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EU-based Production and Exploitation of Alternative Rubber and Latex Sources

Final Report Summary - EU-PEARLS (EU-based Production and Exploitation of Alternative Rubber and Latex Sources)

Executive Summary:

4.1.1 Executive Summary.
The EU-PEARLS project has shown the feasibility and proof of principle for setting up the entire production and application chain of alternative sources of natural rubber from non-endemic crops Russian dandelion and guayule. At the end of the project medicinal gloves and car tyres were produced and tested. The car tyres produced performed similar to normal rubber tree based tyres and in some properties (e.g. wet grip) even behaved superior.
The EU-PEARLS project has greatly increased our knowledge on the alternative sources of natural rubber Russian dandelion (Taraxacum koksaghyz or TKS) and guayule (Parthenium argentatum), has yielded valuable European germplasm, scientific knowledge, dry rubber and latex samples and as proof of concept medicinal gloves and car tyres. Newly produced samples can be used in subsequent RTD projects and for commercial enterprises, as has been summarized in the impact section of the final report. The scientific output ranges from germplasm properties, agronomic experience, genes and proteins involved in rubber biosynthesis, genetic maps and sequence data, properties of latex and rubber, assay methods, and processing methods. Furthermore, regular updates of the scientific literature and patent landscape have been carried out for all elements indicated, while desktop studies gave insight in biorefinery aspects, and in socioeconomic issues (policies, new crops, market, external factors).

Project output. The most important exploitable outputs of the project are:
1. Methods to channel metabolites to rubber biosynthesis
2. Analysis of rubber biosynthesis (proteome, particles, genes)
3. Guayule germplasm, agronomy, large-scale biomass production
4. NIRS method to measure rubber content
5. New TKS germplasm, characterization and method to preserve TKS seeds
6. TKS hybrids and EMS mutants
7. Genetic maps of TKS (QTL, AFLP, COS, EST, sequence data)
8. Guayule latex and dry rubber production and properties
9. Production of guayule latex gloves
10. Development of extraction method for TKS rubber
11. Assessment of TKS rubber physical and material properties
12. Development of prototype car tyres (TKS and guayule)
13. Economic assessment of the entire production chain including main (latex, rubber) and by-products (inulin, resins, extracted biomass).
14. Dissemination activities: more than 15 Peer-reviewed publications by the EU-PEARLS Consortium, 15 Presentations and abstracts, 20 Interviews, articles in magazines and newspapers, 9 Website links, 4 television documentaries (TV5, EuroNews, EenVandaag, CNN), 5 PhD thesis (including those in preparation), Mid-term Workshop (Montpellier, FR, October 2010), Final Congress (Wageningen, NL, September 2012). A number of other dissemination activities are still in progress or planned.
Partners KeyGene and Apollo Vredestein are high profile companies, experienced in exploiting IP to establish new ventures, while the academic and research institutes are well placed to interact with industry in such ventures, or to establish independent activities.
An End-user group is being set-up, consisting of a number of SMEs and large companies that have approached the EU-PEARLS consortium, and are interested in the application of natural rubber obtained from guayule and/or TKS. On specific request these interested parties can acquire small amounts of latex or dry rubber produced during the EU-PEARLS project for analysis and testing. If larger amounts are required, these companies will have to finance the production of these samples, starting from seed production. A number of End-users have already been included in follow-up initiatives (e.g. the EU proposals EAGLES, G-VALUE and DRIVE4EU).
It must be stressed that the two plants, TKS and guayule were completely new crops within the EU and that they were not (TKS) or only partially domesticated (guayule). TKS is not used commercially anywhere in the world, whereas the production of guayule latex and rubber is already for some years in the testing phase for commercial hypoallergenic products and tyres. The collected body of knowledge and experience on the introduction of new crops suggest that it is a very ambitious undertaking. Experience shows that the re-introduction even of existing crops that are no longer grown in the target area – but otherwise well established in regards to breeding efforts, agronomic experience, processing, product distribution and marketing – was exceedingly difficult. Furthermore, guayule and TKS are crops intended to replace or complement an existing product, dry rubber from Hevea brasiliensis, which is obtained from tropical countries with (relatively) low labor and land costs, and requiring very little processing. Making guayule and TKS competitive crops requires a breeding program, and great efforts on the agronomy and processing to have a chance of success by directly competing on the rubber and latex market. However, the difference with other new crops is that natural rubber is an extremely strategic resource: European economies, transportation, mining, and agriculture cannot do without. Out of a strategic interest, the EU should finance demonstration plants, breeding programs and R&D on agronomy and processing or at least facilitate company investments concerned.

Conclusion. The most conspicuous results achieved near the end of the project were the car tyres produced by Apollo Vredestein, NL, containing Russian dandelion or guayule rubber and the medicinal gloves produced by CIRAD and its subcontractor CTTM, FR. These accomplishments pave the way to fully implement the project objectives. For the creation of full commercial scale activities further investments will be required to resolve current bottlenecks, i.e. the production of improved TKS hybrid lines and the construction of pilot to large scale extraction facilities for both TKS and guayule main and by-products.
Project Context and Objectives:
4.1.2 Summary description of project context and main objectives.
EU-based Production and Exploitation of Alternative Rubber and Latex Sources; KBBE-212827, EU-PEARLS. Collaborative Project within the framework of the EU-FP7-KBBE-2007-3-1-06 Program, Activity 2.3 Life Sciences, Biotechnology and Biochemistry for Sustainable Non-food Products and Processes; 2.3.1 Biological Polymers from Plants.

Background. Natural rubber, obtained from the rubber tree H. brasiliensis, is essential to industry, medicine, transportation and defence. However, increased worldwide demand for natural rubber and latex, fundamental threats to natural rubber production in South-East Asia, such as a possible outbreak of South-American Leaf Blight, climate change, and increasing economic pressure on Hevea plantations (labour shortages, increased wages, competition for land with palm oil) make the development of alternative sources of natural rubber as a strategic raw material necessary. Market prices for raw rubber show a large degree of volatility, but have increased drastically to over US$5 per kg in November 2010 before slightly retreating to about US$4.50 per kg and to about US$3.50 per kg in October 2012.
To respond to the threats, a Research Consortium (EU-PEARLS) has been formed that links many of the European stakeholders involved in the development and sustainable use of P. argentatum (guayule) and T. koksaghyz (Russian dandelion) as alternative rubber and latex sources. To enable the sustainable development and exploitation of these crops, and to develop a full value creation chain, the research included the collection and creation of new germplasm and biochemical and genetic analysis of the rubber biosynthetic pathway. Targeted conventional breeding was initiated in Russian dandelion to eliminate bottlenecks and generate plants with commercially viable rubber yields. These lines were tested for efficient growth and rubber production in the field under different climatic conditions in Europe. Further targets were agronomy and processing, while finally, the technical performance and economic potential of latex or rubber extracted from these plants was evaluated by producing specific prototypes, such as surgical gloves and tyres.
In short, EU-PEARLS has aimed at setting up a full value creation chain for the production of natural latex and rubber in Europe.

Importance of natural rubber and the need for alternative sources. Natural rubber is a unique biopolymer that cannot be replaced by synthetic alternatives in many of its most significant applications and therefore plays a vital role in modern economies/societies. Latex tapped from the Brazilian rubber tree H. brasiliensis (family Euphorbiaceae) is currently the sole commercial source of natural rubber (cis 1,4 polyisoprene), which is being imported into Europe for 100%. The Natural rubber raw materials market is $20 Billion globally and $2.8 Billion in the USA and is critical to the $200 Billion rubber products market where the major application is in tyres. In the USA the importation of crude rubber latex occupies the second position behind that of mineral oil. In Western Europe and other industrialized countries the situation is similar.
Natural rubber cannot be replaced by other biopolymers or synthetic polymers without sacrifices in performance and durability. Other applications than tyres include automotive and industrial components, medical products, footwear and adhesives.

Current world production of natural rubber suffers from a number of threats and drawbacks, including:
1- Limited genetic variability of rubber tree H. brasiliensis resulting in increased sensitivity to pests and disease (e.g. South American Leaf Blight)’
2- Production in narrow climatic (tropical) zones mainly in three South-Asian countries (Malaysia, Indonesia, Thailand);
3- Tendency to replace existing plantations for H. brasiliensis with palm oil trees, especially in Indonesia;
4- A fundamental problem with H. brasiliensis rubber concerns the world wide occurrence of life threatening, IgE mediated, latex allergy caused by the proteins in the latex; protein removal cannot be easily or cheaply accomplished; This risk factor has recently led the American Society for Testing and Materials to define a new standard (ASTM D1076-06), a Category 4 natural rubber latex that must contain less than 200 micrograms total protein per gram dry weight of latex and no detectable H. brasiliensis antigenic protein;
5- Rapid increase in rubber need especially in rapidly developing countries (e.g. China, India) and decreased supply causing a 4-5 fold increase of prices over the last decade (www.mongabay.com/images/commodities/charts/rubber.html);
6- Rubber tapping is laborious, poorly paid and is often performed by children;
7- Variable qualities of crude rubber preparations containing considerable amounts of impurities.
These issues are strong indicators that alternative sources of natural rubber should be further developed. There is an immediate need to address the allergy problem of H. brasiliensis. There is also a strategic need to mitigate the risk of a single feedstock supporting essential applications on a global scale.
In the past few years a promotion campaign has been initiated to get this important subject also on the EU Strategic Research Agendas of the EPOBIO (EU/USA) in 2004 and 2006, EPSO European Technology Platform ‘Plants for the future’ and the EU seventh Framework Programme. A recent EPOBIO report (Van Beilen, November 2006, http://www.epobio.net/pdfs/0611NaturalRubberReport_c.pdf) provides a detailed description on the subject matter.
The EPOBIO report concludes that the generation of rubber producing crops and the development of the entire production chain from germplasm, crop development and agronomy to extraction, processing and application development is urgently needed and possible in Europe, thereby focusing on the two plant species guayule (P. argentatum) and Russian dandelion (T. koksaghyz).
Guayule is considered the most promising crop for cultivation in Mediterranean areas, whereas dandelion is considered the second best crop that should be developed in parallel with guayule in northern and eastern European countries. Both types of rubber may be suitable in applications such as tyres and car parts, whereas guayule rubber may also find specific applications in the area of medicine (absence of IgE latex allergy).

Concept of project to tackle the problem described. This multidisciplinary RTD project aimed at the development of two alternative natural rubber sources for Europe; rubber production from guayule and Russian dandelion.
The concept for Europe is so new that there are no previous EU projects devoted to this subject to build upon. Nonetheless, the project also fits well in other EU initiatives, such as “Plants for the future”.
The plant species guayule has attracted most attention until now. Guayule is an indigenous shrub of Mexico and the southern states of the USA. The rubber from guayule has similar polymeric properties as rubber harvested from the rubber tree and significantly meets the new ASTM standard for low protein rubber. Over the last decades, research and development on guayule rubber has mainly occurred in the USA.
Russian dandelion has shown its potential already during World War II when army vehicles were equipped with tyres made from Russian dandelion rubber, but much RTD efforts are needed to achieve the same technology level currently enjoyed by guayule. Moreover, given the strategic importance of this source of rubber costs and economic feasibility for biomass production and extraction were not of prime importance at that time.
Therefore, within EU-PEARLS the technical and economic feasibility of rubber production from guayule and Russian dandelion have been considered of prime importance, leading to strains of guayule and Russian dandelion with good and still sub-optimal performance in southern and northern Europe, respectively. The entire chain from selection of optimal plant varieties, production and processing to finally rubber applications has been studied. This has been accomplished by a highly dedicated Consortium represented by academia, research institutes, SMEs and a large company from a number of northern, southern and eastern European countries and Kazakhstan. Through the Scientific Advisory Board also strong links have been created with initiatives in the USA.

Within this multidisciplinary and ambitious program the most important aspects (different steps in the chain from genes to processing, material testing and applications) have been addressed as follows:
- For Russian dandelion: Collection and genotyping of novel Russian dandelion varieties (germplasm), mutagenesis and conventional breeding (no GMO) to improve crop vigour and yield, root size, rubber yield, a desktop study on bio-refinery principals and use of waste streams (to enhance the chain’s economic profitability), agronomy, construction of BAC library and further construction of EST library, further completion of the genome map, used for QTL mapping for important agronomic traits and for the identification of relevant genes involved in rubber synthesis and precursor supply (genomics, proteomics, microarrays), study and manipulation of pathways determining the acetyl-CoA pool size, the flux through the mevalonate pathway and rubber polymerization (prenyl- and rubber-transferases), heterologous expression in Arabidopsis and yeast (functional genomics), biochemistry of polymerization, study of latex granule biogenesis, structure and proteinaceous (structural proteins and synthetic enzymes) composition via proteomics. The work also included greenhouse and field trials, rubber extraction, rubber polymeric and material property testing and application development in e.g. car tyres.
- For guayule the situation was different because for this crop methods to increase yield and production of rubber already have been studied and there is a history of several decades on rubber production, extraction and application testing mainly in the USA. However, further optimization of the agronomy, the production strains and dry rubber quality (Molecular weight) were needed. Molecular tools for breeding have not yet been developed and the best approaches remain unclear and under debate. The work planned with Russian dandelion in the EU-PEARLS project, which also included the convenient Arabidopsis and yeast as helper organisms should help to identify these approaches.
However, due to the long history of guayule rubber production, in an early phase of the project large-scale production in Europe has been implemented along with agronomic analysis and further product development at an earlier stage than for Russian dandelion.

