Plant Cultivation and Transformation
The infrastructure group of Dr. Karin Köhl develops and uses reproducible and efficient methods for plant cultivation in phytotrons, a climate-controlled greenhouse, a polytunnel-screenhouse and on the field site. These resources permit to conduct large-scale experiments like genome wide association studies (GWAS) under controlled and variable climate conditions. Furthermore, we perform routine Agrobacterium-mediated transformations of Arabidopsis and Capsella by dipping and Solanaceae, Lotus japonicus and maize by tissue-culture method as a service to the entire institute.
The scientific work concentrates on drought tolerance under field conditions and the development of data management structures for phenotyping and metadata capture.
Improvement of drought tolerance in potato
Potato is the 3rd most important food crop and more than 50 % is produced in developing countries. Potato is water efficient, but not drought tolerant, thus many production systems rely on irrigation or sufficient precipitation. As climate changes and fresh water resources become limiting, maintenance of yield stability under water limitation requires efficient selection for drought tolerance. The gold standard for the breeding of crops that yield stably under drought is yield-based selection in arid environments. This method is effective but slow. Therefore, modern breeding schemes employ genomic selection to speed up the breeding cycle. The training of these selection models needs drought tolerance data for several hundred genotypes that are representing the gene pool for breeding. Towards that aim the MPI-MP collaborates with breeders of the GFPi and scientific institutions in the POMORROW consortium (2025 to 2029): the consortium aims to genotype several thousand and phenotype several hundred potato accessions that are stored in the Genbank of the Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben.
In the POMORROW consortium, the group of Karin Köhl in collaboration with the group of Caroline Gutjahr is tasked with the quantification of drought tolerance and mycorrhiza response of 300 accessions. The accessions are cultivated at optimal and reduced irrigation in a field-like test system (Fig. 2) to determine their relative performance [1, 2]. For a fast assessment of drought tolerance, we quantify canopy development during the first weeks of shoot growth with the automatic phenotyping system Planteye F600 (Phenospex). The gantry-based system moves two laser scanners and multispectral cameras over the canopy and yields data on shoot development, leaf position and canopy colour. The approach relies on concepts from earlier research on German potato cultivars and their segregating offspring. In the projects TROST and VALDIS TROST, we determined significant variation in drought tolerance in both populations based on tuber starch yield measured optimal and reduced irrigation in multi-environment trials [2, 3]. In a collaboration project with the International Potato Center (CIP), we established that yield stability under drought was predictable in both populations from parameters of leaf area development and leaf position, two features that directly affect photosynthetic performance and hence yield production under stress conditions [4, 5]. We are now using these concepts to assess the drought tolerance of 300 potato accessions (Fig. 3) to identify breeding parents. In a collaboration with Prof. Caroline Gutjahr, we are additionally screening the population for their response to fungi that form arbuscular mycorrhiza (AM) under optimal cultivation conditions and drought stress. In collaboration with the groups of the Julius Kühn Institute and the IPK, where the accessions are genotyped, we aim to identify gene regions that contribute to drought stress tolerance and AM response. These regions can then be introgressed into modern cultivars or modified by gene editing to generate potato cultivars for future agriculture.
Phenotyping and data management
Plant breeding and genetics require fast and exact phenotyping that is reliable and reproducible by other scientists independent of the location. Standardised data storage facilitates statistical evaluation and compliance with the FAIR principles. We developed a simple and cost-efficient system, the Phenotyper, which employs mobile devices for on-site data entry and open-source software for data management (Köhl and Gremmels, 2015). We employed the system in the data management pipeline that supported our potato drought tolerance projects TROST and VALDIS TROST (Billiau et al., 2012). The system that was originally designed for Windows CE is presently updated by Jürgen Gremmels from the AG Walther for Android systems to make it usable on modern smartphones.
Past data management projects yielded a plant resource and experiment management that is based on the LIMS software Nautius (Köhl et al., 2008) and a management system for plant transformation (Köhl and Gremmels, 2010) and plant cultivation (details see below). These systems are in continuous use since 2005 or 2009, respectively. Jürgen Gremmels from AG Walther and Karin Köhl are presently updating and modernising the plant cultivation management system from a system based on MS Access to a SQL-database with web frontends for the user (details see below).
