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AIR3-CT94-2480
Crambe abyssinica - Production and Utilization - A Comprehensive Programme |
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Proposal No: | AIR3-CT94-2480 |
| Date Prepared: | July 1998, April 1998, October 1996 | |
| Source: |
Final Report Second Annual Progress Report & Meeting First Annual Progress Report |
Summary
During the three years of the Concerted Action,
trials were carried out on many aspects of seed production (breeding, agronomy) and on
utilisation (hulls, glucosinolates for fine chemistry and pharmacy, use of oil
in the mechanical industries and as an adjuvant for pesticides,). All these
aspects as well as the economic factors were reviewed during a meeting that took
place in San Miniato (Italy) in May 1997. The main conclusions are detailed
here.
Seed production
Selection Heritability studies were performed on the basis of the variations between F3 lines. High values were found for thousand seed weight, moderate values for earliness, plant height and oil seed content and a low heritability for seed yield.
Interspecific crosses The best performing lines derived from interspecific crosses between C. abyssinica and C. hispanica equalled the C. abyssinica parent for seed yield, suggesting that these lines were fully fertile. It is interesting to note that earlier and later flowering lines and lines with significantly higher and lower seed weights than the C. abyssinica parent were observed. These results suggest that introgression of C. hispanica germplasm into C. abyssinica can improve the genetic variation in C. abyssinica.
Evaluation of improved genotypes Recently selected genotypes were evaluated for agronomic performance in three locations (Netherlands, Italy and France). Seed yield varied from 1.5 to 3.5 tonnes/ha according to the year and location. In the Netherlands, the best performing genotype yielded on the average 11% more than the reference variety (BelAnn). Breeders rights were obtained for two lines, Galactica (CPRO 9103-16) and Nebula (CPRO 9110-20). The variety CEB02 is also marketed. The variety Mario selected in Italy regularly gives the best yields in this area. In France, results are very variable depending on the year, but they can be as high as in the other countries.
Experiments in agronomy A decimal scale for scoring plant development was adapted for use with Crambe, from that used for rape. This proved to be very satisfactory. Trials were conducted in each country to determine the best sowing date and plant density. Experiments on nitrogen and sulphur fertilisation were also conducted.
Pre-industrial trials The pre-industrial cultivation of Crambe began in 1996 and reached 80 ha in France and 40 ha in Italy in 1997. The factors likely to depress yield, were investigated. In general the main problems related to plant establishment. It was shown that particular attention has to be applied to soil preparation and to the seed bed. Bad soils must be avoided. If the plant is well established, it branches and can be very competitive with weeds. If it is not well established, the crop can be completely smothered. The plant is less sensitive to insects than many other Brassicaceae, but it can be attacked by flee beetle at emergence and by pollen beetle at flowering. These insects can be combated with Pyrethrinoides. Crambe can also be attacked by fungi, mainly by Alternaria spp. in wet conditions. A treatment with flusilazole plus carbendazime is effective. Harvest takes place in July in Italy and in August in France and the Netherlands. It is always easy since the crop never lodges. Yields of around 2 tonnes/ha are realistic as a goal to provide a medium gross margin.
Utilisation
Use of oil in the mechanical industries The main characteristics of the oil extracted from Crambe seeds were determined. This included fatty acid and triglyceride composition, viscosity, smoke point, density, acidity, hygrogenability, emulsionability, heat removal rates and wetability of materials. These preliminary findings indicated that the oil was well suited for use as a lubricant and as a source of erucic acid for chemical applications. For both areas of application, Crambe oil showed better technical properties than other vegetable oils currently available on the market (i.e. soybean, sunflower, rapeseed, linseed etc.). Moreover, compared with some mineral oils, Crambe oil has other advantages such as high biodegradability, low toxicity and high wettability. However, since biodegradability is associated with a lower resistance to oxidation, this results in a shorter oil life during use.