This project aimed at setting up a production chain for natural latex or rubber in temperate crops in Europe. To guarantee maximal focus, other topics mentioned in the call text, such as development of starch-based plastics, production of animal proteins or microbial polymers in plants were not addressed in this project.

Why did we focus on natural rubber in this project?
A. The strategic reasons; natural rubber is an essential resource for human society just like fuel and iron. Natural rubber cannot be replaced by synthetic materials in many applications, the rubber demand will strongly increase in the near future, probably with a factor 2 within the next decade and security of supply is uncertain on the longer term. Society can do without starch-based plastics or microbial polymers, but cannot be deprived of natural rubber.

B. The project-technical reason is that the establishment of natural rubber production in Europe requires a coordinated and concurrent effort on two different crops and at a number of different levels, which include:
1- A gain in fundamental understanding of metabolic processes leading to rubber synthesis in two rubber producing candidate crops: Russian dandelion and guayule. This is essential for building up of appropriate IP and providing tools for directing breeding;
2- Establishment of novel germplasm for Russian dandelion and guayule for cultivation in Europe;
3- Development of breeding strategies to combine the excellent rubber producing properties of Russian dandelion with the vigour and production capacity of normal dandelion;
4- Greenhouse and field trials to select Russian dandelion germplasm capable of rubber production in the range of 1-1,000 kg;
5- Field trials to select guayule lines best suitable for cultivation in southern Europe and capable of latex and dry rubber production of production in the range 500-1,500 kg;
6- The establishment of cost-effective processing technologies to convert guayule latex and dry rubber into high value medical and consumer products (medicinal gloves and car tyres, respectively) and dandelion rubber into industrial products (car tyres), but also to apply biorefinery concepts for valorization of two other main products from both guayule and dandelion including pulp for liquid fuels and inulin into non-food products;
7- Perform economic and LCA analyses to identify the respective strength and weaknesses of the various production scenarios, including the co- and by-products.

The focus on two potential European rubber crops was essential for the project’s performance and success. Because of their different agronomic properties one rubber crop can be grown in the northern and eastern part of Europe and the other crop in the southern part of Europe. Besides, differences in end-product quality of the two rubbers, this approach allows the development of both high value medical and consumer products as well as medium value products like tyres. Since both crops are quite closely related (both Asteraceae), combining the existing background knowledge of both in a single project, will allow a rapid establishment of two European rubber production chains, active in different parts of Europe and focusing on different markets.
The R&D objectives critical for the establishment of European rubber production chain cannot be sufficiently addressed when the project resources are also allocated to different non rubber-related topics, which would be the case should starch and microbial polymers R&D be included in the same project.

C. The economic-political reason is that Europe, enforced by EU political instruments, will see a substantial increase in the acreage of energy crops. To meet a 10% replacement of gasoline by bio-ethanol the EU would require an additional 1 million ha of a sugar crop with a production potential equivalent to sugar beet. However, also rubber producing crops can act as a source of bio-energy.
The major storage sugar of dandelion is inulin, which can easily be converted into ethanol using state of the art fermentation technology. Starting from the perspective that annual rubber producing crops, like dandelion, can ultimately reach the same biomass yield potential as sugar beet (which still needs considerable breeding efforts), a 1 million ha of Russian dandelion will first be able to provide Europe the required bio-ethanol volume to replace 10% of gasoline and second 1 million ton of latex or rubber, which exactly matches the current European rubber demand. The added advantage of a Russian dandelion crop co-producing rubber and fermentable sugars for bio-ethanol, is the added income related to the presence of high value rubber (1 ton rubber/ ha at an average value € 1,500/ton), which amounts to € 1,500/ha. Added to € 500-600 for a moderate 5-6 tons of fermentable sugar (sugar beet provide 12 tons/ha), Russian dandelion provide a gross income of over € 2,000/ha. In conclusion, a dandelion based production system, providing both rubber and fermentable sugar for bio-ethanol production will ultimately provide sufficient income for the Russian dandelion farmer.
Guayule can produce dray matter biomass quantities between 25-35 tons/ha, yielding 2,0-4,2 tons/ha of high mol. weight rubber (8-12%, corresponding to € 2,250-6,300/ha, in a 2-3 years cultivation cycle). In addition to the latex guayule produces about 10% resins containing terpenes and triglycerides (sold by Yulex at € 1,500/ton, i.e. € 4,650/ha) and a high density residual biomass as co-and by-products from the latex extraction process. If the residual biomass, from crops of similar performance in the EU as in the USA, is converted to ethanol it is estimated that approximately 2,000 gallons of ethanol would be produced per hectare. Resins extracted by supercritical CO2 processes can be used in a host of co-product application such as biopesticides, adhesives and strippable coatings. In addition, during the production of dry rubber from latex a low molecular weight fraction (about 25%) is created as a co-product, which may find specific applications such as kits and coatings. Thus, it is anticipated that the farmer income for guayule would be similar or even superior to the dandelion farmer. However, these rough calculations do not include cost for the extraction process running costs and the investments needed.
The expected impact of the project was a substantial gain in understanding how rubber synthesis is controlled, how this can be translated into improved rubber producing crops, how a European production chain can be built and what rubber products can be made from European rubber crops.

This has led to the following strategic objectives:
1- Development of a rubber producing bio-energy dandelion crop and development of a cost-effective efficient and sustainable dandelion rubber chain including genotypes, extraction, processing and application.
2- Development of a high value rubber producing resins and bio-energy guayule crop in Europe
3- Due to the absence of allergenic proteins which are normally present in H. brasiliensis rubber guayule rubber has a high potential for high value medical applications.

S&T objectives include:
1- New genotypes (Russian dandelion);
2- New breeding lines (Russian dandelion) with high rubber yields and quality;
3- Assessment of optimal agronomic tools (guayule for southern Europe and Russian dandelion for the northern Europe climate);
4- Development of optimal extraction, downstream processing and application protocols.

Challenges for guayule. In the short term, a major bottleneck in realizing the potential of guayule is the processing of the feedstock (EPOBIO, 2006). The problem is that in guayule, unlike dandelion, the rubber is contained within the plant cells. This necessitates cell breakage and extraction protocols to separate the rubber biopolymer from other cellular components. Within the project further improvements for commercial scale production have been investigated.
Therefore, R&D focused on optimisation of processing guayule using the currently available varieties, and integrating this processing capability into a biorefining process such that co-products can be used either as an energy source and, ultimately, as a feedstock for higher value products.
In the mid- to long-term, there is considerable potential for guayule crop improvement, although existing varieties can be cultivated in southern Europe. Targets to be addressed include rubber yield and quality, biomass optimisation, water use and other agronomic traits of relevance for European cultivation.

Challenges for Russian dandelion. Development of a genetic model to understand the molecular processes controlling rubber biopolymer synthesis and storage is likely to involve characterisation of Russian dandelion (T. koksaghyz) (EOPBIO, 2006). Currently, yields and agronomic properties preclude the use of this source on a commercial scale. However, this species has considerable potential for molecular-based research, as well as providing in the longer term the potential for an alternative source of natural rubber to guayule.
Therefore, R&D focused on molecular characterisation of Russian dandelion applying genomics and post-genomic technologies.
Project Results:
4.1.3 Description of the main S & T results / foreground.
S&T results have been addressed in work packages (WPs) 2-7 and are summarized individually below. WP1 was devoted to overall project management and some dissemination activities, including the organization of the Final Congress (Wageningen, The Netherlands; September 2012).

WP2. Rubber pathway analysis and improvement.
WP2 aimed to dissect, analyse and improve the combined activities of pathways involved in the production of natural rubber. Understanding the transcriptional regulation of and potential bottlenecks in the underlying pathways would allow us to improve the biosynthesis of high quality rubber in plants either by genetic engineering or chemical mutagenesis (TILLING). The knowledge generated in WP2 therefore supported and underpinned tasks in WPs 3, 4, 5 and 6 by providing:
a. ideal markers (e.g. genes, proteins) for screening and identifying of appropriate wild isolates of T. koksaghyz and guayule on molecular level (WPs 3 and 4);
b. suitable DNA sequences for mapping studies or gene regulation studies of rubber synthesis genes (Mapping of guayule and dandelion in WPs 3 and 4, respectively);
c. potential target genes for TILLING (WP4) and for gene regulation studies (WP3);
d. modified plants with enhanced rubber biosynthesis or better performance in terms of latex extraction (WPs 5 and 6).
A database containing an inventory of data relevant for rubber biosynthesis in guayule and T. koksaghyz has been constructed and is continuously updated. A database has been constructed (and has continuously been updated) containing an inventory of DNA sequences relevant for rubber synthesis in guayule and T. koksaghyz, including HMGCoA synthase/reductase (HMGR) sequences, and two HMGR and one HMGS genes were cloned from T. koksaghyz.
For dandelion, the entire cDNAs of TbHMGR1 and TbSQS1 were isolated from Taraxacum brevicorniculatum. Comprehensive expression studies were performed for HMGS, HMGRs and SQS. Truncated versions of the HMGR1 from A. thaliana were successfully expressed in the latex of T. brevicorniculatum. First knock-down lines are available for rubber biosynthesis related genes such as cis-prenlytransferases (CPT) and small rubber particle proteins (SRPP). Two TbCPTs were successfully expressed in T. officinale. TbCPT1-3 has been identified as the key enzymes in rubber biosynthesis, while the small rubber particle proteins seemed to be necessary for rubber particle stabilization. A latex cDNA-Macroarray was established and comparative expression studies for rubber biosynthesis and mevalonate (MVA) pathway genes were performed between T. officinale, T. brevicorniculatum and T. koksaghyz. One HMGCoA synthase (HMGS) and three HMGCoA reductase (HMGR) genes were identified from T. koksaghyz 203-1ST and compared to their T. brevicorniculatum homologs. The function of TbHMGRs was confirmed by suppression of the lethal phenotype of an E. coli ispH deletion mutant. Comparative analysis of T. brevicorniculatum with T. officinale mRNA using macroarrays showed no significant differences in the expression pattern of genes expressed in laticifers. In contrast, proteome analysis displayed clear quantitative differences between T. brevicorniculatum and T. koksaghyzand between high and low rubber yielding TKS lines. Especially SRPP1 showed variance in protein expression level between different TKS lines. Also enzymes involved in the mevalonate pathway and in vesicle trafficking and coagulation processes were differentially expressed between T. koksaghyz lines. Furthermore, only CPT2 and CTP3 enzymes were detected, correlating very well with the genomics data. All individual T. koksaghyz lines analyzed so far showed similar size distribution of particles. In addition, efficient methods for the vegetative propagation of new T. koksaghyz lines in the greenhouse were developed and applied.
For Arabidopsis, the principal aim of the project was to find ways to increase the cytosolic acetyl-CoA pool, which is the precursor to rubber and other isoprenoids. To maximize the chances of success, two strategies were explored to achieve this goal; a targeted approach relying on the overexpression of the ATP citrate lyase gene (ACL) and a non-targeted approach relying on activation tagging mutagenesis with a T-DNA. Since it was unclear which, if any, of these approaches were going to be successful, both approaches were initiated at the same time. The strategy to overexpress the ACL subunits in transgenic Arabidopsis lines was shown to be successful in increasing the cytosolic pool of acetyl-CoA. Thus, all efforts were invested into exploiting and improving this strategy. A series of new constructs were designed, such as the creation of chimeric fusion proteins and mutagenised version of the protein (for potential phosphorylation sites), have been done and transformed into yeast and plants to hopefully achieve further increase ALC activity beyond the first generation plants. Finally, three ACL constructs under the control of a latex-specific promoter were transformed into dandelion and were evaluated for increased rubber content.
As a consequence of the success of the overexpression of ACL on increasing acetyl-CoA pool, and of the focusing of efforts into exploiting this strategy to improve rubber yield, the second strategy relying on activation tagging mutagenesis with a T-DNA was put on hold.
Transgenic poly-hydroxybutyrate (PHB) producing Arabidopsis lines to monitor the acetyl-CoA pool size, the main precursor for isoprene biosynthesis, have been established. Using this system, it has now been possible to show that increase in ATP citrate lyase (ACL) activity leads to increase availability of cytosolic acetyl-CoA. Increase ACL activity leads to an increase in PHB synthesis as well as wax deposit on leaves but only marginal changes in sterol content in Arabidopsis. In contrast, increased ACL activity in dandelion lead to a 4-5-fold increase in rubber content in 3-month-old plants, thus showing that ACL activity can be limiting rubber biosynthesis. Proteome analyses in new T. koksaghyz accessions revealed a large biological diversity. Individual proteins could be identified and quantified, and compared between rubber particle fraction of the latex and the latex serum, between different T. koksaghyz accessions, and between progeny of a crossing of two T. koksaghyz accessions. All rubber synthesis enzymes could be quantified, next to other proteins present in the rubber latex (in total 400 proteins). The expression level of all these proteins could be measured in the different samples and could be compared between samples. The results showed, for instance, which rubber synthesis enzymes are more tightly associated with the rubber particles. Correlation studies of the proteome of accessions and progeny lines with rubber yield of these lines revealed candidate proteins that might be related to high rubber yield/quality. Among these target candidate genes are also genes from corresponding metabolic and biological processes of rubber synthesis, such as key enzymes from the mevalonate (MVA) pathway and proteins involved in trafficking of rubber particle synthesis components.
For yeast (and E. coli), key enzymes (including cis-prenyl-transferases, CPTs; small rubber particle proteins, SRPPs) of the T. koksaghyz rubber biosynthetic pathway were identified and expression of CPTs achieved. In yeast also three CPT, one REF, and five SRPP genes from T. brevicorniculatum were expressed and analysed. A major problem that emerged was the fact the CPT genes, after their transfer into yeast did not shown any full length but rather truncated mRNAs. Thus, it appeared extremely difficult to establish the effects of a number of transgenes tested on the abundance of intermediates of the rubber synthetic pathway in yeast. Results on the integrity of expressed proteins and 14C-IPP labelling experiments were inconclusive.
Siliciumoxide column chromatography and RP-TLC were tested and valued good methods to isolate isoprenoids from yeast total lipid fraction after extraction and esterification. For the introduction of the rubber biosynthetic pathway in yeast further candidate genes were selected, codon-optimised and introduced into the yeast genome.

