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, and this is a stated goal of the parent project. 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. One gene, called CBF, is responsible for ‘turning on’ a cascade of genes involved in cold tolerance. CBF 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). Over 400 plants from 40 independent transformation events are currently being maintained under greenhouse conditions. Freeze tests have been conducted using different tissues from 24 independent transformants. 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). More transformants will be evaluated and selected transformants will be used as parents to cross with several southern blueberry cultivars. Transformed plants have also been cold-treated and leaf tissues used to verify expression of CBF and other downstream genes in the CBF regulon. This project will help scientists to develop blueberry cultivars that are better able to withstand winter temperatures. Progress was monitored by the ADODR through phone calls and e-mails to exchange data and discuss research plans.