of over 100 plant species from temperate and tropical origins and the number of cases where they are being tested on a large scale or applied routinely is increasing.
In vitro techniques have numerous applications for collecting, propagation and conservation of plant biodiversity. In vitro collecting techniques allow introducing in vitro under field conditions explants from recalcitrant seed and vegetatively propagated species. In vitro culture techniques ensure the production and rapid, large scale multiplication of disease-free material. The use of in vitro culture techniques is of great interest for the conservation of: i) genetic resources of recalcitrant seed and vegetatively propagated species; ii) biotechnology products such as elite genotypes which are multiplied on a large scale in production laboratories and plant materials with special attributes, e.g. metabolite-producing cell lines and genetically engineered material; and iii) rare and endangered plant species. Medium-term conservation is achieved by reducing the growth of the plant material, thus increasing the intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, -196°C) allows to store plant material without modification or alteration for extended time spans, protected from contaminations and with limited maintenance. Slow growth storage protocols have been developed for numerous plant species. They are routinely employed for medium-term conservation of a large number of materials, both from temperate and from tropical origin. Cryopreservation is well advanced for vegetatively propagated species and there is an increasing number of cases where the techniques are ready for large scale experimentation. Research is much less advanced for recalcitrant seed species due to some of their characteristics including their very high sensitivity to desiccation, their structural complexity and their heterogeneity in terms of developmental stage and water content at maturity. There are, nevertheless, various technical approaches to explore in order to develop cryopreservation techniques for a larger number of recalcitrant seed species. Even though the routine use of cryopreservation is still limited, the number of examples where cryopreservation is used on a large scale is increasing steadily.
Cryopreservation is a method that makes possible the preservation of living material in liquid nitrogen, in principle indefinitely and without any physiological alteration. The frozen material can be recovered and amplified in vitro if needed. The methodology is of great interest for commercial in vitro propagation laboratory, for plant breeders but could also take a wide place to maintain the genetic diversity of endangered species. AFOCEL-Biotechnology have used 2 methods to cryopreserve routinely meristems from "dormant" and non dormant buds of hardwood. The general procedure is referred to as "fast freezing" method or "encapsulation-dehydration pre treatment" for the non dormant buds and to "slow freezing method" for "dormant" buds. Regeneration was successfully obtained from Eucalyptus sp., Ulmus sp., Populus sp. This paper will describe the interest and practical applications of cryopreservation regarding the conformity of the regenerated plantlets and the cost of the methodology.
Shoot tips sampled on in vitro cultured apple plantlets of 6 accessions of #M. domestica$ and one accession of #M. robusta$ were successfully cryopreserved using the encapsulation-dehydration technique. Shoot tips were excised from plantlets which had been submitted to 3 weeks of cold-acclimation at 5°C, 70 d after their last subculture. After preculture at 5°C in media with progressively increased sucrose concentration (0.1 M, 0.3 M and 0.7 M), shoot tips were encapsulated and pregrown in medium with 1.0 M sucrose for 1 d, dehydrated for 4 h under the air current of the laminar flow cabinet, thus reaching a moisture content of around 30% (fresh eight basis) and directly immersed in liquid nitrogen. The regeneration rate of cryopreserved apices varied between 70 and 90%, depending on the accession. Using apcies sampled on plantlets which had been maintained on standard medium without subculture for 6 months, sucrose preculture became unnecessary to achieve regrowth after cryopreservation and the dehydration period was shortened. These experiments showed that the physiological state of the plant material directly affects the results and procedures for cryopreservation of apple shoot tips. (Résumé d'auteur)
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.
The effects of activated charcoal on growth and morphogenesis in plate cultures of different plant cells have been studied. It was shown that medium containing charcoal induced embryogenesis in cultures of Daucus carota in which embryo formation could not be brought about by omitting auxin from the medium. Charcoal-medium also induced abundant root formation in older cultures of Allium cepa, which normally did not produce roots. The growth of cultures of Glycine max and Haplopappus gracilis was totally inhibited by charcoal. It is thought that activated charcoal removes substances from the medium, one of which might be auxin.
The vegetative tissues of most angiosperm species, including Eucalyptus grandis, cannot withstand the excessive loss of tissue water required for successful cryopreservation without incurring extensive damage. Tolerance to water loss may be increased and the critical water content of the material depressed by the application of abscisic acid (ABA) or exposure to osmotic stress. Some plants, however, respond to such treatments by becoming increasingly resistant to water loss. This study demonstrated that the responses of E. grandis in vitro axillary buds tends towards the latter, since ABA pretreatment (5 mg/l ABA for 5 days) resulted in a significantly higher water content and higher survival in ABA-pretreated buds after 20 min drying over activated silica gel (at ambient conditions) than in non-treated buds. E. grandis buds also responded positively to a combination of osmotic preculture (0.4 M, 0.7 M and 1 M sucrose and glycerol), and 5 mg/l ABA, which resulted in the maintenance of viability for significantly longer periods at reduced water contents than sucrose and glycerol preculture alone. The results (including ultrastructural observations of the mersitem dome in treated axillary buds), therefore, demonstrated the intolerance of Eucalyptus in vitro axillary buds to excessive water loss, and explained the recalcitrant nature of the buds to cryopreparative regimes, since the lowering of water content is essential for reducing the formation of potentially lethal ice crystals.