WP3. Guayule germplasm, breeding and agronomy.
The aim of WP3 was to define and improve the potential for the cultivation of guayule in Southern Europe by implementing guayule germplasm and agronomic practices from Arizona, USA.
In short the following progress has been achieved:
- First year (2008-2009): planting of a germplasm collection installed in France and Spain for seeds production and to test cold resistance of USDA cultivars and one Yulex line (AZ2.1) in Europe. Production of nursery plants in France and late planting (October 2008).
European geographic regions have been mapped for soil and climatic conditions suitable for guayule. Successful field trials using the best available guayule lines were conducted in France and Spain. Overwintering of different lines has been assessed using young (a few months old) plants, possibly providing cold-tolerant lines. Agronomic experiments (fertilisation, irrigation and line trials) have been completed.
- Second year (2009-2010): The best available subsets of guayule lines from Arizona germplasm have been selected. Germplasm collection both in France and Spain were used for guayule seeds production for the project. Seven lines were selected: AZ2, CAL-6, 11591, N565, 593, AZ1 and AZ 101. Germination rates of seeds produced by the project varied from 0% to 60% depending on origin, age, line and seeds cleaning.
Growing and overwintering of the different lines of the germplasm have continued to be assessed. The set of 24 accessions planted in Spain and 40 accessions in Montpellier provide a good set of materials to test the genetic diversity in guayule. Clear genetic differences are present. To plan the five lines experimental field in Year 3 (2010), seeds of the most promising USDA lines were collected from May to September 2009 in the germplasm plots in France and Spain. This material also contains crossed material that can be used as a start of a European breeding program in guayule.
Fertigation trials based on the same protocol with three levels of irrigation and 3 levels of fertilization, were implemented in France and Spain in May 2009 with AZ2 seeds received from USDA/University of Arizona. The fertigation experimental fields of guayule plants cover 0.2 ha in Montpellier and in Murcia/El Molinar. To achieve this goal, 5000 nursery plants were produced and then planted in field on schedule. DLO-PRI reported for the fertigation trial in Spain a significant influence of irrigation on the growth of the plants, but less influence of the fertilization. Rubber content measured by CIRAD on guayule plants from Spain by Soxhlet and accelerated solvent extraction (ASE) system was 9% for rubber content for 10 months old guayule plants.
In Montpellier CIRAD reported a high mortality (over 60%) of the plants From May 2009 to April 2010. From May to October 2009, 30% of the plants died for unexplained reasons at the start. An additional 30% of the pants died after the hard wintering (cold weather, high rainfall with snow). One of the main explanations is too high rainfall and high humidity of the soil of the site of Lavalette Montpellier, combined with poor drainage of the ground, and use of plastic film used for weed control. The plastic film keeps the moisture of the soil in summer, but also collects the water when it is raining. These conditions apparently were not suitable to grow a desert plant in a soil not sufficiently well drained. Mortality of plants was found lower for plots that received less water in the fertigation experiment.
The fertilisation and irrigation (fertigation) trial with AZ2 line were implemented in France and Spain. The effects of fertilisation and irrigation on biomass production and rubber content in plants in two different weather conditions and type of soils were monitored. Thus, one ha of guayule biomass has become available for WP5 (Extraction and application testing). Other activities included the production of guayule seeds, the development of a reference laboratory method to measure rubber and resins contents and optimization of the reference method through the Automatic Solvent Extraction method (ASE/Dionex). A PhD student has worked on the development of a Near Infra-Red Spectroscopy method (NIRS) analysis on dried guayule crushed pellets.
- Third year (2010-2011): The varietal lines trial was postponed until 2010 when enough seeds were collected from the two collections in France and Spain during the during 2009 summer to be implemented in the 2010 varietal line experiment. A method for seed cleaning was reported by CIRAD and DLO-PRI. The germplasm collection has suffered during the wintering season in Montpellier. The two collections were done to produce seeds of the standard accessions of guayule which also have been studied in other countries. The seed production achieved permitted to plant the additional 0.4 ha of guayule varietal lines in May 2010.
A varietal lines experiment with 6 guayule lines was carried out in France and Spain. Guayule yields were above expectations in Spain (Murcia area), but in France (Montpellier) yields were moderate, because of plant death during winter (2010 particularly cold with snow). Selections were made in Montpellier of plants surviving the extreme low temperatures during winter that can form the basis for more cold tolerant guayule varieties.
It was the combination of low temperature and water logging just after melting of the snow cover that caused most damaged. The soil in Montpellier was not well draining.
The development of the NIRS method has been continued with the establishment of a dedicated database. The first calibration curve on moisture, resins and rubber content has become available. A method to quantify the purity of the solvent extract was developed, using FTIR and SEC-MALS (refractive index / RI), to correct the NIRS calibration equations.
Crop characteristics and biomass and latex production under European culture conditions were monitored. For this, biomass and samples were produced by DLO-PRI and CIRAD in WP3 and further analyzed in WP5. A NIRS method for latex and rubber content determinations was developed to allow measurements under field conditions. Together these resulted in data packages that enabled the economic assessment of guayule cultivation in Europe and application testing in WP5. Secondly, samples of guayule from different tissues, developmental stages and different guayule lines were produced as a basis for studying the genetic basis of guayule rubber synthesis in WP2. Results of WP2 were used to investigate aspects of genetic regulation of rubber synthesis in guayule by DLO-PRI, and analysing within the available germplasm in the project differences between guayule lines in order to select the lines with the most adapted rubber synthesis regulation for Europe.
- Fourth period (2011-2012): During this period work has focussed on the production and analyses of biomass in Spain in order to provide guayule latex and dry rubber for the production of gloves (by sub-contractor CTTM) and car tyres (by Apollo Vredestein), respectively.
To quantify rubber and resin production in plants, three gravimetric methods using Soxhlet, ASE and Polytron have been tested at CIRAD. One of the three methods was used as reference method to set a calibration curve for NIR Spectroscopy measurement of rubber content in the plants from the field. Rubber content of guayule plants in Montpellier increased from 0.5% in June 2009 to 2.9-3.2%. in February 2010 and from 4.5% to 7.8% for resin content. In Spain, CIRAD found for rubber content of samples harvested in March 9% and 10% for resin content by Soxhlet measurements. The rubber content measured was very encouraging and it turned out that biomass with enough rubber or latex content has become available for WP5 tasks.
Accessions planted in Spain provided a good set of materials to test the genetic diversity in guayule. Clear genetic differences are evident. The best line tested in Spain produced 20-25 tons of dry biomass and about 1800 kg of rubber (7.2%) per hectare (extrapolation). Methods for extraction and quantification of rubber and resins content in guayule based on near infrared spectroscopy (NIRS) has been developed at used for quantification of rubber and resins content at laboratory level. Latex and rubber extraction have been performed on laboratory and 10 kg batch extraction process with the production of 32 L of two grades of latex LP and HP based on DRC content and 1,3 kg of raw rubber with a Molar mass (Mw) of 16.000- 22.000 kg/mole higher than Yulex dry rubber received (400-600 kg/mole).

Spain field trials with guayule: 1. fertigation trial with AZ2
The fertigation trial in Spain was established in June 2009. Plants were obtained from local nurseries supplied with seed from the project. A lettuce type bed system was used, with wide ridges of about 1 m on top of which two lines of drip irrigation were placed. On each bed, two rows of guayule plants were planted at a distance resulting in 50,000 plants/ha. This is higher than standard practices with guayule in the USA, were densities of 25,000 plants/ha are used and rarely up to 47,000 plants/ha.
The higher density proved to be a great success as it shortened the period during which intensive mechanical weeding was necessary. Also, yield formation started early.
Detailed yield data for each harvest time and for plant parts are presented in the Deliverable Reports on WP3. Here, a summary is given of the overall results.
Five fertigation treatments were used: 3 irrigation levels (I100 = just non-limiting water supply, I66= 66 % of I100 and I33=33 % of I100) and 3 fertilizer levels (F100 = according to expected uptake of nutrients by the crop, i.e. soil nutrient status should not become lower after all crop material is removed at harvest, F50 = 50 % of F100 and F0 = no fertilizer added).
To establish the growth curve of guayule, biomass was harvested at 8 different times (October 2009 until March 2012). Dry biomass yields of roots, stems and leaves were measured and the content of rubber and resin was determined using the NIRS-method developed at CIRAD (Sunisat et al. 2012).
Here, some final results from the last harvest are presented. The highest total biomass was found at the I100 treatments, at 40 tons dry weight, on average. No statistically significant differences were found between the fertilizer treatments. Increasing water shortage reduced the biomass yield considerably, but at the lowest irrigation level the biomass yield was still 21 tons/ha. The proportion of stem was about 70 to 73 % of total biomass.
Rubber content in stem ranged from 9.9 to 13.1 %, giving rubber yields in the stem of 3,948 kg/ha after 32 months in the I100/F100 treatment and only 1,845 kg/ha at the lowest irrigation level. At the F50 and F0 treatments, rubber yields were 3,400 kg/ha. Rubber content in the root was about 9 %, and rubber yield in the roots was ranged from 260 to 430 kg/ha. Resin content in the stem was about 10 to 11 % with no large effect of the treatments. Resin yields in the stem ranged from 1,700 (at I33/F100) to 3,450 kg/ha (at I100/F100).
Using a value of 40 Euro/ton for lignocellulose (the bagasse) and 2.5 Euro/kg for rubber and 1.5 Euro/kg for resins, a total chain value of the guayule crop was calculated. With the I100/F100 treatment, this total chain value was 7,900 Euro/ha (for the 32 month growth period) at the lowest irrigation level and 16,000 Euro/ha for the highest irrigation and fertilization level, or 2,600 Euro/ha to 5,500 Euro/ha per year.
At the highest irrigation level, the total water availability was 880 mm per year on average and with the lowest irrigation level only 540 mm per year. On the basis of the water availability and the obtained yields, relationships between water availability and yields were determined that can be used in advising farmers on the economically optimal level of irrigation. The water use efficiency for biomass production ranged from 2.5 to 3.8 kg/m3, for rubber yield from 0.21 to 0.33 kg/m3 and for economic yield from 4.0 to 5.6 Euro/m3 of water available. Given the average water costs in Spain of 0.1 Euro/m3, the highest water use efficiency for biomass production was obtained at the lowest irrigation level, but for economic yield the highest water use efficiency was obtained at the intermediate irrigation level.

Cultivar trial in Spain
In June 2010, a variety trial was planted using plants sown in nursery in April 2010 with six varieties (AZ1, AZ101, AZ2, 593, 11591 and CAL6). These varieties differ widely in yield and rubber and resin content. The irrigation treatment and fertilizer application was aimed at achieving a situation without water and nutrient shortages (same strategy as the I100/F50 treatment in the fertigation trial.
Ranking of total biomass yields was CAL6 < 11591 < 593 < AZ2 < AZ1 < AZ101 with yields ranging from 29 to 62 tons/ha (dry weights). The yield of AZ2 was similar to that obtained after the same time in the fertigation trial with AZ2. The ranking of rubber content was: AZ101 < AZ1 < AZ2 < CAL6 < 593 < 11591 (from 4.5-5.0 % for AZ1 and AZ101, 7.3 % for AZ2 and 8-10 % for 593 and 11591). Rubber content of AZ2 was similar to that of AZ2 in the fertigation trial after the same growth period. Resin content was not highly variable (about 8 %).
The results show that high biomass yield and high rubber are not combined in the same variety. AZ101 shows the highest rubber yield (2,014 kg/ha after 24 months), 593, AZ1 and AZ2 have similar rubber yields at about 1,800 kg/ha, and CAL6 and 11591 have the lowest rubber yield (about 1,400 kg/ha). Resin yields follow the same pattern as the total biomass yields (on average 1,900 kg/ha).
Economic yield (total chain value as described above) has the same cultivar ranking as the total biomass. This total chain value ranges from 6,000 to 12,000 Euro/ha for 24 months, or 3,000 to 6,000 Euro/ha per year. This is very close to the total chain values calculated for the fertigation trial.

Gene expression studies
From AZ2, gene sequences (DNA and RNA) were obtained from several genes known to be important for the rubber production: five SRPP genes were found (small rubber particle proteins) with several variants of each and three classes of cis-prenyl-transferases (CPT1, CPT2 and CPT3). The CPTs have a very high similarity to the CPTs of T. kokzaghys. CPT3 was found to be expressed in stem tissue only and not in leaves. The guayule CPT3 has a deletions of 21 nucleotides compared to CPT1 and CPT2, in the same place as the T. kozaghys CPT3. As AZ2 is a tetraploid line, it was expected that if only one locus is present per CPT, maximally four different variants might be present, when AZ2 would be fully heterozygous for these loci (which is possible as AZ2 shows apomictic reproduction mainly). Additional sequencing of the CPT genes revealed that indeed at least four variants occur of CPT3, but CPT1 and CPT2 show less variation.
Further, mRNA was collected from stems and leaves of six cultivars for an RNAseq analysis (Illumina Hiseq 2000 sequencing of the whole transcriptome). The results of the RNAseq analysis have not been fully analysed yet, but at this moment the transcript analysis has resulted in a set of 160,000 different transcripts which are now being annotated using BLAST on the NCBI-sequence database (over 30,000 transcripts have been annotated so far). The RNA-sequences will be used to identify single nucleotide polymorphisms between the six varieties (from the cultivar trials) as a starting point for molecular marker analysis in segregating population to help find QTLs for biomass, rubber and resin yields and other genetically variable traits in the future.

