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A – recovery of cryopreserved embryogenic tissues of Pinus nigra (cell line E 196) one month after thawing. B – bipolar somatic embryos observed in cryopreserved embryogenic tissue (cell line E 196) Bar= 100 µm. C -developing precotyledonary somatic embryos in recovered tissue on maturation medium (cell line E 184) Bar= 1 mm. D – cotyledonary somatic embryos in recovered embryogenic tissue after 8 weeks on maturation medium (E 184) Bar= 1.2 mm. E – plantlets regenerated from cryopreserved embryogenic tissue (E 184) Bar= 7 mm.
Source publication
Six different embryogenic cell lines of Pinus nigra Arn. have been cryopreserved in liquid nitrogen using cryoprotection with sucrose (18%) and DMSO (7.5%). Post-thaw growth and tissue proliferation have been observed in five cell lines. The survival levels after storage in liquid nitrogen reached values between 62.5 and 100%. Growth of recovered e...
Contexts in source publication
Context 1
... the six cell lines started to grow after one week of culture. Initially, growth was very slow and massive proliferation occurred only after two weeks of cultivation ( Fig. 2A). Although all six cell lines under investigation resumed growth after cryopreservation, cell line E 190 stopped growth during the second week of culture and no further proliferation could be observed. We evaluated the effect of cryoprotection and cryopreservation on the recovery of tissues in terms of percentage of cultures that ...
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... cell lines showed features typical of embryogenic tissues, including white color and mucilaginous consistency. Also, microscopic observation revealed that the somatic embryo morphology after cryostorage was not altered due to cryopreservation; bipolar somatic embryos consisted of an embryonic "head" and a suspensor with long vacuolated cells (Fig. 2). The regrowing tissues were also similar in appearance compared to the non- cryoprotected, non-frozen control (control 2). Both showed intensive proliferation and vigorous growth as observed under a dissection microscope. Growth parameters such fresh and dry mass increase two weeks after inoculation were very similar and no ...
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... nigra Arn. at day 14. The values (%) are calculated from day 0 (=100%). Two repetitions each containing four or five Petri dishes. For somatic embryo maturation studies only recovered cell lines that contained well structured somatic embryos (as shown in Fig. 2B) were selected (E 184, E 193, E 196). On the maturation medium the somatic embryos gradually developed and as a result precotyledonary (Fig. 2C) somatic embryos could be observed around the fifth week of culture. They occurred all over the surface of the regenerated tissue. Around the 7 th week of culture cotyledonary somatic embryos ...
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... from day 0 (=100%). Two repetitions each containing four or five Petri dishes. For somatic embryo maturation studies only recovered cell lines that contained well structured somatic embryos (as shown in Fig. 2B) were selected (E 184, E 193, E 196). On the maturation medium the somatic embryos gradually developed and as a result precotyledonary (Fig. 2C) somatic embryos could be observed around the fifth week of culture. They occurred all over the surface of the regenerated tissue. Around the 7 th week of culture cotyledonary somatic embryos characterized by several cotyledons appeared (Fig. 2D). Cotyledonary somatic embryos developed in all the three cell lines although in different ...
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... 196). On the maturation medium the somatic embryos gradually developed and as a result precotyledonary (Fig. 2C) somatic embryos could be observed around the fifth week of culture. They occurred all over the surface of the regenerated tissue. Around the 7 th week of culture cotyledonary somatic embryos characterized by several cotyledons appeared (Fig. 2D). Cotyledonary somatic embryos developed in all the three cell lines although in different quantity. The amount of embryos calculated per Petri dish varied, depending on the cell line; 24 ± 3.3 for E 193, 50 ± 4.9 for E 196 and 54 ± 4.6 for E 184. The cotyledonary embryos were allowed to germinate in the dark and after hypocotyl and ...
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... germinated plantlets were transferred to light. The germination frequencies reached 70.5%, 10.8% and 61.6% for cell lines E184, E193 and E196, respectively. After germination, normal plants were regenerated which showed the same phenotype compared to the plants obtained through somatic embryogenesis without an intervening cryopreservation phase (Fig. 2E). ...
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Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and...
Embryogenic tissues of a conifer species Pinus nigra Arn. have been cryopreserved by a slow-freezing method. The effect of cryoprotective compounds such as maltose, sucrose or
sorbitol (each 0.5M) combined with 7.5% (v/v) DMSO has been tested. After thawing, the following parameters have been investigated:
tissue regrowth 6weeks after thawing, and...
