Optimization of Oil Content And Specific Fatty Acids Traits of Crambe And Camelina As Industrial Oil Crops.

Transcription

1 Optimization of Oil Content And Specific Fatty Acids Traits of Crambe And Camelina As Industrial Oil Crops. Erwin Fajar Hasrianda (Reg. no: ) Master Thesis Submitted to the Laboratory of Plant Breeding at Wageningen University In Partial Fulfilment Of The Requirements For The Degree Of Masters of Science in Plant Sciences Specialization Plant Breeding and Genetic Resources November 2016

2 Abstract Currently, our modern society is using high amount of petroleum oil every day. However, petroleum oil is a finite natural resources and causing a lot of problem such as air pollution and global warming. Green oil from plant production can be an alternative oil because green oil is more environmental friendly and can produce high quality oil composition. Green oil itself already has a potential market, especially for oleochemicals industrial sector. In European Union, industrial sector is demanding high quality oil with specific fatty acids composition. Erucic acid and gondoic acid are among the most valuable fatty acid in European Union market. These fatty acids has potential market as the feedstock material for plasticizers, paintings, cosmetics, coatings high quality nylon and lubricants industry. Crambe and camelina were chosen as the new crop species to produce erucic acid and gondoic acid. To answer this societal challenge, European Union consortium launched COSMOS (Camelina & crambe Oil crops as Sources for Medium-chain Oils for Specialty oleochemicals) project. This project is aiming to improve economical value of crambe and camelina, for producing specific fatty acids production and the derivative products of crambe and camelina. As a part of COSMOS project, this research is focused on gaining information of oil content and oil composition in current crambe and camelina cultivars from various cultivation site (Italy, Poland and Netherlands) by using GC test, NMR measurement and solvent extraction method. This research also collecting information about biomass and seed production (kg/ha) of crambe and camelina field trial. Percentage oil content of crambe and camelina was significantly influenced by cultivation site and species factor while fatty acids composition in crambe and camelina oil were influenced country and species factor and interaction of those factor. For biomass (stem, leaves, pod, seed parameter) production in crambe and camelina, cultivar and harvest time were significantly influencing the biomass parameter. The information from this research is expcted to be used to design a better plant breeding project for crambe and camelina. Keyword : Crambe, camelina, gondoic acid, erucic acid, biomass production, GC test, NMR measurement, solvent extraction.

3 1. INTRODUCTION 1.1 Alternative oil source outside fossil oil for Europe Petroleum is playing major role in the modern society. Small amounts of it are used for chemical and petrochemical as their feedstock (raw material), while most of them (about 90%) is taken to energize transportation and electricity as heat source. Industry is using chemical feedstock from petroleum to make feedstocks, lubricants, synthetic rubbers, solvents, plastics, detergents, fibers, and others (Carlsson, 2009). Since petroleum can create environmental problems (increasing CO2 level in atmosphere), it is also a finite resource and an alternative yet more sustainable material which need to be explored so it can gradually replace petroleum-based material. Alternative oleochemicals came from plan oil sources can be a good option for feedstocks in petroleum-material based industry. With annual production of 129 metric tonnes, plant oil is important commodities in agriculture sector (FAO, 2008; Carlsson 2009). Current industrial feedstock is using approximately 15% of total plant oil production. It has diverse use in global market, such as soap, lubricants, detergents, solvents, chemical feedstocks, surfactants, paints, and cosmetics production (Carlsson, 2009). Although its very diverse function in industry sector, six main fatty acids are dominating the composition of most seed oil: oleic, linolenic, stearic, linoleic, and palmitic acid. They have 16, 12 and 18 carbon chains length (ibid.). Currently, high number of plant oil sources are coming from coconut and palm which grow in tropical area (tropical oil) and unable to grow in European Union (EU) environment. Attractive and promising approach come by developing wild plant oil species which produce great diversity of fatty acids. Those oil crops could be developed with modern plant breeding technique (Carlsson, 2009). In near future, these oil plat species are expected to produce a higher oil content with better fatty acids composition to fullfil the European needs of feedstock from oil crop. EU answered the question by launching COSMOS (Camelina & crambe Oil crops as Sources for Medium-chain Oils for Specialty oleochemicals), a project for developing potential oil crops which can be grown in diverse European soils and climate condition (Blaauw, 2014).

