. Introduction

In Poland, the first six plant species were placed under strict protection in 1919, and in 1946, the first species protection ordinance listing 110 plants was issued. The Ministry of the Environment’s regulation issued on October 9, 2014 (Journal of Laws, 2014), is currently in effect. A total of 415 species (including 270 seed plants) are strictly protected and 301 species (including 121 seed plants) are partially protected.

There are two basic strategies for preserving plant genomic reserves: in situ and ex situ conservation. Plants can be protected outside of their native habitats with a gene bank through traditional methods such as field collection, seed banks, and botanical gardens, as well as in vitro techniques using micropropagation and storage cultures. Many centers have tissue culture laboratories for the multiplication of plants that are difficult to propagate by conventional horticultural techniques.

Tissue culture permits mass production of cloned plants. Plants obtained in vitro can be of great value for research, living collections, and plant reintroduction programs if considered appropriate. Different morphogenetic pathways such as organogenesis, that is, the multiplication of shoots from axillary buds, the formation of adventitious organs, and somatic embryogenesis are used for in vitro production of native, protected, partly protected, endangered, critically endangered, and extinct species.

Australia is an example of a country that has successfully used in vitro cultures to protect endangered plant species for years (Ashmore et al., 2011). This country is currently implementing the Threatened Species Strategy 2021–2031 (Australian Government, 2021).

In vitro techniques were suggested for ex situ propagation of native endangered plant species in Poland for the first time by Professor Krystyna Kukułczanka from the Botanical Garden of the University of Wrocław. His paper, entitled “The role of in vitro cultures in the conservation of rare and threatened plant species” (Kukułczanka, 1987), referenced a seminar that took place ten years earlier in the Royal Botanic Gardens, Kew, England. At that time, the important role of tissue cultures in the protection and preservation of plant species that are difficult to reproduce using traditional methods was indicated. The author emphasized that plants could be multiplied in vitro by initiating a culture from a small number of seeds, organs, or tissues, and the resulting cultures could be maintained under optimal conditions for a long time and serve as tissue banks.

Currently, biotechnological methods based on in vitro techniques are being used as plant protection tools for various purposes. The most important are vegetative reproduction in vitro and protection of the morphogenetic potential of cells, tissues, and organs (Rybczyński & Mikuła, 2006).

For almost 30 years, Polish scientists from universities, research institutes, and botanical gardens have been working on developing micropropagation protocols for endangered species in Poland. However, only a few species of Polish flora are protected using in vitro techniques. The number of endangered plant species is growing, hence, propagation studies must be intensified in the near future and all possible techniques must be combined to cover various aspects of biodiversity protection.

The aim of this study is to review the most significant achievements of Polish scientists in the application of in vitro culture and biotechnology for the protection of native taxa. There are ten notable research institutions in Poland involved in this field, including eight academic centers and two botanical gardens (Table 1). These research centers investigate the possibilities of employing in vitro and biotechnological techniques to protect the endangered species native to Poland.

Table 1

The most notable research institutions involved in this field of in vitro conservation of protected species in Poland.

InstitutionPlant species
University of Agriculture in KrakowLeucojum vernum, Lilium maratagon, Cypripedium calceolus, Dactylorhiza maculate, Epipactis helleborine, Goodyera repens, Gymnadenia conopsea, Biscutella laevigata, Gentiana pneumonanthe, Hepatica nobilis, Primula farinose, Staphylea pinnata, Daphne cneorum
University of GdańskDrosera anglica, Cypripedium calceolus, Dactylorhiza majalis, Epipactis atrorubens, Epipactis palustris, Orchis morio
Nicolaus Copernicus University in ToruńCarlina onopordifolia, Cirsium pannonicum, Inula germanica, Leontopodium alpinum, Taraxacum pieninicum
Poznań University of Medical SciencesEryngium maritimum, Eryngium alpinum, Eryngium planum, Linnaea borealis, Rubus chamaemorus
Botanical Garden of the University of WrocławAsplenium adulterinum, Asplenium cuneifolium, Drosera intermedia, Drosera rotundifolia
Polish Academy of Sciences Botanical Garden – CBDC*Osmunda regalis, Lilium maratagon, Gentiana cruciate, Gentiana punctata
Jagiellonian UniversityPulsatilla vulgaris, Viola ulginosa, Viola stagnina
Adam Mickiewicz University in PoznańMatteucia struthiopteris, Osmunda regalis
University of Life Sciences in LublinAldrovanda vesiculosa, Salix lapponum
Warsaw University of Life SciencesDaphne mezereum

* Center for Biological Diversity Conservation in Powsin.

