Expression of “Rooting” Genes iaaM and rolB Under Regulatory Promoters in Tobacco

Abstract

Hybrid aspen is a major source of fiber in the north-central United States. One major problem for utilizing genetically improved aspen, and a variety of other woody species, is the difficulty in rooting hardwood cuttings. The objective of this project was to construct and confirm the function of genetic transformation vectors with two rooting genes (iaaM from Agrobacterium tumefaciens and rolB from A. rhizogenes) using three promoters (CaMV35S, soybean heat shock-inducible and poplar wound-inducible promoters). The gene constructs can be used to transform aspen for improvement of rooting hardwood cuttings and to elucidate rooting mechanisms. Each of these six gene constructs was inserted contiguously upstream from the promoter-less-glucuronidase (GUS) gene in the plasmid pBI 101. The engineered plasmids were transformed into A. tumefaciens strain LBA 4404 by electroporation. The functions of the genes were confirmed by transforming Nicotiana tabacum with these constructs. In plants transformed with iaaM and rolB under the CaMV35S promoter, the GUS gene expressed constitutively. These plants were also morphologically abnormal, with callus (and sometimes root) formation on stem tissue. The plants transformed with heat shock and Win6 constructs had no root formation and little or no GUS expression, without induction. After induction with heat shock (42°C for 1 hr per day for 7 days) or wounding (wounding the plants daily for 7 days), root formation occurred and GUS expression increased significantly with each gene, respectively. No roots developed in the non-transformed control shoots. The transformed plants with the regulatory promoters appeared morphologically normal. These constructs are currently being tested in hybrid aspen.