A First Approach for the In Vitro Cultivation, Storage, and DNA Barcoding of the Endangered Endemic Species Euonymus koopmannii

Abstract

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies.

Keywords: DNA-barcoding; Euonymus koopmannii; direct regeneration; micropropagation; slow growth storage.

Figure 1

Effect of different concentrations and combinations of cytokinins and auxins on shoot regeneration from E. koopmannii in tissue culture. (A) Frequency of direct regeneration on MSDR I, II, and III medium; (B) Direct shoot regeneration from internodes and nodal explants. Data represent three independent experiments’ mean ± standard deviation (SD) Different letters on the bar indicate statistically significant differences at p < 0.05 Tukey’s (HSD), scale bar 1 cm.

Figure 2

Dynamics of growth and development of E. koopmannii roots during in vitro culture. (A) Frequency of rhizogenesis of E. koopmannii regenerants on MSR I–XII media; (B) 8-week-old plants; (C) 14-week-old plants. Data represent three independent experiments’ mean ± standard deviation (SD). Different letters on the bar indicate statistically significant differences at p < 0.05 Tukey’s (HSD), scale bar 1 cm.

Figure 3

Effect of subculture on elongation of shoot length development and shoot formation of E. koopmannii on MSDR I, II, and III medium: (A) average shoot length, cm; (B) number of shoots per explants, pcs.; (C) seeds; (D) 4-week-old seedlings; (E) 12-week-old plants. Data represent three independent experiments’ mean ± standard deviation (SD). Different letters on the bar indicate statistically significant differences at p < 0.05 Tukey’s (HSD), scale bar 1 cm.

Figure 4

Micropropagation of regenerants obtained from E. koopmannii internodes on MSDR I, II, and III medium: (A) average shoot length, cm; (B) number of shoots per explants, pcs. Data represent three independent experiments’ mean ± standard deviation (SD). Different letters on the bar indicate statistically significant differences at p < 0.05 Tukey’s (HSD).

Figure 5

Development of shoot length (cm) during six months of slow growth on MSP I–IX medium. Data represent the mean ± standard deviation (SD) of three independent experiments. Different letters on the bar represent statistically significant differences at p < 0.05 Tukey’s (HSD).

Figure 6

The phylogenetic tree of E. koopmannii is based on the maximum likelihood method and the Kimura 2-parameter model. (A) matK gene. (B) ITS region. (C) psbA-trnH region. (D) rbcl gene.