Abstract
Apple (Malus × domestica Borkh.) genotypes originating from different plant collections (field collection, in vitro plant collections undergoing or not undergoing cryopreservation) were screened and characterized by SSR markers. Shoot tips excised from plants grown in vitro were successfully cryopreserved by encapsulation-dehydration. The highest regrowth frequency (69%, cultivar Goldrush) of cryopreserved apices was achieved after 24 h of osmoprotection in 0.5 M sucrose, 3 h of desiccation, and 24% water content of alginate beads. No differences in morphological characteristics including shoot length and number and length of roots were observed between controls and plants recovered after cryopreservation. SSR markers were used for calculation of genetic similarities between plants from the field collection, in vitro-micropropagated plants, or plants regenerated after liquid nitrogen storage. The set of microsatellite markers showed a low level of polymorphism among the studied genotypes, which could be distinguished by a specific combination of alleles generated by CH03g07, CH05c02, CH05d11, and CH05e03 primers. The CH03g07, CH05c02, CH05d11, CH05e03, GD96, GD147, and GD162 SSR markers exhibited low levels of polymorphism, while CH04AE07, CH04g10, GD100, and GD142 were nonpolymorphic. The Dice coefficient confirmed the effectiveness of SSRs for distinguishing between plants from ex situ collections and preserved plants. No major differences between ex situ plants, micropropagated plants, and plants recovered after cryopreservation were observed.
Highlights
Germplasms of temperate perennial fruit species are usually conserved as whole plants in the field
Cryopreservation and changes in the water content of alginate beads during desiccation Due to low regrowth percentages obtained with 1.00 M, only 0.25 M, 0.50 M, and 0.75 M sucrose concentrations were considered in the following experiments (Figures 2a– 2f)
A desiccation time of 4 h following osmoprotection in 0.50 M sucrose led to high regrowth rates for cultivars Rebra (64% at 20% Moisture content (MC)), Idared (61% at 21% MC), and Apple chlorotic leaf spot virus (ACLSV) apple stem grooving (ASGV) ASGV apple proliferation phytoplasma (ApP) apple stem pitting virus (ASPV)
Summary
Germplasms of temperate perennial fruit species are usually conserved as whole plants in the field. Ex situ preservation of plant genetic resources plays an important role in the maintenance of biodiversity, preservation of field collections carries the risk of infections, pests, diseases, or environmental disasters (Panis and Lambardi, 2005). To secure the maintenance of plant genetic resources over the years, alternative conservation approaches have been developed. To overcome the difficulties of grafting in woody species, tissue culture techniques have been applied for mass propagation, including various tissue types such as shoot tips (Butiuc-Keul et al, 2010; Feng et al, 2013), dormant buds (Höfer, 2015), or in vitro axillary buds (Condello et al, 2011).
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