The achievements presented in this study include fern and monocotyledon species (Table 2), herbaceous dicotyledons (Table 3), and several woody species (Table 4).

Table 2

Selected species of native fern and monocotyledonous species protected in Poland with the use of in vitro techniques.

Plant speciesInitial explantsMorphogenetic pathwayPlant acclimatization*Cytometric/Molecular analysis* *References
Asplenium adulterinumAsplenium cuneifoliumsporesin vitro spore germination, gametophytes and sporophytes obtained+Marszał-Jagacka et al., 2005
Matteucia struthiopterisrhizomes, dormant buds from rhizomesindirect organogenesis--Zenkteler, 2006
Osmunda regalissporesgametophyte cultures, young sporophytes, saprophyte production--Zenkteler, 1999; Makowski et al., 2016
Amaryllidaceae family

Leucojum vernumscales, leaves from bulbs, ovaries and fruitsindirect somatic embryogenesis-CAPtak, 2010
Liliaceae family

Lilium maratagonseedling explants, adventitious bulblet scalesorganogenesis, indirect somatic embryogenesis-CARybczyński & Gomolińska, 1989; Kędra & Bach, 2005; Pawłowska et al., 2007
Orchidaceae family

Cypripedium calceolusseeds, capsules, young ovariesseedling growth protocorm-like bodies obtained--Znaniecka & Łojkowska, 2004; Pindel & Pindel, 2004
Dactylorhiza maculatacapsules, young ovariesprotocorms, protocorms with shoots and roots development--Pindel & Pindel, 2004
Dactylorhiza majalisseedsseedling growth--Znaniecka & Łojkowska, 2004
Epipactis atrorubens
Epipactis palustris
Epipactis helleborine
Goodyera repens
Gymnadenia conopsea
capsules, young ovariesindirect organogenesis--Pindel & Pindel, 2004
Orchis morioseedsseedling growth--Znaniecka & Łojkowska, 2004

* + acclimatization were conducted, - plants were not acclimatized.

** CA: cytometric analysis.

Table 3

Achievements of Polish scientists in the protection of native, herbaceous, dicotyledonous species by means of in vitro techniques.

SpeciesExplantsMorphogenetic pathwayPlant acclimatization*Cytometric/Molecular analysis* *References
Herbaceous plants
Asteraceae family

Carlina onopordifoliashoot tips from seedlingsorganogenesis: axillary shoot multiplication and rooting--Trejgell et al., 2012a
Cirsium pannonicumshoot tips from seedlingsorganogenesis: axillary shoot multiplication and rooting+-Trejgell et al., 2012b
Inula germanicashoot tips from seedlings, fragments of cotyledons, hypocotyls and rootsorganogenesis: adventitious shoot induction, multiplication and rooting+-Trejgell et al., 2018
Leontopodium alpinumshoot tips from seedlings, fragments of cotyledons, hypocotyls and rootsorganogenesis: axillary and adventitious shoot proliferation and rooting--Trejgell et al., 2010
Taraxacum pieninicumshoot tips from seedlingsorganogenesis: axillary bud proliferation, synthetic seed production and storage in slow growth conditions--Kamińska et al., 2021
Apiaceae family

Eryngium maritimum
Eryngium alpinum
Eryngium planum
apical and axillary budsorganogenesis: multiplication and rooting of shoots+CAKikowska et al., 2014; Kikowska et al., 2020; Thiem et al., 2013
Brassicaceae family

Biscutella laevigatain vitro shootsorganogenesis: shoot multiplication+-Muszyńska et al., 2017
Droseraceae family

