Robotics is entering cellular addition.

The hope is that the cell propagation method will alleviate the shortage of spruce seedling cultivation. The Natural Resources Institute Finland (Luke) and the Southeastern Finland University of Applied Sciences Xamk are seeking ways to mechanize the process.

Spruce seedlings are not always available in sufficient quantities because spruce trees do not produce cones every year, and good cone years occur only about every twenty years. Consequently, genetically improved seedlings cannot be produced in the quantities needed.

Relief for availability issues may be on the horizon. The Natural Resources Institute Finland (Luke) and the Southeastern Finland University of Applied Sciences Xamk are currently developing a method for the cell propagation of spruce seedlings, which involves multiplying the embryos of trees.

Leading researcher Tuija Aronen from Luke explains that cell-propagated seedlings hold significant potential.

"They are top-notch trees that exhibit all the best breeding benefits, such as better growth or resistance to various diseases. These seedlings are suitable for forest owners aiming for the highest possible yield from their forests."

Currently, cell propagation is done manually, which is slow and expensive. Luke sells a batch of cell-propagated seedlings consisting of seedlings from 12 different crosses, totaling 120 different genotypes or genetic types. The price of cell-propagated spruces suitable for nursery cultivation starts at 30 cents and can rise to several euros for specialized seedlings.

Researchers extracting embryos from spruce seeds collected in green cones. This is the last intermediate step before planting them outside. (Photographer: Timo Kilpeläinen)

The ongoing project aims to develop tools and robotics to enable future propagation on a large scale and in an automated manner. This would significantly reduce the cost of seedlings.

According to Aronen, cell-propagated seedlings are not intended to replace seed and seedling production but to become one method alongside others. Cell-propagated seedlings would be particularly suitable for growing specialty trees.

"For example, for Christmas tree cultivation if you want lush Donald Duck spruces. Nowadays, all seedlings grow so well that they don't become lush without pruning."

Another good target is parks, plantings, and other green constructions. Cell propagation could be used, for example, to create spherical spruces, sinuous serpent spruces, or yellow-tipped golden spruces, which are rare and difficult to propagate using other methods.

"Currently, stores sell ornamental trees imported from Central Europe that cannot withstand Finland's climate conditions. Often, the branches turn brown the following spring," says Aronen.

Leading researcher Tuija Aronen and researcher Saila Varis freezing cell mass in liquid nitrogen at a temperature of -196 degrees Celsius. (Photographer: Timo Kilpeläinen)

Response to climate change?

In tissue culture, we start with green cones collected from the top individuals in forest breeding. Cones are taken from many different trees to ensure the preservation of genetic diversity.

The first challenges are already emerging at this stage. Even if the seeds have good parents, the characteristics of the offspring can never be certain. Not all traits are inherited as they are when it comes to reproduction.

After collecting the cones, seeds are extracted from inside the cones. Embryos are then extracted from the seeds and multiplied to produce new pre-embryos. The embryos are matured at room temperature in a dark environment.

According to Norwegian research, the maturation temperature of embryos affects their later climate resilience. This brings new opportunities for tree breeding.

"We can entertain the idea of artificially adapting seedlings to a specific climate already in the laboratory," says Aronen.

In theory, seedlings could be modified to withstand the effects of climate change.

Eventually, embryos will grow into small seedlings that are planted outdoors. In Luke's experimental cultivation area, 97 percent of tissue-cultured seedlings have survived.

Luke has also experimented with adding embryos to spruce seedling buds. The technique is almost the same as with cones, says Luke researcher Saila Varis, who specializes in bud initiation.

"So far, we have tested bud initiation from tissue-cultured seedlings from 40 different lines. Out of these, initiation was successful in two, and embryos-producing tissue started growing from the buds."

The future goal is to perform bud initiation not from seedlings but from large trees.

