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United States Department of Agriculture

Agricultural Research Service

Research Project: MANAGEMENT OF TEMPERATE FRUIT NUT AND SPECIALTY CROP GENETIC RESOURCES Title: Genetic Stability of Cryopreserved Shoot Tips of Rubus Germplasm

Authors
item Reed, Barbara
item Castillo, N.R.F. -
item Wada, Sugae -
item Bassil, Nahla

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 25, 2009
Publication Date: January 20, 2010
Citation: Reed, B.M., Castillo, N., Wada, S., Bassil, N.V. 2010. Genetic Stability of Cryopreserved Shoot Tips of Rubus Germplasm. In Vitro Cellular and Developmental Biology - Plants. 46:246-256.

Interpretive Summary: Scientists want to know if plants are genetically stable when stored in liquid nitrogen. This study examined the genetic stability of blackberry and raspberry shoot tipsthat were stored in liquid nitrogen for over 12 years, then rewarmed and regrown. We analyzed the genetic stability of the plants after regrowth in tissue culture, transplanting to the greenhouse and transfer to the field. No visual differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants were more vigorous than mother plants as is common for plants derived from tissue cultures. Two genetic analyses were performed on shoots immediately after recovery from cryopreservation and on shoots tissue cultured for seven months before analysis. The analysis revealed slight differences in three of the four berry types cultured for seven months while no change was detected in those analyzed immediately after recovery from cryopreservation. Greenhouse and field-grown plants from the shoot tips that showed genetic changes while in culture had no differences when compared to their corresponding mother plants. Only long-cultured in vitro shoot tips displayed differences and they were no longer detected when the plants were grown in the greenhouse or field. The temporary nature of these genetic changes shows that for genetic stability is maintained by storage in liquid nitrogen.

Technical Abstract: Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study followed the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for over 12 years, then rewarmed and regrown. We analyzed the genetic stability of R. grabowskii, two blackberry cultivars (‘Hillemeyer’ and ‘Silvan’) and one raspberry cultivar (‘Mandarin’) after regrowth in vitro, transplanting to the greenhouse and transfer to the field. No phenotypic differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants were more vigorous than mother plants as is common for plants derived in vitro. SSR and AFLP analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for seven months before analysis. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants irrespective of subculture. AFLP revealed polymorphisms in three of the four Rubus genotypes subcultured for seven months while no polymorphism was detected in those analyzed immediately after recovery from cryopreservation. Greenhouse and field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.

Last Modified: 12/20/2014
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