Drosera anglicaleaf segmentsdirect organogenesis--Kawiak et al., 2003
Drosera intermedia
Drosera rotundifolia
seedsseed germination and seedling growth, storage in slow growth conditions--Kukułczanka & Cząstka, 1987; Kukułczanka et al., 1991
Aldrovanda vesiculosashoot fragmentsorganogenesis: shoot multiplication--Parzymies, 2021
Gentianaceae family

Gentiana pneumonantheseedsdirect organogenesis: axillary shoot multiplication, somatic embryogenesis, synthetic seeds-CAPawłowska & Bach, 2003; Bach & Pawłowska, 2003; Bach et al., 2004
Gentiana cruciataseedlingssomatic embryogenesis--Mikuła & Rybczyński, 2001; Mikuła et al., 2005a,b
Gentiana punctatazygotic embryossomatic embryogenesis--Mikuła et al., 2004
Primulaceae family

Primula farinosaseedlingsorganogenesis: axillary shoot cultures and rooting+CASitek et al., 2020
Ranunculaceae family

Hepatica nobilisseedlingssomatic embryogenesis+-Szewczyk-Taranek & Pawłowska, 2015
Pulsatilla vulgarisseedlingssomatic embryogenesis (direct and indirect), organogenesis - adventitious shoot, rooting+CA, ISSRŻabicka et al., 2021
Violaceae family

Viola uliginosafragments of leaf and petioleorganogenesis (direct and indirect), shoot rooting+CA, AFLPŚlązak et al., 2015
Viola stagninafragments of leaf and petioleorganogenesis (direct and indirect)+CA, ISSRŻabicki et al., 2019, 2021

* + acclimatization were conducted, - plants were not acclimatized.

** CA: cytometric analysis, ISSR - inter simple sequence repeat, AFLP - amplified fragment length polymorphism.

Table 4

Achievements of Polish scientists in the protection of native, woody species (including dwarf shrubs) by means of in vitro techniques.

SpeciesExplantsMorphogenetic pathwayPlant acclimatization*Cytometric/Molecular analysis* *References
Caprifoliaceae family

Linnaea borealisnodal segments and shoot tips with apical meristemsorganogenesis: shoot multiplication on solid and liquid media, rooting-CAThiem et al., 2021
Rosaceae family

Rubus chamaemorusshoot tipsorganogenesis: shoot multiplication, rooting+CAThiem, 2021; Thiem & Śliwińska, 2003
Salicaceae family

Salix lapponumshootsorganogenesis: shoot multiplication, rooting+CA, ISSRParzymies et al., 2020
Staphyleaceae family

Staphylea pinnatadormant budsorganogenesis: axillary shoot propagation--Szewczyk-Taranek & Pawłowska, 2016
Thymelaeceae family

Daphne mezereumaxillary shootsorganogenesis: shoot multiplication, rooting-ISSR, RAPDPacholczak & Nowakowska, 2019; Nowakowska & Pacholczak, 2020
Daphne cneorumin vitro rootsroot cultures, tolerance to Thielaviopsis basicola--Hanus-Fajerska et al., 2014

* + acclimatization were conducted, - plants were not acclimatized.

** CA: cytometric analysis, ISSR - inter simple sequence repeat, RAPD - randomly amplified polymorphic DNA.

This study does not include cryopreservation. Detailed information on cryopreservation was presented by Mikuła et al. (2022).

. Endangered national ferns and monocotyledons propagated by tissue culture

In vitro cultures of the native ferns Asplenium adulterinum, Asplenium cuneifolium (Marszał-Jagacka et al., 2005) and Osmunda regalis (Zenkteler, 1999; Makowski et al., 2016) were initiated by spore germination, which is the most common method for this group of plants. Attempts have also been made to grow Matteucia struthiopteris cultures from rhizomes and dormant buds on rhizomes and to induce callus formation and proliferation (Zenkteler, 2006). The mineral salt content of the medium plays a significant role in fern in vitro culture. For the growth of A. adulterinum and A. cuneifolium gametophytes, half strength nutrient Murashige and Skoog (MS) medium was used (Murashige & Skoog, 1962; Marszał-Jagacka et al., 2005). Similarly, in research carried out in the Botanical Garden in Powsin, reducing the content of mineral salts in the MS medium to half or even a quarter had a positive effect on the proliferation of gametophytes Osmunda regalis. The maximum sporophyte production in this fern requires 1/8 MS mineral salts (Makowski et al., 2016). M. struthiopteris in vitro culture could be initiated in the Modified Fern Multiplication Medium (Miller & Murashige, 1976).