4 Carlsson (2009) states there are several requirements for future oil plant producers: ability to produce high quality and quantity oil per hectare and demand only low amount of agricultural resources; and high compatibility with current agriculture infrastructure and less probability of oil crops admix with oil food species (for example, through cross-breeding or mixing in supply chain process). Long-time preservation character of this new oil crop final and broad range of the industrial application are two strong points for plant oil. Furthermore, availability of compatible modern plant breeding technique which may be applied to these new oil crops can be a big advantage to improve production in a long run. 1.2 Fatty acids market for industry in EU In general, oil from plants can be used for two main products: food purpose and non-food (industry) purpose. It is necessary to keep oil plants for industrial purpose are not mixed with its counterpart. In a review paper from Carlsson (2009), it is explained that the main reason for this is because industrial-purpose oil contains special properties carbon chains (specific fatty acids) which have unique molecular structure. The technical oil qualities are essential for industrial feedstock, but might have poisonous effect and indigestible for human organs. The example of this is various types of unusual, unsaturated (triple, mono, conjugated), and branched molecules structure. Furthermore, carbon chains with different length types (short-, medium-, or very long-chained) as well as functional groups or added side chains are also important characteristics in industrial-purpose oil. Plants with these oil characteristics should be specifically dedicated for industry sector usage. Two promising fatty acids in oil plant are erucic (C22:1) and gondoic acids (C20:0). Both are broadly used in industry sector as their derivative products can be essential feedstocks in lubricant industry, paintings, cosmetics, plasticizers, nylon industry, coatings, and lipochemical industrial preparations (Blaauw, 2014). By improving the production of erucic (C22:1) and gondoic acids (C20:0) in crambe and camelina, it can be alternative for filling the demand of EU industrial fatty acids market in more environmental friendly manner, compared to using fatty acids from fossil oil. In order to gain more stable oil plant supply for EU fatty acid market, it will be beneficial if the oil crop can be suitably planted at multiple locations in EU areas and have good result in terms of seed yield, compatibility level with the current cultivation practices, ease of

5 harvesting, oil content, and low resource inputs (Blaauw, 2014). Potential oil crops for this purpose are crambe (Crambe abyssinica Hochst.) and camelina (Camelina sativa). 1.3 Crambe Crambe has already been well-adapted to grow in temperate climate and does not have probability to cross with any other present oil crop. Crambe oil can contain erucic acids around 60% while oil content is about 60% in its seeds (38% when including the pod). It produces a single seed inside a pot with oil yields up to 1 ton/ha (Carlson, 2007). Since its oil content is dominated by erucic acids, crambe has already been categorized as non-food oil plant producer. Other major fatty acids in crambe oil are palmitic (3%), stearic (5%), linoleic (9%), and oleic acid (16%) (Warwick and Gugel, 2003). The challenge in improving crambe as potential oil crop is its lack of genetic diversity (Mastebroek et al., 1994). Currently, breeding project for improving crambe genetic diversity is held (Blaauw, 2014). In taxonomy, crambe belongs to cruciferous family. Depending on plant s density and plantation season, its height is around 1-2m. Commonly, its flowers are yellow or white. A thousand of crambe seeds weigh about 6-10g with its greenish brown colour inside little capsule. One capsule can only contain one seed. The diameter of crambe seed is o.8-2.6mm. Crambe species are natively from Ethiopia highland and able adapting very well with Europe s cold weather. It can grow as winter crop in Mediterranean climate and spring crop in Northern Europe climate (Falasca, 2010). As explained by Castleman et. al., (1999), Crambe has great tolerance to frost and drought. It has short life cycle as it can be harvested in 90 days and bloom in 35 days. Mechanical harvest is possible in crambe since it has uniform maturity. Generally, the commercial cultivars of crambe take days before the seed is ready to harvest. Meanwhile, Wang et al., (2000) state that Crambe in Chengdu area in China needs much longer time ( days) before ready to be harvested. In addition, Falasca (2010) argues that crambe can be sowed in March or April (as winter crop after soybean) because its possibility in mechanized harvesting and its low cultivating costs,. Now, soybean farmers in Brazil are showing great in cultivating crambe in their field.