WP4. Germplasm, breeding and agronomy of Taraxacum koksaghyz (Russian dandelion, TKS).
WP4 aimed to improve the rubber production and the agronomy of T. koksaghyz in order to turn it into a valuable rubber crop for Central and Northern Europe.
Molecular analyses by AFLP fingerprinting of Russian dandelion accessions available from Seed Banks and botanical gardens (labelled as T. koksaghyz) showed that the available lines were all genetically identical triploid apomictic clones, subsequently identified as T. brevicorniculatum.
With permission of the local government new germplasm of T. koksaghyz was collected in Kazakhstan, its centre of diversity, and subsequently characterized for rubber properties and genetic diversity.
Sexual dandelions of the section Ceratoidea (newly recognized and described during the early project stage), which includes T. koksaghyz, were collected during the expeditions to Kazakhstan and unambiguously identified as T. koksaghyz. The collection sites were analyzed for climatic and soil characteristics. AFLP genetic diversity analysis showed that these collected samples were highly polymorphic and rubber concentrations showed considerable variation. The rubber molecular weight was comparable to that of rubber produced by H. brasiliensis.
Plants collected were propagated in experimental gardens in the Czech Republic and in Spain, and were distributed to eight major Seed Banks. Further material was cultivated in Kazakhstan. During the four seasons of the project, over 10.000 individual (flower head) crosses (over 2.000 in 2012) were made between T. koksaghyz and other Taraxacum species and hybrids with different ploidy levels and breeding systems. Plant performance was tested. There are about 150 promising hybrid lines showing agamospermy (full seed set), robust vigorous growth and variable rubber contents.
A dense genetic map of T. koksaghyz was produced, based on a cross between a high and a low rubber producing T. koksaghyz plant, including 703 markers (AFLPS and COS / EST markers). The eight linkage groups corroborate with the haploid chromosome number of T. koksaghyz (2n=16). The first eleven quantitative traits were mapped. Conditions for in vitro propagation of T. koksaghyz were optimized and several in vitro lines were established and distributed for e.g. BAC library production and experiments. The mapping population was also propagated by in vitro culture. A T. koksaghyz BAC library with 8x genome coverage was constructed. The average insert size was 130 Kb and first BACs containing cis- prenyl-transferases (CPTs) and Small Rubber Particle Protein (SRPP) genes have been isolated.
TKS seeds propagated by ECER in the Turkestan Botanical Garden in 2010 were sown for further seed multiplication in Germany (WWUM), Spain (NEIKER) and the Netherlands (DLO-PRI). Unfortunately the percentage of true TKS was very low; most of the emerging plants turned out to be the poor rubber producer, T. brevicorniculatum. These field trials were therefore abandoned. WWUM continued with seed propagation of true TKS by hand pollination in the greenhouse finally resulting in some 30.000 true TKS seeds which were used for the field trials in 2011. NEIKER and IBOT also have produced smaller amounts of true TKS seeds.
More than 60 COS and SSR markers were added to the parental AFLP genetic maps by NEIKER and 41 could be integrated in the integrated map. Three QTLs were detected for rubber Molecular Weight, explaining more than 40% of the variation. Individuals from the mapping population were replicated and grown in the common garden by NEIKER. Quantitative field traits were measured, and 38 new QTLs were detected for 11 traits of interest. Most field QTLs mapped to different chromosomal locations than greenhouse QTLs, suggesting a strong Gene x Environment interaction.
KeyGene isolated and sequenced 31 TKS and 20 T. officinale BACs containing all cis- prenyl-transferases (CPTs), Small Rubber Particle Protein (SRPP) and Rubber Elongation Factor (REF) genes. TKS is a good rubber producer, whereas T. officinale is not. Comparisons may give clues about the critical genes for good rubber production. TKS has three CPT genes, whereas T. officinale only has a single CPT gene. The number of SRPP genes in TKS is six, compared to five in T. officinale, all in a single locus. Both species have only one REF gene. The different numbers and types of CPT genes may explain the differences in rubber production between the two Taraxacum species. Genetic mapping of the biosynthesis genes was complicated due to the repetitive nature of these genes. Two of the rubber biosynthesis genes (CPT3 and REF) could be successfully located on the genetic map.
To increase the agronomic performance of T. koksaghyz genetic material of vigorous relatives has been introgressed. QTLs for rubber yield and quality and rubber synthesis genes have been integrated with a genetic map of AFLP and COS markers. The rubber metabolism has been engineered by inducing mutations at targeted genes (TILLING), based on knowledge generated in WP2. Induced favourable mutations and high quality germplasm were combined in a molecular breeding program. A parental and an integrated linkage map based on AFLP data were established. The integrated linkage map consists of 773 markers, which include 70 COS or SSR markers. QTL analyses were performed with the mapping population using field and greenhouse data. The traits considered morphological and physiological traits as well as rubber characteristics. A total of 86 QTLs for 24 different traits were located in the integrated map.
In order to produce reduce latex coagulation and to improve latex harvesting in T. koksaghyz, more than 3.000 wild T. koksaghyz plants were mutagenized with EMS. Transgenic down-regulation of the latex-specific polyphenol oxidase (PPO) gene had been shown to increase the fluidity of the latex in T. koksaghyz. Despite the fact that the mutation population had a high background level of natural polymorphisms, six putative amino acid mutations of the latex-specific PPO gene could be discovered by Next Generation Sequencing. Two of these were confirmed by Sanger sequencing.
Agricultural performance was investigated in the greenhouse and small field plots. Different agronomic parameters have been evaluated in order to optimize T. koksaghyz cultivation. An NPK fertilization trial was established in the field and the optimal fertilization conditions for T. koksaghyz cultivation were determined. A total of 2l different T. koksaghyz accessions were evaluated in field trials and greenhouse using different planting dates. Morphological characteristics, biomass production, latex and rubber concentrations were evaluated in the different accessions showing a remarkable degree of variation. An irrigation trial was performed to determine optimal water regimes for T. koksaghyz cultivation.
For the final field trial, 30.000 T. koksaghyz seeds were produced in the greenhouse. Seedlings were planted into the field in Germany in early May 2011 and resulting plants were harvested in October 2011. About 80 kg of cleaned dried roots were prepared and sent to the rubber extraction unit of DLO-FBR for further processing and application testing (prototype car tyres by Apollo Vredestein) in WP6.

WP5. Extraction of rubber, application testing and development of products from guayule.
WP5 determined if latex produced from EU-grown guayule is of comparable quality to USA-grown guayule of the same genetics, and provided the necessary data to permit profitable site selection for commercial guayule farms, as well as best growing and extraction practises. The plan also allowed any necessary changes to be made to generate the performance properties required in commercially-acceptable dipped and foamed latex products. Bulk (or solid) rubber applications were also evaluated from practical and economic perspectives. The data, together with those from the closely related WP3 permitted a complete economic analysis of the opportunity for profitable guayule production in the EU. WP5 was strongly linked to WP6 which had further built on the guayule experience.
To summarize, the work focused on latex and rubber extraction techniques, the design of extraction facilities and on manufacturing of gloves from guayule latex and on manufacturing car tyres from guayule dry rubber by Apollo Vredestein. Rubber analysis methods have been set up for guayule. Work on Type I latex allergy has been addressed. For guayule a lab scale extraction process and a pilot production of 10 kg fresh biomass per batch has been developed and used for the production of 36 liters of latex (DRC from 16 to 32%) and for 1.3 kg of raw rubber. By-products of resins, containing a variety of terpenoids (isoprenoids) and fatty acids, and bagasse that can be used for energy production were also produced.
CIRAD has studied at laboratory level conditions to prepare latex from guayule plants available in Montpellier. Crushing and grinding fresh plants with the blender procedure described in the literature have been used successfully to extract latex in small quantities. The efficiency of latex extraction at laboratory level with fresh biomass has varied from 34 to 50%. With stored biomass, efficiency of latex production is reduced. Guayule plants harvested need to be processed very quickly.
For comparison CIRAD worked on the characterization of various rubber samples from guayule, dandelion, Hevea and synthetic polyisoprene. Properties of rubber of different samples were identified by Infra-red (IR/ATR), NMR proton and 13C NMR, DSC for glass transition and purity, and SEC-MALS. Spectra obtained showed that guayule and dandelion rubber is are both true 1,4-cis-polyisoprene. The microstructure properties of dandelion rubber appeared to be very dependent on its process of production. Molecular weight (Mw) of dandelion rubber appeared to be much higher than Hevea and initial samples of guayule rubber. Dandelion rubber is also characterized by a high gel content. Molar mass characteristics of various guayule samples depending age of the plants were studied by SEC-MALS.
Under partly optimized conditions the initial laboratory procedure (100 g of biomass), allowed to extract 70% of the poly-isoprene (as hexane extract) present in the biomass, corresponding to a yield of “dry latex” (DRC) of 3% related to the starting (dry) biomass.
In order to produce latex at larger scale, several pieces of equipment have been tested at 1 kg pilot scale. First trials showed a loss of efficiency compared to lab scale, but nevertheless the production could be done to get sufficient material for other partners to perform their tasks. Options for improving the process included enzymatic softening of the biomass, use of different grinders, continuous centrifugation and ultrafiltration.
At a later stage the content of poly-isoprene from biomass harvested weekly in Montpellier, which was about 4% when starting in 2010, could be doubled before the end of the project. The poly-isoprene content in biomass harvested in Spain was even higher. Thus, even without substantial improvements on the technical side, the productivity per batch was already > 8%, only owing the improvement of the biomass quality.
For large scale production, 100 kg of biomass from the guayule experimental fields in Spain was harvest in May 2011 and processed with equipment available at INP/ENSIACET/INRA.
Work focused on latex and rubber extraction techniques and the design of extraction facilities. Rubber analysis methods have been set up for guayule as well as Russian dandelion. Work on Type I latex allergy has been initiated. Analysis shows that Russian dandelion rubber is highly comparable with Hevea rubber based on molecular mass, molecular mass distribution and (absence of) branching. For both plant species different extraction methods and equipment are used. For Russian dandelion, on lab scale, proof of principle was obtained on an efficient extraction process for rubber. Within this process there is room for the extraction of valuable by-products (inulin). Also for guayule a lab scale extraction process and a pilot production of 10 kg fresh biomass per batch has been developed and used from the production of 36 liters of latex (DRC from 16 to 32%) and for 1.3 kg of raw rubber. By-products of resins, containing a variety of terpenoids (isoprenoids) and fatty acids, and bagasse that can be used for energy production were also produced.
Latex gloves (16 pairs) were produced and tested for several industrial latex formulations with guayule latex produced by CIRAD for the EU-PEARLS project and with Yulex corporation latex from the USA as the reference. Wet behavior of Hevea and guayule latex are very similar. The behavior of the guayule film was very different from the Hevea film which behaves like a “thermoset” polymer, while guayule film behaves like a “thermoplastic” polymer, which is coherent with the gel content (lower in guayule polymer than Hevea polymer) and also the molar mass of the polyisoprene (low for Yulex latex film). The main difference between EU-PEARLS latex and Yulex latex is the solid content (DRC, 14 to 38% instead of 60-65%). Mechanical properties of EU-PEARLS gloves are good, better than those from Yulex latex but not as good as those from Hevea latex. Guayule hypo-allegenicity characteristics was also studied. A method was developed and optimized to quantify the extractable proteins from different types of materials. Extraction and quantification was done with a modified form of the AFNOR norm NF 455-3 of the Lowry method. Protein content in guayule films is 115 lower than Hevea films. Prototype gloves made with guayule latex produced at CIRAD contained 9 times higher levels than the recommended limit due to an inadequate leaching of latex before glove manufacturing, while the protein content from prototype gloves with Yulex guayule latex was very low concentrations, lower than 1 ng/g and in guayule gloves at low concentrations (2.2 ng/gmax). The proteins extracted are not Hevea allergens because the allergen amount is 1.46 ng/g, lower than the cut-off defined in literature: 0.15 µg/g (150 ng/g). The preliminary results on proteins show that guayule extracts contained Hevea-like proteins. Guayule latex film extracts did not contain considerable amounts of proteins when washing of guayule latex is sufficient and about 100 times lower than Hevea latex film extract.
Apollo Vredestein has processed raw rubber samples from Russian dandelion and guayule in tyre formulations and has produced the first sets of car tyres for testing purposes. The alternative rubber samples were part of the tread and with respect to properties like wet grip the tyres behaved equally, if not superior to the conventionally produced winter tyres (http://prod-euronews.euronews.net/2012/11/07/a-tyre-revolution-rolls-around/).

