Transgenic poplars: expression of chimeric genes using four different constructs

Abstract

Leaf or stem explants of a hybrid poplar clone (Populus tremula X Populus alba), sensitive to Agrobacterium tumefaciens, were co-cultivated either by an octopine or a nopaline disarmed A. tumefaciens modified strain. Transformed poplar shoots were readily regenerated from explants. The protocol was improved using the nopaline disarmed strain C58/pMP90 with the binary vector pBI121. This protocol was then used to test three other vectors. The first one, possessing a nptII gene fused to the CaMV 19S promoter, permitted regeneration of transformed shoots in presence of 50 to 100 mg/l kanamycin. The two other vectors carried an additional nptII gene under the control of the CaMV 35S or CaMV 35S promoter with a double enhancer sequence (CaMV 70). CaMV 70 promoter provided consistently higher level of gene expression than the other promoters in both callus and leaf tissues.

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

“…To test the role of CYP79D6 and D7 genes in planta, we created RNAi-mediated knockdown lines with reduced levels of CYP79D6v3 and CYP79D7v2 transcripts after wounding (see Supplemental Figure 5 online). Since there is no established transformation method for P. trichocarpa, we produced these lines in a P. 3 canescens background, the hybrid usually used for poplar transformation (Leplé et al, 1992). A volatile collection made from herbivore-damaged trees of the two independent CYP79D6 and D7 knockdown lines, a line transformed with the empty vector, and the P. 3 canescens wild type showed a significant reduction of aldoximes, benzyl cyanide, and 2-phenylethanol emission in the knockdown lines in comparison to the controls, while the emission of indole, a nitrogenous volatile not related to the above-mentioned compounds, was not affected ( Figure 5; see Supplemental Table 4 online).…”

Transformation as a Tool for Genetic Analysis in Populus

“…The reasons for the extraordinary genotype specificity of regeneration and transformation procedures remains unknown, but imposes a major obstacle to efficient transformation of all plants. Most of transformation procedures using A. tumefaciens follow a classical protocol upon which there are many variations (e.g., Leple et al, 1992;Han et al, 2000;Confalonieri et al, 2003) (Fig. 2).…”

“…For example, transformation protocols have been developed for P. alba (Okumura et al, 2006), P. tremula (Fladung et al, 1997;Tzfira et al, 1997), P. tremuloides (Cseke et al, 2007), P. tremula × P. alba (Leple et al, 1992), P. tremula × P. tremuloides (Fladung et al, 1997), Populus canescens × P. grandidentada and P. tremuloides × P. davidiana (Dai et al, 2003). Successful transformation have been reported for species and hybrids from sections Aigeiros (cottonwood) and Tacamahaca (balsam poplar) including P. deltoides, P. trichocarpa "Nisqually-1" Song et al, 2006), P. trichocarpa × P. deltoides (De Block, 1990;Han et al, 2000), P. deltoides × P. nigra (Heuchelin et al, 1997), P. alba × P. grandidentata (Fillatti et al, 1987) and P. nigra × P. trichocarpa (McCown et al, 1991), P. sieboldii × P. grandidentata (Matsunaga et al, 2002), P. nigra "Italica" (Nishiguchi et al, 2006) and P. ciliata (Thakur et al, 2005).…”

“…Successful transformation have been reported for species and hybrids from sections Aigeiros (cottonwood) and Tacamahaca (balsam poplar) including P. deltoides, P. trichocarpa "Nisqually-1" Song et al, 2006), P. trichocarpa × P. deltoides (De Block, 1990;Han et al, 2000), P. deltoides × P. nigra (Heuchelin et al, 1997), P. alba × P. grandidentata (Fillatti et al, 1987) and P. nigra × P. trichocarpa (McCown et al, 1991), P. sieboldii × P. grandidentata (Matsunaga et al, 2002), P. nigra "Italica" (Nishiguchi et al, 2006) and P. ciliata (Thakur et al, 2005). The most widely used transformation "laboratory rat," has been the Populus tremula × alba hybrid clone known as INRA 717-1B4 (Leple et al, 1992).…”

Wood production, wood technology, and biotechnological impacts

“…The Bt gene has been introduced into several other tree species like walnut and white spruce , Dandekar et al 1998. As a different approach to engineer insect resistance, proteinase inhibitors have been expressed in poplar (Leplé et al 1992, Klopfenstein et al 1997. Increased resistance against the fungal pathogen Septoria musiva has been achieved in poplar by expression of chitin binding peptides .…”