The reviewed publications indicate that in vitro techniques have been used to protect the greatest number of Orchidaceae species to date, even though only two papers published in 2004 investigated the species mentioned here. The development of protocols for protocorm culture and further plant regeneration for native orchid species is particularly challenging. Pindel & Pindel (2004) initiated cultures of five orchid species from green capsule sections. Znaniecka & Łojkowska (2004) established the cultures of five other native orchid species by focusing mainly on the observations of seedling germination and development. Both teams worked on Cypripedium calceolus; but the most advanced development of protocorms with shoots and roots was achieved in Dactylorhiza maculata (Pindel & Pindel, 2004). MS medium supplemented with naphthaleneacetic acid (NAA), 6-benzyl-aminopurine (BAP), and activated charcoal was the best medium for the development of protocorms (Pindel & Pindel, 2004). In contrast, media containing peptone, yeast extract, and casein hydrolysate were the most effective for germination and development of orchid seedlings (Znaniecka & Łojkowska, 2004).

However, Polish scientists have rarely investigated bulbous plants plants. The first research results on the induction of adventitious bulbs in Lilium martagon were published over 30 years ago by Rybczyński & Gomolińska (1989). They cultured bulb scales as explants in MS medium containing NAA and BAP. Significant progress in the propagation of bulbous plants was made at the University of Agriculture in Krakow, where the protocols for adventitious organogenesis and indirect somatic embryogenesis were developed for Leucojum vernum and L. martagon, thus considerably increasing their propagation rate (Kędra & Bach, 2005; Ptak, 2010). In L. martagon, the formation of adventitious bulbs was observed in seedling bulb explants on MS medium in absence of plant growth regulators or in the presence of BAP. The best explants for embryogenic callus initiation were adventitious bulb scales in L. martagon and fruit tissues in L. vernum. The medium for culture initiation, callus propagation, and somatic embryo induction of L. martagon and L. vernum was enriched with 4-amino-3,5,6-trichloropicolinic acid (picloram) and BAP. The abscisic acid and polyethylene glycol added to the medium stimulated somatic embryo maturation in L. vernum (Table 2).

. Endangered national dicotyledon herbaceous species and woody plants propagated by tissue culture

In the case of dicotyledonous species, the research mostly involved herbaceous species, 21 of which belonged to eight botanical families. Woody plants were represented by only six species belonging to five families (Tables 3 and 4).

Drosera species are among the first protected herbaceous plants propagated in vitro. The study was initiated by Professor Kukułczanka (Kukułczanka & Cząstka, 1987) and continued by researchers from the University of Gdańsk (Kawiak et al., 2003).

The in vitro cultures of Drosera are usually initiated from seeds, but also, more rarely, from leaf fragments (Kawiak et al., 2003). The seeds of the protected taxa are easily available, their harvesting does not destroy the mother plants, and furthermore, the seed-initiated culture contributes to maintaining the species variability and biological diversity. In vitro cultures of many other herbaceous taxa have been established from seeds and seedling fragments and used for further propagation. Such procedures have been implemented for species belonging to the Asteraceae family, for example, Carlina onopordifolia, Cirsium pannonicum, Inula germanica, Leontopodium alpinum, Taraxacum pieninicum, from the Gentianaceae family (e.g., Gentiana pneumonanthe), Primulaceae family (e.g., Primula farinosa or Hepatica nobilis), and from the Ranunculaceae family (Pulsatilla vulgaris) (Table 3). In some taxa of herbaceous plants, such as Eryngium maritimum (Kikowska et al., 2014), Eryngium alpinum (Kikowska et al., 2020), Viola uliginosa (Ślązak et al., 2015), Viola stagnina (Żabicki et al., 2019, 2021), and all investigated tree taxa (except for Rubus chamaemorus, which was initiated from seeds), the cultures were initiated from vegetative explants (dormant buds) (Table 4). The most recent advances within this family involved the successful application of nanosilver to maintain sterility in Aldrovanda vesiculosa cultures (Parzymies, 2021).

