Researchers publish hormone-free method for plant regeneration

Some plants can grow a root, a leaf or even an entire plant from a single plant cell. “We call this process regeneration,” explains Jana Wittmer, cell biology researcher at Wageningen University & Research. “This works because a specific cell, such as a root or leaf cell, can be converted into an undifferentiated state – in other words, a stem cell. From this stem cell state, the cell can specialise all over again and develop into a root or leaf, or even into a completely new plant.”

“Regeneration is widely used in agriculture, for example in plant breeding,” says Wittmer. “Regeneration ensures that, when you create new plants, the genome of the original plant is passed on identically to the next generation. In this way, you can maintain the genetic make-up of a plant or plant variety over successive generations. Until now, breeders have used plant hormones in the regeneration process. They add these hormones to the growth medium in which the plant, or part of it, is placed. This often involves young plant tissues, because older, more differentiated tissues respond less well to hormone treatment. By adjusting the hormone regime – which controls plant development and growth – we can steer the development of stem cells so that they grow into roots or shoots, for example.”

Drawbacks of hormone treatment

“However,” Wittmer continues, “regeneration with the help of hormones also has its drawbacks and limitations. The process is very labour-intensive and time-consuming. You first have to determine experimentally which treatment works best for the plant in question. Every plant species needs a different hormone regime, and even within a single species these regimes can vary. On top of that, for quite a few crops, such as pepper and cucumber, we currently still do not have a hormone regime that allows us to carry out the process in a reproducible way. Hormone-based regeneration does not always work, and even when it does, it takes a lot of time and effort – and that also means high costs.”

Alternative method

The researchers therefore set out to find an alternative way to achieve regeneration without using hormones. In doing so, they were inspired by a Nobel Prize-winning method that has been applied in animals. Wittmer: “This technique is also known as induced pluripotent stem cells. In plants, regeneration proceeds via a transient, or temporary, stage in which root stem cells are created. Our group has been working on root stem cells for many years, so we know the genes that are important for these stem cells. The next step is to test different combinations of these genes to see whether you can ‘reprogramme’ cells into a stem cell. From there, the cell can develop into any type of organ.”

Through a series of experiments, the researchers succeeded in regenerating plants without adding hormones. The most surprising outcome, according to Wittmer, was that they needed only two genes to trigger regeneration in the cells. “After that, you do not have to intervene at all – the plant cells organise themselves. From a block of cells, an entire plant develops again. We have demonstrated that this works in the model plant Arabidopsis, but also in crops such as tomato, lettuce and bell pepper. In principle, the technique can therefore be applied to a wide range of plant species, including species like bell pepper that do not respond to hormones, or for which we have not yet found a suitable hormone treatment.”

Saving time and effort

“Eliminating the need for hormones in regeneration can save breeders a great deal of time and work,” says Wittmer. “In addition, our method makes it easier to introduce or switch off genes, because that process also relies on regeneration. This could help make plants more resilient to diseases and pests, for example. In turn, that can have positive effects on crop yields and on the environment.”

Searching for a GMO-free approach

At the same time, Wittmer stresses that the study is only a first step in developing the technique. “It is not yet ready to be used in practice. In the lab we changed the plants’ genetic material. Bringing genetically modified plants to the market in Europe is a particularly costly and therefore hardly feasible route. We now need to find a way of activating the regeneration genes without using genetic modification. One option, for instance, would be to deliver the proteins into the cells that are encoded by the regeneration genes. If that proves possible, the method could be used immediately. But we first need to investigate and develop this further. That could easily take several years.”

“For science, this induction system opens many doors,” Wittmer concludes. “We can now study the regeneration process in greater depth, and in a much simpler way. It also raises some fascinating follow-up questions. Why does regeneration work well in some cell types and plant species, but not in others, for example? It is also exciting to explore how we might maintain the stem cell stage. At present, we can activate genes that trigger stem cell formation, but these cells then regenerate directly into a plant. If you could maintain a stem cell, you could steer it towards a specific plant cell type – for instance, cells that produce particular pharmaceutical compounds or other valuable molecules. But that is work for the future.”

Photo on the right: cross-section through an Arabidopsis root that regenerates somatic embryos after induction of PLT/WOX. The green signal along the outer edge is GFP expression of an embryonic gene.

Read more

• Scientific paper in The Plant Cell: https://academic.oup.com/plcell/article/37/11/koaf252/8293131
• The text of this news item is also available in Dutch: Regeneratie planten zonder hormonen
• KeyGene’s technology innovation in Cell- and Tissue Technology
• Licensing KeyGene innovations in Reproduction and Cell & Tissue Technologies
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