Citations
... It may also concern unfavourable biotic [9] and abiotic [64,65] factors affecting trees. In previous experiments altogether, 25 cell lines of P. nigra were selected with the aim to test their regeneration ability after cryopreservation [66,67]. Subsequently, the regenerated tissues were used in maturation experiments using abscisic acid (94 µM) and maltose 6 to 9% [45]. ...
Background
Cryopreservation makes it possible to preserve plant biodiversity for thousands of years in ex situ storage. The stepwise dehydration method is a simple and versatile cryopreservation technique based on the vitrification phenomenon. However, the commonly used dimethyl sulfoxide (DMSO) in this cryopreservation technique is considered harmful for plant material, thus alternative methods are needed to be applied.
Results
In this study, the possibility of cryopreservation of embryogenic tissues (ETs) of Abies alba x A. numidica and Pinus nigra was investigated. Before freezing, ETs were partially dehydrated in the presence of increasing concentrations of sucrose (from 0.25 to 1.0 M) for 7 days, followed by desiccation of the tissues over silica gel for 2 and 2.5 h, respectively. After these pretreatments, the plant material was frozen in liquid nitrogen (LN; –196 °C). For both coniferous trees the ET survival rate was high and reached 84.4% for A. alba x A. numidica (28 days) and 86.7% for P. nigra (35 days) after recovery of the tissues from liquid nitrogen (LN). The regenerated tissue of A. alba x A. numidica was characterized by more intense growth after storage in LN compared to tissue that had not been cryopreserved (control). The tissue of this tree also undertook relatively rapid growth after thawing from LN. In turn, the ET growth of P. nigra was significantly lower after thawing compared to the other treatment.
Conclusions
The present study demonstrated, that the stepwise dehydration method could be successfully applied to the cryostorage of ETs of both studied trees. To the best of our knowledge, this is the first report on ET cryopreservation based on this method for Abies and Pinus genus representatives, which may be the alternative way for efficient, long-term preservation of germplasm in LN.
... The choice of the technique depends on the material type and characteristics. Among these methodologies, the most recommended for the cryopreservation of an embryogenic callus suspension culture is the two-step, slow-freezing method, which has been widely reported for species of conifers [7,8,35], bananas [36], and herbaceous and hardwood species [5]. No works have been reported for bamboos species, emphasizing the importance of researching cryopreservation strategies for this group of plants. ...
... Indeed, the time of exposure varies among species, and optimization is necessary for the success of the cryopreservation procedure. Salaj et al. [35] reported a 60 min incubation for embryogenic cultures of Pinus Nigra using a cryoprotective solution with 18% sucrose and 7.5% DMSO, followed by slow-freezing, while Lambardi et al. [5] proposed that 30 min of incubation presented the highest callus regrowth when compared to 60 and 90 min of incubation in the two-step freezing cryopreservation of Cypress embryogenic masses using a solution containing 180 g·L −1 of sucrose and 7.5% DMSO; they also reported that conversion rates were not affected when compared to the control. Based on our phytotoxicity test, we used 60 and 120 min incubation times in our further cryopreservation procedure. ...
... Additionally, the highest viability was observed 60 days after regrowth, suggesting that our protocol allowed for the successful cryopreservation of ECs of G. chacoensis. The FDA test is commonly used to verify cell viability during cryopreservation [7,18,34,35]. In this assay, cell wall esterase cleaves fluorescein diacetate and fluoresce green. ...
This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs’ ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.
... For conifer embryogenic tissues successful cryopreservation has been reported in Abies nordmanniana (Norgaard et al., 1993a), A. cephalonica (Aronen et al., 1999;Misson et al., 2006;Krajňáková et al., 2011), A. alba (Krajnaková et al., 2013, A. fraseri (Pullman et al., 2016), hybrids Abies alba x A. cephalonica and Abies alba x A. numidica (Salaj et al., 2010;Salaj et al. 2016), Araucaria angustifolia (Fraga et al., 2016), Picea abies (Gupta et al., 1987;Norgaard et al., 1993b;Vondráková et al., 2010), P. glauca engelmanni (Cyr et al., 1994), P. mariana (Klimaszewska, 1995), P. sitchensis (Find et al., 1993;Gale et al., 2007), Pinus caribaea (Laine et al., 1992), P. nigra (Salaj et al., 2007), P. patula (Ford et al., 2000), P. radiata (Hargreaves et al., 2002), P. sylvestris (Häggman et al., 1998), P. roxburghii (Mathur et al., 2003), P. pinaster (Marum et al., 2004;Lelu-Walter et al., 2006;Alvarez et al., 2012), Pinus elliottii x P. caribaea (Nunez et al., 2017), Torreya taxifolia (Ma et al., 2012), Taxus x media, T. floridana (Skrlep et al., 2008), Tsuga canadensis, T. caroliniana (Merkle et al., 2014), Chamaecyparis thyoides (Ahn et al., 2017). ...
Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.
... For conifer embryogenic tissues successful cryopreservation has been reported in Abies nordmanniana (Norgaard et al., 1993a), A. cephalonica (Aronen et al., 1999;Misson et al., 2006;Krajňáková et al., 2011), A. alba (Krajnaková et al., 2013, A. fraseri (Pullman et al., 2016), hybrids Abies alba x A. cephalonica and Abies alba x A. numidica (Salaj et al., 2010;Salaj et al. 2016), Araucaria angustifolia (Fraga et al., 2016), Picea abies (Gupta et al., 1987;Norgaard et al., 1993b;Vondráková et al., 2010), P. glauca engelmanni (Cyr et al., 1994), P. mariana (Klimaszewska, 1995), P. sitchensis (Find et al., 1993;Gale et al., 2007), Pinus caribaea (Laine et al., 1992), P. nigra (Salaj et al., 2007), P. patula (Ford et al., 2000), P. radiata (Hargreaves et al., 2002), P. sylvestris (Häggman et al., 1998), P. roxburghii (Mathur et al., 2003), P. pinaster (Marum et al., 2004;Lelu-Walter et al., 2006;Alvarez et al., 2012), Pinus elliottii x P. caribaea (Nunez et al., 2017), Torreya taxifolia (Ma et al., 2012), Taxus x media, T. floridana (Skrlep et al., 2008), Tsuga canadensis, T. caroliniana (Merkle et al., 2014), Chamaecyparis thyoides (Ahn et al., 2017). ...
Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the
accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the
cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.
... Somatic embryogenesis technology is usually associated with cryopreservation, which offers an appropriate tool to overcome these problems since all the metabolic and physical processes are arrested and require minimal equipment and maintenance [112]. Additionally, cryopreservation can establish dormancy and help enable massive clonal propagation [18,95]. ...
The peculiar characteristics of conifers determine the difficulty of their study and their great importance from various points of view. However, their study faces numerous important scientific, methodological, cultural, economic, social, and legal challenges. This paper presents an approach to several of those challenges and proposes a multidisciplinary scientific perspective that leads to a holistic understanding of conifers from the perspective of the latest technical, computer, and scientific advances. This review highlights the deep connection that all scientific contributions to conifers can have in each other as fully interrelated communicating vessels.
... The present investigation reveals a primary role of the genotype on culture response to cryopreservation. Differences among genotypes could be due to differences in the nature of embryogenic tissues related to the proportion of hyperhydric vacuolated cells within them [37], to the physiological conditions of tissues [38], or to the physiological response triggered by cryopreservation, which may involve the activity of specific cell wall-plasma membrane proteins [39] or the activation of enzymes protecting against oxidative stress [40]. ...
Olive somatic embryos have been successfully cryopreserved using the droplet-vitrification method on aluminum foil strips. Although acceptable recovery rates have been obtained after rewarming, the influence of this cryopreservation protocol on the somatic embryogenesis process is unknown. To evaluate the effect of cryopreservation on olive somatic embryogenesis, the behavior of cultures established from cryopreserved somatic embryos was compared with that of control, non-cryopreserved cultures in the different phases of the somatic embryogenesis process. In order to analyze the influence of the genotype, this investigation was carried out in two independent lines. During the proliferation step, only the line T1 was affected by cryopreservation, with higher fresh weight increases. Although similar total embryos were produced per culture, freezing in liquid nitrogen significantly improved the maturation pattern in the line P5. Better germination results were also found in this embryogenic line. The genotype plays a key role, largely determining the effect of cryopreservation on olive somatic embryogenesis. A specific genotype-dependent response was found depending on the culture step. Variations observed could not be associated to differences in the embryogenic lines’ instability to maintain their morphogenic competence after cryopreservation. Embryogenic cultures established after rewarming retained their regeneration capacity, with no evident negative effects affecting their regeneration capacity.
