Experimental propagation study establishes in vitro protocol and outdoor collection of Dalmatian pyrethrum clones

An experimental propagation study established an in vitro micropropagation protocol for the Dalmatian pyrethrum, Tanacetum cinerariifolium, and used the resulting one-seed-derived clones to establish an ex situ collection in Bulgaria. The study addressed bacterial contamination, tissue necrosis and survival during repeated subcultivation, followed by acclimatization and outdoor cultivation of four selected clones.

Dalmatian pyrethrum is a perennial herb in the Asteraceae family, endemic to the eastern Adriatic coast from Italy to northern Albania and the mountainous regions of Croatia, Bosnia and Herzegovina, and Montenegro. It is cultivated in many countries because it contains pyrethrins used as contact insecticides. The experiments began with seeds from a private ex situ collection in Bogdan, Bulgaria, and subsequently used seeds from a natural Croatian population assigned accession MAPO2821 in the Croatian Plant Genetic Resources Database.

For the initial culture experiments, the researchers compared agar-solidified Murashige and Skoog (MS) medium without plant growth regulators with two formulations containing kinetin and indole-3-butyric acid: 1.0 mg/L kinetin with 0.5 mg/L indole-3-butyric acid and 0.2 mg/L kinetin with 0.1 mg/L indole-3-butyric acid. Nineteen one-seed-derived clones were multiplied for up to eight subcultivations. The regulators stimulated shoot formation and elongation, but bacterial contamination and necrosis caused substantial losses. On the medium containing 0.2 mg/L kinetin and 0.1 mg/L indole-3-butyric acid, 81.3% of the plantlets died from microbial contamination. Losses were also high on the other media, and by the eighth subcultivation all plantlets on regulator-free MS medium were affected by necrosis or contamination.

For the Croatian seeds, the investigators tested MS and B5 media alongside MS media containing 100, 200 or 300 mg/L Medaxone, whose active compound is ceftriaxone sodium. Germination percentages were similar across the five media. Seeds began germinating on control MS medium after 12.5 ± 1.7 days, compared with approximately day 15 on the antibiotic-containing media, while onset on B5 occurred after 16.8 ± 2.2 days. No infected seedlings were observed at the first subcultivation on MS media containing 200 or 300 mg/L Medaxone, and the two higher concentrations also yielded the highest percentages of surviving seedlings.

Increasing the antibiotic concentration was associated with more necrosis, so MS medium containing 200 mg/L Medaxone was selected for further work. After eight subcultivations, 671 well-shaped, rooted plantlets survived on that medium, compared with 58 at 100 mg/L, 15 on antibiotic-free MS, three on antibiotic-free B5 and 114 at 300 mg/L. The 300 mg/L formulation appeared to inhibit shoot multiplication. A temporary immersion system using liquid medium in RITA containers produced faster-growing plantlets with long roots but did not improve the propagation coefficient, while the proportion of necrotic plants remained high; agar-solidified medium was more suitable for these cultures.

The researchers subsequently added 30, 75 or 120 mg/L calcium as calcium carbonate to MS medium containing 200 mg/L Medaxone. Propagation coefficients remained between 1.9 and 2.1 across the treatments, while calcium concentration influenced the frequency of necrosis. The lowest percentage of necrotic plantlets occurred with 75 mg/L added calcium, and the coefficient of variation in multiplication rate declined from 6.04 without added calcium to 5.40 at 30 mg/L, 4.80 at 75 mg/L and 4.24 at 120 mg/L. Necrosis increased with continued subcultivation at the two higher calcium concentrations, with the increase statistically significant at 120 mg/L.

MS medium containing 200 mg/L Medaxone and 75 mg/L added calcium produced no further infected plants, reduced the percentage of necrotic plants threefold and yielded 38.06 ± 10.11 new plants per initial shoot over seven months. Several thousand in vitro plants were produced in one year. Of 360 plants from four selected clones that were potted for ex situ adaptation, 88% strengthened during six weeks in a growth chamber as humidity was reduced from 90% to 60%. Almost all survived subsequent adaptation in a room phytotron and an unheated greenhouse.

Sixteen plants, four from each clone, were planted outdoors in October 2019 in the ex situ collection of IBER, with 40 cm between plants and between rows. They flowered in June of the first growing season and produced a smaller secondary flowering in autumn. Stem branching and flower-head numbers varied among clones and among individual plants within a clone. In the second year, Clone 3 produced an average of 328 ± 138 flower heads per individual, approximately six times the number recorded for that clone in the first year; this was not a species-wide estimate. Some plants died over winter, and plants of Clone 4 were adversely affected by shade from a nearby tree.

Outdoor performance was limited by the small number of plants, loamy-sandy soil poor in carbonates, and a rainy spring and summer that adversely affected development. Flower heads were collected from each individual for pyrethrin analysis, but the study did not report analytical results for individual clones. The authors concluded that further field experiments are needed to select productive individuals on the basis of both flower-head number and pyrethrin concentration before selected plants are further propagated for larger plantations.

Subject of Research: Experimental in vitro propagation and outdoor acclimatization of Dalmatian pyrethrum clones

Article Title:  In vitro clonal propagation of Tanacetum cinerariifolium and establishment of an ex situ collection of selected clones

Article References: Ilinkin, V., Traykova, B., & Stanilova, M. (2023). In vitro clonal propagation of Tanacetum cinerariifolium and establishment of an ex situ collection of selected clones. BioRisk, 20, 97-114. https://doi.org/10.3897/biorisk.20.97566

Image Credits: AI Generated

DOI: 10.3897/biorisk.20.97566

Keywords: Dalmatian pyrethrum, in vitro micropropagation, plant tissue culture, tissue necrosis, Medaxone, calcium carbonate, ex situ collection, flower-head production, clone variation, plant acclimatization, vitro, clonal