Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae) is a perennial herb endemic to the eastern coast of the Adriatic Sea. The species is widely cultivated in many countries for its bioactive compounds pyrethrins, which are used as natural insecticides. Plants derived from seeds vary greatly in pyrethrin content; therefore, the vegetative propagation of high-quality individuals is very important for the establishment of agricultural pyrethrum crops. The present study deals with rapid in vitro multiplication of pyrethrum, ex vitro adaptation of selected clones and creation of an ex situ collection, as a first step towards introducing the species into agriculture in Bulgaria. Seeds from a private ex situ collection in Bulgaria and from a natural Croatian population were used as initial material for in vitro cultures initiation. Basal MS medium (Murashige and Skoog 1962) or MS supplemented with different concentrations of kinetin and indole-3-butyric acid were used for seed germination and multiplication of one-seed derived clones by consecutive subcultivations. The propagation effectiveness was evaluated as a number of new plants obtained per initial shoot. Considerable losses were noticed due to both endophytic contaminations and necrosis, especially on media supplemented with plant growth regulators. These problems were overcome by medium optimization: adding an antibiotic and modifying the medium to increase the calcium concentration using CaCO₃. In the best medium variant (basal MS + 200 mg/L Medaxone + 75 mg/L Ca) no more infected plants were observed, and the percentage of necrotic plants decreased threefold, which resulted in formation of 38.06±10.11 new plants per initial shoot for a period of 7 months. Three hundred and sixty plants were ex vitro adapted in a phytotron (88% surviving rate), then 16 plants from 4 selected clones were transferred to the ex situ collection and bloomed twice from the very first growing season (June and September). The number of the flower heads increased in the second year of field cultivation and an average of 328±138 capitula per plant were counted for the best clone. The first trials to establish a pilot plantation of pyrethrum are promising.

Chrysanthemum cinerariaefolium, ex vitro adaptation, in vitro micropropagation, nutrient medium modification, plant acclimatization, pyrethrum

Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip., also known by its synonyms Pyrethrum cinerariaefolium Trev., and Chrysanthemum cinerariaefolium Bocc (Pignatti 1982; Grdiša et al. 2009; Rahmatullah et al. 2010), is a perennial herb of Asteraceae family. It is endemic to the eastern coast of the Adriatic Sea, distributed from Italy to northern Albania and in the mountainous regions of Croatia, Bosnia and Herzegovina, and Montenegro (Staykov and Ilieva 1961; Nikolić and Rešetnik 2007; Grdiša et al. 2009; Grdiša et al. 2013; Šegota et al. 2016). The species was first introduced into agriculture in Armenia (Casida 1980), and is currently widely cultivated in many countries due to the pyrethrins it contains (Staykov and Ilieva 1961; Fulton et al. 2001a; Grdiša et al. 2009; Casida 2012). The use of pyrethrins as an insecticide is thought to have originated in Persia, where Pyrethrum roseum Bieb. and Pyrethrum garneum Bieb. were known long ago (Staykov and Ilieva 1961; Wainaina 1995). According to other authors, dried parts of T. cinerariifolium were used as insecticide in folk medicine in Croatia (Grdiša et al. 2009).

Natural pyrethrins can be characterized as an excellent means of combating insect pests (Jovetic 1994; Hammond 1996; Palmquist et al. 2012). They are very effective against a wide range of insects (Tattersfield et al. 1929; Arnaudov 1930; Richardson 1931; Greenhill 2007; Cai et al. 2010) acting as a contact insecticide ( Grdiša et al. 2009), and the risk of developing resistance is low.

Seed germination of T. cinerariifolium is not high, especially in natural populations (Singh and Sharma 1989; Fulton et al. 2001b). In our previous study on seeds gathered from an ex situ collection in Bulgaria, it was found that seeds quickly lost their vitality and their germination rate dropped dramatically after several years of storage (Ilinkin et al. 2020). Seeds can also be purchased from pyrethrum breeding programs (British Oxygen Company) with 80% germination rate (Fulton et al. 2001b). However, agricultural crops of T. cinerariifolium are usually established by vegetative propagation as plants derived from seeds vary greatly in pyrethrin content (Casida 1973; Ikahu and Ngugi 1988; Lindiro et al. 2013).

