Tony Chen

My research focuses on biotechnology and stress and postharvest physiology.

The research in my lab focuses on three areas: 1) cloning and characterization of regulatory genes contributing to barley cold tolerance; 2) improving cold tolerance by metabolic engineering of glycinebetaine synthesis and by over-expression of CBF genes; and 3). improvement of potato cold tolerance by over-expressing cDNAs of Arabidopsis CBF genes.

Characterization of Regulatory Genes Contributing to Barley Cold Tolerance A major cold tolerance QTL cluster has been identified on barley chromosome 7 (5H). Recent studies in Arabidopsis and other dicots have demonstrated that CBF/DREB genes act as key regulators of plant cold tolerance and other stress responses. We have cloned 15 barley CBF gene family members to date and mapped 13 of the barley CBF genes directly adjacent to the major cold tolerance QTL region. Assignment of map positions for the remaining barley CBF genes, as well as saturation of this area with additional markers, is in progress. The size of the barley CBF gene family and response of each member to environmental stress (cold, drought, etc.) is being characterized. A barley BAC clone has been mapped to a position directly under the cold tolerance QTL peak, and overlapping BAC clones identified as a first step towards a chromosome walk across this region. A set of 59 DM near-isogenic lines is being densely genotyped to define the segments of chromosome 7(5H) that play a central role in conveying the low temperature tolerance phenotype. Determination of the genes/regions conferring superior cold tolerance will allow development of winter hardy barley varieties that retain the superior malting traits of current spring varieties.

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants and flowers from chilling damage Tomato plants normally do not accumulate glycinebetaine (GB) and are susceptible to chilling stress. Exposure to temperatures below 10°C causes various symptoms of chilling injury and greatly decreases fruit set (the number of fruits per plant) in most tomato cultivars. Conventional breeding efforts have failed to produce tomato cultivars with satisfactory cold tolerance and, with this in mind, we transformed tomato (Lycopersicon esculentum Mill. cv. 'Moneymaker') with the codA gene of Arthrobacter globiformis, which encodes choline oxidase. This enzyme catalyzes the conversion of choline to GB. Transgenic tomato plants expressed the codA gene and synthesized choline oxidase, accumulating up to 0.23 mol GB/g fresh weight in their leaves. GB-accumulating transgenic tomato plants were more tolerant to chilling stress than wild-type plants at various stages of growth and development from the germination of seeds to the production of fruit. At the reproductive stage, the GB-accumulating transgenic tomato plants yield on average 30% more fruits than wild-type plants after an episode of chilling stress. Our results demonstrate that introduction, by metabolic engineering, of the biosynthetic pathway to GB in tomato is an effective strategy for improving fruit production under cold-stress conditions.

Improvement of potato cold tolerance by over-expressing cDNAs of Arabidopsis CBF genes We transformed potato (S. tuberosum, ST) and a related species, S. commersonii (SC), with the Arabidopsis CBF1, 2 and 3 genes driven by either the CaMV35S promoter (a constitutive promoter) or the Rd29A/COR78 promoter (a cold-inducible promoter) and characterizing the stress tolerance of the transgenic plants. Freezing tolerance had been evaluated for 10 transgenic lines of S. commersonii and 3 transgenic lines of S. tuberosum transformed with 35S::CBF1. Under non-acclimated conditions, there was a maximal increase of 4°C in freezing tolerance in transgenic SC lines, and of 2°C for ST lines. After two weeks of cold acclimation at 2°C, transgenic SC lines showed a further increase of 3°C in freezing tolerance, whereas there was no further increase in freezing tolerance in ST lines. We have also evaluated the freezing tolerance of transgenic ST lines transformed with Arabidopsis CBF1, CBF2, and CBF3 driven by the Rd29 promoter. ST lines transformed with an Rd29::CBF3 construct had a 4°C increase in freezing tolerance.