Regeneration is the ability of plants to develop a new root, leaf, or even a whole new plant from a single cell. This technique is commonly used in science and breeding, for example in the production of many ornamental plants. Researchers use it when modifying plants. They do this for example when investigating the impact of a particular gene.

Until now, this has only been possible by first stimulating these cells to de-differentiate with plant hormones and then regenerate. However, hormone treatment is species-, genotype- and tissue-specific, meaning it is time-consuming to determine the ideal treatment for a given plant. Many species, particularly edible crops, also regenerate poorly under these conditions. Jana Wittmer, Renze Heidstra and their colleagues at Wageningen University & Research (WUR) and KeyGene have now found a way to initiate regeneration without hormones.

Stem cells

However, hormone-free regeneration was not the goal of the Wageningen research. ‘The actual goal was to generate stable stem cells,’ says Renze Heidstra, Associate Professor of Stem Cell Specification and Regeneration at WUR. But those stem cells took the next step independently and developed into plants via somatic embryogenesis. ‘We hadn’t expected that,’ says Heidstra.

The team, who have extensive knowledge of root meristem (undifferentiated cells capable of cell division) regulation, identified opportunities for obtaining stem cells while researching animal stem cells. They had previously succeeded in developing animal cells into stem cells and maintaining them in that state by overexpressing four essential genes.

To investigate whether this could also be achieved in plant cells, the researchers selected eight genes known to induce cell dedifferentiation or be involved in root meristem regulation. However, because these genes are involved in multiple steps in plant development, constant overexpression of these genes disrupts that process. To prevent this, the researchers created a construct that makes these genes inducible, which they then used to transform Arabidopsis thaliana (mouse-eared cress).

Two genes

After inducing the selected genes in seedlings, the researchers were surprised to observe regeneration. ‘Normally, we control this from the outside, but now it turns out that the plant itself knows very well what is needed. That came as a real surprise to me,’ says Bjorn Kloosterman, Team Leader of Plant Regeneration at KeyGene.

But that was not the only surprise of the project. Of the eight induced genes, only two — PLT1 and WOX5 — turned out to be necessary. These two alone were enough to start the whole process.

Arabidopsis, the lab rat of the plant world, is relatively easy to transform and regenerate. To see if this method would also bear fruit with plants of interest to breeding companies, the researchers tested it on lettuce, tomatoes and peppers. Lettuce proved to be the most responsive to the hormone-free method of the three, but tomatoes and peppers, which are more resistant, were also successfully regenerated from stably transformed tissues after the temporary expression of PLT1 and WOX5.

Applications

It is therefore still necessary to first transform the plants with inducible PLT1 and WOX5 genes before regeneration can be induced in these transgenic plants. In the future, the researchers hope to find a way to activate PLT1 and WOX5 without first creating a transgenic plant.

Even with the current method, however, this represents an improvement on what was previously possible. ‘Now that we know we can use it to induce regeneration, we can answer the question of why some plants succeed and others do not,’ says Heidstra. It is now also possible to analyse the functioning of specific genes – for example, those linked to disease resistance or drought tolerance – in recalcitrant and resistant plants. ‘So, for research purposes, I think it’s a very useful tool’, says Kloosterman.

Wittmer, J. et al. (2025) The Plant Cell 37(11), DOI: doi.org/10.1093/plcell/koaf252