1. Introduction

For thousands of years, people have been looking for ways to increase yield and improve food quality by breeding new crop varieties. The crops we use today are the result of selecting the most valuable genotypes and carefully analyzing the offspring. The introduction of new traits, such as disease resistance, or the creation of more efficient combinations of parental genes in the offspring to ensure better product quality, usually took more than 10 years. Currently, shortening the breeding process is the goal of scientists and breeders. Therefore, breeders, in addition to classical procedures, apply new scientific methods to accelerate the breeding of the desired genotypes (J. Zimny & Michalski, 2019). The most recent method is the direct gene transfer method. It is characterized by high precision and provides detailed information on the traits of the expected final product (Sowa et al., 2021). Conventional breeding methods, in vitro culture, and genetic engineering are complementary methods for creating the desired varieties. Contemporary agriculture has different objectives, depending on its geographical location and economic conditions. In North America, it is the efficiency of production that counts; in Asia, it is the ability to meet the needs of the population; and in Europe, it is the challenge of sustainable agriculture. Owing to innovative technologies, breeding is becoming increasingly precise and fast to respond to the needs of changing markets on local and global scales. Over the past 35 years, genetic engineering, combined with tissue culture methods, has created the possibility of introducing DNA into the cells of various plant species and subsequently regenerating plants with new properties (Junker et al., 1987; Twardowski et al., 2003). Over the last 25 years, genetically modified (GM) crops obtained using recombinant DNA technology have been successfully cultivated by millions of farmers worldwide. The challenges for Polish breeders are competition from foreign companies, level of investment in research and development, and implementation of innovations in breeding. To meet these requirements, it is necessary to apply the latest scientific achievements, together with new breeding techniques. These include various variants of genetic transformation, but most importantly, editing of the genome through directed mutagenesis or modification. The development of new breeding techniques clashes with the problem of asynchronous authorization of the breeding products on a continental and global scale (Woźniak et al., 2020). However, science must develop independently from political turmoil.

The establishment of a very efficient plant regeneration system is the first prerequisite for any type of plant transformation experiment. Among various techniques, the electroporation of protoplasts and plant regeneration from GM protoplasts is the most difficult for dicots and even more for monocots. Before the first transformation could be done on cereal plants, it was necessary to develop in vitro plant regeneration protocol, which would enable the introduction of foreign genes into the cereal genome and the regeneration of plants with new genes and traits. Several varieties of cereal explants suitable for in vitro cultures have been studied by many authors. Even roots have been examined, which in the case of rice proved to be applicable (J. Zimny & Lörz, 1986), but not in the case of wheat (Chin & Scott, 1977; Dudits et al., 1975). In the late 1970s and early 1980s, it was demonstrated that immature embryos are capable of forming embryogenic callus at a fairly high frequency, and many plants can be derived from such calluses (Bajaj, 1986; Carman et al., 1987; He et al., 1986; Heyser et al., 1985; Maddock et al., 1983; Ohnoutkova et al., 1984; Ozias-Akins & Vasil, 1982; T. Shimada & Yamada, 1979). In Poland, cereals were generally considered to be difficult plants for in vitro cultures, and perhaps this was the reason why only a few research centers performed in vitro studies on rye (Rybczyński, 1978a, 1978b, 1980; Rybczyński & Zduńczyk, 1986) and barley (Grünewaldt & Malepszy, 1975; Rybczyński et al., 1986). At the end of the 1980s, in vitro research on wheat started at Plant Breeding and Acclimatization Institute, National Research Institute (IHAR) in Radzików and resulted in the development of a system allowing to induce the somatic embryos from different explants (Menke-Milczarek & Zimny, 1992a, 1992b). This system was used to select plants resistant to deoxynivalenol, a mycotoxin produced by pathogens of the genus Fusarium (Menke-Milczarek & Zimny, 1991). At the end of the 1990s, the wheat somatic embryogenesis system was used by Prof. Jan Rybczyński from Botanical Garden of the Polish Academy of Sciences in collaboration with the University of Nebraska (USA) to achieve high transformation efficiency and long-term embryogenic potential of transformed tissue (Zhang et al., 2000). As far as triticale is concerned, the first works on callus induction and plant regeneration were recorded in the mid of 1980s (Stolarz & Lörz, 1986; J. Zimny & Rybczyński, 1986). These works were carried out in the Botanical Garden of the Polish Academy of Sciences, Powsin and IHAR, Radzików in cooperation with Dr. Horst Lörz from the Max Planck Institute in Cologne.

Research on plant tissue cultures and their uses in genetic engineering aimed to enrich the possibilities of modern plant breeding has been conducted at the IHAR since the 1970s. Here, we describe in detail the achievements of the Department of Biotechnology and Cytogenetics, a special unit of the IHAR, in the field of cereal somatic embryogenesis, genetic modification, GMO detection and identification, and GMO legislation.