Access to high-quality plant material is the main limiting factor for the mass cultivation of T. cinerariifolium; therefore the development of an efficient in vitro protocol for micropropagation of selected high-yielding plants is a task for many researchers (Lindiro et al. 2013). Most studies have been based on agar-solidified media (Staba et al. 1984; Hussain et al. 1994; Keskitalo 1999) and rarely has liquid media been tested (Staba and Zito 1985; Keskitalo 1999). The characteristics of the culture medium can be a major factor in establishing a highly efficient in vitro culture. Some authors (Roest and Bokelmann 1973; Kaul et al. 1990; Hedayat et al. 2009) reported successful propagation of Chrysanthemum species on MS medium (Murashige and Skoog 1962). According to Hedayat et al. (2009) the composition of MS medium is optimal for species belonging to the genus Tanacetum. These authors compared the results obtained on three media differing in their basic composition: MS, B5 (Gamborg et al. 1968), and SH (Schenk and Hildebrandt 1972) and noticed the highest weight of fresh biomass on MS medium. The addition of BAP in the medium stimulated the formation of multiple shoots and led to decrease of their length, which confirmed earlier results obtained for Tanacetum vulgare L. (Keskitalo 1999).

The aim of the present study was to establish suitable conditions for propagation of T. cinerariifolium by in vitro methods and to create an ex situ collection of selected clones, as a step towards introducing the species into agriculture in Bulgaria.

For their normal growth and development, plants need macro salts and microelements, the optimal concentrations of which may depend on the species. Among media for in vitro plant propagation, MS and B5, as well as their modifications, with or without addition of PGRs, are most commonly used. Bacterial contamination could be critical for in vitro culture initiation despite disinfection of primary explants (Hennerty et al. 1987; Misaghi and Donndelinger 1990; Keskitalo 1999). Usually, the composition of media for plant cultures is not optimal for microorganisms or suppresses their development, so their presence in the culture can go unnoticed for a long time (Leifert and Waites 1992; Leifert et al. 1994; Isenegger et al. 2003). In general, bacterial infections appear soon after the cultures are transferred to fresh media, because during subcultivations the roots are removed and the plants are cut into segments, making them weaker, as in the case of our experiments. Antibiotics are habitually added to the medium to control bacterial contamination, but they can alter the morphogenesis of in vitro cultures and slow down their growth (Teng and Nicholson 1997; Eady and Lister 1998; Keskitalo et al. 1998; Teng and Teng 2000; Bergant et al. 2005). The effectiveness of the antibiotics varies depending on the plant species, and sometimes even on the species genotype (Keskitalo et al. 1998; Bergant et al. 2005).

In our opinion, necrosis observed in vitro in pyrethrum cultures was due to an insufficient amount of calcium in the most commonly used media. T. cinerariifolium is not pretentious regarding soil conditions (with the exception of waterlogged soils), but it develops remarkably well on calcareous soils (Hennigsberg 1941; Astadzhov et al. 1980). According to some authors, the natural populations of the species are on carbonate soils and karst terrains (Sladonja et al. 2014). The physiological functions of calcium are related to cell division, normal functioning of cell membranes and activation of enzymatic reactions that positively affect photosynthesis. Calcification strengthens plant cell walls, thereby increasing plant resistance to infections (Gorbanov et al. 2005; George et. al. 2007). In addition, calcium manifests synergism with NO₃, i.e. facilitates its uptake and hinders the absorption of some elements such as K⁺, Na⁺, Mg²⁺, Fe²⁺ and Zn²⁺. Calcium deficiency in the presence of potassium and magnesium can lead to significant changes in the normal functioning and growth of roots and root hairs (Gorbanov et al. 2005). Another negative effect of this insufficiency can be the severe limitation of the shoot tips growth and even their death due to hyperhydricity, which can be avoided by frequent subcultivation (George et. al. 2007). According to some authors (Sha et al. 1985; Singha et al. 1990), the degree of changes may differ among different genotypes of the same species. On the other hand, excessively high carbonate content in soils and high pH of the soil solution can negatively affect plant development and production of pyrethrum flower heads (Sastry et al. 1988). In our experiments, the optimal result was obtained at the intermediate calcium concentration of the three variants tested. It is important to note that calcium is also essential for in vitro morphogenesis, especially when using PGRs such as auxins and cytokinins (George et al. 2007). This may explain the faster death of plants subcultured on media containing PGRs, without antibiotic and supplemental calcium. In in vitro cultures, the addition of calcium as CaCl₂ is not preferable, as it may even lead to a significant increase in chlorine content to levels of chlorine toxicity (George et al. 2007). On the other hand, addition of calcium as CaCO₃ and subsequent lowering of pH, which usually occurs in in vitro cultivation, could also lead to an increase in chlorine content. This may be a likely explanation for the presence of credible trends (at concentrations of 75 and 120 mg/L Ca) for an increase in necrotic plants with increasing cultivation time.