The findings have encouraged private companies to collaborate with the Concerted Action. Interesting niches have been found in steel casting and hardening, as lubricant for metalworking as well as hydraulic fluids. Two stroke engines (boats, motorcycles ) working with Crambe oil maintain their power output and in addition, pistons remain clear and smoke is considerably reduced which help maintain air quality.
In lipochemistry, Crambe oil showed potential reflecting its high erucic acid content (around 60%). This is significantly higher than that of all the other animal and vegetable oils on the market. Pre-industrial tests are under way in the Netherlands, Germany, England and Italy. The possibility of synthesising new oils by esterification with neopentylpolyols both of total Crambe fatty acids and of pure erucic acid were also studied. The possibility of producing oils with different technological characteristics has been proved. The most important property is the improved resistance to oxidation.
Use of the oil and its derivatives as adjuvants for pesticides Various derivatives of Crambe oil were evaluated as adjuvants for herbicides and fungicides. Their effectiveness was compared with that of adjuvants already in use. The performance of the novel products were broadly similar to commercially available mineral and plant oil based products as regards retention, penetration and overall activity. Commercial oil adjuvants have been "optimised" and with sufficient research and development inputs, Crambe oil adjuvants could match existing products. However, results to date suggests that Crambe oil adjuvants are unlikely to be significantly superior to existing oil adjuvants.
Characterisation and properties of cellulose isolated from Crambe hulls. Hulls were fractionated by steam explosion and organosolv treatments in order to isolate the cellulose component. After bleaching and alkali treatment, the cellulose was characterised by X Ray, FTIR and CP-MAS 13C-NMR spectroscopy. The profile of the cellulose I allomorph showed a fairly low degree of crystallinity and relatively small crystal dimensions. Hence, it is suitable for use in the synthesis of derivatives such as carboxymethyl cellulose or cellulose esters. Mixing Crambe cellulose with Kraft fibres at 10 to 20% resulted in paper with reasonable physico-mechanical properties.
Glucosinolates Seeds of Crambe and defatted protein meals derived from them contain glucosinolates (GLs) at concentrations of around 4 and 6 % respectively. The most abundant is epi-progoitrin (E-PRO) at around 90 %. At neutral pHs, myrosinase (MYR) catalyses hydrolysis of E-PRO, producing 5-vynil-oxazolidine-2 thione (VOT) whereas, at acidic pHs, the corresponding nitrile (NI) and epithionitrile (ENI) are formed, although in presence of Fe(2+) and an epithiospecifer protein, also contained in Crambe seeds. Investigations on Crambe myrosinase (CMYR) carried out during the third year of activity gave some original and interesting results. We established that in Crambe defatted meal, CMYR is mostly insoluble, particularly active and overall specific for E-PRO. This activity is about seven times higher than that determined with sinigrin as a substrate. The physicochemical properties of CMYR are also quite singular. The observations in this area led to the hypothesis that Crambe seeds contain a thermolabile activator that is able to bind with CYMR to form a super active enzyme complex. This complex appears to be very specific for E-PRO but not for the R isomer progoitrin. VOT and NI were produced by MYR-catalysed reactions and evaluated for anti-tumour, anti fungal and anti-germination activities. A small amount of EBI was produced from Crambe meal, then purified by chromatography and used to determine physico-chemical and biochemical properties.
The results of these experiments, apart from confirming that E-PRO is suitable as a starting material for production of VOT, NI and ENI, indicated a middle to low anti-proliferative and anti-fungal activity of the first two compounds, especially if compared to the other GL degradation products tested. No conclusive results were obtained concerning the biological activity of ENI, though its strong chemical reactivity suggests that it should be biologically active. The anti-germination experiments confirmed the low activity of VOT and demonstrated that solutions of NI at 0.25 mg per ml strongly inhibits (around 50%) the germination of lettuce seeds.
