FAIR-CT96-1914 Origanum sp. and Salvia sp. integrated breeding research to improve homogeneity and quality of multifunctional secondary plant products

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Crops for Biological Control Products : Crops for Flavours/Fragrances : Crops for Pharmaceuticals/Cosmetics : Essential Oil : FAIR Area 4.3 - Diversification : Flavours/Fragrances : Integrated Crop Protection & Biological Control : Pharmaceuticals/Cosmetics : Plant Genetics

	Proposal No:	FAIR-CT96-1914
	Date Prepared:	June 2000, September 1999
	Source:	Final Report Executive Summary Annual Progress Report

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Final Report Executive Summary

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Annual Progress Report

Introduction

The general objective is to improve homogenous quality in marjoram (Origanum majorana L.), in oregano species (Origanum sp.) and two sage species (Salvia officinalis L. and Salvia fruticosa Miller), to secure seed production of marjoram and to estimate the genetic variability of antimicrobial and antioxidant properties of marjoram, oregano and sage. This will contribute to a substitution of plants collected from natural habitats by an economically more efficient systematic and controlled production of aromatic and medicinal plants.

Activities Tasks for the reporting period were as follows.

Task 1 Hybrid variety system in marjoram (Origanum majorana L.):

• development of male sterile inbreed lines
• development of pollinator inbreed lines
• controlled crossings for testing combining abilities
• testing of general combining ability
• essential oil content and composition
• sensorial quality and identification of off-flavour types
• morphometry of leaves and colour of dried herb
• RAPDS
• technological machinability

Task 2 Seed production of marjoram.

• Annual versus perennial seed production and optimal harvest time for seeds
• Seed physiology

Task 3 Breeding oregano and sage pure in chemotype using selfings and search for male sterility, using:

• Salvia officinalis
• Origanum vulgare ssp. hirtum (Link) letswaart, O. vulgare ssp. gracile (Koch) letswaart, O. vulgare ssp. glandulosum (Desfontaines) letswaart, O. vulgare ssp. virens (Hoffmann & Link) letswaart, O. vulgare ssp. vulgare
• Salvia fruticosa Miller, Origanum vulgare ssp. hirtum (Link) letswaart, O. dictamnus L., O. onites L. and Origanum x intercedens letswaart, O. microphyllum (Bentham) Vogel, Origanum x minoanum Davis
• GC-Analysis
• mode of inheritance and microsporogenesis of male sterility
• RAPDS

Task 4 Antioxidant and antimicrobial properties of marjoram, oregano and sage

• Antimicrobial properties
• Antioxidant properties

Results

The main results were as follows:

Task 1 - Marjoram Hybrid System Cms (generation 2) and pollinator (generation 2) genotypes have been prepared. The combinations between cms and poll have been made with sufficient seed set for the combining ability testings.

• Development of male sterile inbreed lines (cms) - after some generations maintainer and cms-genotypes differ only in flowering time. The variability even within the populations is still high. The five cms-lines established differ in their heigth, ramification and resistance to lodging.
• Development of the pollinator inbreed component of the hybrid - within the 9 genotypes selected for the preparation of pollinator inbreed lines, variation in all traits is quite high and selection for higher yield is going on.
• Controlled crossings for testing combining abilities - with the fertile pollinator-lines sufficient seed set for the subsequent combining ability-tests could be obtained.
• Testing of general combining ability (gca) - regarding herb yield, no significant hybrid effect is visible, but good combinations were identified.
• Essential oil content and composition - essential oil content. the plants proved to be extremely heterogeneous in their essential oil content again.
• Essential oil composition - the populations tested in 1997 were not significantly different in their essential oil, although the variability was high.
• Sensorial quality and identification of off-flavour types - this year, there were clear differences of the test combinations regarding their sensorial parameters with some combinations off-flavour (bitter) and some combinations very promising with better sensorial qualities than the standard.
• Morphometry of leaves and colour of dried herb - regarding the morphometry, clear genotypic differences were found. Objective colour measurement parameters could be correlated to (subjective) sensorial colour evaluations.
• RAPDS - a broad DNA polymorphism between the individual plants was revealed, demonstrating the heterogeneity of the plant material within accessions, whereas the accessions themselves showed a low degree of polymorphism.
• Technological machinability - in contrast to last year's report, the important production parameters 'leaf content' and 'tiny parts' showed high variation with good chances to detect and select highly efficient combinations.