Dormant vegetative buds from diverse species can be preserved using cryopreservation. Sakai (1960) provided one of the first studies showing that winter twigs of poplar (Populus sieboldi) and willow (Salix koriyanagi) could survive low temperatures if slowly cooled prior to immersion in liquid nitrogen. A later study demonstrated that this simple methodology was also applicable to twigs of several fruit species (Sakai and Nishiyama 1978). With rising interest in the preservation of genetic resources, methodologies were further developed for fruit, nut, forest and ornamental species that can cold acclimate. Although dormant buds from cold hardy herbaceous perennial species might also be useful for cryopreservation, there are few studies, with the exception of garlic, that addressed the use of cryopreservation to preserve dormant buds from herbaceous species. It should also be emphasized that in this chapter, we use the term “dormant” in a broad sense to include buds that are dormant due to either endogenous (endodormancy) or to a variety of environmental conditions (ecodormancy).
The Food and Agriculture Organization of the United Nations’ (FAO’s) international definition of a forest, as presented in the Global Forest Resources Assessment 2000 (GFRA-2000), sets the canopy cover requirement of 10% as the threshold value between forest and other types of land. According to the latest preliminary information of GFRA published in 2005 by the FAO, there are around 3.9 billion ha of forest in the world making up about one third of the total land area. Between 1990 and 2000 the loss of forest land was 8.9 million ha per year and between 2000 and 2005, a little less than 7.3 million ha per year (GFRA-2005; Finnish Statistical Yearbook of Forestry 2005). The area of natural forests, which accounts for 36% (i.e. 1423 million ha) of the total forest area is decreasing around 6% per year (GFRA-2005).
Today forests are managed for many different purposes. Approximately one third are used to produce construction timber, pulp, paper and other forest products. Maintaining biodiversity was the priority in 11% (i.e. 443 million ha) of the forests, and the area of these forests has increased by 96 million ha since 1990. Around 9% of the global forests are intended for different protection purposes, such as soil, groundwater or coast protection, to prevent desertification or to control avalanches (GFRA-2005). However, in Africa 545 million m3 of wood is annually consumed for fuel which is more than six times the amount of wood consumed for all the other purposes (FAO 2005).
In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength
MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each
step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h.
After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved
shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and
cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved
shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected
survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot
elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength
MS supplemented with 1 mg l−1 BA and 0.1 mg l−1 NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and
cv. Superior, respectively.
Cryopreservation is a very important tool for the long-term storage of plant genetic resources for future generations, requiring
only a minimum of space and maintenance. With increasing interest in the genetic engineering of plants, the preservation of
cultured cells and somatic embryos with unique attributes is assuming greater importance. Recently, cryopreservation was reported
to offer real hope for enhancing the preservation of endangered and rare plants (Touchell 1995; Touchell and Dixon 1996).
This chapter describes protocols for successful vitrification using plant vitrification solution 2 (PVS2), and highlights
some of the factors contributing to high levels of post-LN recovery. The development of a simple and reliable method for cryopreservation
would allow the widespread storage of cultured cells, meristems, and somatic embryos. Vitrification involving vitrification
solutions (Langis et al. 1990; Sakai et al. 1990; Yamada et al. 1991) and encapsulation-dehydration techniques (Fabre and
Dereuddre 1990) were developed in the 1990s, and the number of cryopreserved species has increased markedly since then (Sakai
1995, 1997; Engelmann and Takagi 2000). A vitrification procedure using an ethylene glycol-based vitrification solution and
French straws was presented by Steponkus and colleagues. They reported successful cryopreservation of Dianthus and Chrysanthemum (Langis et al. 1990; Schnabel-Preikstas et al. 1992), and potato (Lu and Steponkus 1994). This chapter will outline the development
and uses of PVS2 developed by Sakai and associates.
Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the
encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure
to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium
containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips
(83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties
and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step
freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment
of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose
and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar.
Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.
Embryogenic cells of asparagus (Asparagus officinalis L.) were successfully cryopreserved by vitrification and subsequently regenerated plants. The cells were cryoprotected with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C for 10 min and then transferred to 1.8-ml plastic cryotubes. The cryoprotected cells were dehydrated with a new vitrification solution (designated PVS3) at 0°C for 20 min prior to a plunge into LN2. PVS3 contains 50% (w/v) glycerol and 50% (w/v) sucrose in water. The vitrified cells in LN2 were rapidly warmed in a water bath at 40°C and then expelled into LS medium supplemented with 1.2 M sucrose. The cell survival evaluated by fluoresceine diacetate and phenosafranin amounted to 80–90% of untreated, unfrozen controls. Revived cells resumed growth within 3 days after plating and regenerated plantlets via embryogenesis. Plant regeneration efficiency was nearly the same as that of the untreated, unfrozen control. Even an 80% concentration of PVS3 diluted with water produced 80% survival.
This paper presents preliminary results from a study to develop methodology for the cryopreservation of axillary buds from an in vitro hybrid Eucalyptus grandis (W. Hill ex Maiden.) x Eucalyptus camaldulensis (Dehnnh.), maintained for use in a genetic modification programme. Axillary buds were encapsulated in an alginate gel, precultured on media containing elevated levels of sucrose, or a combination of sucrose and glycerol. Encapsulated buds were then dehydrated by evaporation prior to a two-step freezing process in liquid nitrogen. Eighteen percent of shoot explants survived freezing when sucrose alone was used as a protectant against dehydration and cryopreservation. Significantly higher survival (49%) was obtained with the incorporation of glycerol into the protocol. Following cryopreservation, shoots appeared to develop normally, with no evidence of adventitious meristems.
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