Conclusion
For three years, eleven teams carrying our research funded by national programmes were coordinated through the Concerted Action. This activity has confirmed the potential for use of products derived from Crambe through new outlets in a wide range of applications:
However, farmers will grow Crambe only if it is profitable for them. This depends not only on the technical performance of products, but also on the potential income, which in turn depends on productivity, the value of the crop and any premium that industrial crops may receive. Although new varieties have been identified, more adapted to European soil and climatic, yields are still very variable. Hence, more work has to be done to commercialise this crop, focused on breeding and agronomy.
Plant Breeding -Interspecific crosses
In a two replicated
trial, 2 lines derived from interspecific crosses between C abyssinica and
C hispanica were evaluated for agronomic performance. The best
performing lines equalled the abyssinica parent for seed yield,
suggesting that these lines were fully fertile. Significantly earlier and later
flowering lines and lines with significantly heavier or lighter seed weights
than the abyssinica parent were observed. These results suggest that
introgression of C. hispanica germplasm into C abyssinica can
improve the genetic variation in C abyssinica.
Plant breeding - Evaluation of genotypes
Dutch experiments
In a joint experiment of CPRO-DLO and CEBECO Zaden, advanced genotypes
were evaluated for agronomic performance. The experiment consisted of two
treatments (+ or -fungicides) and four replicates. Significant differences were
found for each characteristic evaluated. The best performing genotype yielded
3510 kg hulled seeds/ha with an oil content of 39.3%, 60.4% erucic acid in the
seed oil and a moisture content of 8 percent in the seeds. The non
treated trial showed a 5% lower seed yield than the treated trial. The non
treated trial ripened one week earlier, probably due to infection bv Sclerotinia.
No infection by Alternaria was observed. Due to wet weather
conditions harvest was delayed which caused shattering in the non treated trial.
Significant interaction between genotypes and treatment was found for earliness.
Lack of significant interaction for seed yield, seed weight and oil content,
suggest that the genotypes reacted similarly on fungicide treatment. Attack by
caterpillars reduced the seed yield of pubescent genotypes by 10 to 20%.
French experiments
The experiment took place in 2 sites in
Burgundy with deep soils. Quetigny is a clay soil and Rigny is a more loamy. 50%
of the seeds were lost and gave no plant. No fungicide was sprayed and in both
locations, the crop was infected by Alternaria and by black leg. Sclerotinia
was observed only at Rigny. 91W-100 is to early and is not adapted to the
Burgundy area. In Rigny, Cebeco 02, Cebeco 04, 9103-16 (Galactica), 9110-20
(Nimbola), were significantly different fro the control. In Quetigny, there were
no significant differences between the cultivars.
Italian experiments
In Italy, Galactica, Mario and Indy gave
better yield than the control but were not submitted to analysis of variance
because of the scarce amount of seeds.
Agronomic experiments
Crambe can be grown in France with as
good results as in the other countries. Nevertheless, all the soils are not
suitable for Crambe (it is very sensitive to dry and shallow soils) and the soil
must be prepared very carefully. Soil preparation is the key to success. Three
experiments failed because of over light soils or of a bad seed bed. It is now
clear that the best sowing date is the beginning of April after the last frosts.
I in very good soils, 60 plants /m2 are sufficient to ensure a good
yield. In light or badly prepared soils, the plant density must be increased to
150 to 200 plants /m2. The yields can reach 3t/ ha in very good
conditions, while the use of fungicides can increase the yield.
Pre-industrial trials
5 ha were grown in Burgundy (France) in
deep organic soils yielding 2.8 t/ha and 40 ha were cultivated in North
Italy.The yield varied from 1.4 to 2.6 t/ha. The results are considered
potentially promising and therefore, cultivation trials on a larger surface were
programmed for 1997.
Industrial utilisation.
Hulls were submitted to steam
explosion, were fractionated, delignified and treated in a Organosolv reactor.
