The potential migration and gene flow from genetically modified or invasive plant species to their wild counterparts have raised significant public and regulatory concerns. To tackle this challenge, a spectrum of strategies has been developed, ranging from identifying naturally sterile plants to achieving engineered sterility through gene editing techniques.
As our understanding of the molecular biology of flowering has advanced, innovative methods targeting conserved floral genes have emerged, encompassing RNA interference (RNAi) and CRISPR technology. Efforts to induce early flowering in trees have included manipulating photoperiods and hormonal treatments, with genetic-accelerated flowering predominantly relying on stable transformation. Nevertheless, these methods have faced challenges such as variable responses in different plant clones and unintended impacts on overall plant form.
The ultimate challenge lies in effectively controlling genetic flow without negatively affecting the fundamental floral form and vegetative structures of typically flowering trees.
In June 2023, Horticulture Research unveiled a research paper titled “Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering.”
This study focused on optimizing early floral induction in 717 Populus tremula x Populus alba and 353 Populus tremula x Populus tremuloides clones. By utilizing four different promoter and gene combination constructs (HSP:AtFT, 35S:PtFT1, 409S:AtFT, 35S:AtFT), the research aimed to induce early-flowering in selected clones.
The results showed that the HSP:AtFT construct yielded the highest rate of flowering in both clones 717 and 353, with the female clone 717 demonstrating a lower flowering rate compared to the male clone 353.
The research further induced early-flowering in selected CRISPR-modified trees from both clones, using either constitutive or heat-inducible floral constructs. Events with bi-allelic mutations in LFY or bi-allelic changes in both AG genes were selected from the male clone 353 and female clone 717. These events were subsequently subjected to two experimental approaches: one using the constitutive 35S promoter driving AtFT expression, and the other utilizing a heat-inducible floral construct.
In the former approach, conducted on male clone 353, no flowering occurred in wild-type control trees. However, retransformation of certain LFY events (DL106) produced flowering rates of up to 83.3%. The latter approach involved retransforming selected events in both clones using a heat-inducible floral construct HSP:AtFT.
For clone 353, 22.4% of all trees flowered, while for clone 717, the flowering rate was 34.1%. Notably, the vegetative performance, measured by tree height, remained consistent across both flowering and non-flowering trees in both clones. Nevertheless, female clone 717 exhibited some stress indicators, such as yellowing of lower foliage after heat induction, whereas male clone 353 generally appeared healthy.
Regarding floral morphology, HSP:AtFT control trees typically produced standard terminal catkins, whereas retransformation events resulted in a wide array of atypical catkins and floral structures. Particularly, AG knockout trees displayed diverse replicated floral organs, varying in size and color.
In conclusion, this study effectively employed heat-induced FT overexpression to expedite the assessment of floral phenotypes following CRISPR knockout of key floral genes, LEAFY and AGAMOUS, in poplar trees. These genetically modified poplar trees exhibited a broad range of inflorescence and floral forms.
This research not only underscores the potential horticultural value of these modifications but also offers an early glimpse into the impact of knocking out specific genes on floral characteristics and apparent sterility. These findings pave the way for potential applications in controlling plant breeding and addressing concerns related to gene flow.