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QLK5-1999-01505
HARMONIA: Hemp as Raw Material for Novel Industrial Applications |
| Contract No: | QLK5-1999-01505 |
| Source: | Final Progress Report April 2004 |
| Third Annual Progress Report March 2003 | |
| Second Annual Progress Report March 2002 | |
| First Annual Progress Report 2001 |
Summary
Objectives
The RTD part of the proposal aims at identifying the basis of the excellent fibre quality of the Chamaeleon hemp variety. This will be done by chemical and physical analysis of the fibre. In addition, genes involved in cell wall metabolism, that might determine the good fibre quality, will be isolated and transgenic hemp prototypes containing these genes will be produced. To produce fibre material for the demonstration project cultivation methods of the crop will be optimised and various methods to refine the hemp fibre will be tested.
In the demonstration part of the project whole plant material and refined fibres will be applied in various high added value applications. Steam exploded fibres will be used for applications in composites and yarn. Fibres and whole plants will be used to produce pulp as a basis for speciality papers and biodegradable CNC cellulose for diapers. The project will produce prototypes containing fibres from the bright hemp and give insight in the economical perspectives of these industrial applications.
Results and Milestones
Year 1
The project started with 15 kg of seed of the Chamaeleon variety. This breeder's seed was used by Vandijke SEMO to produce basis seed. One multiplication took place in the Netherlands and yielded 90 kg of Chamaeleon seed. Seed processing conditions were adapted and optimised to improve seed quality. In order to have sufficient seed for the second year of the project, a winter multiplication was initiated in Australia. This enabled the production of sufficient seed to produce Chamaeleon fibre in the second year of the project.
Due to the limitations of the Chamaeleon seeds available it was not possible to drill seed for fibre production. The project management committee has decided that the partners will analyse the quality of the European reference variety Felina 34 in the first year of the project. In the subsequent years the Chamaeleon fibre will be included in the trails. At Hemcore a field trial with 10 hemp varieties including Chamaeleon was carried out, The application of Nitrogen resulted in increased yield in dry matter. Retted and not retted stem material of Chamaeleon and Felina 34 was processed. Under the applied conditions processing behaviour of both varieties was similar. Retted material c ould be decorticated without any problem. It was not possible to process unretted material,
The development of hemp-based yarns proved very difficult with the available hemp varieties. With the use of fibres produced by the short staple process (by Hemcore) it was almost impossible to produce a yarn. The coarseness and stiffness of the fibre and the wooden impurities made it very difficult to process the fibre into a sliver. Under exceptional conditions William Ross was successful in producing a yarn though this was of a very coarse quality. This yam will be used as the reference yarn for Chamaeleon based yams.
IAF Reutlingen supplied the partners with a water-retted fibre that was processed by Zignago Tessile. The material was relatively clean and quite stiff. The hackling behaviour was very heterogeneous. Hackling tow were sent both to William Ross & Co. and JAF. After roving various chemical routes were carried out to obtain a good dissociation level for good spinnability. The treatments gave high degree values of dissociation but different white degrees and spinning behaviours. By wet spinning a Nm 24 yarn was produced. The characteristics of the yarns obtained from the different rove chemical treatments were assessed.
The hackled tow was processed further by William Ross & Co. on a comb and a blending/drawing drawframe, With specific setting for the draft and the twist factor on the spinning frame it was possible to spin a Nm 3.6 hemp yarn.
Funder demonstrated the usability of hemp in the fibreboard wet process, hemp board samples were formed in a wet sheet former and pressed in a laboratory press size 600 x 600 mm.
Additionally, hemp fibres were processed together with the shive contend in trial test-run on a semi-industrial fleece forming and dewatering machine. The challenge of this test was the study of the behaviour during mixing of short wood fibres with long hemp fibres. The machine was specially designed with two radial flow distributors to get an optimal dosing percentage and a good mixing effect and a maximal fibre connection.
For the analysis of hemp by IAF and Plant Research International a trail field was sown. The field consisted of the varieties: (Chamaeleon, Felina 34, Green Sibling and Koinpolti TC. Samples from the stems were taken to analyse the chemical composition by microscopical and spectroscopical means and to isolate DNA and RNA. Chemical analyses of 205 samples were performed to determine acid detergent fibre (ADF), acid detergent lignin (ADL) and neutral detergent fibre (NDF).
Chamaeleon stem material of three developmental stages was used to construct three cDNA libraries. cDNAs were randomly picked for the libraries and the base sequence was determined. Based on sequence homology it is expected that several of these genes will have a function in cell wall metabolism. The expression patterns of these genes will be monitored by DNA microarrays. In order to further elucidate the function of the genes, a number of genes will be tested by transformation to hemp. For this a transformation protocol will be developed within the project. With specific hormonal treatment it was possible to develop shoots from hemp callus. The transformation of hemp callus could also be achieved.
