Ely Zayova

Comparative study of in vitro, ex vitro and in vivo grown plants of Arnica montana – polyphenols and free radical scavenging activity

Abstract Arnica montana L. is an endangered species rich in sesquiterpene lactones, phenolic acids and flavonoids with high pharmaceutical value. The polyphenolic content and free radical scavenging activity of plants that had passed all stages of cultivation: micropropagation and rooting (in vitro), adaptation in greenhouse (ex vitro) and mountain conditions (in vivo) were evaluated. Four surface flavonoid aglycones [scutellarein 6-methyl ether (hispidulin), scutellarein 6,4’-dimethyl ether (pectolinarigenin), 6-OH luteolin 6-methyl ether and kempferol-6-methyl ether] were detected in the acetone exudates of the studied samples bymeans of thin layer chromatography.No differences in the accumulation of surface flavonoids were found among the tested leaf extracts of in vitro, ex vitro and in vivo samples. However, the extracts from the flowers were richer in surface flavonoids than extracts from the leaves. The methanol extracts of the samples from ex vitro and in vivo grown A. montana plants had significantly higher radical scavenging activity and polyphenolic content than the extracts of in vitro samples. The observed differences in the contents of these biologically active compounds were related to different growth conditions and stages of plant development. The biotechnological method of A. montana established holds promise for the future production of antioxidants.

INDIRECT SHOOT ORGANOGENESIS OF EGGPLANT (SOLANUM MELONGENA L.)

A protocol for indirect shoot organogenesis of Solanum melongena ‘Larga Negra’ and ‘Black Beauty’ was established using hypocotyl and cotyledon derived calluses. The maximum morphogenic callus induction was observed from cultured cotyledons of 30-days old seedlings on Murashige and Skoog (MS) medium containing 2.0 mg/l α-naphthalene acetic acid and 0.5 mg/l 6-benzylaminopurine. The highest percentage of shoot regeneration and the highest mean number of shoots/callus were obtained on hormone-free MS medium. In terms of callus induction and subsequent plant regeneration, cotyledon explants were more responsive than hypocotyl explants. Regenerated shoots (2-3 cm) were rooted on MS hormone-free medium or medium containing 0.1 mg/l indole-3-butyric acid. About 90% of regenerated plantlets survived under field conditions after hardening in the glasshouse. Several somaclones exhibiting useful variation would to be proposed as initial plant material for eggplant breeding programs.

Morphological evaluation and antioxidant activity of in vitro - and in vivo -derived Echinacea purpurea plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L−1 6-benzylaminopurine, 0.1 mg L−1 α-naphthalene acetic acid and 0.2 mg L−1 gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L−1 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L−1 α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L−1 indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.

Biotechnological approaches for cultivation and enhancement of secondary metabolites in Arnica montana L.

Arnica montana L. (Asteraceae) is a valuable medicinal plant with anti-inflammatory and cicatrizing properties attributed to the sesquiterpene lactones, flavonoids and essential oil produced in the flower heads. In many European countries, the populations of A. montana are close to extinction in their natural habitats because of uncontrolled eradication and indiscriminate collection of the plants. Various approaches for in vitro propagation of the species and also, biosynthesis of secondary metabolites in tissue and cell cultures are assessed in the current review. Special attention is paid to the biological activity and chemical composition of compounds produced by the species as well as the opportunities of in vitro methods to isolate high-productive clones. The influence of different factors on the micropropagation, callusogenesis, genetic transformation and identification of certain biologically active substances is discussed in detail. By the reference to the available issues we concluded that biotechnology applied to A. montana cultivation may improved the plant preservation and increased the production of sesquiterpene lactones and other secondary metabolites.

Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes

The induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.

Comparison of antioxidant activity of the fruits derived from in vitro propagated and traditionally cultivated tayberry plants

BACKGROUND Tayberry is a hybrid between Rubus fruticosus L. and Rubus idaeus L. These fruits contain valuable vitamins and antioxidants. An effective protocol for micropropagation of tayberry plants is here described. Different concentrations of cytokinins (6-benzylaminopurine, zeatin, and 6-(γ,γ-dimethylallylamino)purine) were added in Murashige and Skoog, 1962 (MS) medium to micropropagation using stem tip and nodal explants. RESULTS The highest propagation rate was recorded on MS medium containing 2 mg L(-1) zeatin, where the shoot formation resulted in 3.4 shoots per stem tip explant after 4 weeks of culture. It was found that half-strength MS medium with 0.1 mg L(-1) indole-3-butyric acid was the best for plant rooting. For ex vitro acclimatization of plants, the mixture of peat, soil, and perlite (1:1:1 v/v/v) was the most suitable planting substrate for hardening. The micropropagation protocol described in this study might be useful for the production of healthy plant materials. Tayberry fruits from in vitro propagated plants and adapted to the field conditions possessed higher antioxidant capacity in comparison to traditionally cultivated plants. CONCLUSION Fruit extracts of micropropagated tayberry plants and adapted to field conditions can be used as a rich source of natural antioxidants.

Developmental and Environmental Effects on Sesquiterpene Lactones in Cultivated Arnica montana L.

The amount of sesquiterpene lactones and the lactone profile of Arnica montana L. in flowering and seed formation stages in vitro and in vivo propagated from seeds of German, Ukrainian, and Austrian origin and grown in two experimental fields were studied. It was found that in vitro propagated 2‐year plants in full flowering stage accumulated higher amount of lactones in comparison to in vivo propagated 3‐year plants and to the seed formation stage, respectively. Helenalins predominated in in vivo propagated 2‐year or in vitro propagated 3‐year plants. 2‐Methylbutyrate (2MeBu) was the principal ester in the samples with prevalence of helenalins, while isobutyrate (iBu) was the major one in the samples with predominance of 11,13‐dihydrohelenalins. The results revealed that the environmental conditions on Vitosha Mt. are more suitable for cultivation of A. montana giving higher content of lactones.