2. Somatic Embryogenesis as a Prerequisite for Genetic Modification

In the 1980s, obtaining somatic embryos from monocots, especially cereals, was a major challenge for researchers worldwide (J. Zimny & Michalski, 2019). Scientists from IHAR, in collaboration with other Polish and German institutions, conducted pioneering studies that led to the description of somatic embryogenesis in triticale and rye. In 1985, an article on somatic embryogenesis in rye appeared at the Eucarpia meeting (Rybczyński & Zimny, 1985). One year later, the world’s first reports on somatic embryogenesis in triticale were published (Stolarz & Lörz, 1986; J. Zimny & Rybczyński, 1986). These studies identified and described the structures emerging from calluses as somatic embryos. Histological analysis performed with electron microscopy revealed that these somatic embryos had all the characteristics of the zygotic embryos (J. Zimny & Lörz, 1989). Both induction of somatic embryogenesis and efficient plant regeneration had to be optimized. This was performed in the late 1980s by developing appropriate protocols. In subsequent years, we focused on triticale as a cereal of growing economic importance in Poland. Embryogenic lines derived from single plants of different genotypes were selected during several growing seasons. The best results were obtained with the MAH 1590 winter triticale line, in which high somatic embryogenesis efficiencies were recorded for all plants tested in subsequent experiments. Some lines from the other two genotypes showed a trend of decreasing efficiency in the following years. The question of what caused the observed variation within the stabilized genotypes must be answered. One possible explanation is the lack of genetic stability in evolutionarily young species such as triticale. Therefore, we developed a method to obtain homozygous lines by inducing haploid embryos from microspores. Statistical analysis showed no significant variation in somatic embryogenesis potential among MAH 1590 individuals with a very high level of somatic embryogenesis efficiency (>83%). At the same time, significant variability was observed among plants of the other genotypes tested. The MAH 1590 line, showing the highest embryogenesis efficiency, was at the same time the most uniform in terms of in vitro responsiveness. Therefore, this genotype was used in subsequent studies on the genetic modification of triticale. In a short time, MAH 1590 was registered by Dr. Bogusław Łapiński as ‘Bogo,’ a new high-yielding winter triticale variety. ‘Bogo’ has been successfully grown for many years in Poland, and after 30 years since our discovery, it continues to serve as a model genotype used in studies on in vitro plant regeneration and transformation of triticale (Hensel et al., 2012; Michalski et al., 2021; Oleszczuk et al., 2004; Sowa et al., 2005).

3. Genetic Modification of Cereals

Genetic engineering is currently used in both research and practice (Czaplicki et al., 2005; J. Zimny & de Vries, 2005). At the beginning of the new millennium, it was difficult to predict; however, the introduction of GM plants into the environment would face strong opposition from European society (Bock & Zimny, 2005; Twardowski et al., 2003). In our laboratories, the first studies on plant transformation were carried out using triticale plant material developed at IHAR, but our methods were later used for the transformation of cereal plants in other laboratories. The list of transgenic plants is long, but their reports are often vague and misleading. According to Potrykus (1991), proving an integrative transformation requires meeting several conditions, including complete Southern Blot analysis (signal in the region of high molecular weight), the presence of a complete gene, the absence of DNA contamination (negative control), and data allowing to distinguish false and true transformants. Fulfillment of these requirements by the transgenic triticale plants developed in IHAR was a result of cooperation with leading European laboratories in 1993. These triticale plants contained a gene conferring resistance to the Basta herbicide. This was the world’s first transgenic triticale (J. Zimny et al., 1995). The Polish Academy of Sciences awarded this achievement in 1996. In the following year, other cereals, such as rye, were also successfully transformed (Sowa et al., 2000).

4. Multi-Generation Analysis of Transgenic Triticale Plants

Transgenic lines have been stabilized by the regeneration of homozygous androgenic lines (Oleszczuk & Lukaszewski, 2014). Several generations of transgenic plants have been tested over the past 29 years. The most interesting finding was the localization of transgenes on individual chromosomes, which indicated the completely random nature of the insertion of the foreign gene into the recipient genome (Pedersen et al., 1997). This was the starting point for many studies, not only on genetic transformation but also on the potential negative impact of transgenic cereal plants on the environment. These studies were carried out in various Polish laboratories after 2000, and among many others, included a project on feeding successive generations of experimental mice with transgenic triticale (Baranowski et al., 2006; Jaszczak et al., 2008; Krzyżowska et al., 2010). The latter was conducted by IHAR in cooperation with the Institute of Genetics and Animal Biotechnology, Polish Academy of Sciences, Jastrzębiec and with the Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW.