Task 2 - Seed production of marjoram Some additional parameters influencing seed production were examined.

• Annual versus perennial seed production and optimal harvest time for seeds - the quantity of seed yield in respect of the planting time was about double for the first transplanting date compared to the second. Concerning stand density, only the lowest stand density showed inferior seed yields. The time of cutting and harvesting in the first year had high influence on the seed yield of the second year. Irrigation during seed setting enhances seed yield. Water requirement of the crop can be satisfied with a partial restoring of the field capacity.
• Seed physiology - the average seed weight did not differ significantly among the seedlots. A 13 year old commercially obtained marjoram seedlot gave an almost 50% germination. No significant germination differences were observed among seeds from the three harvest dates.
• The study of the temperature dependence of germination showed optimum values at 15° and 20° C while at 25° C some decrease occurred. The germination is particularly fast. The role of light seems rather minor; germination occurred in continuous darkness or under white or far-red light.

Task 3 - Breeding oregano and sage populations pure in chemotype using selfings and search for male sterility The chemotypes have been established and a second generation is already growing.

• Salvia officinalis L. - flowering of the second generation plants is expected for the early spring. Furthermore the special requirements for flower induction were studied. Sage needs a treatment below 10° C for more than 6 weeks. Then flowering starts 15 to 26 days later accompanied by a notable change of the leaf colour. If young plants are exposed to this cold treatment, they, however, did not start to flower afterwards. There seems to be a certain age of the plants necessary to proceed into the generative phase. The first selfing generation revealed heterogeneity at alpha-pinene, 1,8-cineol, alpha-thujone, bornyl-acetate, beta-caryophyllene and an unknown compound.
• Origanum vulgare spp. - the male sterility of some plants was examined under the microscope. Three different causes for male sterility depending on the genotype were found.
• Salvia fruticosa, Origanum spp - in S. fruticosa selfing was successful and 800 plantlets established. In Origanum selfings were successful but yielded not as many seeds as expected. The natural variability of Origanum microphyllum was studied and showed four clearly distinguishable chemical groups. The examination of the natural variability of Origanum dictamnus was started.
• GC-analysis - with the exception of the Salvia officinalis analysis, the work is on schedule. The analyses are in time.

Task 4 - Antioxidant and antimicrobial properties of marjoram, oregano and sage A good activity of essential oils especially of oregano could be observed.

• Antimicrobial properties - antibacterial activity was demonstrated using a new improved antibacterial assay, Origanum oils were shown to be even more effective. Also essential oils of Salvia officinalis proved to be effective inhibitors and once again no discrimination was shown by bacterial cell wall type. Oils from Salvia fruticosa killed Clostridia cultures completely, but zones of inhibition on the remainder of the test organisms were less dramatic in comparison with oils from Origanum species. The essential oils of two Origanum species and one Thymus species were compared in their activity with ten known antibiotics challenging 25 bacterial genera. Origanum was by far more effective than Thymus vulgaris and all of the antibiotics tested. Antibacterial assessment of oils from O. majorana has shown that some of these oils have considerable bioactivity with maximum inhibition. Methicillin-resistant, Staphylococcus aureus was susceptible to Origanum essential oil while Vancomycin-resistant Enterococci were less susceptible to these essential oils.
• Antifungal activity - the Origanum essential oils were very active. Also Salvia officinalis was active but not as potent as Origanum oils. There was not enough oil from Salvia fruticosa to complete testing but from those that were tested Aspergillus parasiticus was the most resistant and A. ochraeus the most susceptible (in contrast to the oils of S. officinalis).
• Antioxidant properties and activity - the most potent oil was obtained from leaf and flower of 0. x intercedens.
• Comparing three Origanum oils and Thymus vulgare showed that all three Origanum species prevented more lipid peroxidation, than thyme oil, with carvacrol-rich 0. syriacum exhibiting greatest activity followed by thymol-rich 0. vulgare. Antioxidant analysis of Salvia officinalis oils exhibited that the most effective was population #4, while two populations showed no activity at all. From the oils produced from Salvia fruticosa, colour retention and intensity was much poorer than that produced by the oil from Salvia officinalis.
• Composition and antioxidant activity of methanolic extracts: there were marked differences found between the populations of Salvia officinalis and Salvia fruticosa regarding the compounds responsible for the antioxidant activity (rosmarinic acid, camosol, camosic acid and methyl camosate). The Origanum samples varied greatly.