Experiments on abiotic stress tolerance require metadata on atmospheric and edaphic conditions. We therefore run two weather stations – one on the field, one in the polytunnel – to continuously measure light intensity and light quality, total radiation, air temperature and humidity, windspeed and soil temperature to provide metadata for our experiments under variable climate conditions. Since 2021 we are using Lorawan soil sensors to automatically measure soil temperature and soil humidity in the bigbags that are used for drought stress trials. Data are automatically transmitted to a server hosted at the institute.
Services
The plant cultivation facility is run by Karin Köhl together with the 'greenteam', a group of gardeners and technicians. This unit of skilled people and controlled environment facilities forms the basis of our research on higher plants. The main research objects are Arabidopsis thaliana, Lotus japonicus, maize, tobacco and tomato; special aspects are studied in rice and potato.
Cultivation
The greenteam service runs plant cultivation on 1600 m² fully climate-controlled greenhouse, 600 m² phytotrone area plus a summer greenhouse and a 5 ha field side. Construction work for the extension by an additional 500 m² of phytotron space are ongoing. We routinely cultivate Arabidopsis thaliana, Lotus japonicus, tobacco, tomato, potato, rice, maize and Capsella species 365 d per year. This includes controlled production of seeds for various Arabidopsis accession. The service includes the development of cultivation protocols for new model system like Lotus japonicus, including the establishment of hydroponics protocols. The service runs a modern integrated pest management system that controls pest like Western Flower Thrips predominantly by hygiene and biological control systems.
Transformation
The greenteam routinely performs Arabidopsis and Capsella transformation by the floral dip method with a capacity of ten transformations per week. We use a rockwool-based system to select for BASTA and antibiotic resistance of both species. Agrobacteria-mediated nuclear transformation for tobacco, tomato, potato, Lotus japonicus and maize is performed in tissue culture, the rice transformation system is under development. Scientist hand over transformed Agrobacteria, receive feedback on the vector quality and finally obtain T1 plants from the service.
Data Base System
The plant transformation and cultivation group developed a plant data base system based on a LIMS (Köhl et al. 2008). The system stores information on all transformations and all plants cultivated in the Max Planck Institute. It allows easy identification of plants, pedigree overviews, and location tracking of plants and seeds. The system fulfills the regulatory requirements for storage and cultivation of GMOs. It furthermore stores the information on plant import and export to provide the documentation required as a registered plant producer/importer and with regard to the Nagoya protocol. A document management system links files entries to LIMS entries. Flexible and easy-to-use search tools on web pages facilitate the information retrieval.
Additionally, standard operations procedures (SOP) in plant cultivation and plant transformation service are documented in database format (Köhl & Gremmels 2010). The system also documents the composition of all media and generates work schedules for current transformations. On the basis of the SOP system, we developed a resource management system for work and space in the plant cultivation area. The work organiser produces standardised job schedules that make work peaks visible and facilitate handover between technical staff to facilitate efficient team work. The same system is used to optimise use of the limited and expensive space in the cultivation facilities and to ensure the legally required documentation on pest monitoring and pesticide use. The system is presently updated to a modern database system with web frontends by Jürgen Gremmels from AG Walther.
1. Köhl KI, Mulugeta Aneley G, Haas M, Peters R: Confounding factors in container-based drought tolerance assessments in Solanum tuberosum. Agronomy 2021, 11(5):865.
2. Haas M, Sprenger H, Zuther E, Peters R, Seddig S, Walther D, Kopka J, Hincha DK, Köhl KI: Can metabolite- and transcript-based selection for drought tolerance in Solanum tuberosum replace selection on yield in arid environments? Frontiers Plant Sci 2020, 11.
3. Sprenger H, Rudack K, Schudoma C, Neumann A, Seddig S, Peters R, Zuther E, Kopka J, Hincha DK, Walther D et al: Assessment of drought tolerance and its potential yield penalty in potato. Func Plant Biol 2015, 42(7):655–667.
4. Köhl KI, Mulugeta Aneley G, Haas M: Finding phenotypic biomarkers for drought tolerance in Solanum tuberosum. Agronomy 2023, 13(6):1475.
5. Mulugeta Aneley G, Haas M, Köhl K: LIDAR-based phenotyping for drought response and drought tolerance in potato. Potato Research 2022, 66:1225 –1256.
