CAMELINA SATIVA: A POTENTIAL OILSEED CROP FOR BIOFUELS AND GENETICALLY ENGINEERED PRODUCTS
Chaofu Lu
January, 2008

Camelina [Camelina sativa (L.) Crtz.] is an ancient crop belonging to the Brassicaceae family. It has been cultivated for oil production since prehistoric times, and it was extensively grown in Europe in the 19th century. However, Camelina fell into disfavor when more productive crops such as wheat and rapeseed began to be produced, and camelina production gradually declined and almost vanished after World War II. It became a common weed in Europe known as false flax (contaminating flax fields) and by its Roman name, Gold-of-Pleasure.

The recent interests in camelina are inspired by its unique oil composition. About 90% of fatty acids in camelina oil is unsaturated. The nutritionally essential polyunsaturated fatty acids (linoleic acid, 18:2n-6 and α-linolenic acid, 18:3n-3) constitute over 50% of total fatty acids, and linolenic acid is the most predominant fatty acid (35-40%). Therefore camelina oil has great potential as an excellent source of omega-3 fatty acids1, which have been recommended in the diet to achieve essentiality and cardiovascular benefits. Non-food uses of camelina oil have also been exploited for the production of soaps, varnishes, and cosmetics2, 3. Because camelina has lower fertilizer and pesticide requirements, the production cost is substantially lower than many other oil crops such as rapeseed, corn, and soybean; therefore camelina is an attractive potential crop for biodiesel and many other industrial applications2.

Currently, the lack of a clear utilization pattern of camelina oil limits its uses and large-scale production despite its adaptation to a wide range of climates2. Camelina oil contains about 15% eicosenoic acid (20:1), which is unique among many other vegetable oils. The usefulness or disadvantage of eicosenoic acid is not clear; however, it may present a hurdle to obtaining food approval1. The high percentage of polyunsaturated fatty acids makes camelina oil more susceptible to oxidation and thus is undesirable for fuel and other industrial applications. Therefore it is necessary to modify camelina oils to find a role for this potential crop in the world oilseed market. Since 2002, researchers at Montana State University have initiated a research program to evaluate and scale up production, improve camelina characteristics, and explore the utilization of camelina oil and meal. In this report I will present an overview on our recent efforts to improve fatty acid compositions of camelina oils, and to develop camelina as a potential platform for production of biotechnological products through genetic engineering.

Screening camelina mutants for desired fatty acid composition
We conducted an experiment to screen camelina mutants that contain desired fatty acid compositions. To induce mutations, camelina seeds were treated with ethane methyl sulfonate (EMS). The seeds from individual M2 plants were subject to analyses using high-throughput gas chromatography. This method, as described previously4, has a capacity to analyze about 600 samples a day. We screened over 50,000 M2 lines and identified a number of mutant lines that contain altered contents of different fatty acids, e.g., oleic (C18:1), linoleic (C18:2), linolenic (C18:3), and eicosenoic (C20:1) acids. A mutant with significantly increased oleic acid content is given as an example in Fig. 1.

Oleic acid is the most predominant fatty acid resulting from fatty acid synthesis in plant cells. Much of the oleic acid will be desaturated or otherwise modified during storage oil biosynthesis. In camelina, about 80% of C18:1 will be desatured to C18:2 and C18:3, or elongated to C20:1 and C22:1. Typically, camelina oils contain over 50% polyunsaturated fatty acids, which may render oil instability during storage and applications at elevated temperatures. Therefore high-oleic camelina oil is desirable for food and non-food uses.

An efficient method of camelina genetic transformation
Camelina is a low-cost oilseed crop that is not at present a major crop for food oil production; therefore it has great potential to become as a biotechnological platform for genetically engineered products. There have been limited research activities on camelina biotechnology. However, like many cruciferous oil producing plants such as Arabidopsis thaliana and Brassica napus, camelina is amenable to transformation. We have recently developed a simple, in planta transformation method for Camelina sativa5. Instead of a lengthy and laborious tissue-culture based method (United States Patent No. 20040031076), we generated transgenic camelina plants by vacuum-infiltration treatments of flower buds using Agrobacterium. Greater than 1% transformation efficiency can be achieved.

To demonstrate that camelina can be effectively used to produce genetically engineered products, we transformed camelina seeds with a castor fatty acid hydroxylase (FAH12) gene4 (Fig. 2a). We used a fluorescent protein selection marker DsRed (Clontech, Mountain View, CA, USA) driven by the constitutive cassava vein mosaic virus promoter to facilitate screening transgenic seeds. The transgenic fluorescent seeds (Fig. 2b) could be visually detected using a pair of red-lens sunglasses by illuminating a sea of seeds with a green LED flashlight. The transgenic events were verified by PCR analyses using primers for the DsRed and FAH12 genes (Fig. 2c). Fatty acid methyl ester analyses by gas chromatography (Fig. 2d) indicated that all red seeds analyzed accumulated novel fatty acids, which had been previously identified in transgenic Arabidopsis as ricinoleic acid (18:1OH), the major component of castor oil, and three other hydroxy fatty acids: densipolic acid (18:2OH); lesquerolic acid (20:1OH); and auricolic acid (20:2OH)6. This result clearly confirmed that the red fluorescent seeds were successfully transformed and expressed the castor FAH12 gene.

Conclusion
Camelina is an under-exploited crop species of great potential economical importance. We believe that camelina oils with improved fatty acid compositions will be useful for a variety of food and non-food uses. Our transformation system would provide a useful tool to rapidly improve many agronomic charactersistics, as well as a model system for biological and biotechnological studies. In this report, we demonstrated that novel hydroxy fatty acids were produced in camelina oils by seed-specific expression of a castor fatty acid hydroxylase. The low-cost oilseed crop, Camelina sativa, has great utility as an economical platform for a plethora of genetically engineered industrial and pharmaceutical products.

References

1. Leonard C. (1998) Camelina oil: a-linolenic source. INFORM9, 830-838

2. Putnam DH, Budin JT, Field LA, and Breene WM. (1993) Camelina: A promising low-input oilseed. InNew Crops, J.E. Simon, Editor. Wiley: New York

3. Zubr J. (1997) Oil-seed crop: Camelina sativa. Ind Crop Prod6, 113-119

4. Lu C, Fulda M, Wallis J, and Browse J. (2006) A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J45, 847-856

5. Lu C and Kang J. (2007) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep, in press

6. Broun P and Somerville C. (1997) Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsisplants that express a fatty acyl hydroxylase cDNA from castor bean.Plant Physiol113, 933-942

Chaofu Lu, Jinling Kang, David Sands, Alice Pilgeram
Department of Plant Sciences and plant Pathology
Montana State University, Bozeman, MT 59717-3150 USA
clu@montana.edu