At IAF, hemp stems have been investigated by quick quality measurements like determination of the water content, colour of the stems and NIR measurements. For Chamaeleon the maturity can also be measured by the colour (CIELAB) of the stems and an evolution of the fibres can be obtained. It is easy to separate Chamaeleon from other varieties but it is also possible to differentiate between Felina and Green + Kompolti. Within the plant the core and bast and maturity can be distinguished by UV/VIS/NIR and IR-spectroscopy due to the plants morphology and their chemical composition. Maturity can be determined by measurements of the cell wall thickness. Some selected samples are analysed with an IR-Microscope on a 10 to 100 micron lateral resolution.
Year 2
In the second year a field trial was also sown to provide material for spectroscopical, molecular and chemical studies The field consisted of 4 different varieties: Chamaeleon, Felina 34, Green sibling and Kompolti TC, The first harvest took place six weeks after sawing on the 24th of June. In order to focus more on the transition from green to bright, subsequently, material was harvested at week 9, 11, 12, l3, 14. and 16. Samples were used for microscopical analysis or separated into core and bast parts for isolation of mRNA or spectroscopical and chemical analysis.
From the three different cDNA libraries 1308 cDNA inserts have been sequenced to determine the 5' DNA sequence. For 76% of the clones homology was found with proteins with a known function, 18% of the sequences could be identified as an expressed protein of which the function is still unknown and the remaining 6% of the sequences could not be identified. Within the cDNA clones about 56 genes could be identified as coding for proteins involved in cell wall metabolism.
To study the expression profiles of genes of interest cDNA microarrays have been printed containing the sequenced clones mentioned above and an additional 1700 randomly picked clones. A first series of analysis was carried out with mRNA isolated from the Chamaeleon bast samples from the 2000 material. The expression profiles indicate that genes belonging to the same pathway in cell wall biosynthesis have different expression profiles. An extended hybridisation experiment was carried out with plant material of the year 2001. In this case bast samples were taken from the middle part of Chamaeleon and Felina 34.
Within the first year of the project a regeneration protocol for hemp has been developed. In the second year the regeneration protocol was used as a basis for the transformation experiments. In three transformation experiments shoots were obtained within three weeks. GUS analysis on a leaf of one of the shoots showed expression of the beta-glucoronidase gene, confirming the transgenic nature of the shoot. In addition a second transformation protocol based on meristem transformation has been applied.
Spectroscopical analysis (Quick Quality Measurements) carried out on fresh stem material showed that in the UV/VIS range Chamaeleon is easy to distinguish from the other varieties.
Principal component analysis of near infrared spectra of dried first year samples showed that the first principal component can be attributed to the maturity. A differentiation between bast samples of maturity I to 3 and samples of maturity 5 to 7 is possible. The second principal component can be attributed to the different parts of the stems (top, middle, bottom). In the second year samples a separation due to the maturity, as can be seen for the dried first year samples in the NIR was not observed.
To predict mechanical properties dried samples of the second years harvest have been analysed with the FOSS VlS/NIR. spectrometer system. Since they reflect different chemical information, the VIS and the NIR region of the spectra are analysed separately. In most cases, Chamaeleon samples significantly differ from the other samples. Therefore, the sample set is, additionally, divided in two sub-sets, one only with and one without Chamaeleon. The best results are achieved by analysing the first derivative of the spectra both for the VIS and NIR region, In contrast to the original spectra, the 1st derivative produces a much better correlation between the spectroscopic and mechanical measurements.
The spectra obtained in the UV/VIS area were analysed by means of principal component analysis (PCA) and partial least squares (PLS I). Due to the fact that Chamaeleon samples significantly differ from the others sample sets only with or without Chamaeleon were analysed separately. Omitting Chamaeleon from the analysis, did not allow to distinguish the different other varieties nor the different origin of the samples. Generally, a separated analysis leads to better results than the PLS of the whole set of samples. This indicates, that a careful selection of the samples for the analysis results in a better correlation and a more robust calibration model to correlate the measured spectra with mechanical properties.
