Genes that could help save the endangered Brazilian pine are identified

Embryonic formation of the piñon, the seed of Brazilian pine, is the target for a new molecular approach able to help preserve the species.

By Diego Freire

Agência FAPESP – Researchers from the University of São Paulo (USP) have identified 24,181 genes linked to formation of the embryo of araucaria (Araucaria angustifolia), a tree native to Brazil, also known as Brazilian pine, and its seed, the piñon.

Araucaria wood is highly valued in the market, and the researchers’ discovery could help establish a system for the in vitro propagation of the species, which is critically endangered according to the International Union for Conservation of Nature (IUCN).

By identifying these genes, it will be possible to have greater control over the process of somatic embryogenesis, or, in other words, the formation of an embryo without fertilization, starting from non-reproductive cells.

Somatic embryogenesis is one of the most promising plant embryo production biotechnology techniques, allowing cryopreservation (conservation through freezing) and mass cloning. In the case of araucaria, the process is complicated because the seeds do not survive long storage periods and become unviable.

“Given this difficulty, it is critical that the functioning of these genes be fully understood. Only with in-depth knowledge of the biochemical, physiological and genetic factors that control the development of the zygotic (in vivo) embryo will it be possible to conduct in vitro embryonic development,” explained Eny Iochevet Segal Floh, coordinator of the Laboratory of Plant Cell Biology (BIOCEL) at the USP Biosciences Institute (IB) and responsible for the project “Gene expression analysis during somatic and zygotic embryo development in Araucaria angustifolia (Bert.) Kuntze,” conducted with FAPESP funding.

The work at BIOCEL, carried out in partnership with the Laboratory of Plant Molecular Biology (LPMB), also at IB-USP and coordinated by Maria Magdalena Rossi, has focused on the analysis of transcriptomes, the set of the cell’s messenger RNA (mRNA), for the purpose of determining the genes involved in the process of araucaria embryo formation.

RNA sequencing was carried out at the Centralized Multi-user Laboratory of Functional Genomics applied to Agriculture, Animal Husbandry and Agroenergy, a facility at the Luiz de Queiroz College of Agriculture (ESALQ) at USP with funding from FAPESP’s Multi-user Equipment Program (EMU).

The analysis used large-scale sequencing technology (RNA seq), which allows the exploration of mRNA diversity and the profile of genes expressed during embryonic development.

“The use of this technology, new to most molecular biology groups in Brazil and still little used in plant systems, has provided important information about the regulation of the piñon’s embryonic development,” Floh emphasized.

The more than 24,000 genes identified will allow scientists to understand and describe the behavior of metabolism during the formation of the embryo. “In addition to its significance for plant biology, the data generated will allow us to obtain markers for enhancing the technique of micropropagation, which consists of utilizing somatic embryogenesis to produce hundreds of clones starting from a single cell or piece of plant tissue,” said Floh.

The findings could also help in establishing conservation strategies for A. angustifolia, including germplasm banks, the preserved genetic diversity of the plants, and genetic improvement programs that utilize biotechnology tools.

Threat

According to the IUCN survey, 97% of the original area of araucaria has already been lost, drastically compromising its genetic variability and causing it to be endangered. These trees once covered nearly 40% of the mixed ombrophilous forest, a type of forest that makes up the Atlantic Forest biome.

“Demand for the establishment of sustainable management and conservation programs are becoming more urgent by the day and seek the recovery and replacement of endangered species, in addition to ensuring that the resources they represent are maintained,” Floh asserted.

Gymnosperms, land-dwelling plants that live in cold or temperate environments, the group to which araucaria belongs, represent more than half of the planet’s forest reserves. In addition to providing wood, fiber and energy to the forest industry, they are an important source of biofuels and attenuate the effects of global warming.

Furthermore, the life cycle of gymnosperms is considered to be long: one tree can live for centuries, requiring approximately 15 years just to achieve reproductive maturity. The formation of seeds is similarly time-consuming and could take up to four years, which according to the researcher, demands biotechnology alternatives to the natural reproductive process.

“In light of this, in vitro mass cloning via somatic embryogenesis associated with cryopreservation and selection assisted by molecular markers has been added to the genetic improvement programs and germplasm conservation of endangered gymnosperms,” she said.

The initial findings were published in the journal Plant Cell, Tissue and Organ Culture and target strategies to make species preservation biotechnology viable. In addition to funding from FAPESP, the studies also received funding from the National Council for Scientific and Technological Development (CNPq) and Petrobras.