Genetic stability of cryopreserved shoot tips of Rubus germplasm

Abstract

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars ("Hillemeyer" and 'Silvan'), and one raspberry cultivar ("Mandarin") as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from "Marion" and "Meeker" microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and 'Silvan' displayed the same AFLP fingerprints as their corresponding mother plants. Only longcultured in vitro shoot tips displayed polymorphisms in vitro, 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.

Phenotypic and Molecular Characterization of <i>Phaseolus vulgaris</i> Plants from Non-Cryopreserved and Cryopre-served Seeds

“…Similar results were found in many plant species using a variety of different molecular markers including SSR [18,19].…”

Fundamental Cryobiology and Basic Physical, Thermodynamical and Chemical Aspects of Plant Tissue Cryopreservation

“…However, differences in band intensities among some but not all bands might indicate structural rearrangements in DNA caused by different types of DNA damage (Danylchenko and Sorochinsky, 2005) that might not be readily detected using the given system. The RAPD technology has previously been used successfully to detect occurrence of genetic alterations (Finkle et al, 1985;Harding, 1997;Aronen et al, 1999;Ahuja et al, 2002;Urbanova´ et al, 2005;Castillo et al, 2010), but this approach possesses limits with reproducibility, and it is currently being replaced by techniques such as Amplified Fragment Length Polymorphisms (AFLPs) and/or Simple Sequence Repeats (SSRs, microsatellites). These techniques are now being used to consider more carefully the issue of genetic fidelity after cryo-procedures, especially in the breeding of long-living conifers (Salaj et al, 2010), where genetic changes might be substantially expressed only later on, in mature trees.…”

“…Currently, efforts are being made to simplify this process by exploring new methods of cryopreservation in both angiosperms and gymnosperms (Touchell et al, 2002;Gale et al, 2008;Popova et al, 2009;Yin and Hong, 2010). DMSO was found to cause genetic alteration under some conditions by Vannini and Poli (1983) and DNA damage and/or rearrangements in some cases viz., Abies cephalonica (Aronen et al, 1999), Solanum tuberosum L. (Harding, 1997) and Rubus grabowskii (Castillo et al, 2010) This effect of DMSO is usually explained by its effect on membrane permeability and function, thermostability of chromosome structure, or inhibition of DNA synthesis. The addition of abscisic acid to in vitro stock plants has been found to improve cryopreservability for cold hardy species (Ryyanen, 1998) but in general, cryopreservation procedures have been shown to be species specific.…”

<b>Cryopreservation of the mangaba tree (<em>Hancornia speciosa</em> Gomes): a protocol for long-term storage

“…Storage in liquid nitrogen is safe and economically viable (SANT et al, 2008) and allows for long-term in vitro conservation (CASTILLO et al, 2010;REED et al, 2011;CEJAS et al, 2012).…”