Initial microscopic analysis showed that the transition zone from bast to core seems to be less tight for Chamaeleon than for Felina stems. This finding has to been repeated in the 2002 material but this difference may be important in decortication and other technical behaviour. Selected cross sections of Chamaeleon and Felina stems were analysed by IR microscopy. Due to the small measuring spot (10 to 100 microm in diameter) the spectra are more pronounced. Therefore it is possible to measure a small proportion of wax in the epidermis and this can also be seen in the change from bast to core and in the bast region. Characteristic lignin peaks are obvious in core spectra which are not found in spectra of the transition zone between core and bast. By lR- and Raman spectroscopy the existence of calcium oxalate crystals in the hemp samples was shown.
From the seed multiplication in 2000 and the winter multiplication in Australia 2000/2001 in total 448 kg of Chamaeleon seed was obtained. It was distributed to Hemcore UK), Funder (A) and Kenderfono (Hu) for fibre production The remaining seed was used in basic seed production to build up the seed bank for future development of the variety.
Autumn weather conditions were not favourable in Europe in 2001 and the harvest of the Chamaeleon fields was not easy, finally most of' the seed harvest was done by hand. In spite of these difficult climatic conditions in France as in Austria different harvest techniques were tested and good experience for subsequent years was obtained.
By adapting existing cleaning systems and by combining the different cleaning methods, a suitable system for upgrading of seed quality has been established. The total yield of cleaned and good seed was 510 kg.
For the second year Chamaeleon was compared with six other industrial hemp varieties. Yield of Chamaeleon in the trail is comparable to the existing French and Hungarian varieties. Straw from the Plant Research International trials in 2000 were analysed for decortication behaviour. It is clear from this work that Chamaeleon has less resistance to decortication than the reference varieties. Also the blond fibre of Chamaeleon makes is aesthetically more attractive to Industry and it is stronger than well retted fibre.
Chamaeleon and the reference variety was grown commercially on 4 hectares, Unfortunately the seed was delivered rather late and the crop was not sown until 12th June. Both crops grew extremely well and we saw the characteristic of Chamaeleon exhibited in its colour change prior to harvest from green to yellow. Unfortunately after harvest we experienced a great deal of rain, so that the crops were not baled until November. It was noticed that the Chamaeleon lost most of its fibre in the field, possibly due to the easy separation from the woody core characteristic of this variety.
For the industrial partners the objective of the second year was, as a new project strategy, the production of yarn and composite materials not only from the new bright variety hemp but also from a reference variety (Felina 34) cultivated in the same areas and processed in the same factory. For the spinners the first operation schedule was carried out in a Hungarian hemp scutching mill in order to overcome problems linked to retting and scutching procedures. For Fonder the production took place in Austria but was also lost due to weather conditions. Unfortunately due to the extremely bad weather conditions no material was obtained suitable for the dedicated process. It was only possible to carry out a quality comparison between material coming from the new variety and Felina 34. Chamaeleon exhibited a better cleanliness than Felina reference possibly due to the easier scutchability. Initial experiments were carried out using fibre material from outside the project.
Year 3
Material from the 2001 field frail was further analysed by Plant Research International and IAF Reutlingen. Gene expression analysis of 47 samples was carried out. The data will be analysed statistically and added to the results database.
Microscopical analysis at LAF revealed that there is a significant difference between the lumen of Chamaeleon and Felina. This could be one of the causes of the different technical behaviour of Chamealeon. With Multivariate Curve Resolution (MCR) and Fluorescence imaging the evolution of molecular markers during development of Chamaeleon has been elucidated. The results obtained by PRI and lAF will be integrated and statistically analysed. The large amount of data produced in both laboratories will be further analysed in the coming year.
In 2002 the production of long fibre has been carried out in Hungary and Hemcore (UK). In Hungary the harvest was lost due to bad weather conditions. After field retting the UK Chamaeleon crop was harvested manually and decorticated on flax scutching line, The resulting fibre had excellent characteristics especially its softness, lack of entanglement and wooden residues. Regrettably, the fibre was uneven. The best yarn obtained by wet spinning was Nm 19,5. For dry spinning short fibre has been treated by a modified steam explosion (LISTEX) for analysis by Partner 7.
Seed production of Chamaeleon has been carried out in 3 European countries. Only France had previous experience with hemp seed production . In other countries the seed growing has to be developed. This was reflected in a lower seed yield than expected. However, sufficient seeds have been produced for further multiplication and cultivation by Hemcore and Kenderfono. In 2002 Hemcore grew 2 ha of Chamaeleon and produced almost 4 tons of fibre for the project partners. At Funder the fibres were tested in wet and dry processes for the production of fibre boards. In general Chamaeleon performed comparable to the reference hemp.
In the applied part of the project it is clear that especially for the textile route Chamaeleon seems to be advantageous. This has to be confirmed in future years.
