AnAGAMOUSintron‐driven cytotoxin leads to flowerless tobacco and produces no detrimental effects on vegetative growth of either tobacco or poplar

Abstract

SummaryFlowerless trait is highly desirable for poplar because it can prevent pollen‐ and seed‐mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the β‐glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non‐floral‐specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT‐A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel‐less) and carpel‐less. Further, the vegetative growth of these transgenic lines was similar to that of the wild‐type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild‐type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT‐A transgenic poplar plants was similar to that of the wild‐type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT‐A gene may be useful for producing flowerless poplar that retains normal vegetative growth.

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

“…To optimize the time needed for robust transient T-DNA expression, tobacco leaf explants inoculated with Agrobacterium (containing the pCAMBIA1305.1 vector) were incubated in the dark at 25 °C for 2, 3, 4, 5, or 6 days. Half of the leaf discs (six) from each treatment were stained for GUS activity 55 . The remaining explants were transferred to solid MS medium containing 150 mg/L timentin to suppress Agrobacterium growth.…”

Transgenic Reduction of Cytokinin Levels in Roots Inhibits Root-Sprouting in Populus

“…A number of molecular confinement strategies have been reported that may block seed and pollen-mediated transgene flow in tree species (Klocko et al, 2018). We have also recently demonstrated that an AGA-MOUS intron-driven expression of a cytotoxin gene can be used to produce flowerless tobacco (Nicotiana benthamiana), and this technique may also be effective in reducing pollen-and seed-mediated transgene flow in poplar (Li et al, 2016). Further, we have reported a transgene deletion technology called the "Gene Deletor" that might be useful in eliminating transgenes from pollen and seeds of poplar, possibly obviating the need for containment (Luo et al, 2007).…”

“…Transgenic technology has been effectively used to produce poplar trees with a variety of useful traits, including herbicide resistance, biotic and abiotic tolerance, improved growth rate, higher nutrient use efficiency, and improved processing and end-use characteristics (Yang et al, 2009;Chang et al, 2018). Although many transgenic field trials have been carried out in the United States, China, and elsewhere, no transgenic trees have been commercially adopted to our knowledge (Li et al, 2016;Klocko et al, 2018).…”

Identification of candidate genes for leaf scorch in Populus deltoids by the whole genome resequencing analysis

“…The qRT-PCR was conducted on the Applied Biosystems 7300, and the volume of reaction was 20 μl, including 10 μl of SYBRR Premix Ex Taq™ (Perfect Real Time; TaKaRa), 0.3 μl (10 pM) of each primer, 1 μl of 10-fold-diluted cDNA and 8.4 μl of sterile double-distilled water. The program for the qRT-PCR were as following: 95 °C for 3 min, 95 °C for 25 s with 35 cycles, 62 °C for 25 s, and 72 °C for 40 s. The UBQ gene was used as control gene to normalize gene expression 52 . The relative expression level of genes was analyzed with the 2 −ΔΔCt method, which represented the difference of the cycle threshold (Ct) between the control UBQ products and target gene products.…”