The micropropagation of protected and endangered plants is mainly based on organogenesis, and research has focused primarily on determining the influence of growth regulators on shoot multiplication and in vitro rooting. The research also focused on the selection of micro- and macro-elements in the nutrient medium; for example, the medium according to Reinert and Mohr (1967) was the best for Drosera rotundifolia seedling development (Kukułczanka & Cząstka, 1987) and the liquid Fast medium (Fast, 1981) was the best for the proliferation of Drosera anglica shoots (Kawiak et al., 2003).

In contrast, in studies on C. onopordifolia, the MS medium was modified by replacing ferric ethylenediamine-tetraacetic acid (FeEDTA) with ethylenediamine di-2-hydroxy-phenyl acetate ferric acid (Fe-EDDHA). These modifications had no effect on the rate of axillary shoot proliferation, but they enhanced leaf chlorophyll content (Trejgell et al., 2012a).

In addition to MS medium, in vitro propagation of woody plants has also been achieved on QL medium (Quoirin & Lepoivre, 1977) and Woody Plant Medium (WPM) (Lloyd & McCown, 1980) for Staphylea pinnata (Szewczyk-Taranek & Pawłowska, 2016) and WPM medium for Daphne mezereum (Nowakowska & Pacholczak, 2020).

During axillary shoot multiplication of S. pinnata, the identification and elimination of endophytic bacterial contamination was studied. VITEK®;2, a rapid bacterial identification system, and the 16S rRNA gene sequencing method allowed for the identification of Acinetobacter johnsonii strain ATCC 17909 and Methylobacterium rhodesianum strain DSM 5687. The addition of gentamicin to the medium was the most effective in eliminating bacteria (Szewczyk-Taranek et al., 2020).

The cultures were maintained under fluorescent white light. Szewczyk-Taranek et al. (2017) showed that LED light may be an alternative light source for the in vitro shoot proliferation stage of S. pinnata (Szewczyk-Taranek et al., 2017).

To protect endangered plants, Polish researchers have also used the process of somatic embryogenesis for plant propagation. This process was used by Mikuła and Rybczyński (2001) in Gentiana cruciata, and by Bach and Pawłowska (2003) in G. pneumonanthe. Plantlet regeneration from somatic embryos was achieved in both studies. Supplementation of MS medium by 2,4-D (dichlorophenoxyacetic acid) was a key factor for somatic embryogenesis induction. In G. cruciata, embryogenic callus formation was observed in seedlings (best performance on hypocotyl and cotyledonous seedlings, 39–46 somatic embryos were formed per explant). In G. pneumonanthe, the explants were leaves or apical meristems derived from shoots multiplied in vitro (25–37% of the explants formed embryogenic callus). Additionally, embryogenic callus in marsh gentians were observed in media containing picloram. Embryo development was possible after lowering the auxin level in the culture medium, and maturation occurred in the media without growth regulators.

Moreover, Mikuła et al. (2005a) demonstrated the huge potential of embryogenic suspension cultures of G. cruciata for the long-term mass production of somatic embryos. Somatic embryogenesis was also performed in two species of the Ranunculaceae family. Żabicka et al. (2021) used shoot tips of P. vulgaris seedlings to obtain embryogenic callus and somatic embryos. A method for secondary somatic embryogenesis was developed for H. nobilis (a species protected since 2004) embryos derived from embryogenic callus formed on seedlings. The most efficient repetitive cyclic process of secondary somatic embryogenesis was observed in MS medium without growth regulators (on 78–87% primary somatic embryos), and the best rate of somatic embryo germination was achieved in MS medium with BAP (52%) (Szewczyk-Taranek & Pawłowska, 2015).

The final stage of micropropagation of protected plants is acclimatization. In vitro regenerated plants of C. pannonicum (Trejgell et al., 2012b), E. maritimum (Kikowska et al., 2014), H. nobilis (Szewczyk-Taranek & Pawłowska, 2015), I. germanica (Trejgell et al., 2018), P. farinosa (Sitek et al., 2020), P. vulgaris (Żabicka et al., 2021), R. chamaemorus (Thiem, 2001), V. stagnina (Żabicki et al., 2019), V. ulginosa (Ślązak et al., 2015) and Salix lapponum (Parzymies et al., 2020) were successfully acclimatized to greenhouse conditions. Studies of plant adaptation to ex vitro conditions in a greenhouse also included observations of plant performance in the field (e.g., for Biscutella laevigata) (Muszyńska et al., 2017).

