Development of molecular markers for assessment of juvenility during micropropagation of Silver birch (Betula pendula Roth)

Fundamental and Applied Research Study No. lzp-2019/1-0387

The scientific objective id to develop a technology (molecular markers) for determining juvenility during the micropropagation process of Silver birch (Betula pendula Roth) based on expression changes of miRNAs and their target genes, and to investigate factors affecting juvenility of birch genotypes with different in vitro morphogenic ability.

New knowledge will be obtained as a result of research development – determination of factors affecting birch rejuvenation during micropropagation, identification of conserved miRNAs, correlation of miRNA (also miR156/157, miR172) and their target gene expression with juvenility state, as well as verification of rejuvenation model developed during this research using grafted seedlings and on selected birch genotypes with different morphogenesis ability in vitro. Development of this technology will improve the propagation success rate for high quality and value selected birch genotypes, increasing the efficiency of breeding programs and the value of forest stands over the rotation period. Using the obtained knowledge, an effective birch micropropagation technology will be developed and used in birch breeding programs to produce genetically valuable birch reproductive material.

Research tasks:

1. High-throughput sequencing of microRNAs and expressed genes in seedlings (juvenile control), mature silver birch, and in birch in vitro shoots with different morphogenic potential.
2. Development of microRNA and target gene molecular markers for the determination of Silver birch juvenility state.
3. Evaluation of morphological, anatomical and physiological traits of birch in vitro shoots during rejuvenation.
4. Investigation of the rejuvenation mechanism in grafted birches using the developed molecular markers.
5. Testing of developed juvenile markers on 10–15 birch in vitro genotypes with different morphogenetic ability.

Work plan:

1. Sequencing and data analysis.
2. Plant material and extraction of RNA for further analyses.
3. Marker development and validation.
4. Evaluation of shoot morphology, anatomy and physiology during birch in vitro shoot rejuvenation:
   4.1. Morphological evaluation;
   4.2. Anatomical evaluation;
   4.3. Physiological evaluation.
5. Assessment of the developed markers on 10–15 selected birch genotypes in vitro and grafted birches.
   5.1. Analysis of miRNAs and their target gene expression levels in 10–15 birch genotypes with different in vitro morphogenic ability.
   5.2. Analysis of miRNAs and their target gene expression levels in grafted birches.