Some authors recommended for in vitro shoot rooting MS medium free of PGRs or B5 supplemented with 2 mg/L NAA (Rostiana and Seswita 2007; Hedayat et al. 2009). Information on ex vitro adaptation and outdoor acclimatization of regenerated pyrethrum plants is scarce. Survival of about 2/3 of the plants was reported, with the remark that the process can be greatly improved using a phytotron with tightly controlled environmental parameters (Catalano et al. 2011). Our results proved the positive effect of the strict control of the ambient conditions in the growth chamber during the first weeks of ex vitro adaptation, when in vitro plants are most vulnerable to changes in air humidity and temperature. Soil characteristics are of importance for seed germination and plant development. A pot experiment involving several different soil types proved that Rendzic Leptosol was the most suitable for pyrethrum seed germination (Ilinkin 2019). The soil mixture used for ex vitro adaptation in pots (Light mix Biobiss, France) was suitable for pyrethrum plants and they survived several years in the unheated greenhouse and flowered. The field plot conditions were less appropriate as the soil was loamy-sandy, poor in carbonates. Plants in the ex situ collection bloomed twice a year; however during the third growing season some of them remained small. Sunlight proved to be crucial for the survival and vigorous growth of T. cinerariifolium plants. Plants of this species should be planted at a greater distance from each other to avoid their death. Wandahwa et al. (1996) also reported the importance of the edaphic conditions for successful cultivation of the species and capitula yield. These authors propagated selected clones vegetatively in soil for 3–4 months, and then seedlings were planted in a permanent place of cultivation at 60 cm inter-row spacing and 30 cm between plants in the row. Up to 10% of the plants died and were replaced with new seedlings to maintain the plantation. Planting on ridges was recommended by Kroll (1963) to provide better soil aeration and avoid waterlogging. In our case, the dead of some plants was probably due to the rainy spring and summer and the shadow of the neighboring tree.

Data on the influence of nitrogen, phosphorus and potassium fertilization reported for different pyrethrum growing countries are conflicting. Clone-specific responses to nitrogen and phosphorus fertilization were also noted (Ngugi and Ikahu 1989). Our results related to some features of the clones we selected, such as the number of capitula, are consistent with the observations of these authors and confirm the assumption that the study should be conducted at individual level.

A protocol for in vitro micropropagation of Tanacetum cinerariifolium has been established, starting with seeds, and several one-seed-derived clones have been obtained by multiple consecutive subcultivations. The optimization of the nutrient medium composition was of crucial importance for the successful in vitro cultivation. Shoot loss due to both endophytic bacteria and necrosis was overcome by adding an antibiotic and modifying the calcium concentration in the medium, according to the specific requirements of pyrethrum. The first attempts to establish a pilot plantation of pyrethrum are promising, as ex vitro adaptation of the plants was easy and the outdoor acclimated plants bloomed twice from the very first growing season. However, field cultivation conditions need to be improved, as pyrethrum plants are shade intolerant and direct sunlight is crucial for their survival. The number of flower heads increased during the second year of cultivation in the ex situ collection, and some differences were found between the clones tested. Flower heads were sampled from each individual for analysis of pyrethrin content. More field experiments are needed to select highly productive individuals in terms of both number of flower heads and concentration of pyrethrins. Selected individuals should be further in vitro propagated to produce seedlings for pyrethrum plantation.

This work was supported by the National Research Programme “Healthy Foods for a Strong Bio-Economy and Quality of Life” at the Bulgarian Ministry of Education and Science (DCM Nº 577/17.08.2018)