Year 4
At Plant Research International Agrobacterium tumefaciens transformation of hemp only GUS spots at the cut surface were seen indicating that deeper cell layers within the meristem could not be reached. After regeneration this leads to chimeric shoots. To hit more internal meristematic cells particle bombardment with the pPG5 construct was applied on different types of explants from the cultivars Felina or Chamaeleon. In various tissues GUS spots were obtained, but no transgenic shoots developed.
By means of experimental design, a set of stem samples has been collected during growth at different maturities and different location of the plant. A total of 63 microarray experiments were performed with 47 biologically different stem samples Data from all microarray experiments were used for principal component analysis and the results showed a clear difference in gene expression of certain genes in core tissue compared with bast tissue of both Chamaeleon and Felina 34. We searched for gene expression, where levels were over 2-fold increased in core tissue compared to bast tissue or the other way around. A total number of 184 EST's, encoding 67 different proteins, showed this 2-fold increased expression in core tissue. A large number of these up-regulated ESTs, together covering about 28 contigs, appear to encode proteins involved in C1 metabolism and pathways leading to lignin biosynthesis (Van der Broeck et al. in prep). A total number of 131 EST's, encoding 62 different proteins, showed a over 2-fold increased expression in bast tissue. Among these are arabinogalactan proteins, proline rich proteins, lipid transfer proteins, lipoxygenases and endoxyloglucan transferases. With multivariate analysis such as Partial Least Squares (PLS) and Nearest Shrunken Centroid. and univariate analysis such as Residual Maximum Likelihood (REML) a set of genes that is most descriptive for the differences between the two varieties has been identified (Maliepaard et al. submitted, Reijmers et al. submitted).
Straw samples from the cultivation trials of the years 2000 to 2003 have been investigated by means of technical methods by IAF Reutlingen. The major topics were: fibre yield. decortication behaviour and fibre properties such as tenacity, fineness, fibre length and processability of fibers. The varieties analysed are: Chamaeleon, Felina, Green Sibling and Kompolti. Chamaeleon and Kompoiti show almost similar fibre contents which are higher than the other samples. In the total yield (yield after a first refining step) Chamaeleon tends to show higher values than Kompolti while Kompolti possesses more coarse bast residuals, which may adulterate the values.
By varying the production process based on the steam explosion technology, defined fibres for different industrial applications can be produced. For the dry spinning process applied by William Ross & Co. it is of importance that the fibre bundles are loosened, without fragmentation or a significant shortening effect. Thereby the fineness should possibly correspond to flax fibre fineness, which can be seen as a standard. So the process has to be a compromise between refining and keeping of the necessary fibre length for the drawing process. Ideally, the bundles are processed in such a way that by the first carding step an easy mechanical refining can take place.
Large-scale seed production of commercial seed and trial productions were set up for harvest 2003 by Vandijke Semo. Large-scale production contracts were made with different organizations in France, Hungary and Austria (total acreage about 60 hectares), Due to the fibrous character of the plants at harvest time mechanical harvest with the machinery used is very difficult. Enough seed was produced to meet the demands for trials within the Harmonia project and to secure future seed production. Small scale seed production trials were organized in Italy, Spain and Maroc. In the Netherlands further research was done on optimizing growing and harvesting techniques.
The 2003 field trails of Hemcore showed that the French varieties Fedora 17, Futura 75 and Felina 34 performed best on straw yield with Chamaeleon yielding 91% of control. The commercial crops of Chamaeleon (236ha) performed very much better than the trial plots. In yield terms Chamaeleon performed similarly to Felina 34 and Fedora I7. However, Chamaeleon's key characteristic of easier decortication from less retted stems was to prove a real bonus in the 2003 season,
Kenderfono officially joined the HARMONLA project on 17 July 2004, In March 2004 Kenderfono drilled I ha Chamaeleon, I ha Felina 34 and 1 ha of the local variety Tiborsalaci on two location in the Szegvar region. The summer of 2003 was warm and dry and reduced the stem yield. After ten days water retting the fibres of Chamaeleon and Felina 34 were scutched on the Kenderfono scutching line. Fibre yield was almost 800 Kg for Chamaeleon and 950 Kg for Felina 34. Samples were send to IAF Reutlingen and Zignago for further trials. The fibres appeared dark grey coloured and fineness of the fibre seemed good both for Felina and Chamaeleon.
The fibre processing of annual plants for the production of air laid dry fibre mats require an expensive decorticating process and need a high amount of artificial fibres to reach the required product properties. By processing hemp straw in a thermo-mechanical refiner the shives can be separated from the fibres and ground to produce nice wooden fibres. At the same time fibres fibrillise. The refiner process permits simultaneously the gluing of the fibres to increase the quality property after moulding of the fibre mat. This enables the proportion of artificial polypropylene or other plastic fibre to be reduced, resulting in a reduction in the production costs.