WP6. Extraction of rubber, application testing and development of products from Russian dandelion.
This WP encompassed the very important part of the chain, from isolation of the rubber, via determination its basic polymer properties into finally making derived prototype products. This provided insight in the relation between the extraction methods and the final rubber material properties obtained. WP6 had strong cross-links with WP5 (extraction and application of guayule rubber) and WP4 (germplasm breeding and agronomy of dandelion).
A full literature update has become available on Russian dandelion rubber with support from WP7. This literature study gives insight in the several options for rubber extraction from T. koksaghyz; latex extraction, rubber extraction from dried roots and rubber extraction in wet processes. Although information on yields and purity of extracted rubber can be found, limited information on the methods how yield and purity was determined is available. Since most literature is old, there is limited information on the characterization of T. koksaghyz using advanced techniques like GPC Malls. Still, literature indicates that T. koksaghyz produces high quality rubber.
The literature study was completed with relevant papers that became available for the project consortium in the last year. In the first project period T. brevicorniculatum was the sole biomass source available for extraction trials. Although the quality of the rubber that can be extracted from this species is good (high Mw) yields are very low since under the conditions tested T. brevicorniculatum produces low quantities of rubber. This severely complicated rubber extraction and the production of samples and therefore parties working on Russian dandelion commenced using T. kokzaghyz.
Initial extraction trails were run using conventional methods on roots from Russian dandelion grown in Spain and data on root composition (polyisoprenes, proteins, inulin) was obtained.
Regarding extraction methods and trials the first 1.5 years of the project extraction trials were performed using T. brevicorniculatum. However, it appeared that T. brevicorniculatum is not suitable for industrial rubber extraction (and production). Although the plants and roots are larger than in T. koksaghyz, rubber production is low (about 1%). Moreover, (apart from latex extraction) the common extraction methods are based on the rubber threads that are formed in T. koksaghyz. Since rubber threads are not found in T. brevicorniculatum these common extraction methods are not suitable for this species. Based on these results it was concluded that WP6 needed to proceed with T. koksaghyz.
Analysis of 12 representative plants of T. koksaghyz has shown a large variation in plant and root sizes rubber contents and rubber molecular weight distribution. Based on the analysis results of these 12 plants it was estimated that the rubber yield per hectare (240.000 plants) would be around 70 kg.
Estimations on rubber production and rubber contents are most efficiently performed using ASE and within WP6 a method was developed. Still ASE extractions are expected to underestimate the actual rubber content because of the limited solubility of T. koksaghyz rubber. In this respect T. koksaghyz rubber is highly similar to Hevea rubber that commonly contains an insoluble gel fraction. The similarity is also found using GPC and DSC. On laboratory scale a ball mill method was investigated (based on the Eskew process) and compared to the Buranov method (grist mill) and latex extraction. The ball mill method outperformed the Buranov method and latex extraction methods in yield, purity and efficiency.
During the last project period it was proven that the higher rubber concentration in T. kokzaghyz leads to the formation of rubber threads and that these rubber threads are essential in (mechanical) extraction processes. Also latex extraction of this species is possible and an advanced latex extraction process was tested. Furthermore, despite the limited amount of biomass available within WP6 good progress was made in the development of an extraction process based on a process developed by Buranov. For the experimental design of the extraction process and to determine the expected yield of the 1 ha. field trial the variation (in rubber production, plant size, etc) of the wild species was studied using various analyses techniques. The proof of principle on dry rubber extraction using a modified Buranov process, the limited yield during latex extractions and the variation in the wild species were used to set-up the experimental design for the development of an extraction process.
The analyses have shown that Russian dandelion rubber is highly comparable with Hevea rubber based on molecular mass, molecular mass distribution and (absence of) branching. For both plant species different extraction methods and equipment are used. For Russian dandelion, on lab scale, proof of principle was obtained on an efficient extraction process for rubber. Within this process there is room for the extraction of valuable by-products (inulin).
Apollo Vredestein has achieved the development and testing of passenger car tyre prototypes emplying either Russian dandelion or guayule dry rubber.
During the last project phase in spring 2012 dry rubber samples (about 1 kg) have become available that were analysed, shipped to Apollo Vredestein (in addition to guayule dry rubber samples from CIRAD) and incorporated into a small scale tyre production process (in treads only). Given the relatively small rubber quantities obtained a modification was required of equipment and compounds to allow production of hand-made tyres. In the Apollo Vredestein laboratory a method has been developed to make treads with a very limited amount of compound. In regular manufacturing at least 600 kg of compound is required. In the new method where thin strip of compound was used only a few kg of compound was required. The tread and the tyre had to be made completely by hand. The new developed method has been validated against the regular way of manufacturing and the tyre results (indoor and outdoor) were comparable. This method has been used for manufacturing the EU-PEARLS tyres.
A method for preparing steel cord belts of natural rubber compound is developed together with Bekeart. In this method a very limited amount of steel cord wires are rubberized and later assembled to a steel cord belt that can be used in tyres. The method is available however not used due to the very limited amount of NR compound available.
With the experimental technique tyres Vredestein Quatrac Lite were produced in the size 155/65R14 T rated. Tyres were made with in the tread TSR 20 (reference), dandelion NR (evaluation 2, Germany/Netherlands) and Guayule (evaluation 2, Spain/ France). The winter car tyres produced were subjected to standard wear and durability test and have shown good to excellent properties. Especially, the so-called wet grip performance outcompeted that of Hevea rubber.
In short, in WP6 clearly significant results were obtained especially during the final project period, including:
- Up-scaling with a factor 100 of the extraction of dandelion rubber from laboratory scale (10g roots per batch ≈ 0.5g rubber per batch) to pilot-scale (1 kg roots per batch ≈ 50g rubber per batch), while maintaining good quality rubber with a high yield. - Total extraction of 1.5 kg dry rubber from TKS grown in Europe, whereas the guayule rubber derived from WP5 (CIRAD).
- Production of the first prototype winter tyres where the natural Hevea rubber in the tread was replaced by dandelion rubber from TKS and guayule grown in Europe.
- Without optimisation of TKS or guayule lines, agronomy, extraction process and tyre formulation, the tyre properties were only marginally different from those using Hevea rubber.

WP7. Dissemination & exploitation.
WP7 aimed at the communication, dissemination and technology transfer of the project results to the relevant stakeholders including policy makers, industry and society including the management of IPR. WP7 was also responsible for internal communication, training, and the development of desktop studies to inform the other WPs and the extended audience on biorefining and socioeconomic issues relevant to guayule and Russian dandelion cultivation, processing and commercialization.
Background scientific knowledge and IP has been provided to all WPs during the entire Project via the Project website or other means of internal dissemination, while material for external dissemination has been actively acquired from and shared with all WPs. Exploitation and management of IP involved close collaboration with WP1, and with the individual partners in each of the WPs.
Major results were obtained as described in individual sections below.

Updates of scientific state of the art and IP positions.
A full database (in Endnote) contains collected information (papers, patents, news items, reports, etc., presently over 3500 entries). In most cases, abstracts are included. PDFs are named such that they can be retrieved based on their Endnote entry (Author-Year-Keyword(s).pdf or PatentNumber-(Year)-Keyword(s).pdf), and are made available in a searchable format if possible in view of the quality of the PDF. The Endnote files, recent PDFs, and PDFs of papers and reports that are not readily available through SCOPUS, WOS, and other database access tools have been placed on the EU-PEARLS Teamsite, or are for project partners available on request.

Project websites for internal and external communication.
Online since January 2009 (www.eu-pearls.eu update 2013: www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects.htm Teamsite: portal.wur.nl/sites/eu-pearls/default.aspx).

Communication and dissemination plan for EU-PEARLS.
The plan is based on EU-PEARLS project proposal.
Dissemination and communication have played a central role in EU-PEARLS. As such, they were the responsibility of all participants, aided by the Dissemination and Exploitation Officer (DEO). The DEO coordinated issues related to Dissemination and Exploitation of results, take the lead in the construction and maintenance of a website, accessible to internal and external parties, and in producing or organizing various publications, patents, press releases, training activities, and workshops.
Current and new information networks of the participants involved in this Project ensured that both Project participants and those outside the Project were informed about latest developments of science, application development and technology in the area of Rubber research, production and applications.
A web-based system, hosted by Wageningen UR, was used to support information exchange within and management of the Project. This web-system provided the facility to accommodate all documents related to the Project, including scientific, technical, management and dissemination activities, in one central domain underpinning the joint taskforce image. Access to confidential information was restricted by different levels of authorisation. Furthermore, different modules provided tools for optimal exposure of results to the general public and interested parties.

Internal dissemination.
The dissemination of knowledge has been a task of all participants in EU-PEARLS. Primary information was collected, catalogued and kept by each Workpackage. This material was used to generate reports, summaries, newsletter contributions, material for press releases, and other documents in electronic format and incorporated into the dedicated Project website. A large part of the internal communication was handled via access-restricted parts of the EU-PEARLS website (Teamsite).
Extensive use was made of e-mail, conference calls, Skype, and online file-sharing (Teamsite). Experience has demonstrated the need to maintain the impetus of projects in between formal events such as the Workshops. Regular meetings were organized as detailed in the description of work.
In complex projects sufficient emphasis should be given to reproducibility and transferability of results. To ensure efficient research, standards and protocols were agreed on early. Implementation of these standards and protocols was enabled by inter-group training modules organized by appropriate partners.
Other training activities, e.g. within the Leonardo and university student frameworks, were used to increase involvement of additional researchers and to promote the flow of information between the Contract Partners.

External dissemination tasks.
The external dissemination tasks included management of the Extended Audience through the activities listed below. The extended audience included companies such as BASF, the Chemical Company, BASF Plant Science, DOW Benelux, Pirelli, researchers in academic and government institutes, the farming community, the biorefining and processing industry, plant breeders, end-users (tyre companies and producers of gloves, medical equipment), professional associations and organisations, NGOs, and the general audience.
An important tool for external communication was the public Website 2009 and the update 2013.
This web-system provided the facility to accommodate all documents related to the Project, including scientific, technical, management and dissemination activities. The Project website also acknowledges the EC contribution.
Different levels of authorization restrict access to confidential information. Furthermore, different modules provide tools for optimal exposure of results to the general public and other interested parties. This also ensures that the products and applications developed by the Consortium are of wider, recognized benefit for the environment and for the consumer.
The following external dissemination activities were addressed:
1- Congress papers. Project results were be made available to various target groups by presentations to professional events at the national and international level. Depending on the nature of the results, major international events in appropriate areas (rubber products, plant science, agronomy, processing, bio-refining, etc.) have been addressed.
2- Articles in refereed scientific journals. Various target groups were informed about project results through publications in national and international professional journals. These journals include specialized journals such as the European Rubber Journal, Industrial Crops and Products, Phytochemistry and Biomacromolecules. Other output has been published in science journals specialized in appropriate fields (plant science, agronomy, processing, etc.), depending on the focus of the project results.
The output of EU-PEARLS in the form of scientific papers has been monitored, as well as their impact based on citations and inquiries.
3- Newsletters and other information material. To avoid the delay resulting from publication cycles of professional journals, selected research information has been released via nationally or internationally circulated newsletters or bulletins. A number of specialized websites such as www.biomatnet.org www.europabio.org www.european-bioplastics.org www.rubberworld.com and www.rubber-stichting.info are also well suited to publicize progress or announce events. Other tools that have employed are press releases, fact sheets, and flyers to be distributed at conferences.
4- Dedicated congress, seminars and workshops. In addition to the meetings of the EU-PEARLS consortium, the project partners have organized meetings aimed mainly at dissemination of selected project information to audiences representing various target groups (potential customers, processors, producers, NGOs). The following major events were organized:
4.1- The international EU-PEARLS Midterm workshop, entitled “The future of Natural Rubber in Europe” has taken place in Montpellier at Agropolis International, from October 14-15, 2010. CIRAD has organized the Midterm Workshop in collaboration with the WP7 leader and the overall project coordinator. Most research groups and companies interested in alternative sources of natural rubber have attended, and presented their research goals and results. Abstracts, presentations, a brochure and further information are available on the meeting website (http://www.eu-pearls.eu/UK/Midterm+Meeting update www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects/News-and-agenda/Show-16/2010-Meeting-The-future-of-natural-rubber.htm). The workshop has been attended by about 80. Topics included:
- Natural rubber, the European dimension and economic aspects
- Isoprenoid biosynthesis
- Processing
- Agronomy and genetics
- Chemistry and products
- Policy and regulatory issues
The Abstracts / Presentations are available through the website indicated.
4.2- Final Congress 2012: BioRubber for Europe in Global Perspective, organized by the EU-PEARLS consortium to mark the end of the FP7 EU-PEARLS (www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects/News-and-agenda/Show-16/2012-Congress-BioRubber-for-Europe-in-Global-Perspective.htm).
The workshop has been attended by about 80. Topics included:
- The economic prospects of natural rubber in a changing world
- Plant breeding and genetics, isoprenoid biosynthesis, analytical methods
- Agronomy, processing and biorefining
- Applications of latex/rubber and co-products from (alternative) sources of natural rubber
The Abstracts / Presentations are available through the website indicated.
5- Direct contact to members of the extended audience. Companies, organizations, and other stakeholders were approached directly when a strong interest could be expected.

Monitoring of EU-PEARLS output.
Papers describing the EU-PEARLS project were presented at the ACS-meeting in Pittsburgh (October 12-15, 2009), 9th International Plant Molecular Biology (IPMB) Congress (October 25-30, 2009), and a various local meetings.
Papers reporting EU-PEARLS scientific output and/or work by consortium partners prior to the start of EU-PEARLS were published during the fourth reporting period
A number of newspapers, magazines, and other media (radio, local TV) have reported on the EU-PEARLS project (as a result of the CORDIS press release, and press releases of the individual partners. These media reports are collected on the EU-PEARLS Teamsite.