... Similar attempt was reported by Latta in 1971 for Daucus carota cells however there was an additional cryogenic storage step after cooling the cells to -40°C at a cooling rate of 2-4°C min -1 . The slow freezing technique which then involves a slow cooling of plant tissues to a prior defined freezing temperature which then followed the immersion in liquid nitrogen was optimized for vast number of species (Panis et al., 1990;Salaj et al., 2007;Salaj et al., 2010). Depending upon the rate of cooling and the pre-freezing temperature, different amounts of water will leave the cell before the intracellular contents solidify. ...
The Food and Agriculture Organization predicts that the global population
will reach 9.2 billion by 2050, and there will be high demand for a large
quantity of foods. With limited farming land and freshwater resources,
traditional systems cannot cater to the increasing demand. Adaptation of
modern technology is one of the alternatives that can be used by the
farmers to increase their production in a sustainable manner. However, the
poor knowledge on the modern technology is one of the limitations for
technology adoption. Therefore, the main purpose of this chapter is to
introduce modern technologies that can be used in the farming to improve
the productivity and sustainability of the agricultural production.
Technologies developed in the last century have created to huge
improvement in land preparation, crop management, harvesting and
breeding. Modern technologies like nano technology, sensor technology,
molecular markers, remote sensing, wireless sensor networks, geographic
information system (GIS), Global Positioning System (GPS), drones,
photovoltaic technology, and information and communication technology
etc are discussed in this chapter.
... After thawing in a water bath of 40°C, cells are plated out on semi-solid culture. This protocol (or adaptations to it) is still widely being used for the long-term conservation of embryogenic cultures of a wide range of unorganized plant tissues such as embryogenic cell suspensions banana (Panis et al., 1990) en embryogenic calli of Abies (Salaj et al., 2010) and Pinus (Salaj et al., 2007). The main reason why this method is less successful with organized plant tissues is that it results in a relatively severe dehydration leading to breakage of the plasmodesmata. ...
... Similarly, during the post-thaw period the structure of somatic embryos was recovered and somatic embryos showing similar organization prior to cryopreservation were observed in regrown tissue. The cryopreserved tissue was capable of maturation and somatic seedling development (Salaj et al., 2007b). No correlation was found between maturation capacity of cell lines and their cryotolerance . ...
Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.
... Although SEC has been successfully cryopreserved using various cryogenic procedures, two-step cooling was the most often applied cryoprocedure (Table 2). In this strategy, a solution containing sucrose (0.3-0.5 M) or sorbitol (0.3-0.8 M) and DMSO (5.0-7.5%) was frequently used to treat samples (Salaj et al. 2007(Salaj et al. , 2010(Salaj et al. , 2011Á lvarez et al. 2012). Use of maltose (0.4-0.5 M) (Marum et al. 2004;Salaj et al. 2011;Latutrie and Aronen 2013), or 0.4-0.8 ...
... M sorbitol (Touchell et al. 2002;Gale et al. 2008) or polyethyleneglycol (PEG, 10-20%) (Marum et al. 2004;Á lvarez et al. 2012) or 10 lM ABA (Hazubska-Przybył et al. 2013) was found to improve recovery rates of SEC (Latutrie and Aronen 2013). Cryoprotected samples were usually pre-frozen to -30 to -40°C, followed by immersion in LN for cryostorage (Maruyama et al. 2000;Marum et al. 2004;Gale et al. 2007Gale et al. , 2008Salaj et al. 2007;Latutrie and Aronen 2013). In encapsulation-dehydration cryopreservation, embryogenic masses were encapsulated into calcium alginate beads (4-5 mm in diameter), followed by preculture in sucrose-enriched medium (0.4-0.75 M) for hours (18 h) to days (3 days) and subsequent desiccation to 17-35% water content, prior to a direct immersion in LN for cryostorage (Fernandes et al. 2008;Gale et al. 2008). ...
... The nurse tissue consisted of a vigorously growing cell line of Pinus radiata that was genetically distinct from all of the thawed lines. Genotypes (Gale et al. 2008;Salaj et al. 2007Salaj et al. , 2010Salaj et al. , 2011Á lvarez et al. 2012; Latutrie and Aronen 2013), developmental stages (Valladares et al. 2004;Gale et al. 2008), culture age (Find et al. 1998;Gale et al. 2008;Latutrie and Aronen 2013) and cell density (Find et al. 1998;Marum et al. 2004) of SEC were found to affect success of cryopreservation. A comprehensive study was conducted by Gale et al. (2008), who tested encapsulationdehydration and vitrification for cryopreservation of four genotypes (A1-A5, C1-C5, D1 and D5) of somatic embryos at the globular and torpedo stages of Picea sitchensis. ...
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.