The fourth year of research at Funder showed that refining of hemp straw stem is possible and the fibre length distribution is optimal, that means that the short fibre amount is under control and is adjustable with the processing conditions. The result is a confirmation that hemp straw can processed in a refiner and in further consequence the fibres can be glued in the blow line of the refiner in front of the dryer.
The differences between Felina and Chamaeleon have been determined in an industrial sized demonstration test. Felina and Chamaeleon fibres where processed solely in a production line and all quality parameters where metered in each production step up to the finish fibre mat. Metered were mainly the fibre shortening. production lost (dust and waste) as well as the product properties of the fibre mat. This production run demonstrated that Chamaeleon fibres had a better moulding strength performance in the fibre mat as well as the fibre shortening was not as strong as at the Felina fibres. Last not least, the colour of the Chamaeleon mat was brighter and had a smoother consistent surface. The optical properties of the fibre (Chamaeleon and Felina) Hempcore supplied had a significant colour difference. The mechanical touch feeling of Chamaeleon was much softer than Felina.
A real life test of the on line quality control has been carried out at the Linificio/Zignago.production line. Measurements had been conducted during 4 weeks of consecutive production. Different product qualities can be distinguished at various stages of the production line.
Artificially water retted stems were scutched at a flax scutching line and at the Kenderfono scutching line and further processed at Zignago.
The long scutched Chamaeleon fibres exhibited better quality than reference for cleanness, softness, evenness and hackling behaviour. Mechanical preparation was carried out without any particular problems except some gills ruptures due to the length, strength and stiffness of the fibres by comparison with flax fibres, Felina 34 fibres showed better fineness than Charnacleon as well as better rove evenness. This was probably due to the greater number of doublings applied for Felina than Chamaeleon, in order to reach the same rove count. A specific dissociation process was developed for the hemp fibre in order to increase dissociation and at the same time to maintain a yarn colour more similar to the raw fibre. Chamaeleum was better than Felina not only for dissociation degree but also for the yield of the process and bleachability
Treated materials were suitable for spinning Nm 13 yarns with the same spinning frame settings utilized for linen. Nm 19.5 was obtained with more restrictive settings with a modified drafting system. With a special treatment of the material and adjusted spinning settings a Nm 27 yarn could be spun. Reference hemp showed equal (Nm 13) or better spinning performances attributable to the better rove evenness and finer fibres but not always better tensile properties Wet spinning of light steam exploded hackled tow of Chamaleon could be processed by Willam Ross & Co. to produce a count 5 hemp yarn. This type of yarn has economical value although the current process can not be economically operated.
Benefits and Beneficiaries
The project was successful in reaching a large number of deliverables and milestones. A production chain for the new hemp variety has been established. Applications for the fibre have been investigated and developed and Vandijke Semo, Hemcore and Funder will incorporate the results of the project into their production processes. Due to problems with fibre supply in the beginning of the project the research to textile applications was not as successful as hoped. The partners William Ross & Co. Ltd.and Linificio Canapiflcio Nazionale - Zignago SpA will require additional research to implement the project results into their production line.
The properties of the Chamaeleon variety have been well researched. On a scientific level a lot of insight into the molecular and technical behaviour of fibre development has been obtained. This is visualised by the large list of scientific publications presented at international conferences and in scientific papers.
Partners have given presentations at various congresses and meetings to inform hemp industry and researchers about the aims of the HARMONIA project.
Abstract
Material from the 2001 field trail was further analysed by Plant Research International and AF Reutlingen. Gene expression analysis of 47 samples was carried out. The data will be analysed statistically and added to the results database.
Microscopical analysis revealed that there is a significant difference between the lumen of Chamaeleon and Fetina. This could be one of the causes of the different technical behaviour of Chamealeon. With Multivariate Curve Resolution (MCR) and Fluorescence imaging the evolution of molecular markers during development of Chamaeleon has been elucidated. The results will be integrated and statistically analysed. The large amount of data produced in both laboratories will be further analysed in the coming year.
In 2002 the production of long fibre has been carried out in Hungary and Hemcore (UK). In Hungary the harvest was lost due to bad weather conditions. After field retting the UK Chamealeon crop was harvested manually and decorticated on a flax scutching line. The resulting fibre had excellent characteristics especially its softness, lack of entanglement and wooden residues. Regrettably, the fibre was uneven. The best yarn obtained by wet spinning was Nm 19.5. For dry spinning short fibre has been treated by a modified steam explosion (LISTEX) for analysis by Partner 7.