Researchers Tuija Aronen, Mikko Tikkinen, and Saila Varis examining cell-propagated spruce seedlings planted four years ago. Nearly all seedlings have survived, but there are significant differences in height growth between different crosses. (Photographer: Timo Kilpeläinen)

From craftsmanship to robotics:

Petri dishes, familiar from chemistry research, are used as growth substrates for the cells and embryos. Different growth substrates containing sugars, nutrients, and growth hormones are applied to them at different stages of cell culture. Maintaining the cultures largely involves manual work, as embryos are transferred from one substrate to another using tweezers and a microscope.

Xamk is currently developing bioreactors, or cultivation vessels, that can automatically dispense nutrient solutions and vary them according to the growth stage.

Existing commercial bioreactors are not very suitable for spruce cell culture because they are designed for the cultivation of deciduous trees. The problem is that spruce embryos are only a few millimeters in size, whereas the shoots of deciduous trees are measured in centimeters.

In addition to bioreactors, Xamk is designing robotics that can identify high-quality embryos and transfer them undamaged to a new substrate.

"Teaching a machine to see things is not easy. From among the embryos, one should be able to select symmetrical individuals with a proper rudimentary root and at least four cotyledons," says Xamk researcher Markku Kuosa.

The goal is also to develop robotics so that tiny seedlings can be trained to grow mechanically in peat in a greenhouse.

Markku Kuosa, a researcher at Xamk, explains that interdisciplinary research is interesting. He develops, among other things, bioreactors, which are cultivation vessels for cell propagation. (Photographer: Timo Kilpeläinen)

Learning from Sweden and Canada:

In Sweden, research on spruce cell culture has been ongoing since the 1980s, but cell-cultured seedlings are not yet in commercial production there. Similarly, in Canada in the early 2000s, the possibilities of white spruce propagation were explored, and cell-cultured seedlings are already commonly used.

Luke began researching spruce cell culture in 2011. Pine cell culture had been studied earlier, but it was considerably more challenging.

Although there is foreign literature on spruce cell culture, not all information is applicable to Finnish conditions.

"The reasons may be the differences between spruce species and perhaps the materials adapted to our northern climate," reflects Aronen.

Particularly problematic is the limitation of research literature to individual stages. Mikko Tikkinen a researcher at Luke involved in cell culture development, explains that literature usually focuses on either laboratory procedures or planting seedlings.

"We try to integrate the entire chain. The most challenging stage is transferring seedlings germinated on petri dishes in the laboratory to further cultivation in the nursery."

The Luke and Xamk project has received funding from the European Regional Development Fund. The final results of the project are expected in about a year.

Cell culture is meticulous work.

Markku Kuosa, a researcher at Xamk, discusses the challenges of teaching machines to recognize good-quality embryos and transfer them intact to a new substrate. (Photographer: Timo Kilpeläinen)
The embryos are placed onto a cell propagation substrate, where they are nourished with nutrients and sugars. The cell propagation substrate is kept at room temperature in a dark place. Within two to eight weeks, the embryos begin to grow pale-colored cells around them. Within these cells, new embryonic structures form. (Photographer: Timo Kilpeläinen)
The cellular material is placed into small plastic tubes containing growth medium and anti-freezing agents. Subsequently, the cellular material is deep-frozen in liquid nitrogen. Deep freezing allows the cell cultures to be stored for years without a decrease in their capacity for embryo production. (Photographer: Timo Kilpeläinen)
The cellular material is removed from the freezer, thawed, and transferred to a new culture medium, which is kept in darkness at room temperature. By adjusting the growth hormones, the early embryos are induced to mature into fully developed embryoids in approximately eight weeks. (Photographer: Timo Kilpeläinen)
The embryos are germinated by gradually increasing the amount of light. Once germinated, the seedlings are transplanted into peat and taken to grow in the nursery. (Photographer: Timo Kilpeläinen)
In the spring, the seedlings can be planted in the ground. (Photographer: Timo Kilpeläinen)

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