Exploitation.
In the first phase of the project exploitation issues and targets have defined. EU-PEARLS had a strong industrial component with a major industrial participant: Apollo Vredestein as a tester and end-user of the product, i.e. car tyres, and an SME KeyGene with a specific interest in developing Russian dandelion as a platform crop. The eight academic partners and research institutes have had critical roles in the acquisition of germplasm and breeding, in metabolic engineering, the development of processing technology, and product development. This has created opportunities for each of the partners as reflected by their interest in the results of EU-PEARLS.
In the first year a first draft of an Exploitation Business Plan has been developed, which gives an overview of potential exploitable results, already foreseen by the participants, potential markets, and foreseen profits for the potential end-users. This Exploitation Business plan has been further developed and sharpened during the project. In the final year of EU-PEARLS concepts for follow-up projects have been developed. These may be demonstration projects, further scientific and development programs, or programs aimed at specific issues coming up during EU-PEARLS all aiming at the exploitation of EU-PEARLS projects results in European practice. An Exploitation Business plan and a Plan for management of intellectual property and other foreground have been prepared.

By-products from guayule and Russian dandelion and H. brasiliensis.
This desktop study deals with biorefining co-products from guayule and Russian dandelion to feed WP5 and WP6 on new developments in biorefining and co-product valorization.
Guayule (P. argentatum) and Russian dandelion (T. kok-saghyz, TKS) may step up as alternative sources of natural rubber to supplement the current source H. brasiliensis. However, at current yields and estimated costs for cultivation and processing, the demand for natural rubber must significantly and structurally exceed supply (i.e. much higher natural rubber prices) to allow these alternative sources to enter the market. In general, new crops for existing products must be highly competitive and/or benefit from significant credits from by- or co-products to improve their economics.
In the case of guayule, the resin fraction is usually considered a promising by-product, although the complex and variable mixture of chemicals presently complicates applications other than its calorific value. However, during the past few years a strong industrial interest has emerged for specific products such as the anti-termite and anti-fungal activities present and the unsaturated fatty acids that may find usage in drying paints. The current selling price of guayule resins is about $2.5 (which is not far below the rubber price) and will help to provide economic feasibility for the entire rubber production chain. Guayule bagasse will most likely be used to generate heat and energy carriers, as it appears less suitable for material applications.
In the case of TKS, inulin is a promising by-product, which, in principle, could be used as a food-additive. However, the market size for food-grade inulin is currently limited to about 100,000 t/y, while the production of inulin syrup (fructose sweetener) in Europe is under a quota of 300,000 t/y in the framework of the European sugar regime. Therefore, conversion of the inulin (either isolated or as part of the bagasse) to an energy carrier (ethanol or methane) may be the most viable option.

Desktop study ‘Production of natural rubber from new crops in Europe: a socio-economic. evaluation’. This desktop study supported WP5 and WP6 to judge the effect of external influences affecting the economics of producing natural rubber from guayule and Russian dandelion. The focus is was on the collection of information and developing sub-studies, including:
- economic analysis for H. brasiliensis and alternative sources
- an overview of relevant EU-policies (CAP, non-food crops, energy crops, rural development, sugar regime, etc.)
- historical overview to understand the failure of earlier projects
- introduction of new crops (with focus on industrial crops such as sugar beet, fibers crops, oil crops)
The economic analysis indicates that under current circumstances rubber and latex from guayule and Russian dandelion are not yet competitive, mainly because of processing costs, land rent, and cultivation costs are significantly higher than for Hevea rubber. Also, the risks posed by peak oil and climate change are very large, and it is even possible that an oversupply of natural rubber will develop. A preliminary conclusion from the sub-report on EU-policies is that specific support policies for bio-renewable polymers such as natural rubber are lacking, but required to enable the expensive and risky early development.
The above analyses indicate that the strategic value of natural rubber is key to a successful development of alternative sources of natural rubber in Europe: the 4 years available to the EU-PEARLS consortium were not enough to bring breeding, cultivation and processing forward to the point that natural rubber and latex from guayule and Russian dandelion have become a mature and competitive industry on the world market. However, the project has provided proof of principle for all chain segments at the end leading to prototype products medicinal gloves and car tyres. To guarantee that early stages of commercial development take place, natural rubber must be recognized as a strategic raw material. It cannot be replaced in many crucial industrial applications, and is as important to the EU-economy as the rare metals and minerals covered by the EC Raw Materials Initiative. Therefore, the Consortium has responded to the public consultation aiming to convince the Commission to include natural rubber in its list of strategic materials, and develop appropriate policy to support the develop of alternative sources of natural rubber. The entry can be found on the EC-website (http://ec.europa.eu/enterprise/policies/raw-materials/files/pc-contributions/org-100-eu-pearls-universite-de-lausanne_en.pdf.
Potential Impact:
4.1.4 Potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and the exploitation of results.
EU-PEARLS has been fully in line with the objectives of several major EU technology platforms, action plans, and strategies, such as ‘Challenge 3’ of ETP Plants for the Future ’to develop advanced plant-based raw materials and pharmaceuticals (green) materials)’, the EU strategy on biotechnology, the EU Biomass Action plan, the Community strategic guidelines on rural development, and the broader aims of the Lisbon agenda.

Impact on knowledge base.
Putting in place two complete novel production chains for the domestic production of rubber in place can only be accomplished through the thorough characterization of both crops with respect to breeding, cultivation, rubber biosynthesis, rubber properties, and processing.
WP2 was aimed at obtaining in-depth knowledge on the two new crops guayule and Russian dandelion (T. koksaghyz, TKS) to increase yield and quality of natural rubber. In the case of TKS, which was not well-characterized at all, enabling technologies included a thorough genetic characterization to identify and manipulate relevant genes, pathways and enzymes involved in rubber synthesis and precursor supply), and the study of latex granule biogenesis. It also aimed at identifying genes for important agronomic traits. This knowledge will be applicable for the production of other biopolymers and compounds derived from common precursors.
First steps in the domestication of Russian Dandelion were the collection and genotyping of germplasm to provide a solid base for breeding to improve crop vigour, yield, rubber content, agronomic properties, and processability. Here, the Consortium was set back by the discovery that germplasm obtained from botanical gardens appeared to be T. brevicorniculatum, a different dandelion species. This meant that genotyping, selections, and initial breeding experiments could only start after legal transfer of the germplasm collected in two expeditions in Kazakhstan (end of 2009), leaving only 2.5 years for this work. Further proposed work included greenhouse and field trials, rubber extraction, rubber properties testing and application development (WP4&6). In spite of the lacking germplasm early in the project, all of these steps have been accomplished, providing a major first step in the development of TKS (it must be recognized that crops that are in regular large-scale use in agriculture typically have a history of many centuries or even millennia of selection and breeding).
In the case of guayule, the Consortium established that the USDA variants and available germplasm are suitable to develop guayule as a crop for Europe (WP3). Rubber extraction from guayule is currently energy-intensive, consuming solvents and contributing to carbon dioxide emissions. EU-PEARLS has assessed an alternative extraction method using scCO2 to reduce these negative environmental impacts (WP5).

Development of guayule and Russian dandelion will reduce natural rubber imports and contribute to bio-energy production. At the time of writing the EU-PEARLS proposal (2007), multiple economic and political reasons to carry out the EU-PEARLS project were identified. Rubber produced in Europe could replace imports, especially in case of a production bottleneck in Asia. However, alterative natural rubber (ANR) crops were also proposed as sources of significant amounts of bioenergy and other co-products.
Europe expected to see a substantial increase in the acreage of energy crops, and indeed biofuels production strongly increased until 2010. However, the EU energy crop premium was dropped in 2008. In principle, bagasse or inulin (easily converted to ethanol) from ANR crops could to help meet the 10% mandate for biofuels in 2020. However, this mandate is now under heavy fire as many biofuels do not contribute to the fight against climate change. This shows that detailed LCA studies are required to determine the best use of guayule or TKS bagasse or other co-products such as TKS inulin. Uncertainties in inputs, processing, cultivation and other costs, and the value of the (co)-products (which depends on the processing methods), still preclude such calculations.
In 2007 set-aside was seen as an opportunity for ANR crops, as it would have allowed the production of crops for specified non-food uses, subject to certain conditions. However, set-aside has been abolished as well, meaning that ANR crops, especially TKS will have to compete with existing food crops for arable land. Guayule will not compete for land with food crops, as it can be grown in semi-arid regions, but it will have to compete with other crops for water: it survives but is not very productive without irrigation. However, guayule could replace irrigation-intensive crops such as cotton and rice, which will not only reduce water use, but also gives greater flexibility to respond to droughts. The latter issue is becoming even more pressing as climate change progresses, and southern Europe increasingly suffers from heat waves and droughts. The argument that Mediterranean countries are economically weak and could benefit from a new crop has been borne out by recent economic developments.
As a multiannual crop guayule has considerably higher establishment costs than annual crops. Support for establishment costs is possible under EU rural development regulations. Regulations also allow Member States to grant national aid of up to 50 percent of the costs of establishing multiannual crops.
The two desktop studies on biorefining and socio-economic aspects of rubber production from guayule and Russian dandelion (WP7) indicate that the value of current co-products is not likely to be very high (they do not provide a critical advantage over Hevea rubber, which benefits from rubberwood as a co-product). However, the resin fraction from guayule (co-extracted with the latex fraction and currently sold for 2 US$ per kg) may yield much more valuable end-products and applications. The major driving force for establishment of ANR crops remains its strategic value. Accordingly, the EU-PEARLS Consortium has answered the call for input on the Raw Materials Initiative, and recommended to include natural rubber as a strategic resource (WP7).
The EU-PEARLS proposal also emphasized that policy support for new crops is crucial in establishing ANR crops, especially as ANR crop cultivation and the processing facilities will constitute a locked chain similar to that for sugar beet. However, current CAP and the on-going CAP reform do not provide any specific support for non-food crops (except for energy or agro-fuel production), and it is not likely that such support can be introduced, as many conflicting forces act on the CAP reform.

Sustainability: environmental impacts of guayule and Russian dandelion. Guayule grown as a new crop in semi-arid regions of Southern Europe may help to fight the important problem of soil erosion in these regions due to its extensive root system, which is harvested only every 4-6 years. Guayule has no relatives in Europe with which it can hybridize, and the chance that it will become a weed is marginal.
TKS as an annual root crop is similar to sugar beet and potatoes for its impact on soil erosion. TKS was found to be much smaller than the common dandelion (T. officinale), a prolific weed, and caused no problems for crop rotation in the USA rubber emergency program during WWII: TKS hardly ever reappeared on the fields in the next year. It also did not establish itself in the wild after the program was ended. The experience of botanical gardens worldwide has shown that TKS is easily overgrown by more robust dandelions, such as T. brevicorniculatum (WP3). However, this also means that establishment of the crop is tricky, as it is easily overgrown by weeds.

Successful development of guayule and Russian dandelion will lead to the establishment of a new agro-industry business. To process guayule and TKS local processing industries must be developed, providing new commercial opportunities for farmers, as well as the potential to create and sustain employment both in the farming sector and in rural areas. Because ANR crops and the necessary processing plants would constitute locked chains, as is the case for sugarbeet, and the germplasm will be owned by breeding companies, it is likely that farmers and processing facilities will be tightly linked, perhaps as cooperatives, but more likely as new companies using contract farming and licensed germplasm.
The costs of the required facilities depend very much on the processing method, and also at the end of EU-EARLS the required investments can only be roughly estimated. Concerning TKS, true TKS (T. koksaghyz) germplasm was only available from November 2009, and significant amounts of biomass only from November 2010. Therefore, many processing experiments were carried out with T. brevicorniculatum material containing much less rubber, and processing experiments with TKS had to be scaled down and delayed. Therefore, further R&D is necessary, both for guayule as well as for TKS.

Sustainability: Exploiting alternative sources for natural rubber will contribute to reduce the need for synthetic rubber, which is made from a non-renewable resource. To reduce the use of fossil fuels, synthetic rubber could be replaced by NR, e.g. from a low input crop such as guayule. However, much of the demand for NR depends on the tyre market, which in turn heavily depends on the state of the economy, the availability and cost of gasoline, and climate change policies (WP7, socioeconomic study). Therefore, it is not at all certain that NR demand will increase any further. Also, detailed information on processing methods is necessary to be able to judge whether ANR really reduces environmental impacts.

Social and ethical aspects. The EU-PEARLS proposal stated that the development of a competitive alternative European source of natural rubber could have a negative impact on workers in the established Hevea rubber business. On the other hand, the expansion of guayule cultivation and processing could readily take place in conjunction with developing countries. Guayule is well suited to the climates of many developing countries and cultivation and processing in those geographic regions would also be driven by the shortage of supply issues and growing markets. Similarly, TKS is well suited for eastern European countries and may provide new sources of income to rural areas here.

Consumers and health. Established rubber production from guayule and Russian dandelion may lead to new products and new markets. Hypoallergenic latex from guayule was already considered for medical and consumer products. However, low-protein Hevea NRL gloves, better hospital management, reduced use of powdered gloves and better alternatives have strongly reduced the number of Hevea latex allergies, reducing the size of this market niche to already sensitized patients and workers.