Seed production of Chamaeleon has been carried out in 3 European countries. Experience with hemp seed production only exists in France. In other countries seed growing experience has to be developed. This was reflected in a lower seed yield than expected. However, sufficient seeds have been produced for further multiplication and cultivation by Hemcore and Kenderfono. In 2002 Hemcore grew 2 ha of Chamaeleon and produced almost 4 tons of fibre for the project partners. At Funder the fibres were tested in wet and dry processed for the production of fibre boards. In general Chamaeleon performed comparable to the reference hemp.
In the applied part of the project it is clear that especially for the textile route Chamaeleon seems to have advantages. The coming years work will be used to confirm these observations.
Benefits and Beneficiaries:
For partners the first two years of the project generated information for the development of novel applications based on hemp. Scientifically, insight in chemical and molecular processes underlying fibre quality has been generated and will be used for improving fibre quality. The European Commission has received a report describing the progress of the project n the first and second year. In year 3 it has been shown that for hemp yarn production Chamaeleon has special features. However, cultivation of the hemp crop has been hampered by, for example, poor weather conditions. The strong points of Chamaeleon became apparent and applications related to these areas will be developed. On the scientific site a large amount of data has been produced. In the coming year the data will be analysed to give a more detailed description of fibre development in hemp and the differences between Chamaeleon and the standard variety.
Partners have given presentations at various congresses and meetings to inform hemp industry and researchers about the aims of the HARMONIIA project.
In 2003 the consortium applied for the addition of a new partner in the project: Kenderfono es Zsineggyarto Kft, that will develop technology for the production of fibre for the textile partners
Abstract
Year 2 A field trial was also sown in the second so as to provide material for spectroscopical, molecular and chemical studies. The field consisted of 4 different varieties: Chamaeleon, Felina 34, Green sibling and Kompolti TC. The first harvest took place six weeks after sawing on the 24th of June. In order to focus more on the transition from green to bright fibre, material was harvested at week 9, 11, 12, 13, 14. and 16. Samples were used for microscopical analysis or separated in core and bast parts for isolation of mRNA or spectroseopical and chemical analysis. From the three different cDNA libraries 1308 cDNA inserts were used to determine the 5' DNA sequence. For 76% of the clones homology was found with proteins with a known -function. Eighteen percent of the sequences could be identified as an expressed protein of which the function is still unknown and the remaining 6% of the sequences could not be identified. Within the cDNA clones about 56 genes could be identified as coding for proteins involved in cell wall metabolism.
To study the expression profiles of genes of interest cDNA microarrays have been printed containing the sequenced clones mentioned above and an additional 1700 randomly picked clones. A first series of analysis was carried out with mRNA isolated from the Chamaeleon bast samples from year 2000 material. The expression profiles indicate that genes belonging to the same pathway in cell wall biosynthesis have different expression profiles. An extended hybridisation experiment was carried out with plant material from year 2001. In this case bast samples were taken from the middle part of Chamaeleon and Felina 34.
As indicated above, within the first year of the project a regeneration protocol for hemp was developed. In the second year this regeneration protocol was used as a basis for the transformation experiments. In three transformation experiments shoots were obtained within three weeks. GUS analysis on a leaf of one of the shoots showed expression of a glucoronidase gene, confirming the transgenic nature of the shoot. In addition a second transformation protocol based on meristem transformation has been applied.
Spectroscopical analysis (Quick Quality Measurements) carried out on fresh stem material showed that in the UV/VIS range Chamaeleon is easy to distinguish from the other varieties. Principal component analysis of near infrared spectra of dried first year samples showed that the first principal component can be attributed to the maturity. A differentiation between bast samples of maturity 1 to 3 and samples of maturity 5 to 7 is possible. The second principal component can be attributed to the different parts of the stems (top, middle, bottom). In the second year samples a separation due to the maturity, as observed for the dried first year samples in the NIR, was not observed.
To predict mechanical properties dried samples of the second years harvest have been analysed with the FOSS VIS/NIR spectrometer system Since they reflect different chemical information, the VIS and NIR regions of the spectra were analysed separately. In most cases, Chamaeleon samples significantly differ from the other samples. Therefore, the sample set was divided in two sub-sets, one only with and one without Chamaeleon. The best results are achieved by analysing the first derivative of the spectra, both for the VIS and NIR region. In contrast to the original spectra, the lst derivative produces a much better correlation between the spectroscopic and mechanical measurements. The spectra obtained in the UV/VIS area were analysed by means of principal component analysis (PCA) and partial least squares (PLS 1). Due to the fact that Chamaeleon samples significantly differ from the others, sample sets only with or without Chamaeleon were analysed separately. When Chamaeleon was omitted from the analysis, it was not possible to distinguish the other varieties nor the origins of the samples.