Actualities

1. 30.01.2020. Participation in the 78th Scientific Conference of the University of Latvia with presentation: Krivmane B., Girgžde E., Samsone I., Ruņģis D. Development of molecular markers for assessment of juvenility during micropropagation of Silver birch (Betula pendula Roth).
2. 23.01.2020. Participation in the career development support event "Many sided and interesting forest science through the eyes of a researcher" for 7th–9th grade students of the Riga 75th secondary school. The aim of the event is to inform pupils about the field of forest science (with a focus on molecular genetics), the most current problems and research in forest science, to introduce with the researcher's daily life and to find out its role in the development of forest science.
3. 23–25.09.2020. Participation in the event "Forest Science Afternoon", organized by LSFRI "Silava" with activities for Salaspils 1st secondary school pupils "Forest in a toy box".
4. 25.09.2020. Participation in the event "Forest Science Afternoon" organized by LSFRI "Silava" with activities "Find out and trace what we are researching? " and creative activity "Wooden Story" for all visitors.
5. 10,16.02.2020. An educational and informative excursion with practical work in DNA extraction was leaded at the Genetic Resource Centre’s Molecular Genetics Laboratory of the LSFRI "Silava" for 3rd grade pupils of 1th Salaspils Secondary School.
6. 26.11.2020. Presentation for master’s students of the University of Latvia Faculty of Biology within the course of plant physiology "Plant tissue culture". The presentation is about Plant tissue culture’s laboratory and their arrangement, goals, directions of activity and active projects. The project "Development of molecular markers for the assessment of juvenility during micropropagation of Silver birch (Betula pendula Roth)" has been described in more detail – about the topicality of the topic, goals, tasks, about obtaining the necessary data, the first results and further tasks.
7. 26–27.01.2021. Participation in the poster section of the conference "Knowledge-based forest industry" organized by LSFRI "Silava", Forest Sector Competence Center, JSC "Latvijas Finieris" and JSC "Latvia's State Forests" in cooperation with other forest industry partners with a presentation "Development of molecular markers for assessment of juvenility of silver birch in vitro cultures". The presentation is about the topicality and novelty of the research, the goal, the main tasks, the obtained results and conclusions.
8. 28.06–01.07.2021. Participation in the Plant Biology Europe 2021 conference (program), organized by the Federation of European Societies of Plant Biology (FESPB) and the European Plant Science Organization (EPSO), with the poster presentation: Girgžde E., Samsone I., Gailis A., Ruņģis D. Effect of exogenously applied sugars on silver birch (Betula pendula) in vitro shoot rejuvenation
9. 30.04.2021. Participation in the European Researchers Night 2021 "Scientific achievements for the future". LFSRI "Silava" scientists created the quiz puzzle "Genetics and Research", where by correctly answering all the questions of the game about genetics and research in the genetic Resource centre, it was possible to put together a puzzle. In addition, it was possible to watch a video presentation and poster presentation Development of molecular markers for assessment of juvenility during micropropagation of Silver birch
10. 24.10.2022. Participation in the COST action CA21157 European Network for Innovative Woody Plant Cloning (COPYTREE) meeting of Management Committee. Participants were informed about the study "Development of molecular markers for assessment of juvenility during micropropagation of Silver birch (Betula pendula Roth)". Participation in the Management Committee of COST action will give the opportunity for the next four years to participate in discussions, development of methods and protocols, workshops, seminars, etc., in order to improve knowledge and experience in the field of molecular genetics and in vitro cultures.
11. 30.09.2022. During the "Forest Science Afternoon" organized as part of the European Researchers’ Night, a lecture "The role of microRNAs in the maturation of silver birch in vitro cultures" was presented to 11th-grade pupils of Salaspils 1st secondary school. During the lecture, pupils gained general knowledge about molecular biology, as well as about current research and had the opportunity to examine in vitro cultures in different developmental phases. After the lecture, pupils visited the molecular genetics laboratory and were informed about the laboratory and equipment used for this and other research activities. More information here.
12. 22.07.2022. Colleagues from Oxford University visited the Molecular genetics laboratory of the LSFRI "Silava" Genetic Resources centre and were informed about current studies, including "Development of molecular markers for evaluation of juvenility in silver birch (Betula pendula Roth) in vitro cultures during micropropagation".
13. 2021–2023. Training of a Biotechnology and Bioengineering bachelor's study program’s student and conducting research experiments at the LSFRI "Silava" molecular genetics laboratory in order to develop a bachelor's thesis within the framework of the research "Development of molecular markers for evaluation of juvenility during micropropagation of outdoor birch (Betula pendula Roth)".
14. 1,06.04.2022. As part of the Shadow day (work experience day), 4th, 6th and 10th grade students from Ulbroka Secondary School, Ogre 1st Secondary School and Riga State 3rd Gymnasium were introduced with the LSFRI Silava Centre for Genetic Resources and current research, including "Development of molecular markers for evaluation of juvenility in silver birch (Betula pendula Roth) in vitro cultures during micropropagation". More information here.
15. 26.11.2022. Presentation of the lecture "Insights into plant molecular genetics" to participants in the School for Young Biologists.  During the lecture, grade 9–12 pupils were informed about the role of microRNAs in silver birch maturation in in vitro culture.
16. 30.11.2022. Grade 10–11 biology pupils from the Ādaži Free Waldorf School were, presented educational and informative lessons on "Plant cloning", "Genetic resources" and "Molecular biology" with practical work in DNA extraction and micropropagation at LSFRI "Silava" Molecular genetics laboratory and Plant Physiology laboratory. During the lessons, pupils gained general knowledge about molecular biology and plant micropropagation methods and were informed about current research activities in the laboratories.
17. 28.12.2022. Developed protocol for quantification of expression of miR156 precursors for evaluation of juvenility in silver birch 
18. 17.–19.04.2023. Participation in the conference "Innovative Woody Plant Cloning", held in Santiago de Compostela (Spain), organized by the COST action CA21157 European Network for Innovative Woody Plant Cloning (COPYTREE) with the poster presentation "Differentially expressed conserved microRNAs in mature and rejuvenated silver birch in vitro propagated tissues".
19. 6.06.2023. Kaiva Solvita Ruņģe has been developed and defended the bachelor’s thesis "MiRNA Expression in Plant Vegetative Phase-Change of Birch Betula pendula in vitro Cultures” within of University of Latvia and Riga Technical University academic Bachelor's study programme "Biotechnology and Bioengineering". Supervisor – Baiba Krivmane.