A strategic impact on policy makers.
In view of the strategic nature of natural rubber, EU-PEARLS addresses a topic that has implications beyond RTD, agriculture and rural economies. EU-PEARLS has ensured a close linkage to policy makers by inviting them in to meetings organised by the Consortium; reporting of project results at appropriate European meetings; publication of interim and final reports; and targeted dissemination and science communication activities throughout the project, with updates sent to key figures in EC and national policy-making and national funding agencies. EU-PEARLS has also created direct links to associations and stakeholder groups.
As policy changes will strongly affect (determine) the economic viability of guayule and TKS, close links will be maintained to ensure that policy makers are informed about the requirements of RTD (and in future programs, also growers and processors). Based on the conclusion that the strategic interest in NR is the main driver of scaling-up, we have focused on this aspect.

There is great added-value in carrying out the work at the European level. Extensive activity in the development of industrial use of plant-based resources is fragmented across the Member States of the European Union. In the case of EU-PEARLS, European partners from all regions of Europe were brought together. This was essential in view of the complexity of the problem: development of two new crops (to be grown in entirely different geographic areas), concurrent with the development of processing industries to have the different components of the supply chain in place from the start. The project has shown that the plants can be cultivated in Europe, and that the rubber can be isolated from the two potential crops. Large-scale processing has not been set-up due to delays discussed above, and also because of the need for significantly improved germplasm (TKS basically is a wild plant, while guayule has been improved by selections only to a limited extent)
EU-PEARLS has reduced the large gap between EU and North American countries (USA, Mexico) in developing alternative rubber producing crops, and built new cooperations with international partners in the US, Kazakhstan and elsewhere. As such, this project has a strategic component providing competitiveness, social and industrial independence, and a secure supply of the strategic material rubber.
EU-PEARLS as brought together the key players involved in natural rubber biosynthesis, processing and utilization, which will catalyze developments in the field. The current team will provide the core for further development enabling other interested parties to enter the field. Many of the participants have not collaborated before. These new alliances may lead to new, unforeseen, exploitable ventures.

Dissemination.
A project website has been constructed (http://www.eu-pearls.eu/UK/) and updates have been prepared of the scientific state of the art and IP positions, desktop studies 'Biorefining co-products of guayule and Russian dandelion' and 'Production of natural rubber from new crops in Europe: a socio-economic evaluation' have been issued and a Midterm Workshop has been organized in Montpellier, France, in October 2010 (http://www.eu-pearls.eu/UK/Midterm+Meeting/). As an element of the desktop studies on by-products and socio-economics a summary and comparison of current insights on the economics of guayule and Russian dandelion rubber production with the conventional production from the Rubber tree is now available. The valorisation of by-products from both crops will strongly increase the economic feasibility. In Russian dandelion the inulin co-extracted from roots can be used as a building block for chemicals and in feed applications. Furthermore, the leaf material may be used as cattle feed. Remaining resins and bagasse from guayule can be exploited for chemicals and biofuel production.
The installation of an End-user group, which intends to produce and test latex and rubber samples in
parallel with the EU-PEARLS Project, is currently underway. Industrial partners involved in this End-user group will test the latex and rubber in their product portfolio.
Many dissemination activities have been performed and several publications have appeared or are in preparation. The complete list is shown elsewhere.
Major events were:
1- 2010 Midterm Workshop, Montpellier, FR: The future of natural rubber; (www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects/News-and-agenda/Show-16/2010-Meeting-The-future-of-natural-rubber.htm)
2- The Mid-term review organized by the EC in Brussels on 11 November 2010 yielded a number of valuable project related suggestions from the evaluation panel in addition to qualifications "impressive" for scientific progress and "excellent" for project management and how the consortium dealt with set-backs encountered.
3- 2012 Final Congress: BioRubber for Europe in Global Perspective, organized by the EU-PEARLS consortium to mark the end of the FP7 EU-PEARLS (www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects/News-and-agenda/Show-16/2012-Congress-BioRubber-for-Europe-in-Global-Perspective.htm).
The Abstracts / Presentations of 1- and 3- are available through the websites indicated.

Major results and deliverables.
As final results the project is expected to yield a number of important results and deliverables, including:
- Analysis of rubber biosynthesis (proteome, particles, genes)
- Methods to channel metabolites to rubber biosynthesis
A) Russian dandelion
- True T. koksaghyz lines submitted to international seed banks,
- New hybrid lines between T. koksaghyz and other Taraxacum species
- Genome maps, libraries and markers for rubber quantity and quality
- Rubber proteome
- Method to preserve TKS seeds
- TKS hybrids and EMS mutants
- Genetic maps of TKS (QTL, AFLP, COS, EST, sequence data)
- Field trial and agronomical data
- Laboratory scale latex/rubber extraction process and design for large scale extraction
- Assessment of TKS rubber physical and material properties
- Results of processing and compounding for prototype development
- Prototypes: car tyres
B) Guayule
- Selection of best guayule lines for cultivation in Spain and France
- Field trials, agronomical data and large-scale biomass production
- NIRS method to measure rubber content
- Laboratory scale latex / rubber extraction process and assessment of properties
- Results of processing and compounding for prototype development
- Prototypes: car tyres and medicinal gloves

These studies will be complemented with final reports on socio-economic impact and the wider societal implications and an implementation plan involving an End-user group (now under construction).



Exploitation of results.
This section summarizes the main results of the project and discusses how these results can be exploited.
The impact section of the Final Report describes the expected impact of the EU-PEARLS project. It looks back on the reasons to initiate the project, and finds that almost all justifications for the project still stand, except that the latex allergy issue (high prevalence of latex allergies in the 1980s and 1990s due to the HIV epidemic and inferior gloves) has become far less urgent. The great strategic importance of NR, and the risk of NR shortages, consequently escalating prices, and cartel formation by producers (and local consumers) remain threats to the supply of NR and NR latex to Europe.
The expected impacts listed in the project proposal were – for the most part – accomplished in the sense that a great body of scientific knowledge and experience with guayule and TKS has been gathered (as specified in the Deliverables), an excellent scientific network has been established, with many contacts to research institutes, companies and organisations outside the EU-PEARLS consortium and outside the EU. This has built a solid basis for further R&D, recognizing that the available germplasm, crop and rubber yield and processing technology will only allow commercialization with continued financial support.

Next steps
A) TKS germplasm. At the start of EU-PEARLS, our knowledge of T. koksaghyz (TKS) was limited to that available from the scientific literature and reports from the 1930s and 1940s. This means that no modern methods had been applied to the species prior to the EU-PEARLS project. Some breeding was carried out, indicating potential yield increases, but all plant material (roots, seeds, germplasm, hybrids, selections) was lost after the various countries stopped researching TKS after WWII.
After collecting true TKS germplasm in Kazakhstan by partners IBOT and ECER, and legal transfer of the collected material to the EU and the EU-PEARLS partners, it turned out that the plants labeled as TKS in all botanical gardens investigated actually belonged to another dandelion species (T. brevicorniculatum): work carried out before the start and during the first two years of EU-PEARLS was carried out with this species, which contains much less rubber than TKS. This left only 2.5 years to start with the domestication of TKS. It also hindered the development of processing methods as the efficiency of such methods strongly depends on the rubber content.
At the end of the EU-PEARLS project, true TKS germplasm deposited in seed banks is now available to the scientific community. First hybrids and selections have been made, and together with the knowledge on rubber biosynthesis genes and proteins, rubber particles, and the rubber molecules, acquired during EU-PEARLS, a solid basis has been prepared for a breeding program to start converting the wild TKS species to a crop. Such a breeding program has many targets, identified during EU-PEARLS. To name a few:
1- Root morphology: TKS roots are tangled (large clumps of soil are caught between the roots) and thin, instead of straight, smooth, fleshy and bulbous (e.g. like sugar beet)
2- Plant robustness: TKS plants are small and easily overgrown, instead of robust, fast growing, and high-yield
3- Yield: currently only a few hundred kg per ha
4- Rubber content: currently highly variable between individual TKS plants
Partners KeyGene and IBOT have expressed an interest in breeders’ rights, and wish to continue to work with TKS to improve its properties, and to use specific traits of TKS that could be used in other crops.
A wet, mechanical processing method has been developed to separate rubber from dried TKS roots. The method does not involve solvents, but it is a water-based extraction method that allows the simultaneous extraction of inulin, a potentially valuable co-product, from the remaining bagasse. The separation method constitutes possible IP although patents have not been applied for. Extraction of inulin and its use as a chemical building block from the bagasse (not a deliverable of the EU-PEARLS project) is one example of possible future R&D.
Discussions with potential R&D partners (e.g. the PENRA consortium in the US, or the Kazakhstan government) are taking place, but commercialization is not envisaged within the next 5 years.

B) Guayule. The main aims of the deliverables drawn up for guayule were to establish that guayule can be grown in Europe, to determine yields in response to location, irrigation and fertilisers, to set up processing, to develop screening and assay methods, to determine the properties of rubber, latex and by-products isolated from guayule, and to test gloves and tyres made from guayule rubber and latex. These goals have all been accomplished, as described.
Guayule lines were obtained from the USDA by CIRAD early in the EU-PEARLS project, while public domain information adequately describes methods for the extraction of rubber or latex from guayule. Whether Yulex technology is more advanced is unknown, but Yulex has been able to produce at least 20,000 to 30,000 L of latex concentrate. Two barrels of latex (each 200 L) were sent by Yulex by July 2011. Prof. Cornish of Ohio State University also offered to send latex and dry rubber.
Guayule grown in Spain had excellent biomass yields of 20 to 25 tons per ha per year, containing 8 to 13 percent of rubber. In principle, these yields are high enough to rival the rubber tree, where annual rubber yields of 2 tons per ha and year are considered excellent. However, only a part of the rubber and/or latex can be extracted using current methods.
Guayule grown in Montpellier had to cope with wet and very cold winters, which killed many plants. Some lines did not survive at all. Nevertheless, some lines survived well, and could be used as a starting point for breeding guayule lines that tolerate the occasional cold and wet winters in the Mediterranean. Seeds from the cultivation program in France and Spain will be stored and kept for further research.
Experience has been gained with the application of processing methods described in the scientific literature and in patents. Supercritical CO2 (scCO2) extraction was used for the extraction of polar compounds, and allows the production of purified natural rubber. This experience has been documented and is available as a basis for further R&D.
The development of a NIRS (Near Infrared Spectroscopy) method to determine the rubber and resin content of biomass samples, and the calibration of this method with solvent extractions, now allows the fast measurement of rubber and latex content in the lab. It is envisaged that the method can also be applied in the field.
Commercialization of guayule latex and rubber in the EU is envisaged within the next 5 years only, if investors can be identified to design, build and operate a large-scale extraction facility. More likely is the construction of a small demonstration plant, which will need support from the EU or other funding sources.



Exploitation plans of individual Consortium Partners.
1a- Stichting DLO, Food & Biobased Research (DLO-FBR)..
DLO-FBR will attempt to further develop its expertise acquired in the field of genes involved in the flux through the mevalonate pathway and in the polymerization of IPP units toward small, medium and high molecular weight isoprenoids.
In addition, the knowledge gained on the water-based extraction process of dry TKS roots will be further developed and integrated into the design of pilot or large-scale extraction facilities. This process allows the extraction of both dry rubber and inulin.
Close collaboration between various scientific and industrial partners from EU-PEARLS and our track record enabled us to submit a project proposal for Dutch governmental funding (Topsectoren) together with these partners. Furthermore, we are coordinating a project proposal for EU FP7 call on ‘Life sciences, biotechnology and biochemistry for sustainable non-food products and processes with a consortium consisting of several partners from the EU-PEARLS consortium. Contacts are on-going with non-European governments for setting up research projects on natural rubber from TKS and, if possible for climatic reasons, guayule. Together with DLO-PRI and KeyGene a new initiative is underway to implement the EU-PEARLS accomplishments in Kazakhstan: KZ-PEARLS.

1b- Stichting DLO, Plant Research International (DLO-PRI).
The exploitation plans of the results and knowledge obtained from the EU-PEARLS project for Plant Research International (DLO-PRI) are in the field of gaining (funding for) new research projects on both TKS and guayule as new (European) rubber crops. Dissemination of the obtained results and expertise has led to a track record in research on TKS rubber biosynthesis and guayule agronomy.

The collaboration between several industrial partners from EU-PEARLS together with new industrial partners enabled us to submit a proposal on Russian dandelion for Dutch governmental funding (Topsectoren) as a consortium. We coordinated a project proposal for EU FP7-KBBE call on Support for demonstrating the potential of biotechnological applications with a consortium of several EU-PEARLS partners in combination with new industrial and scientific partners, called DRIVE4EU, on demonstrating the potential and application of Russian dandelion as new natural rubber and inulin crop. This proposal has been granted and will start at the end of 2013.
The protein expression data obtained by proteomics analysis of Russian dandelion rubber biosynthesis will be used for biomarker development for molecular breeding of TKS. The proteomics results will be published in international scientific journals. DLO-PRI has carried out guayule agronomy studies in Spain, showing that high biomass yields and high rubber content can be obtained. Furthermore, genomic and gene expression data on rubber genes was collected. The results will be published in peer-reviewed journals and reported at conferences. Seeds and data will be used in new projects, provided that funding is secured.
- The agronomy trials in Spain will result in scientific journal publications. One manuscript co-authored with CIRAD on NIRS is in review.
- The cultivar trial in Spain, including the genomic and expression data on rubber genes: peer-reviewed publication and conference paper
- The offspring of seed multiplication in Spain will be used in new projects. Outcrossing offspring could be the start of a European breeding program and the start of a segregating population (mapping population) for the study of the genetics of yield, rubber and resin traits.
- Data on irrigation and fertilisation can be used in new projects, and to advise potential growers
- Expression data on rubber genes in 6 guayule cultivars can be used to develop a set of SNPs for genetic mapping studies in new projects
- Participation or coordination of a new EU-project on guayule biomass, resin and rubber, in which results of EU-PEARLS will be brought in as background.
- The guayule plantation in Cartagena, ES, is still maintained for the production of seeds and biomass delivery.


