on biodiversity and geneflow of selected biofuel crops Klaus Ammann Delft University of Technology, [email protected] January - February 2007, 10 studies for the working groups of www.epobio.net Avena, Beta vulgaris, Brassica napus, Crambe, Linum, Miscanthus, Nicotiana tabaccum, Populus, Salix, Triticum 2 Contents Contents ............................................................................................................................................. 2 Figures................................................................................................................................................ 4 Avena, Oats 11 1. Taxonomy ..................................................................................................................................... 11 2. Biosafety considerations............................................................................................................... 14 3. Transgenic oats ............................................................................................................................ 16 4. Management and mitigation of gene flow..................................................................................... 20 Gene Flow Assessment for Avena ................................................................................................... 26 Beta vulgaris, Beet 28 1. Taxonomy ..................................................................................................................................... 29 2. Reproduction biology.................................................................................................................... 40 4. Biosafety considerations............................................................................................................... 44 Brassica, Oilseed Rape 56 1. Taxonomy ..................................................................................................................................... 56 2. Biosafety considerations............................................................................................................... 69 Crambe, Seakale 80 1. Taxonomy ..................................................................................................................................... 80 2. Biosafety considerations............................................................................................................... 87 Linum, Flax 90 1. Taxonomy ..................................................................................................................................... 90 2. Biosafety considerations............................................................................................................... 94 Miscanthus 99 1. Taxonomy ..................................................................................................................................... 99 2. Reproduction biology.................................................................................................................. 105 3. Biosafety considerations for Miscanthus .................................................................................... 106 Nicotiana tabacum, Tobacco 111 1. Taxonomy .............................................................................................................................. 111 2. Biosafety considerations............................................................................................................. 114 3. Mitigation .................................................................................................................................... 120 4. Summary Gene Flow Nicotiana, draft......................................................................................... 121 Populus, Poplar 123 1. Some general remarks about regulation .................................................................................... 123 2. Introduction ................................................................................................................................. 124 3. Taxonomy and its relation to biodiversity ................................................................................... 124 4. Genomics of poplars................................................................................................................... 127 5. Reproduction biology.................................................................................................................. 134 6. Risk assessment of transgenic poplars:..................................................................................... 139 3 Salix, Willow 154 1. Taxonomy ................................................................................................................................... 154 5. Reproduction biology.................................................................................................................. 169 6. Risk assessment of transgenic salices....................................................................................... 173 Triticum, Wheat 179 1. Introduction ................................................................................................................................. 179 2. Taxonomy ................................................................................................................................... 179 2.1. Evolutionary history of wheat................................................................................................... 180 2.2. Reproductive biology ............................................................................................................... 183 3. Biosafety considerations............................................................................................................. 185 Triticum, Wheat risk assessment scheme...................................................................................... 194 7. Cited literature ............................................................................................................................ 197 4 Figures Fig. 1 Neighbor-joining tree inferred from rDNA ITS sequences in 'core genera' of Aveneae subtribe Aveninae analysed with the distance matrix method. Numbers above branches are bootstrap values. Branch lengths are proportional to distance. Outgroup: Lolium perenne (Poeae). Asterisk designates sequence data taken from (Hsiao et al., 1994, 1995a, b), out of (Grebenstein et al., 1998). 12 Fig. 2 Phylogenetic tree inferred from ITS sequences of 15 genera of tribes Aveneae and Poeae generated by the neighbor-joining distance matrix method. Numbers above branches are bootstrap values. Branch lengths are proportional to distance. Outgroups: Bromeae (Bromus inermis) and Triticeae (Secale cereale). Asterisk designates sequence data taken from (Hsiao et al., 1994, 1995a, b) 13 Fig. 3 Mitotic metaphase squash of line D1.3, probed with labelled pUBA (green) and pACT1-F (red) showing that both transgenes have integrated at the same site (arrowhead). (b) The transformed chromosome from (a) enlarged, showing the independent signal from: (i) the pUBA probe (green); (ii) the pACT1-F probe (red); and (iii) the combined signals from both probes. 17 Fig. 4 Maps of plasmids used to produce transgenic oat lines 3830 (pNGI) or 11929 (pH24 and pScBV 3m). Restriction sites used in the analysis are shown. From (Makarevitch et al., 2003) 20 Fig. 5 Summary of sequence analysis of transgene locus clones isolated from transgenic line 3830. a. Linear map and structural components of pNGI used in producing transgenic line 3830. Colors indicate the structural components of pNGI. b. Structures of locus 3830-1 and ten lambda clones isolated from line 3830 corresponding to the main transgene locus 3830-1. Colors indicate transgene sequences that are identical to structural components of pNGI. White boxes indicate unknown sequences that are presumably oat genomic DNA. Black vertical arrows denoted by ‘J’ indicate junctions between noncontiguous transgene fragments or between transgene and genomic DNA. Red vertical arrows denoted by ‘J’ indicate the deduced position of junctions between noncontiguous genomic DNA fragments. Black bars above loci represent the regions of extensive scrambling that were PCR-amplified and sequenced from total genomic DNA for the structure verification. Red bars under loci represent regions of genomic DNA that were used as probes for hybridizations. c. The structure of lambda clone 7 isolated from line 3830 and corresponding to one of the minor transgene loci designated as 3830-2. A matrix attachment region is designated as MAR. Colors, bars, and arrows are coded as in b. From (Makarevitch et al., 2003) 20 Fig. 6 Effect of different cultivation regimes on the build-up of wild oat (Avena fatua). Aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides are applied each year achieving 90% mortality of susceptible plants. The starting seed bank consists of 100 newly shed seeds rn-5 there is a mutation rare of 10 per generation to resistance; 0.293 of ovules are cross-pollinated. 21 Fig. 7 Effect of continuous application of aryxophenosyproprionate AOPP) and cyclohexanedione (CHD) herbicides vs. rotation of two or three modes of action on the build-up of wild oat (Avena fatua) herbicides causing 90% mortality (with the exception of AOPP/CHD herbicides, which do not kill resistant plants). The starting seed bank consists of 100 newly shed seeds m-2; there is a mutation rate of 1O-6 per generation to resistance; 0.2% of ovules are cross-pollinated. 22