Half-high blueberry plants from bioreactor culture display elevated levels of DNA methylation polymorphism

Blueberry (Vaccinium spp. L.) plants exhibit high potential of regeneration via adventitious shoot formation on a semi-solid medium followed by shoot elongation in a liquid medium under bioreactor systems. To find out whether DNA methylation plays a role during shoot elongation, we compared DNA methylation level in the regenerants of two in vitro-grown half-high blueberry (V. corymbosum L. × V. angustifolium Ait.) cultivars Patriot and Chippewa on a semi-solid medium (SSM) in glass bottles and in a liquid medium in temporary immersion bioreactors (TIB), via methylation-sensitive amplification polymorphism (MSAP) technique. The SSM was separately fortified with various combinations of two plant growth regulators, zeatin and thidiazuron for shoot regeneration but elongation was carried out using the same medium under both SSM and TIB systems with only zeatin. Zeatin at 9.2 µM produced the maximum shoots in SSM and TIB for both cultivars, which varied from 30 to 33 in SSM and from 28 to 29 in TIB for Patriot and Chippewa, respectively. However, shoots proliferated in TIB were more vigorous. Noticeable changes in the methylation profiles were detected using MSAP in the regenerants grown in each type of culture system. The TIB system exhibited a significant increase in total methylation percentage (47–50%) in comparison to SSM (25–32%) in both cultivars. Similarly, the plants regenerated from TIB system were more polymorphic than those from SSM system. We describe here the effects of culture in vitro on DNA methylation that induced during the process of shoot elongation in two half-high blueberry cultivars. In vitro shoot culture was achieved in half-high blueberry cultivars using a semi-solid and a liquid medium; plantlets developed from liquid medium showed higher level of DNA methylation alterations.