2- KeyGene N.V. (KeyGene).
KeyGene intends to breed elite plants that produce at least 3-4 times more rubber and inulin than the wild types and that are less costly to grow. Because of the specific in house expertise on dandelion biology and plant molecular breeding, its contacts, as well as its technologies, KeyGene is in a unique position to breed improved rubber dandelions. The improved plants will be protected by Breeders’ Rights in UPOV countries and in other places by cytoplasmic male sterility. Plants can also be protected by making agreements with the root processing industry, which will contract farmers for the growing of rubber dandelions (a closed production chain). Given the strategic importance of natural rubber, the tyre industry may also be interested in organizing the production chain of which elite cultivars will be a crucial factor. Because of the higher yields and lower cultivation costs the farmers or the root processing industry will be interested in buying KeyGene's dandelion seeds for a higher price. A similar business concept has been suggested by BASF for the Amflora potato (together with Avebe).

3- Westfälische Wilhelms-Universität Münster (WWUM).
The laboratories of Prof. Prüfer intend to further investigate isoprenoid (e.g. rubber) and carbohydrate (e.g. inulin) biosynthesis in different crop and non-crop species such as TKS. The gained knowledge will be used to assist breeding activities and to develop novel production chains and products. The results will be evaluated for patentability and published in international scientific journals. Moreover, the achieved expertise will be used to attract new research grants from either companies or national and international public funding agencies.

4- Université de Lausanne (UNIL).
The Prof. Poirier laboratory intends to further research the control of acetyl-CoA flux to industrially useful metabolites, including isoprenoids and NR. Depending on the data obtained, IP will be sought when appropriate and contacts with European companies will be established depending on the metabolite in question. Such contacts may lead either to further research grants (within the context of Framework Programmes) or to specific research contracts with companies.

5- Czech Academy of Sciences, Institute of Botany (IBOT).
A wide range of hybrids between T. koksaghyz and related triploid or tetraploid dandelions were obtained during the project. Many of these hybrids have agamospermous (non-sexual) reproduction and robust growth, while the rubber content does not exceed 7 %. The latest hybrid products are being analysed and promising lines will be registered and protected by Breeder's Rights in UPOV countries. IBOT will continue the breeding programme, either with KeyGene (preferably) or with a tyre industry partner (negotiations in progress), as another two years are needed to reach a line suitable for commercialization.

6- Basque Institute for Agrarian Research and Development (NEIKER).
Within the EU-PEARLS Project NEIKER has received diverse germplasm accessions of different TKS populations and related species and also characterized the different populations with respect to morphological and physiological characteristics, biomass production, latex, rubber and inulin content. Numerous crosses have been performed and superior genotypes with improved agronomic performance and morphology have been obtained.
On the other hand within EU-PEARLS several useful tools, markers and knowledge was developed which can be exploited and applied for marker-assisted breeding, such as the knowledge about the rubber biosynthesis pathway or the high density TKS linkage map and the results of QTL analyses.
Taking into account the new germplasm resources, which are available NEIKER will try to apply classical breeding through crossings and selection over several generations, in order to obtain agronomical interesting genotypes which can be converted into commercial varieties. For this purpose NEIKER is looking for appropriate calls for research funds, but are also sensibilising the Basque government to fund these activities.

7- Establishment Center Ecological Reconstruction (ECER).
High genetically diverse germplasm is a requirement for the success of every breeding program for improved rubber properties and yields, but also for the breeding of resistance against all kinds of pests and for the adaptation to diverse environments.
During the initial project phase it turned out that the Russian dandelion samples available at a number of seed banks all belonged to another species called T. brevicorniculatum that differs from T. koksaghyz with respect to morphological characteristics (cornicula are shorter) and to its rubber content which is much lower than in T. koksaghyz. Therefore, two (May 2008, May 2009) field collection trips have been made to the Tien Shan mountains in the Republic of Kazakhstan, where T. koksaghyz was originally discovered in 1931, where the diversity centre of the whole group is found and where ECER has discovered a new population of T koksaghyz recently. The collections were made by the Kazakhstan Establishment Center “Ecological Reconstruction” (ECER) and the Czech Academy of Sciences, Institute of Botany (IBOT). ECER is a leading centre for biodiversity research in the Republic of Kazakhstan. IBOT is the world’s leading expert group for the taxonomy of Central Asian dandelions. This collaboration ensured that true germplasm of T. koksaghyz was collected. Also other, related Taraxacum species were collected which were used in hybridization and introgression studies, in order to improve the vigor of rubber producing dandelions. A small part of the collected seeds were distributed among the other partners directly after collection and used for the other tasks. In addition, a description has been made of climate and soil conditions at the collection sites.
Root fragments and seedlings of the collected accessions were grown and propagated in the Republic of Kazakhstan by ECER at the Turkestan Botanical Garden. The conditions here for growing T. koksaghyz are very good since in this region the species was grown as a crop at a rubber Sovkhoz until 1948. Plants from a single accession were grown together in a single plot, isolated by distance from other such plots. This has maximized the crossing between plants from a single accession and has minimized the crossing between accessions, ensuring maintenance of maximum genetic diversity from the original collections. Multiplied seeds from these accessions were donated to the Kazakhstan National Germplasm Bank. Sub-samples were donated to other Germplasm Banks in Europe.
The EU-PEARLS consortium has received the formal permission from the Kazakh government to export T. koksaghyz wild type isolated and use these for scientific purposes.

8- Centre de coopération internationale en recherché agronomique pour le développement (CIRAD).
Although the EU-PEARLS has ended, guayule biomass is still available in Spain, and at a smaller scale (germplasm) in France, for more production of latex and crude rubber and for supplying seeds of the best cultivars to potential users. Contacts are continued with public and private research organisations and with rubber goods producers, for providing additional samples for further testing (contract terms are under discussion).
Guayule seeds can be made available by CIRAD on demand, but preferably within a framework of cooperation or consultancy with CIRAD and DLO-PRI for any private developers.
CIRAD’s objective is to continue guayule development in South Europe within the framework of a new European project. More detailed objectives are:
- to develop guayule cultivation in emerging countries, in Maghreb countries (Morocco, Tunesia), in West Africa (Benin, Burkina Faso), in East Africa (Madagascar) and in Central America (Guatemala), with close links with EU-based rubber users;
- to stimulate innovation in substitution of petrochemicals by bio-based products, and in resource efficiency;
- to develop NR materials markets in Europe and globally;
- to ease safe and sustainable access to raw materials as a central economic issue, because the global demand of natural rubber is to grow, given the expected increase in demand for transportation. Supply shortage in some sectors may have consequences which will have effect on societal important sectors;
In addition, CIRAD aims to continue its work on extraction and valorization of biomolecules of guayule biomass, to improve the efficiency of latex recovery, on the characterization of the non-allergenic guayule latex with clinical tests on allergens reactivity, on more efficient solvent extraction of the resins, on bagasse valorization for energy applications and on the development of an effluent treatment process to recycle the water used in the latex extraction process. In the field of agricultural research activities, CIRAD aims to produce and transfer guayule seeds now available through the EU-PEARLS guayule fields in France and Spain to potential developers of guayule. CIRAD will sign contracts with large or small enterprises willing to test guayule rubber properties produced by CIRAD with the EU-PEARLS guayule biomass available in Spain and France. CIRAD plans also to work on the further development of existing USDA lines to create European guayule lines that will fit better with European climate, mainly to improve cold resistance.
The future CIRAD research activities should prove that commercialization of a guayule rubber sector can be competitive to Hevea rubber and that natural rubber can be produced in Europe to reduce the risk of supply of an important raw material and to improve competitiveness of the European rubber goods industry. To prove the feasibility of a European guayule industry, CIRAD will look for industrial partners to build an experimental plant for latex and bulk rubber production with a capacity to process 1 to 5 tons of wet biomass per day. Latex and co-products of the plant will be tested by European SMEs of the rubber, pharmaceutical, green chemicals and wood industries to develop new products for new markets at a competitive price.
Foreground produced during the EU-PEARLS project includes guayule seeds, living guayule plants, harvested biomass, latex and dry rubber samples.

9- Apollo Vredestein (VRB). VRB intends to continue its activities towards the application of TKS and guayule rubber in different tyre formulations.
End-users. The End-user group is an initiative intended to establish a production and application chain for guayule (and later on TKS) latex and rubber and thus support the Implementation phase of EU-PEARLS. The chain comprises all steps from the establishment of a guayule seed collection, cultivation, harvest, extraction, utilization of side streams (resins, bagasse), compounding and a variety of applications, and their economical assessment.
At the start of the project it was envisaged that the chain would become active on such a scale (e.g. 1-5 ha) that sufficient samples would have become available to allow in house (initial) testing by the genuine End-users. As a matter of fact, several independent issues that were out of the control of the EU-PEARLS Consortium led to delays and a scaling down of production. Thus, sufficient material was available only to partners of the Consortium (VRB) or Subcontractors of CIRAD (CTTM, AFSSAPS). The Consortium was not in a position to supply samples of TKS and guayule latex and dry rubber to potential end-users, a number of which formed an end-user group.
The incentive for any company to join this end-user group was seen as the opportunity to source NR or latex material from different suppliers or with different properties, also with a view at the possibility that plant diseases such as South American Leaf Blight threaten the supply out of Asia. As long as this is not the case, the European supply will have to compete on the market with Hevea latex and NR both on quality and price. For commercial success, the EU-PEARLS Consortium must determine if they (eventually) can produce TKS or guayule latex or NR at commercially attractive prices and with a quality that meets the performance needs for the different market segments.
The main use of an end user group was to give feedback on the usability of the latex or NR in the different market segments. In exchange for the time/money invested on this evaluation, they were to be rewarded with a commercial advantage. This advantage could be any of the following:
1- Reduced price for a certain volume of product
2- First right of refusal to exclusivity of product use in their specific segment / geographical area
3- Time limited exclusivity in a segment / geographical area against a commitment of volume purchase.

Financing of samples. Near the middle of the EU-PEARLS project, a price of 50-100K€ for a 5 kg sample was quoted. However, this was seen as unrealistic by members of the end-user group. Companies are not willing to pay this price, knowing that in a few years samples may be offered for free for testing as the “guayule/TKS latex/NR extraction companies” will promote their product on the market, provided that yields and costs can be optimized. Thus, the cost of sample production would have had be borne by the project and, should certainly be part of a potential follow-up project to EU-PEARLS. These costs should be recuperated from the profit made once the product starts to sell on the market (i.e. from the company, which commercializes guayule/TKS latex/NR). As samples have not been provided to possible end-user during the project, this issue will have to be clarified in a possible follow-up project.
The following questions need to be answered :
1- what samples can be provided at what (reasonable) price?
2- what commercial incentive can be given to an end-user to pay for these samples and incur costs for testing them?

Conclusion. At the end of the EU-PEARLS project no samples were supplied to end-users, because latex and NR were produced later in the project, and also in smaller quantities than originally envisaged. Therefore, material was supplied only to the internal end-users of the Consortium: VRB and CIRAD’s Subcontractors CTTM and AFSSAPS.

List of Websites:

4.1.5 Public Web address:
1- www.EU-PEARLS.eu/UK
2- www.wageningenur.nl/en/Research-Results/Projects-and-programmes/eu-pearls-projects.htm.

Relevant contact details of
Partners:
1- (coordinator), Stichting DLO, Food & Biobased Research, DLO-FBR, NL, www.fbr.wur.nl.
Contact: Dr. Hans (A) Mooibroek, email hans.mooibroek@wur.nl phone: +31 317 840214.
2- Stichting DLO, Plant Research International, DLO-PRI, NL, www.pri.wur.nl.
Contact: Dr. Ingrid van der Meer, email: ingrid.vandermeer@wur.nl.
3- Keygene N.V. Keygene, NL, www.keygene.com.
Contact: Dr. Peter van Dijk, email: peter.van-dijk@keygene.com.
4- Westfälische Wilhelms-Universität Münster, WWUM, DE, www.uni-muenster.de.
Contact: Prof. dr. Dirk Prüfer, email: dpruefer@uni-muenster.de.
5- Université de Lausanne, UNIL, CH, www.unil.ch.
Contact: Prof. dr. Yves Poirier, email: Yves.Poirier@unil.ch.
6- Czech Academy of Sciences, Institute of Botany, IBOT, CZ, www.ibot.cas.cz.
Contact: Prof. dr. Jan Kirschner, email: kirschner@ibot.cas.cz.
7- Basque Institute for Agrarian Research and Development, NEIKER, ES, www.neiker.net.
Contact: Dr. Enrique Ritter, email: eritter@neiker.net.
8- Establishment Center Ecological Reconstruction, ECER, KZ, www.nip.kz.
Contact: Prof. dr. Issa Baitulin, email: risology@mail.ru.
9- Centre de coopération internationale en recherché agronomique pour le développement, CIRAD, FR, www.cirad.fr.
Contact: Ir. Serge Palu, email: serge.palu@cirad.fr.
10- Apollo Vredestein B.V. VRB, NL, www.apollovredestein.com; www.apollotyres.com.
Contact: Ir. Nico Gevers, email: nico.gevers@apollovredestein.com; nico.gevers@apollotyres.com.