Generally, a separated analysis led to better results than the PLS of the whole set of samples. This indicated that a careful selection of the samples for the analysis yields a better correlation and a more robust calibration model to correlate the measured spectra with mechanical properties. Initial microscopic analysis showed that the transition zone from best to core seems to be less tight for Chamaeleon than for Felina stems. This finding has to been repeated using the 2002 material but this difference may be important in decortication and other technical behaviour.
Selected cross sections of Chamaeleon and Felina stems were analysed by IR microscopy. Due to the small measuring spot (10 to 100 micro m in diameter) the spectra are more pronounced. Therefore it is possible to measure a small proportion of wax in the epidermis and this can also be seen in the change from bast to core and in the bast region. Characteristic lignin peaks are obvious in core spectra which are not found in spectra of the transition zone between core and bast. By IR- and Raman spectroscopy the existence of the calcium oxalate crystals in the hemp samples was shown.
From the seed multiplication in 2000 and the winter multiplication in Australia 2000/2001 a total of 448 kg of Chamaeleon seed was obtained. It was distributed to Hemcore (UK), Funder (A) and Kenderfono (Hu) for fibre production. The remaining seed was used to produce further seed for use in future development of the variety. Autumn weather conditions were not favourable in Europe in 2001 and the harvest of the Chamaeleon fields was not easy, so finally most of the seed harvest was done by hand. In spite of these difficult climatic conditions in France and Austria it was possible to test different harvesting techniques, providing valuable experience for subsequent years. By adapting existing cleaning systems and by combining the different cleaning methods, a reasonable up-grading of seed quality was achieved. The total yield of cleaned and good seed was 51 0 kg.
During the second year Chamaeleon was compared with six other industrial hemp varieties. Yield of Chamaeleon in the trail were comparable to the existing French and Hungarian varieties.
Straw from the Plant Research International trials in 2000 were analysed for decortication behaviour. It is clear from this work that Chamaeleon has less resistance to decortication then the reference varieties. Also the blond fibre of Chamaeleon makes is aesthetically more attractive to industry and it is stronger than well retted fibre.
Chamaeleon and the reference variety was grown commercially on 4 hectares. Unfortunately the seed was delivered rather late so the crop was not sown until 12th June. Both crops grew extremely well and it was possible to observe the characteristic colour change of Chamaeloon, prior to harvest, from green to yellow. Unfortunately after harvest there was a great deal of rain, so that the crops were not baled until November. It was noticed that the Chamaeleon lost most of its fibre in the field possibly due to the easy separation characteristic of this variety. These crops are being processed and yield information will be presented in the next reporting period.
For the industrial partners the objective of the second year was, as a new project strategy, the production of yarn and composite materials not only from the new bright variety hemp but also from a reference variety (Felina 34) cultivated in the same areas and processed in the same factory. For the spinners the first operations were carried out in a Hungarian hemp scotching mill in order to overcome problems linked to retting and scotching procedures. For Funder the production took place in Austria, but was lost due to weather conditions. Unfortunately, due to the extremely bad weather conditions no material was obtained suitable for the dedicated process. It was only possible to carry out an quality comparison between material coming from the new variety and Felina 34. Chamaeleon exhibited a better cleanliness than the Felina reference material, possibly due to the easier scutchability. Initial experiments were carried out using fibre produced from outside the project.
Beneficiaries
The first two years of the project generated information for the development of novel applications based on hemp. Scientifically, insight into chemical and underlying molecular processes has been generated and will be used for improving fibre quality. The European Commission has received a report describing the progress of the project in the first and second year. Partners have given presentations at various congresses and meetings to inform the hemp industry and researchers about the aims of the project.
Objectives
The RTD part of the proposal aims at identifying the basis of the excellent fibre quality of the Chamaeleon hemp variety. This will be done by chemical and physical analysis of the fibre. In addition, genes involved in cell wall metabolism, that might determine the good fibre quality, will be isolated and transgenic hemp prototypes containing these genes will be produced. To produce fibre material for the demonstration project cultivation methods of the crop will be optimised and various methods to refine the hemp fibre will be tested. In the demonstration part of the project whole plant material and refined fibres will be applied in various high added value applications. Steam exploded fibres will be used for applications in composites and yarn. Fibres and whole plants will be used to produce pulp as a basis for speciality papers and biodegradable CNC cellulose for diapers. The project will produce prototypes containing fibres from the bright hemp and give insight in the economical perspectives of these industrial applications.