. Selected achievements of biotechnological research

Experiments on G. pneumonanthe (Bach et al., 2004), T. pieninicum (Kamińska et al., 2021) and E. alpinum (Kikowska et al., 2020) have investigated the possibility of storing plant material in the form of synthetic seeds. Sodium alginate coatings were used for lateral shoot tips derived from gentian, cloudberry, and eryngo propagated in vitro and for shoot tips taken from dandelion seedlings germinated in vitro.

In vitro cultures have also been used to investigate the protected plants with respect to their resistance to fungal pathogens, such as Thielaviopsis basicola attacking Daphne cneorum (Hanus-Fajerska et al., 2014), or tolerance to heavy metals such as Pb and Cd (B. laevigata) (Muszyńska et al., 2017).

To assess the usefulness of the micropropagation protocol for species protection, it is important to test the genetic stability of the regenerated plants. This is typically performed using molecular markers and flow cytometry. Recently, the genetic stability of the in vitro regenerated plants was analyzed using the following markers: inter simple sequence repeat (ISSR) in S. lapponum, D. mezereum, V. stagnina and P. vulgaris; randomly amplified polymorphic DNA (RAPD) in D. mezereum; and amplified fragment length polymorphism (AFLP) in V. uliginosa (Ślązak et al., 2015; Nowakowska & Pacholczak, 2020; Parzymies et al., 2020; Żabicki et al., 2019, 2021). Cytometric analysis of the regenerated plants was performed for E. maritimum (Kikowska et al., 2014), E. alpinum (Kikowska et al., 2020), G. pneumonanthe (Bach & Pawłowska, 2003), L. vernum (Ptak, 2010), L. martagon (Kędra & Bach, 2005; Pawłowska et al., 2007), Linnaea borealis (Thiem et al., 2021), P. farinosa (Sitek et al., 2020), P. vulgaris (Żabicka et al., 2021), R. chamaemorus (Thiem & Śliwinska, 2003), S. lapponum (Parzymies et al., 2020), V. uliginosa (Ślązak et al., 2015) and V. stagnina (Żabicki et al., 2019). Majority of the studies found no differences between in vitro regenerated plants and mother plants, indicating that the developed micropropagation protocols are suitable for use. In some cases, as suggested by Ślązak et al. (2015), more direct methods are preferred for micropropagation, even when the process is less effective. The authors showed that the genetic uniformity of the regenerated V. uliginosa plants with the mother plants, as detected by AFLP analyses, clearly depended on the type of organogenesis.

Notably, biotechnological research also focuses on the use of in vitro cultures of protected species as a source of therapeutically important substances. For this purpose, various types of in vitro cultures have been performed (e.g., shoot, callus, root, and suspension cultures), and various treatments have been used to stimulate the biosynthesis of secondary metabolites (Kikowska et al., 2022). This issue has been discussed in a separate publication (Pietrosiuk et al., 2022).

. Conclusion

Although studies on the implementation of in vitro multiplication and conservation procedures for protected plant species are actively carried out in Poland, they are concerned only with a small percentage of endangered species of the Polish flora. As the number of endangered plant species is constantly increasing, these studies should be intensified in the future. However, this claim has considerable limitations. First, the protected species come from various botanical families, and the development of micropropagation procedures is highly individualized and, therefore, a slow process. Second, due to a lack of legal regulations in Poland on plants propagated in vitro, the reintroduction of such species is not common.

Despite the wide range of possibilities provided by well-equipped laboratories and experienced researchers in Poland, the potential of in vitro cultures for biodiversity conservation has not been fully utilized. Currently, the use of in vitro cultures is usually restricted to storing plant material in in vitro gene banks and long-term storage under in vitro conditions. Future studies should focus on using in vitro cultures as a source of explants stored in liquid nitrogen (cryopreservation).