5. Legal Situation and Risk Assessment of Transgenic Plants

After the first transgenic plants appeared in the United States in the 1990s, an intense debate began on the potential negative impact of all GMOs, especially GM plants (J. Zimny & de Vries, 2005). Polish law on GMOs came into force in 2001, and we were involved in preparing the legislation in the form of workshops and publications under the project GF/1200-98-84 financed by the United Nations Environment Program (Anioł et al., 1998, 1999a, 1999b, 1999c; Zimnoch-Guzowska et al., 2004). In 2006, the cultivation of GM maize MON 810, resistant to European corn borers, was introduced in Poland. In 2007, it was grown on 300 ha, and a year later, on approximately 3,000 ha. However, placing GM seeds in the market was later banned in Poland, so farmers had to buy seeds from other countries of the European Union (EU). MON 810 maize was rapidly gaining popularity, while at the same time raising voices claimed that these new biotechnological products had never been tested under Polish conditions. In response to this outcry, we began preparations for a special grant proposal because we already had some experience in this area resulting from similar projects, conducted together with Prof. Andrzej Anioł.

The grant proposal was successful, and in the years 2008–2011, after receiving funds from the National Centre for Research and Development, we carried out the research project (No. PBZ-MNiSW-06/1/2007) titled “Environmental and economic aspects of the authorization of the cultivation of genetically modified plants in Poland.”

6. Gene Flow and the Coexistence of Genetically Modified, Conventional, and Organic Farming Systems

7. National Reference Laboratory for GMOs Supporting Official Control of GMOs in Poland and the EU

The experience gained in genetic modifications and molecular analyses of plants allowed us to establish the GMO Controlling Laboratory at the IHAR as an element of the biosafety system in Poland. In 2004, when Poland joined European Union, this laboratory became a member of the European Network of GMO Laboratories (ENGL). The latter is a consortium of official enforcement laboratories designated by the EU Member States. The primary objective of ENGL is to provide scientific and technical support to the European Union Reference Laboratory facing the challenges of proper detection, identification, and quantification of GMOs (Żmijewska et al., 2016; Żurawska-Zajfert et al., 2016). These tasks are required by Regulation (EC) 1829/2003. Plenary meetings are held annually to share the experiences gained by member countries. ENGL serves as a platform for technology transfer between ENGL members and the Joint Research Center (JRC), promoting the exchange and training of scientists. ENGL experts have prepared guidance documents on topics of interest for executive laboratories (Corbisier et al., 2017; Trapmann et al., 2020). The GMO Controlling Laboratory at the IHAR was nominated as one of the National Reference Laboratories supporting the European Union Reference Laboratory for GM Food and Feed under Regulation (EC) 1829/2003 and the National Reference Laboratories under Regulation (EC) 2017/625. National Reference Laboratories collaborate with the JRC of the European Commission providing scientific and technical support in areas relevant to health and consumer protection (Żmijewska et al., 2017). The GMO Controlling Laboratory is accredited by the Polish Center for Accreditation according to the ISO 17025 standard, ensuring a high quality of analysis and flexible scope of accreditation.

8. New Genomic Techniques – Legal Problems and Challenges for Identification

We actively participate, through publications, lectures at scientific conferences, and as members of appropriate bodies, in the debate on the legal aspects of new genomic techniques in plant breeding, which takes place in the EU (Sowa et al., 2021; Twardowski et al., 2017; T. Zimny & Sowa, 2021; T. Zimny et al., 2019). Genome editing is becoming an efficient tool that is being increasingly used in plant breeding. However, in Europe, owing to legal restrictions, these new research methods are currently used exclusively for scientific purposes (Menz et al., 2020). After the judgment of the European Court of Justice (Eriksson & Zimny, 2020), which is widely interpreted as classifying products of most new genomic techniques as regulated GMOs, the European Commission decided that the current regulatory framework requires amendments to address problems that may be caused by the development and use of such products (European Commission, 2021).

Being aware of all these challenges, we continuously master genomic techniques through scientific research and must be ready to produce improved and safe breeding materials, now by optimizing the recent CRISPR/Cas9 method (Michalski et al., 2021).

9. Final Remarks

From the beginning, scientific work at the IHAR was carried out using the latest available methods and in cooperation with the best research centers in the country and abroad. Our colleagues continuously follow the ongoing events related to biotechnological innovations in the European market through active participation in both European (European Network for GMO Laboratories at the JRC, and EFSA Scientific Network for Risk Assessment of GMOs) and national bodies (Committee on Biotechnology of the Polish Academy of Sciences, Commission on GMM and GMO at the Ministry of Climate and the Environment, and Technical Committee of The Polish Committee for Standardization). Over the years, this has built up the authority of the institute and the department. We hope that this high-level of scientific research will be successfully continued.