Successful cryopreservation of Quercus robur plumules |
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Authors: | Chmielarz Paweł Michalak Marcin Pałucka Małgorzata Wasileńczyk Urszula |
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Institution: | (1) Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 K?rnik, Poland;(2) Kostrzyca Forest Gene Bank, Miłk?w, 300, Poland |
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Abstract: | Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant
seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose,
followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and
cooling (in slush at –210°C or in vials filled with liquid nitrogen, LN, −196°C), and were then cryostored for 24 h. High
percentage of survival was obtained after cryostorage (21–67%, depending on pretreatment, assessed in vitro by greening plumules
that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation
(58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5–18%. The extent of plumule
desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5–0.6
g H2O g−1 dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after
cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation
over silica gel and direct cooling in vials with LN (survival 51–76%, regrowth 8–20%). Normal plants developed from the recovered
shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species. |
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