Results
The project started with 15 kg of seed of the Chamaeleon variety. This breeders seed was used to grow material for further seed production. One multiplication took place in the Nethedands and yielded 90 kg of seed. Seed processing conditions were adapted and optimised to improve seed quality. In order to have sufficient seed for the second year of the project, a winter multiplication has been initiated in Australia. This will enable the production of sufficient seed to produce Chamaeleon fibre in the second year of the project.
Due to the limitations of the Chamaeleon seeds available, it was not possible to drill seed for fibre production. The project management committee has decided that the partners will analyse the quality of the European reference variety Folina 34 in the first year of the project. In the subsequent years the Chamaeleon fibre will be included in the trails. At Hemcore a field trial with 10 hemp varieties, including Chamaeleon was carded out. The application of nitrogen resulted in increased yield in dry matter. Retted and not retted stem material of Chamaeleon and Felina 34 was processed. Under the applied conditions processing behaviour of both varieties was similar. Retted material could be decorticated without any problem. It was not possible to process unretted material.
The development of hemp-based yams proved very difficult with the available hemp varieties. With the use of fibres produced by the short staple process (by Hemcore) it was almost impossible to produce a yarn. The coarseness and stiffness of the fibre and the wooden impurities made it very difficult to process the fibre into a sliver. Under exceptional conditions William Ross was successful in producing a yarn though this was of a very coarse quality. This yarm will be used as the reference yarn for Chamaeleon based yarns.
IAF Reutlingen supplied the partners with a water retted fibre that was processed by Zignago Tessile. The material was relative clean and quite stiff. The hackling behaviour was very heterogeneous. Hackling tows were sent both to William Ross & Co. and IAF. After roving various chemical routes were carried out to obtain a good dissociation level for good spinnability. The treatments gave high degree values of dissociation but different white degrees and spinning behaviours. By wet spinning a Nm 24 yam was produced. The characteristics of the yams obtained from the different rove chemical treatments were assessed.
The hackled tow was processed further by William Ross & Co. on a comb and a blending/drawing drawframe. With specific setting for the draft and the twist factor on the spinning frame it was possible to spin a Nm 3.6 hemp yarn.
Funder demonstrated the usability of hemp in the fibreboard wet process, hemp board samples were formed in a wet sheet former and pressed in a laboratory press size 600 x 600 mm.
Additionally, hemp fibres were processed together with the shive content in test-run on a semi- industrial fleece forming and dewatering machine. The challenge of this test was the investigation of the behaviour during mixing of short wood fibres with long hemp fibres. The machine was specially designed with two radial flow distributors to get an optimal dosing percentage and a good mixing effect and a maximal fibre connection.
A trial field was sown for the analysis of hemp by IAF and Plant Research International. The field consisted of the varieties: Chamaeleon, Felina 34, Green Sibling and Kompolti TC. Samples from the stems were taken to analyse the chemical composition by microscopical and spectroscopical means and to isolate DNA and RNA. Chemical analyses of 205 samples are being performed to determine acid detergent fibre (ADF), acid detergent lignin (ADL) and neutral detergent fibre (NDF).
Chamaeleon stem material in one of three developmental stages was used to construct three cDNA libraries. cDNAs were randomly picked for the libraries and the base sequence was determined. Based on sequence homology it is expected that several of these genes will have a function in cell wall metabolism. The expression patterns of these genes will be monitored by DNA microarrays. In order to further elucidate the function of the genes, a number of genes will be tested by transformation to hemp. For this a transformation protocol will be developed within the project. With specific hormonal treatment it was possible to develop shoots from hemp callus. The transformation of hemp callus could also be achieved.
At IAF, hemp stems have been investigated using quick quality measurements such as determination of the water content, colour of the stems and NIR measurements. For Chamaeleon the maturity can also be measured by the colour (CIELAB) of the stems and an evolution of the fibres can be obtained. It was easy to separate Chamaeleon from other varieties but it was also possible to differentiate between Felina and Green + Kompoli. Within the plant the core and bast and maturity can be distinguished by UV/VIS/NIR and IR-spectroscopy due to the plants morphology and their chemical composition. Maturity can be determined by measurements of the cell wall thickness. Some selected samples were analysed with an IR- Microscope on a 10 to 100 micron lateral resolution.
Discussion
The first year of this project has generated information that will aid the development of novel applications based on hemp. Scientifically, the required insight into chemical and molecular processes underlying fibre quality has been generated and will be used for improving fibre quality.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
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