Reducing Pre- and Post-Treatments in Cryopreservation Protocol and Testing Storage at −80 °C for Norway Spruce Embryogenic Cultures

Varis, Saila A.; Virta, Susanna; Montalbán, Itziar A.; Aronen, Tuija

doi:10.3390/ijms232415516

Cited by 5 publications

(3 citation statements)

References 43 publications

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“…Following the preconditioning treatment, the tissue is exposed to a cryoprotective solution usually composed by cryoprotective penetrating substances as DMSO (added to a final concentration of 5 to 15%, Panis and Lambardi, 2006 ), or a mixture of various penetrating [e.g., proline and glycerol, Cryptomeria japonica D. Don ( Taniguchi et al., 2020 )] and non-penetrating [e.g., high molecular mass PEG, Picea abies ( Varis et al., 2022 )] substances. This step lasts from minutes ( Picea sitchensis (Bong.)…”

Section: Cryopreservation Of Embryogenic Materials In Woody Speciesmentioning

confidence: 99%

“…An interesting alternative to the conservation in LN has been proposed by NEIKER group which maintained a collection of over one hundred cells lines of Pinus radiata at -80°C ( Montalbán and Moncaleán, 2017 ). However, attempts to replicate it in Picea abies have been unsuccessful ( Varis et al., 2022 ). As mentioned previously, SE in conifers is not feasible from adult tissues, so cryobanks are still necessary for the deployment of multivarietal forestry, to maintain the cell lines while the somatic trees are evaluated in the field ( Corredoira et al., 2014 ).…”

Section: Cryobanks Of Embryogenic Materialsmentioning

confidence: 99%

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Current status of the cryopreservation of embryogenic material of woody species

Ballesteros,

Martínez,

Sánchez-Romero

et al. 2024

Front. Plant Sci.

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Cryopreservation, or the storage at liquid nitrogen temperatures (-196°C), of embryogenic cells or somatic embryos allows their long-term conservation without loss of their embryogenic capacity. During the last decade, protocols for cryopreservation of embryogenic material of woody species have been increasing in number and importance. However, despite the large experimental evidence proved in thousands of embryogenic lines, the application for the large-scale conservation of embryogenic material in cryobanks is still limited. Cryopreservation facilitates the management of embryogenic lines, reducing costs and time spent on their maintenance, thus limiting the risk of the appearance of somaclonal variation or contamination. Somatic embryogenesis in combination with cryopreservation is especially useful to preserve the juvenility of lines while the corresponding clones are being field-tested. Hence, when tree performance has been evaluated, selected varieties can be propagated from the cryostock. The traditional method of slow cooling or techniques based on vitrification are mostly applied procedures. For example, slow cooling methods are widely applied to conserve embryogenic lines of conifers. Desiccation based procedures, although simpler, have been applied in a smaller number of species. Genetic stability of the cryopreserved material is supported by multiloci PCR-derived markers in most of the assayed species, whereas DNA methylation status assays showed that cryopreservation might induce some changes that were also observed after prolonged subculture of the embryogenic lines. This article reviews the cryopreservation of embryogenic cultures in conifers, fruit species, deciduous forest species and palms, including a description of the different cryopreservation procedures and the analysis of their genetic stability after storage in liquid nitrogen.

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Section: Cryopreservation Of Embryogenic Materials In Woody Speciesmentioning

confidence: 99%

Section: Cryobanks Of Embryogenic Materialsmentioning

confidence: 99%

Current status of the cryopreservation of embryogenic material of woody species

Ballesteros,

Martínez,

Sánchez-Romero

et al. 2024

Front. Plant Sci.

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“…In 2011, the two-day pre-treatment method in liquid mLM media described by Kartha [35] and modified by Find et al [36] was applied to cryopreserve six samples from every 260 cell lines. From 2012 to 2019, a two-day pre-treatment method in semi-solid media was used as described in Varis et al [34], and in 2022, the pre-treatment method was simplified to a one-day dehydration in semi-solid media [37]. After pre-treatment, 1.5 mL suspended ET was placed in a 2 mL sterile cryovial, or 200 mg of ET from semi-solid media was placed in sterile cryovials containing 400 mL of liquid mLM medium with 0.4 M of sucrose without plant growth regulators (PGR) or glutamine, after which 400 mL of preservative mixture containing polyethylene glycol 6000, glucose, and DMSO 10% w/v each were added.…”

Section: Initiation Proliferation and Cryopreservationmentioning

confidence: 99%

How to Capture Thousands of Genotypes—Initiation of Somatic Embryogenesis in Norway Spruce

Varis

Tikkinen

Edesi

et al. 2023

Forests

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Somatic embryogenesis (SE) is considered the most effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). When the aim is commercial production, the process needs scaling up. This includes many initiations to increase the number of available genotypes in the cryo-bank. Numerous genotypes are needed to maintain genetic diversity in reforestation and, at the same time, are a prerequisite for the efficient improvement of breeding traits. Norway spruce is also highly susceptible to Heterobasidion root rot. We analysed the data from the SE initiations of Norway spruce from six different years, including a total of 126 families and almost 13,000 initiations, and used several genetic (including allele PaLAR3B improving Heterobasidion resistance), environmental, and operational variables to explain the initiation success and the number of cryopreserved embryogenic tissue (ET). Overall, the cone collection date was the best and most comprehensive single variable for predicting the initiation success and the number of cryopreserved ET in the logistic regression models. PaLAR3B allele did not interfere with SE initiation or the cryopreservation. In the optimal scenario, according to the current data, Norway spruce cones would be collected in southern Finland during the first two weeks of July (in approximately 800 d.d. accumulation) from the seed orchard or greenhouse and delivered quickly to the laboratory, and the cones would be cold-stored for five days or less before initiations on mLM media. Lower initiation frequencies in some families can be compensated by increasing the number of explants—however, taking operational limitations into account.

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Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

Nzama,

Myburg,

Hills

2024

Plant Cell Tiss Organ Cult

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Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH4+:NO3− medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH4+:NO3− molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH4+:NO3− and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

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Reducing Pre- and Post-Treatments in Cryopreservation Protocol and Testing Storage at −80 °C for Norway Spruce Embryogenic Cultures

Cited by 5 publications

References 43 publications

Current status of the cryopreservation of embryogenic material of woody species

Current status of the cryopreservation of embryogenic material of woody species

How to Capture Thousands of Genotypes—Initiation of Somatic Embryogenesis in Norway Spruce

Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

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