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FAIR-CT98-4333
DiCra: Diversification with Crambe: an industrial oil crop |
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Contract No: | FAIR-CT98-4333 |
| Date Prepared: | March 2001, June 2003 | |
| Source: | Final Report | |
| Source: | Second Annual Progress Report |
Conclusions
The first aim of DiCra was to strengthen the weak position of the novel oilseed crop crambe by various breeding-related investigations. In general the research as such was focused on improvement of seed yield and seed quality and adaptation of the crop to unfavourable environmental conditions.
The evaluation of the germplasm collection has shown that the species C. hispanica has valuable characteristics for crambe breeding with respect to winter hardiness, early emergence, and in particular disease (Alternaria ) resistance. The gene pool of this species also showed significant variation in time of initial flowering.
Introgression breeding so far resulted in promising breeding materials. However one or two generations of multiplication will still be needed to get rid of the sterility problems associated with imbalances in chromosome number. The mutation breeding efforts resulted in genotypes having a content of glucosinolate (epiprogoitrin) in their seeds, ranging from 16.8 to 55.0 micromole per g seed. The average content of epiprogoitrin of 34.8 micromole per g seed is about half the content of the standard varieties BelAnn and Galactica.
As a result of the multilocational study carried out in Italy, France, UK, Netherlands, and Germany, a loy of useful information was collected. On average over a range of genotypes and environments, crambe yielded 2353 kg of seed/ha or 846 kg of seed oil. An average crop had a plant density of 80 plants per m2 with a plant height of 120 cm. The period from sowing to the onset of flowering and seed ripening took 61 and 96 days, respectively. Seeds had on average a weight of 6.5 mg with a content of 35% oil and a content of 65 micromole of glucosinolates per g seeds.
Seed oil contained 57.8 % erucic acid. The range among genotypes for seed yield was 546 kg/ha and for oil yields 326 kg/ha. For all other traits studied the ranges among genotypes were quite substantial. However, analyses of variance showed in general that environment (=combination of Year and Location) is the most prominent source of variation for all traits. Within Environments the factor Genotype was the most prominent and highly significant source of variation. Interaction mean squares of genotype and the other main factors tended to be fairly small. This implies that the ranking of genotypes within environments is relatively stable. It also indicates the presence of little genotypic differences among the set of genotypes in stability of traits over environments.
Correlation studies gave a clear indication for the direction of future crambe breeding. An ideal genotype (a so-called ideotype) for the set of environments in this study should be early flowering, produce tall (vigorous) and highly productive plants having predominantly large seeds. In addition, the seeds should have a high oil and erucic acid content. An additional selection objective could be lowering of the content of glucosinolates in the seeds.
In general the DiCra programme made a significant contribution to the knowledge of crambe selection covering selection methods, the sources of genes and the environmental effects. It is now known that, under favourable conditions, the promising.yields may be obtained. - The studies on crambe myrosinase and its cofactors were the continuation of a previous study started in the framework of the Concerted Action AIR3-PL-2480 "Crambe abyssinica: A comprehensive programme". Crambe myrosinase is present in the seeds mainly in an insoluble form. It has a high specificity for epiprogoitrin, the main glucosinolate in crambe, showing a higher activity with this substrate than progoitrin that differs from epi-progoitrin in the position of one OH group only. It is also the only myrosinase that is so specific for its natural substrate.
The two main quality parameters for crambe are related to the content of erucic acid and glucosinolate. During the DiCra programme, numerous analysis were done on these components, as well as on nitrogen and protein contents. These analyses were necessary since:
In addition, as the number of analyses were very numerous, new methods, rapid and reliable were found. Thus the DiCra programme gave the opportunity to improve the knowledge on routine analysis, and on myrosinase determination.
The results of the fertilisation trials indicate the importance of nitrogen for the development and yield of crambe. Considerable differences in plant development became apparent with the dry matter and the absorbed nitrogen depending on nitrogen fertilisation. There were also considerable differences with the yield, the dry matter and the absorbed nitrogen depending on location and year.
The maximum nitrogen uptake ranged from about 115 kg N per ha without fertilisation to 270 kg N per ha with the fertilisation rate of 150 kg N per ha in Rostock whereas in Quetigny the quantities were much lower with 46 to 156 kg N per ha. This indicates that the nitrogen from soil and fertilisation was not completely absorbed especially in Quetigny. It was found that considerable amounts of nitrogen were lost by the fall of the leaves and therefore could not be utilized for seed production. Another problem was that the crambe plant was not able to absorb all the available nitrogen from soil and fertiliser especially with the higher fertilisation rates. Dry matter was also variable according to location and fertilisation. The maximum dry matter ranged from 3.6 to 7.9 t/ha in Quetigny to 7.3 to 12.5 t/ha in Rostock.
