2012 Annual Report 1a.Objectives (from AD-416): Introduce genes that may play a role in determining cold hardiness levels, such as the blueberry CBF gene isolated by ARS, into southern and northern highbush cultivars to test their function. 1b.Approach (from AD-416): Function of cold-responsive genes, such as the CBF gene, that have been isolated from blueberry flower buds, will be tested for their role in determining cold tolerance in blueberry. Constructs designed to over express and/or knock out expression of certain genes will be introduced into appropriate southern and northern highbush cultivars by Agrobacterium-mediated transformation. Introduction of the gene(s) will be confirmed by GUS assays, PCR, Southern blotting, etc. Expression of genes will be monitored by quantitative RT-PCR. Freeze tolerance of transgenic plants will be determined first by a controlled laboratory freeze together with an electrolyte leakage assay using leaf tissue. Later, as plants mature, freeze tolerance of flower buds will be determined visually by combining the controlled freeze together with dissection of buds and looking for browning. In this way, it will be determined if the introduced gene(s) result in an increase or decrease in freeze tolerance as compared to control levels. 3.Progress Report: There is a need to develop more cold hardy blueberry varieties for the northern and midwestern United States. Scientists at Michigan State University have worked out the methodology for the stable transformation of blueberry. ARS scientists have isolated genes that may be involved in cold acclimation in blueberry and would like to test their function by overexpressing these genes and examining their effects on cold tolerance. The CBF gene, a transcription factor responsible for ‘turning on’ a cascade of genes involved in cold tolerance, was isolated from blueberry by ARS scientists and provided to Michigan State through a Material Transfer Agreement. Michigan State scientists have overexpressed the blueberry CBF gene (from the cold hardy variety ‘Bluecrop’) in the blueberry variety ‘Legacy’ (less cold hardy). Plants from 40 independent transformation events have been maintained under greenhouse conditions, and freeze tests are being conducted on leaf tissue of all of them. To date, significant increases in freeze tolerance have been observed in young and mature leaf tissues (1 - 5ºC) and in dormant floral buds (0-11 ºC) of a few transformants. In addition, one of the transformants that lacks freezing tolerance appears to be reblooming. Selected transformants are being used as parents in crosses with several other blueberry cultivars to verify the heritability of these traits. Transformants showing increased freezing tolerance and reblooming capability are being propagated for exchange and further testing between labs. This project relates to objective 2 of the in-house parent project, developing and utilizing genomic tools in blueberry and strawberry for the identification and characterization of genes associated with increased production of fruit under stressful temperature conditions. This project will help scientists identify genes involved in freezing tolerance and flowering and help scientists to develop blueberry cultivars that are better able to withstand winter temperatures.