Molecular Breeding of Sugar Beet, Transformation, Transgene silencing, Virus and Herbicide resistance

Abstract: The main objectives in sugar beet breeding, are to improve yield, quality and to develop disease resistance. The disease resistance may be developed either by conventional, or molecular breeding techniques. The molecular breeding techniques include, methods to genetically engineer plants for expression of introduced genes. A necessity for the production of transgenic plants, is the development of a screening system, to identify the transgenic shoots as effectively as possible. Multiplex PCR was therefore developed to efficiently screen putatively transgenic shoots. In a basic study, progenies of transgenic Nicotiana tabacum containing the nptII gene, expressed by the 35S-, hsp80- or the hsp80 promoter including the omega enhancer element were evaluated for expression of the transgene in a greenhouse trial. The expression varied depending on different factors such as copy number, positional effects, development and environment. Additionally, the 35S promoter expressed the nptII gene to higher levels compared to the hsp80 promoter even if the omega element was included. However, the omega element enhanced the expression of the nptII gene compared to hsp80 without the omega element. Rhizomania is considered to be the most severe virus disease in sugar beet agriculture and difficult to control by conventional breeding. The disease causing agent is Beet Necrotic Yellow Vein Virus (BNYVV). The coat protein gene from BNYVV was introduced by Agrobacterium tumefaciens mediated transformation into sugar beet. Stable transgenic sugar beet plants showed resistance to BNYVV compared to the non transgenic plants in greenhouse and field trials. No coat protein was detected in the transgenic plants, but RNA-transcripts were found. Thus the resistance was most likely RNA - mediated and not dependent on protein accumulation. Transgenic sugar beet plants were produced containing a gene encoding the 5-enolpyruvinylshikimate-3-phosphate synthase from Agrobacterium sp. CP4, and a gene encoding the glyphosate oxidase from Achromobacter sp. LBAA, to obtain tolerance to the herbicide glyphosate (Roundup®). More than 300 transgenic shoots were produced using several sugar beet genotypes for transformation. The plants were evaluated in greenhouse and field trials. Two candidates were identified that displayed agronomically useful tolerance to glyphosate. It was found that plants containing a single copy of the transferred genes tolerated higher levels of treatment with glyphosate compared to plants containing multiple copies.