Occurrence of tetraploidy in Nicotiana attenuata plants after Agrobacterium-mediated transformation is genotype specific but independent of polysomaty of explant tissue

Abstract

Genotypes of Nicotiana attenuata collected from Utah and Arizona were transformed with 17 different vectors (14 unpublished vectors based on 3 new backbone vectors) using an Agrobacterium-mediated procedure to functionally analyze genes important for plant-insect interactions. None of the 51 T1-T3 transgenic Utah lines analyzed by the flow cytometry were tetraploid, as opposed to 18 of 33 transgenic Arizona lines (55%). Analysis of T0 regenerants transformed with the same vector carrying an inverted repeat (IR) N. attenuata pro-systemin construct confirmed the genotype dependency of tetraploidization: none of the 23 transgenic Utah lines were tetraploid but 31 (72%) of 43 transgenic Arizonas were tetraploid. We tested the hypothesis that the differences in polysomaty of the explant tissues accounted for genotype dependency of tetraploid formation by measuring polysomaty levels in different seedling tissues. Hypocotyls, cotyledons, and roots of Utah and Arizona genotypes contained similar percentages of 4C nuclei (61 and 60; 7 and 5; and 58 and 61%, respectively). Since we used hypocotyls as explant sources and the nonoccurrence of tetraploid Utah transformants does not correspond to the high percentage of 4C nuclei in Utah hypocotyls, we can rule out a direct relationship between tetraploid formation and polysomaty level. We hypothesize that the difference between the Utah and Arizona genotypes results from the failure of polyploid Utah callus to regenerate into fully competent plants. We propose that future work on post-transformation polyploidy concentrate on the processes that occur during callus formation and plant regeneration from callus.

Jasmonate and ppHsystemin Regulate Key Malonylation Steps in the Biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an Abundant and Effective Direct Defense against Herbivores inNicotiana attenuata

“…Expression was quantified relative to that of N. attenuata actin using primers Actin F1 (59-GGTCGTACCACCGG-TATTGTG-39) and Actin F2 (59-GTCAAGACGGAGAATGGCATG-39) with probe Actin P1 (59-TCAGCCACACCGTCCCAATTTATGAGG-39). Flow cytometric analysis as described by Bubner et al (2006) confirmed that both lines were diploid. Growth and fitness were measured in a competition experiment where one IRggpps line 1 plant was paired with one empty vector plant in a 2-liter pot (pRESC empty vector characterized in ; beginning on day 33 after germination, the two plants in a pot were elicited one time/week either with Lan or Lan+MJ (n = 10 pairs) to the leaf one older than the source/sink transition leaf (+1) on rosette-stage and bolting plants or to the base of the stem on elongated plants.…”

Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature

“…Vector construction and the pSOL3 plasmid have been described previously (Bubner et al, 2006). A cross was created between ir LOX2 and ir PI homozygous lines; however, the hemizygous ir PI construct did not silence TPI activity or transcripts, and these plants therefor served as vector controls for comparison with ir PI and had slightly greater residual GLV production than ir LOX2 (see 'Results').…”

A below-ground herbivore shapes root defensive chemistry in natural plant populations

“…Latex in Eppendorf tubes and glass vials was extracted, analysed and quantified as described above. The ploidy level of each accession was furthermore determined by flow cytometry as described by Bubner et al [42].…”