The insoluble cellulosic residue was used for paper making. Paper with 15 % of
Crambe showed good retention of the original tensile strength, reduced air
permeability, slightly decreased tear strength.
Oil in mechanical industry
Research is under way to verify the
possibility of replacing animal fats in some lubricant products by Crambe oil.
The main difficulty is a high ash content. When the minimum 100 t oil necessary
to run an industrial plant is available, tests will be done to rectify the oil
and eliminate ashes. Crambe oil has been tested in mixture with synthetic oils
and additives in outboard motors for 1~0 hours. All the components (pistons,
pistons, rings, cylinders and candles) were in a very good condition. Crambe oil
has been esterified using various alcohols. The resulting esters are highly
variable in character (the viscosity, for instance, varies from 5.8 to 78.7
mm/sec) and a higher resistance to oxidation. The low pour point of some them
could be improved using additives. Tests for lubrication are still in progress.
Some of them have also been tested as phytochemical adjuvants in plant
protection.
Oil in lipochemistry
Trials to substitute fish oil by Crambe in
lipochemistry are also in progress. Additives for plastics and monomers for
polymerisation are under investigation.
Oil as adjuvants in plant protection Crambe oil enhances the spreadability of fungicidal emulsion concentrate formulation and to a certain extent of wettable powder formulations. For instance, the penetration of propiconazole into a wheat leaf increases when Crambe oil at 0.5 % is added to a Tilt spay liquid. 42 % reached the inner leaf tissue. Used alone, Crambe oil has a fungicidal property on Botrytis cinerea. The foliar penetration of herbicides such as phenmedipham into barley, cleavers and wheat was enhanced by glycerol monoerucate.
Glucosinolates
Crambe seeds and their derived defatted protein
meals contain large amounts of epi-progoitrin
(2-hydroxy-but-3-enyl-glucosinolate) (E-PRO), which in these materials is the
main GL. At neutral pHs, MYR-catalyzed hydrolysis of E-PRO produce
5-vinyl-oxazolidine-2-thione (VOT), whereas the corresponding nitrile (NI) and
epithionitrile (ENI) are formed at acidic pHs. The latter needs the presence of
Fe2+ and a epithiospecifier protein, which is also contained in crambe seeds.
The VOT and NI were evaluated as anti tumoral, anti fungal and anti germinative
activity. The results of these experiments, indicated the potential of E-PRO as
a starting material for the production of VOT and NI. The results also indicated
a middle to low anti-proliferative and anti-fungal activity of both as compared
to other GL-degradation products tested. Tests on seed germination confirmed the
low activity of VOT and demonstrated that NI causes about 50 percent inhibition
of germination of lettuce seeds at a concentration of 0.25 mg/ml.
Meeting :
A meeting on Crambe abyssinica, organised as
part of the activity of the concerted action was held in San Miniato (Pisa),
Italy, May 1997, co-ordinated by E D Tittonel (ENESAD Dijon, France) and
organised with Sandro Palmieri (ISCI Bologna, Italy), Luca Lazzeri (ISCI
Bologna, Italy) and Dick Mastebroek (CPRO-DLO Wageningen, The Netherlands). It
was sponsored by Progetto Ricerca sulle Colture Alternative (PRisCA MIRAAF,
Italy) and the following companies: 110 Bourgogne, Cerealtoscana, Unichema
International and Cebeco Handeslraad. Papers, presented either orally or as
posters covered economics, Breeding and Agronomy (section chairman: W J M
Meijer), Processing (section chairman: A Capelle) and Utilisation (section
chairman: S. Palmieri). The meeting opened with the following general
introduction to Crambe abyssinica presented by E D Tittonel. The
abstracts have been published (contact the coordinator for further information)
and are available in the crops section of the NF-2000 Database and web site.
