Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5762112 | Industrial Crops and Products | 2017 | 10 Pages |
Abstract
Clonal breeding programs of the highly important interspecific hybrid Pinus elliottii var. elliottii and Pinus caribaea var. hondurensis require the establishment of robust cryopreservation protocols, with no genetic variability. Embryonal masses of this hybrid were successfully cryopreserved by a fast slow-freezing method. The protocol was optimized by testing the effects of dimethyl sulfoxide (DMSO) based cryoprotectant solutions, sugar cryoprotectants (e.g. sucrose or maltose, 0.4Â M) and different times of pretreatments and pre-cooling storage. The addition of DMSO in a mixture of polyethylene glycol (PEG) 4000 and sucrose (PSD solution), instead of DMSO alone, was beneficial for recovery of cryopreserved cultures. This parameter, together with the time of pre-cooling storage (before plunging in liquid nitrogen), were the factors that most influenced the survival and regrowth rates of embryonal masses. A pretreatment combination of sucrose (0.4Â M) and 5% PSD followed by a pre-cooling storage of 24Â h allowed the cryopreservation and regrowth of embryogenic cell lines without major genetic variations (DNA-ploidy) or loss of embryogenic potential. Eight of the nine tested embryogenic cell lines survived to the cryopreservation procedures, however the genotype and treatment conditions clearly influenced the response. Somatic embryos maturation and conversion from recovered embryonal masses (EMs) took place according to our standard somatic embryogenesis protocol for Pinus, using modified Litvai (mLV) medium. This is the first report for this hybrid, and the proposed protocol is fast, robust, providing high rates of recovery and working with several genotypes. This protocol's suitability for industrial breeding programmes was also confirmed by allowing the regeneration of plants with no apparent variability from cryopreserved embryonal masses.
Keywords
RAPDFWIWPBMVFMLVECLCryoprotectantPPFDPGRCPPUFCMBAPSSF2,4-D2,4-dichlorophenoxyacetic acid6-BenzylaminopurineDMSORandomly amplified polymorphic DNAPlant growth regulatorsGenetic variabilitySomatic embryogenesissomatic embryosDimethyl sulfoxideCryopreservationDispersion indexCoefficient of Variationphotosynthetic photon flux densityFlow cytometryCSFfresh weightPropidium iodidepolyethylene glycolPEG
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Authors
Sandra Nunes, Liliana Marum, Nelson Farinha, Vanessa Tolentino Pereira, Tânia Almeida, Maria Celeste Dias, Conceição Santos,