Ivan Kolak

Molecular and chemical characterization of the most widespread Ocimum species

DNA fingerprinting (AFLP) and chemical analyses of essential oils were utilized to define the extent of variation existing in the genus Ocimum. Research was carried out on 22 Ocimum accessions representing seven species. Concerning the essential oil composition of all investigated accessions, 115 compounds were identified. UPGMA cluster analysis, based on Euclidian distances of essential oil constituents between all pairs of accessions, showed four well-supported clusters (O. tenuiflorum, O. basilicum/O. africanum, O. basilicum, and O. americanum/O. africanum). Relating to the essential oil composition of all of the investigated accessions, 17 compounds were identified as the main ones, and according to them 13 chemotypes were determined. AFLP relationships were determined by neighbor-joining (NJ) cluster analysis based on Dice’s distance matrix and by maximum parsimony (MP) analysis. O. basilicum, O. americanum/O. africanum, O. tenuiflorum, and O. gratissimum represented four clusters supported with high bootstrap values. A neighbor-net diagram allowed the visualization of apparently conflicting data by revealing relationships between genotypes and chemotypes. Concerning the O. africanum species, two distinct chemotypes, geranial/neral (accession 11) and estragol (accession 10), have been established, while all the studied O. americanum accessions belong to the geranial/neral chemotype. This could be additional evidence that O. americanum is one of the parents of O. africanum. Furthermore, the fact that the O. africanum accession (10) as well as O. basilicum ‘Purpurascens’ and O. basilicum ‘Erevanskii’ accessions belong to the estragol chemotype supports the theory that O. africanum is one of the parents of these two O. basilicum accessions.

Efficiency of morphological trait descriptors in discrimination of Ocimum basilicum L. accessions

Abstract Most commercial basil cultivars belong to the species Ocimum basilicum L. Moreover, within O. basilicum species, there are five main botanical varieties (var. basilicum L., var. difforme Benth., var. minimum L., var. purpurascens Benth. and var. thyrsiflorum /L./ Benth.) that are mainly found on the market. High levels of both morphological and chemical variability exist within the species due to the intraspecific hybridization and long-term uses throughout the world. The aim of this work was to study the resolving power of morphological traits for reliable identification of basil accessions. Phenotypic dissimilarities between pairs of accessions were calculated, and the unweighted pair-group method dendrogram was constructed. Six clearly defined clusters have been detected, giving a good representation of traditional taxonomic relationships. To compare the efficiency of the morphological traits in accession identification, confusion probability and discriminating power of each trait were calculated. Our results show that with a careful analysis and stringent selection of traits, morphological markers provide an inexpensive and reliable method for routine screening of a large number of accessions, in order to monitor and manage germplasm collections.

Chemical characterization and genetic relationships among Ocimum basilicum L. cultivars.

Twenty‐seven Ocimum basilicum cultivars were subjected to a chemical characterization of essential oil components by gas chromatography/mass spectrometry (GC/MS) and a genetic characterization using the amplified fragment‐length polymorphism (AFLP) technique. Since the same 27 accessions had previously been classified into six morphotypes, these analyses allowed us to make detailed comparisons of chemistry, genetics, and morphology. The chemical composition and morphology of the studied cultivars appeared to have a strong genetic component. The AFLP analysis revealed a distinction between the green and purple morphotypes. The green morphotypes predominantly utilized the terpene biosynthetic pathway, while most purple morphotypes primarily utilized the phenylpropene biosynthetic pathway. The GC/MS analysis led to identification of 87 volatiles. Among the 27 cultivars, five chemotypes were identified. A detailed characterization of the essential oil constituents indicated the existence of both specific combinations of compounds and ‘private’ compounds with the potential to be used in many aspects of human life. The established relationship between a genetic profile, chemical composition, and morphology represents an important step in future breeding programs and in the cultivation of this species.

Genetic Diversity and Structure of Dalmatian Pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip., Asteraceae) within the Balkan Refugium

Dalmatian pyrethrum (Tanacetum cinerariifolium Trevir. /Sch./ Bip.) is an outcrossing, perennial insecticidal plant, restricted to the eastern Adriatic coast (Mediterranean). Amplified fragment-length polymorphisms (AFLP) were used to investigate the genetic diversity and structure within and among 20 natural plant populations. The highest level of gene diversity, the number of private alleles and the frequency down-weighted marker values (DW) were found in northern Adriatic populations and gradually decreased towards the southern boundary of the species range. Genetic impoverishment of these southern populations is most likely the result of human-related activities. An analysis of molecular variance (AMOVA) indicated that most of the genetic diversity was attributed to differences among individuals within populations (85.78%), which are expected due to the outcrossing nature of the species. A Bayesian analysis of the population structure identified two dominant genetic clusters. A spatial analysis of the genetic diversity indicated that 5.6% of the genetic differentiation resulted from isolation by distance (IBD), while 12.3% of the genetic differentiation among populations followed the pattern of isolation by environmental distance (IBED). Knowledge of the genetic diversity patterns of the natural populations and the mechanism behind these patterns is required for the exploitation and possible conservation management of this endemic and economically important species.

Chemical diversity of the natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir. /Sch. Bip.) in Croatia

Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch.Bip.) is a plant species endemic to the east Adriatic coast. The bioactive substance of Dalmatian pyrethrum is a natural insecticide, pyrethrin, a mixture of six active components (pyrethrins I and II, cinerins I and II, and jasmolins I and II). The insecticidal potential of pyrethrin was recognized decades ago, and dried and ground flowers have traditionally been used in Croatian agriculture and households.

Conservation of Medicinal and Aromatic Plants in Croatia

The use of medicinal and aromatic plants (MAP) in Croatia has a very long tradition. Commercial gathering was economically important source of income in many Croatian regions. However, gathering in the wildness has a negative impact on biodiversity, collected plant material is not uniform and its price is significantly lower. Current production of medicinal and aromatic crops in Croatia is very limited. The MAP cultivation on family farms is usually marginal activity. MAP producers and processors generally agree that marketing opportunities do exist in case of a number of species. Natural MAP populations in Croatia show great biodiversity in morphological, biochemical and genetic level. The assessment of biodiversity is a starting point for efficient conservation of plant genetic resources and its use in plant breeding programmes. The main aim of the management of plant genetic resources is conservation, characterization, evaluation and documentation of the existing genetic biodiversity. For the purpose of conservation of these valuable genetic resources, The Collection of Medicinal and Aromatic Plants has been established at the Faculty of Agriculture, University of Zagreb, Croatia. Characterization and evaluation of accessions in plant genebanks is traditionally based on morphological traits. Currently, the analysis of target species including Dalmatian sage (Salvia officinalis L.), Dalmatian pyrethrum (Tanacetum cinerariifolium/Trevir./Sch. Bip.) and basils (Ocimum spp.) has been carried out on morphological, biochemical and genetic level.