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.
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 scientists at Michigan State University have begun to establish appropriate northern and southern blueberry cultivars in tissue culture to be used for the transformation experiments. The CBF gene, a transcription factor responsible for ‘turning on’ a cascade of genes involved in cold tolerance was isolated recently from blueberry by ARS scientists. This gene was provided to Michigan State scientists through a Material Transfer Agreement for introduction and overexpression in blueberry. This project will help scientists to develop blueberry cultivars that are better able to withstand winter temperatures, which is a need of the blueberry industry. Progress was monitored by the ADODR through phone calls and e-mails to exchange data and discuss research plans.