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United States Department of Agriculture

Agricultural Research Service

Research Project: RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS Title: Cryopreservation of embryogenic cell lines of Araucaria angustifolia (Bert.) O. Kuntze

Authors
item Pieruzzi, Fernanda -
item Dos Santos, Andre -
item Walters, Christina
item Floh, Eny -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 24, 2011
Publication Date: N/A

Technical Abstract: Brazilian pine (Araucaria angustifolia) is native to the Atlantic Rainforest of Brazil and is an endangered species. Mature embryos of this conifer are large (about 3 cm in length), contain more than 1 g H2O/ g dry mass, and are killed by drying. These morphological and physiological traits make it difficult to preserve whole embryos, precluding genetic improvement or ex situ conservation efforts. Cryopreservation of embryogenic cell lines (ECL) presents an alternative strategy for preserving germplasm of tree species. Through somatic embryogenesis, thawed ECL can be used to regenerate hundreds of individuals. In this work, we report the development of an efficient cryoprotection protocol for ECL of A. angustifolia adapted from procedures described by Hargreaves (1995) for Monterey pine (Pinus radiata). The procedure requires optimizing ME2SO (dimethyl sulfoxide) concentrations to minimize its toxic effects and maximize its cryoprotective effects. Toxic effects were evaluated in ECL that were pretreated with 0.4 M sorbitol and then exposed to medium containing 0.4 M sorbitol and 0 to 20% ME2SO for 24 h. Growth of ECL on fresh semi-solid MSG medium was monitored weekly by change of fresh mass. The original mass of ECL exposed to < 10% and > 10% ME2SO increased by about 500% and 100%, respectively, over a 5 week period. Cryoprotectant ability of different concentrations of ME2SO was measured in similarly-treated ECL cultures that were immersed and stored in liquid nitrogen for 72 h then thawed at 40°C for 4 minutes. Only ECL treated with 20% ME2SO showed significant recovery within 5 weeks post-thawing, and growth rate was comparable to counterparts not exposed to liquid nitrogen. Growth of ECL treated with 15 and 20% ME2SO and exposed to liquid nitrogen continued to increase after 6 weeks of culture, suggesting that cryopreservation will delay yield of ECL by a few weeks. Considering the faster recovery following liquid nitrogen exposure, a cryoprotectant protocol using 20% ME2SO seems to be the most promising for the cryopreservation of ECL of A. angustifolia.

Last Modified: 12/21/2014
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