In vitro and molecular approaches for propagation and germplasm improvement of blueberries

Ghosh, Amrita (2021) In vitro and molecular approaches for propagation and germplasm improvement of blueberries. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Blueberries are known as a “super-fruit” and have tremendous commercial importance due to their high antioxidant contents. During in vitro culture, by introducing various plant growth regulators (PGRs) in the culture medium redifferentiation is stimulated in regenerating cells. During organogenesis and somatic embryogenesis (SE), a somatic cell goes through the process of dedifferentiation which is predominantly controlled by various epigenetic factors. I have investigated the effect of different PGRs on SE and established a protocol in half-high blueberry plants, for the first time, using thidiazuron (TDZ) on a semi-solid medium (SSM). I compared the antioxidant capacity of the in vitro grown plants with their donor counterparts to see the effect of SE on the biochemical profile of the regenerants. It was seen that not only the SE process but also the different concentrations of TDZ and the physiological age of the explants significantly affects the antioxidant activity. To get a more detailed insight into the effect of in vitro propagation on differential methylation pattern, I have analysed the global methylation pattern of young leaves and regenerated calli of one hybrid blueberry and three lowbush blueberry clones using methylation sensitive amplification polymorphism (MSAP) technique. Methylation assay results showed that calli regenerated in the SSM supplemented with TDZ are significantly hypermethylated relative to the donor plants, and the level of methylation varies with different concentrations of TDZ. Not only that but different plant genotypes showed differential effect on methylation pattern. These findings further confirm the effect of different aspects of plant tissue culture techniques on altered DNA methylation pattern. Finally, to gain further insight into how various in vitro culture systems affect the global methylation pattern, I performed global methylation analysis on half-highbush blueberry plantlets regenerated from SSM and liquid medium in a temporary immersion bioreactor (TIB) in the presence of TDZ and zeatin. From this experiment I found that significant increase in total methylation percentage and methylation polymorphism were present in plantlets from TIB system in comparison to SSM. Overall, my results indicate that each component of in vitro propagation has strong effects on the epigenetic and biochemical profile of the regenerants.

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