OBJECTIVE
Crambe Abyssinica is a Cruciferae which was grown on 25.000 ha in the USA in 1993 with the aim of selling it in the future on the European market. Experiments mainly in the Netherlands (300 ha) have demonstrated that it can also be cultivated as a spring crop in Western Europe. Sowing dates and rates, plant depth and harvest practices are now well determined. However, pest and weed control are still under evaluation. Other trials will be conducted to increase the cultivation of this new crop especially on marginal and on set-aside lands. New varieties adapted to these situations are under evaluation.
Crambe appears to be a promising crop because of the many possible uses of its seed (pharmaceuticals, detergents, cosmetica, ceramics etc.). In the concerted action, the interest will be focused on oil glucosinolate and fibre.
ACTIVITIES
Plant breeding: Much of the seed used in Europe comes from the United States and may not be adapted to European climatic conditions. Selection trials are being carried out, mainly in the Netherlands where 12 advanced genotypes are being assessed in comparison with the standard variety BelAnn. These showed large variations in emergence, plant development, infection by the fungus Alternaria, oil content and fatty acid composition.
Two genotypes were found to possess a low content of nervonic acid in the seed oil, as demanded for the production of erucamide. Infection by Alternaria was found to be a serious problem in growing Crambe. However, within the set of genotypes evaluated, some less susceptible genotypes were found. After two generations of self pollination, fully fertile plants were obtained from the interspecific cross between C. abyssinica and C. hispanica. Crossed plants were selected for a DNA content comparable to that of C. hispanica but with seeds retaining characteristics of C. abyssinica. These genotypes are probably suitable for introduction of the seed retaining characteristic into C. hispanica. Steps are being taken to register the variety "Mario".
Cultivation: It seems that the best area for Crambe cultivation is the Po Valley in Italy. Experiments are required to establish new areas suited to this crop.
Seed processing: At the moment, the quantities of seeds are not large enough to supply an oil mill. Hence, oil is extracted on the small scale by pressure.
Industrial utilisation: The composition of the hull indicates that, in comparison with other lignocellulosic materials, the alpha-cellulose and ash content of the hulls are higher, whereas the hemicelluloses are lower. The lignin content was comparable to that of annual plants. A steam explosion process was very effective in the separation of cellulose, hemicellulose and lignin. Cellulose properties after steam explosion are promising for dissolving and derivatising and for use in paper production.
Oil in mechanical industries: Contacts have been made with Italian and French companies involved in mechanical processes. Test have been encouraging, using oil in lubrication as well as in mixtures with petrol for two stroke engines.
Oils as an adjuvant in plant protection: Crambe oil per se had no effect on spray retention by plant foliage, although formulated oil enhanced retention thanks to an associated emulsifier. Methyl erucate (the most abundant compound in methylated Crambe oil) performed slightly better than methyl oleate (methylated rapeseed oil) in enhancing the foliar penetration of pherimedipharm into barley. Formulated Crambe oil proved to be as efficient as Actipron, a reference mineral oil, in enhancing the efficacy of the herbicide quizalofop ethyl towards several graminaceous weeds. Crambe oil increased the penetration of the fungicide propiconazole into wheat leaves and influenced positively its translocation in the plant. In spite of trials dealing with 35 herbicides, no compound proved to be efficient in controlling volunteer rapeseed in the Crambe crop.
Glucosinolates: Tyrosinase catalysed hydrolysis of epi progoitrin at neutral pHs produces 5-vinyl oxazolidine 2 thione (VOT) as a derivative of the correspondent isothiocyanate by cyclisation. At acid pHs in the presence of Fe2+, the formation of nitrile (ENI) and epithionitrile is favoured. The latter, however, requires the presence of an epithiospecifer protein (ESP) which can also be isolated from Crambe seeds. Sufficient amounts of enantiomerically pure VOT and NI were prepared by immobilised myrosinase for carrying out tests in various fields of possible utilisation. Finally, VOT and NI were tested as anti-fungal compounds, as well as starting material for synthesising new bioactive molecules. VOT was also tested as an anti-tumour compound.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
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