As a consequence there were also considerable differences in yield depending on location and nitrogen fertilisation. The highest yield was obtained in Bologna (4.13 to 4.66 t/ha) and the lowest yield in Quetigny (1.63 to 2.03 t/ha). At all locations nitrogen fertilisation of 75 kg/ha resulted in a distinct yield increase compared with no nitrogen fertilisation while a fertilisation of 150 kg/ha did not significantly improve the yield compared to 75 kg/ha. The oil content slightly decreased and the raw protein content significantly increased with increasing nitrogen fertilisation while sulphur fertilisation showed no influence on either. The glucosinolate content distinctly increased with sulphur fertilisation. The influence of nitrogen and sulphur fertilisation on the content of erucic acid was not very significant.
The results of trials with sulphur fertilisation show no positive effect on crambe. There was no increase of seed yield while a decrease in seed quality took place due to the increase of the glucosinolate content. Because of the anti-nutritional effect of glucosinolates crambe meal is unsuitable as an animal feedstuff. This is important because the economic viability of growing crambe also depends on the possibilities and constraints of using the by-products.
It has been found that a high nitrogen fertilisation rate together with a high content of mineral nitrogen in the soil in spring increases the risk of pollution with nitrates because it may lead to a higher content of mineral nitrogen in the soil after harvesting. It has also been taken into account that considerable quantities of nitrogen were lost by the fall of the leaves and remains in the field in addition to the quantities after harvesting according to soil analysis. This amount increases with higher nitrogen fertilisation. This confirms the necessity of a well-adapted nitrogen fertilisation strategy to avoid pollution by nitrates.
According to the experiments a nitrogen fertilisation of 75 kg/ha can be recommended for crambe depending on mineral nitrogen in the soil, soil type and the preceding crop. Fertilisation with sulphur is not necessary. Conclusions for breeding based on these experiments are to select genotypes with a high dry matter production, a better ability for absorbing nitrogen from the soil and a late leaf fall.
Objectives
Crambe is a cruciferous species suitable for diversification whose potential outlets are well established. The seed provides oil for lipo chemistry and for mechanical industry. The seeds also provide a meal that must be improved through lowering glucosinolate content. As regards its cropping, crambe can be cultivated in Europe as a spring plant. However, its sensitivity to cold, which hampers its development, must be improved. To encourage the cultivation of crambe in the EU it is necessary to identify those climatic zones to which it is best fitted and develop environmental friendly techniques for its production. Hence, the DiCra programme focused on 3 tasks, breeding, agro physiology and quality control. This third task was introduced as a support of the two previous.
Activities
Task 1: Breeding
The aim of this part of the project is to provide genotypes adapted to the EU pedoclimatic conditions, mainly frost resistant, producing a high level of erucic acid and a low content of glucosinolates (anti-nutritional factors that can affect the value, as animal feed, of the meal after oil extraction). The work was divided into 2 main parts, as indicated below, aimed at selection and evaluation of improved genotypes through the pedoclimatic network.
Selection: Sensitivity of C. abyssinica, C. hispanica and C. glabrata genotypes to diseases (mildew and Alternaria) and to frost were compared. It can be concluded that C. hispanica may provide valuable resistance to infection by Altemaria and to frost, but its susceptibility to mildew infection should also be taken into account. Interspecific crosses between C. hispanica and C. abyssinica were successful. During year 2, F1 plants were raised. The F1 plants were rather fertile and produced a high number of seeds. The seed yield was only slightly lower than the yield of the parental C. hispanica plants. Attempts were also made to produce new dihaploids using microspore culture, however these failed, whatever the technique used.
Pedoclimatic network: The best European areas for growing crambe are under investigation. Twelve accessions were tested in spring sowings in 5 locations: Germany near the Baltic Sea, UK in the Bristol area, the Netherlands, France in Burgundy and central Italy (Bologna). In South Italy, the 12 accessions grew well even when cultivated during winter. Plant growth, yield and chemical composition of the harvested seeds were recorded. Yield was very dependent on the location. Next year, the same accessions will be cultivated in the same network.
Task 2: Agrophysiology
The aim of this activity is to find the best environmental friendly techniques for growing crambe , including aspects of nitrogen and sulphur fertilisation, weed control and germination capacity. The result will be a crop guide to be given to interested farmers covering:
Weed control: The key points for weed management in Crambe identified so far are:
Germination capacity: Immediately after seed ripening, the seed undergoes a dormancy period which lasts about 2 or 3 months. Dormancy can be lifted by a combination of light and temperature. When dormancy is broken, the final germination, in the laboratory conditions, seems to be dependent of the climate during seed maturation. Spores of many fungi including Alternaria are very common on Crambe seeds at harvest. During their germination, these spores produce toxins that can cause necrosis on the leaves. These toxins have been isolated and their characterisation is under way. The possibility of a chemical germination inhibitor in crambe seeds was investigated through experiments conducted during cress and lettuce germination. Through a field experiment it was observed that fungi treatments can significantly improve crambe yield in comparison with the control. However, the dramatic green effect following the use of some of fungicides during plant maturation, did not resulted in a higher yield. These findings have to be confirmed next year.
