Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 23, 2012
Publication Date: March 9, 2012
Citation: Walworth, A.E., Rowland, L.J., Polashock, J.J., Hancock, J.F., Song, G. 2012. Overexpression of a blueberry-derived CBF enhances cold tolerance in a southern highbush blueberry cultivar. Molecular Breeding. 30:1313-1323. Interpretive Summary: Freezing injury in blueberry can damage flower buds, resulting in reduced yield of berries. The blueberry plant’s resistance to cold injury is controlled by a complex process, and the level of resistance differs among blueberry varieties. As part of their growth cycle each year, blueberry plants become more cold tolerant in the fall and winter and less tolerant as growth resumes in the spring. To better understand development of cold tolerance or cold acclimation, we isolated a gene, thought to be involved in ‘turning on’ this complex process, from a northern cold-hardy blueberry variety. We then inserted the gene in a southern cold sensitive blueberry variety in a way that caused it to function at a higher than normal level. We verified that the cold-hardiness gene was present in the normally cold-sensitive variety, that it ‘turned on’ other cold-tolerance genes involved in this complex process, and that it resulted in increased freezing tolerance in flower bud and leaf tissue. This confirmed its involvement in the cold acclimation process, and that cold hardiness of blueberry can be modified by increasing the degree to which this gene functions. This information will be useful to plant breeders working on developing cold tolerant blueberry varieties and to research scientists studying the onset of the complex process of cold tolerance in plants.
Technical Abstract: Blueberry cultivars are subject to freezing damage in the winter and early spring. C-repeat binding factor (CBF) genes are transcription factors known to induce the expression of genes associated with cold acclimation and freezing tolerance. A CBF-encoding gene (BB-CBF) was isolated from the northern highbush blueberry ‘Bluecrop’ (a cold tolerant cultivar) and introduced into the southern highbush blueberry ‘Legacy’ (a more cold sensitive cultivar) to determine the effects on cold tolerance. The BB-CBF coding sequence was linked to the cauliflower mosaic virus 35S promoter and the 35S polyA terminator. This construct was cloned into the pCAMBIA2301 binary vector, which also contained the neomycin phosphotransferase (nptII) gene for selection and the ß-glucuronidase (GUS) reporter gene (gusA). Subsequently, this construct was introduced into ‘Legacy’ using Agrobacterium-mediated transformation of leaf explants. Fifty-seven transgenic events were obtained and were positive for the histochemical assay for GUS and PCR amplification of the transferred CBF. Southern blotting confirmed stable integration of transgenes in the putative transformants tested. Non-acclimated mature leaves, dormant flower buds, and flowers at various developmental stages from transformants and nontransgenic plants were subjected to electrolyte leakage assays over the course of three years, 2009-2011. Transgenic lines showed an increase in freezing tolerance in leaves and dormant buds. Expression of putative downstream components of the blueberry CBF regulon was increased in non-acclimated transgenic lines, and in some cases, to a level similar to that of acclimated control plants. Following low temperature exposure, BB-CBF overexpressing transgenics and controls expressed these genes at similar levels.