Characterisation and Evaluation of Species of the Boraginaceae Family as Source of Gamma-Linolenic Acid for Mediterranean Conditions

Gamma linolenic acid (GLA) is an essential fatty acid that is being used for the treatment of health problems related to deficiencies in essential fatty acids and prostaglandins. Borage plant is an important source of GLA, but the shattering habit of the genus limits large scale seed production. Looking for alternatives to the available borage material and for the possibilities of borage and other plant species belonging to the same botanical family as potential crops in Mediterranean conditions, this work describes: i) the evaluation of the seed quality of European borage population, and further selection of entries with high oil and GLA, and low erucic acid content, ii) the isolation of two borage mutants with improved seed production characteristics, iii) the first results of the anatomical and molecular studies in relation with the reproductive system and the cariotype of normal and mutants plants, and iv) the beginning and characterization of a germplasm collection of Boraginaceae plant species for its fatty acid composition and adaptability to semi arid environments: Anchusa azurea and Echium boissieri highlight as potential new crops for GLA production. INTRODUCTION Gamma linolenic (all-cis-6,9,12-octadecatrienoic) acid (GLA) is an essential fatty acid in demand for clinical and pharmaceutical applications. This fatty acid is precursor of indispensable compounds in the body such as prostaglandins (PGE1) and leucotrienes (LT). Many factors, such aging ageing, stress, diabetes, high alcohol intake and nutritional deficiencies, have been shown to interfere the biosynthesis of GLA from linoleic acid via ∆-6 desaturase. Health problems related to deficiencies in essential fatty acids and prostaglandins are being treated by supplementation of the diet with vegetable oils containing this fatty acid (Gunstone 1992; Horrobin, 1999). The most common commercial sources of GLA for pharmaceutical uses are evening primrose (Oenothera biennis L.) and borage seed oil. Borage seed appears to be the richest known plant source of GLA, with 16 to 28 percent of GLA from a total seed oil content of 27 to 37 percent. Additional advantages of borage in comparison with evening primrose is the annual life cycle and the larger seed of borage that make harvest and oil extraction easier (Muuse et al., 1988; Janick et al., 1989; Galwey and Shirlin, 1990). Large scale commercial production of borage is limited because of the nonuniform seed maturation and shattering habit of the plant, factors that cause a large amount of ripened seed fall to the soil before and during harvest. Janick and co-workers (1989) have attempted to find solutions to the seed-shattering challenge with the development of vacuum harvesters and in vitro production of GLA by zygotic and somatic embryos culture. Galwey and Shirlin (1990) initiated a program of selection in borage for GLA, oil content, and seed production, including mutagenesis with sodium azide and with ethyl methane sulfonate (EMS), but no mutant phenotypes were produced in subsequent generations. In addition to borage, other members of the Boraginaceae family also contain GLA and represent potential sources of GLA (Janick et al., 1989). Among these, are a Proc. XXVI IHC – Future for Medicinal and Aromatic Plants Eds. L.E. Craker et al. Acta Hort. 629, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 232 large number of annuals native to Mediterranean members of the Boraginaceae that grow during the winter, taking advantage of the available rains and developing the flowering shoots when climatic conditions are adequate. Therefore, borage and the above mentioned species of Boraginaceae represent potential new crops for Mediterranean and semiarid conditions. This paper describes the approaches and results obtained by our research group aiming to: 1) test the available European borage germplasm and select genotypes with the best seed quality characteristics, 2) produce new borage lines with improved characteristics for seed and GLA production, 3) progress in the knowledge of the reproductive system of borage, and the modifications produced y the mutants, 4) study the chromosome architecture of borage by developing and applying molecular techniques (fluorescence in situ hybridization, FISH), and 5) collect, characterise, and select other Boraginaceae species that could be used as new sources of GLA in semiarid conditions. MATERIALS AND METHODS Evaluation of Seed Quality of European Borage Populations The germplasm evaluated consisted of: i) white flowered material cultivated as vegetable in the North of Spain (Southern Europe), ii) wild blue flowered roadside populations collected in the South of Spain, and iii) wild and cultivated populations of the North of Europe. Seed weight was determined by weighing 1000 seeds. Oil content was determined by nuclear magnetic resonance. The fatty acid composition of the oil was determined on a bulk of 10 seeds/entry by simultaneous extraction and methylation, followed by gasliquid chromatography (Garcés and Mancha, 1993). Isolation of Two Chemically Induced Mutants of Borage Due to the lack of variation in seed shattering observed in the germplasm collection, a trial to increase the natural genetic variability by inducing chemical mutagenesis with EMS was done. Approximately 15,000 seeds of white flowered borage belonging to a Spanish accession with high self-fertility (RG-001) were exposed to a solution of 1 % (v/v) of EMS for 16 h under continuous stirring. Plants from the M1 generation were bagged to force self-pollination. Seeds of each bagged plant were sown in a single row to produce the M2 generation and, before flowering, five plants of each row were bagged and self-pollinated to obtain M3 seeds. In the M3 generation, 50 pollinated flowers from each mutant type were chosen at random to calculate the variability in the number of petals, sepals, ovules and seeds per flower, and to apply statistical analysis (De Haro and Del Río, 1998). Significant differences between means were determined by Duncan’s Multiple Range Test. Anatomical Studies of Mutants with Retention of Petals and Seeds Fruits in different developing stage of both wild type and mutant type showing petals and seed retention were fixed in formalin, embedded in paraffin, sectioned at 12 μm and stained with tannic acid, iron chloride, safranin, and fast green (Jensen, 1962; Rapoport et al., 2001). Cytogenetic Characterisation of Cultivated Borage by Fluorescence in situ Hybridization (FISH) Seeds were germinated for 3 days at 24 oC. The roots were treated with colchicine in darkness and then fixed in acetic acid/ethanol. The samples was then treated with HCl, placed on acid-carmine solution, squashed and mounted. The selected slides were used for FISH, using the 18/25S rDNA repeat sequence from soybean. The in situ hybridization protocol used was according Cabrera et al. (1999).