Nitrogen and sulphur fertilisation: Three experiments were conducted, in France, central Italy and Germany. The dry matter and the nitrogen content were followed during the crop cycle. The dry matter accumulation was very variable depending on the locations and nitrogen fertilisation. In the best situations, the dry matter exceeds 10 tons per ha. Accumulation of nitrogen begins at stem elongation and lasts to flowering time. Its maximum is about 150 to 200 kg/ha. When the plant is not in good conditions, the nitrogen remains in the leaves and does not migrate to the reproductive part. We could calculate that as much as 100 kg/ha could be lost during cropping by leaf fall. The yield reflected the nitrogen uptake. It seems that sulphur does not play an important part in yield formation. Next year, the same trial will be conducted in order to confirm the results from the first two years.
Task 3 Quality control:
The quality control laboratory analysed all the seeds and the plants from the breeding and agro-physiology actions. It is also involved in the characterisation of analytical parameters related to cold tolerance. Nearly all the samples of the partners were analysed including seeds from the pedoclimatic network, from the fertilisation experiment, from new accessions breeding and from samples during seed ripening for glucosinolate, oil and proteins content, fatty acid profile (erucic acid). Also analysed was green dry matter from the experiment on nitrogen content. The 12 accessions cultivated in the fields were also evaluated for their frost resistance by visual assessment and by electrical conductivity. The results from these two methods showed good correlation. Other experiments indicated an effect of relative humidity on frost resistance.
Results
Genotypes with improved cold resistance and/or with a lower glucosinolate content were investigated. Genotypes resistant to frost were found in C. hispanica. A technique for obtaining doubled haploid plants of Crambe using microspore embryogenesis proved much more difficult than expected. At the end of year two the technique was still not working and was abandoned.
The agronomic performance of 12 genotypes was evaluated in six environments in five countries. The 12 genotypes were grown and the seeds analysed providing information on their characteristics (oil, erucic acid and glucosinolates).
By the end of year 2 crosses between genotypes with cold resistance, a low glucosinolate content and disease resistance and high yielding genotypes had been investigated. Crosses between genotypes with cold resistance are completed. No low glucosinolate plants were found in this experiment. However, seeds from the first generation after crossing became available and subsequently low glucosinolate genotypes were found.
Work on identifying genotypes with stable seed yields in particular environments continues aimed at obtaining an insight into environmental and plant physiology factors determining winter hardiness. Results from work on genotypes with improved quality of the meal indicate that by breeding for low glucosinolate content a substantial improvement is possible.
Progress
Progress over the first 24 months is reported on a task by task basis below:
Task 1.1 Evaluation of genetic variation for important agronomic characteristics. During year one, 11 lines of C. abyssinica, 70 C. hispanica and 13 C. glabrata were compared. 10 lines of C. hisponica resists to -7°C. An experiment made during year 2 confirms this finding.. C. hispanica resisted to -9°C. but C. abyssinica and C. glabrata did not. During year 2, 11 lines of C. abyssinica, 66 C. hispanica and 16 C. glabrata were also compared for their sensitivity to disease (mildew and Alternaria). 28 lines of C. hispanica turned to be not infected by mildew, whereas 4 were only slightly infected by Alternaria. Thus, C. hispanica may be considered as a valuable source for frost and for disease resistance.
Task 1.2 Selection of improved genotypes During year 1, year 1 it became apparent that the microspore technique used for rape could not be adapted for crambe. Many modifications in the protocol were done as well for the growth conditions of the donor as for the composition of the medium without success. Another approach using other model plants such as barley, tobacco and Capsicum, were tried as was modification of the technique by adding phyto hormones, without result. During year 2, such work continued, using starvation media such as used with tobacco cultures, by increasing the medium osmolarity and by using phytohormones. Attempts to stabilise microspore against bursting, also failed as did treatments including the use of Picloram. It was thus impossible to establish a protocol of microspore culture in crambe during two years of research. Since there was not enough time left for breeding, even if a method was found, this work was stopped.
Interspecific crosses, produced during year 1, were analysed for fatty acid composition using 22 interspecific lines and 2 parental lines. The fatty acid composition varied significantly between the lines. However, no lines exceeded the C. abyssinica parent for erucic acid. During year 2, F1 plants of interspecific crosses between C. hispanica and C. abyssinica were raised. The seed yield was only slightly lower than that yield of the parental C. hispanica plants.
Task 1.3: Evaluation of improved genotypes -pedoclimatic network. The same lines were tested in very different pedoclimatic conditions. During year one, no lines were obtained from the trial on resistance to winter in North Europe (France, Great Britain, Netherlands, Germany and even Central Italy). However, crambe grew well in South Italy (Naples area). The yield varied from 657 kg/ha at Long Ashton (U.K.) to 2946 kg/ha at Rostock (Germany). The oil and glucosinolates varied according to the lines and the location. During year 2, we confirmed that crambe is adaptable to North Germany and to Central and South Italy.
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Updated
by CPL Press:
03/07/2007
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