6 Crambe usually grows in the site with rainfall range of 350 to1200 mm, with annual temperature average in the range of 5.7 C to 16.2 C, and with ph soils range of 5.0 to 7.8 [9: Falasca (2010)]. Crambe roots may reach more than 15 cm depth so it provides tolerance in dry periods. Crambe cannot tolerate very wet or waterlogged environment and cannot grow well in the site without enough soil depth or rocky soils. During germination stage, crambe seeds are moderately tolerant to saline soils with a range of moderate soil temperature of 10 C to 30 C (Duke, 1983; IENICA, 2002; Falasca (2010). In the main vegetative stage, crambe grows well in the temperature of 15 C 25 C, even though it can tolerate higher temperature in blooming stage. Crambe requires sufficient supply of water when bloom, otherwise crambe crop might be lost and the oil content reduces due to hydric deficiency effect (Castleman et. al., 1999). Crambe tolerance level of frost is -4 C to -6 C (Castleman et. al., 1999). The lowest temperature for crambe vegetative growing is 6.8 C. High yields of this species may be expected when more cold resistant and early flowering crambe cultivars are planted in autumn-winter period (Meijer, 1996; Falasca, 2010). Regarding oil yield per ha and its seeds unique fatty acids composition, crambe has big potential to develop further as industrial oil producer for Europe regions. Tabel 1. Fatty acids composition in crambe seed w/w % (Bondioli., et al., 1999). Fatty acids % C16:0 1.8 C18:0 0.7 C18: C18:2 8.7 C18:3 5.2 C20:1 3.4 C22:0 2.0 C22: C24:0 0.7 C24:1 1.6 Other 2.5

7 1.4 Camelina Camelina is oilseed that known for its potential for low-input biofuel feedstock and its beneficial fatty acids content (Ghamkhar et al., 2010). There are several discussions on the origin of camelina. Ehrensing and Guy (2008) argue that camelina is native crop in Finland and Romania. On the other hand, Ghamkhar et al., (2010) believe Russia and Ukraine are the most possible origin of camelina in regard of high genetic diversity in both countries area. Additionally, Zohary and Hopf (2000) in Ghamkhar et al., (2010) state that central Europe is the where camelina originally came from. Dating back to bronze age of northern Europe, camelina oil was collected by crushing and boiling the seeds. The oil was used by people as medical use, lamp oil, and food. In many area of Europe, camelina is also known by the name of gold-of-pleasure or false flax (Ehrensing and Guy, 2008). Camelina is commonly planted as summer annual oilseed crop, although it can be sown as winter annual crop in milder climate. It has short-season crop which matures in days after sowing. The plant is tolerant in very frost seedling environment and can germinate in low temperature. Camelina needs only very few agricultural inputs, compared to other oil plant. It is often cultivated on marginal land. It has also good tolerance level against drought environment in regions with low rainfall level which usually is not suitable for most of oil crop (Ehrensing and Guy, 2008). Camelina will grow until 1-3 feet tall and have branched stems which will be woody when the plant is mature. The leaf s size is 2 to 3½ inches long, with arrow-shaped and smooth edge. This plant produces seed pods which resemble flax bolls. The seeds have rough surface and the size is quite small (1,000-seed weight is comparable to grams). In addition, Camelina has no seed dormancy. The colour of its mature pod is brown or dark tan. Different from mustard family s other members, the already mature camelina can hold its seeds tightly in branch and not easily fall to the ground. It makes seed shattering not a common problem in camelina (Ehrensing and Guy, 2008). Camelina has good response to phosphorus, nitrogen, and sulphur fertilizer application (Ehrensing and Guy, 2008). From research by Ghamkhar et al., (2010), it is showed that

8 additional phosphorus and nitrogen fertilizer can significantly increase camelina yield. Specifically, phosphorus fertilizer plays important role in increasing oil content while nitrogen fertilizer is essential in increasing camelina seed yield. Until recently, very less plant breeding effort or crop production development have been done for camelina. It means the full potential of camelina has not been explored yet. Due to its ability to be cultivated in marginal condition with less input cost, farmers in Montana, United States, are currently trying to produce camelina in large scale as low-input-cost oilplant by using dryland production system (Ehrensing and Guy, 2008). Camelina can produce yields range from 336 to 2240 kg of seeds per hectare in the end of plantation (Ehrensing and Guy, 2008). The seeds have oil content range between 30-45% which contain approximately 50% highly polyunsaturated fatty acids with linolenic acid and linoleic acid as the dominant fatty acids (Imbrea, 2011). The fatty acids composition in camelina compared to other oil crop (Ehrensing and Guy, 2008) is showed in Table 2. Tabel 2. Fatty acids composition in camelina compared than other oil crops in percentage. Oil source C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:1 Camelina Canola Lineseed Sunflower Camelina can produce up to 907 litters per hectare, lower than rapeseed plant but higher than oil produced from sunflower and soybean plant. With its high diversity level and combination with modern genetic improvement, camelina can produce higher amount of oil compared to rapeseed plant (Ehrensing and Guy, 2008). In addition, its good adaptability level and its less agricultural input requirement make camelina very promising as the future industrial oil producer in temperate regions such as Europe. 1.5 Oil content and oil composition measurement Oil is the most valuable part from crambe and camelina. Oil content and oil composition are important traits in oil production from plant. Oil content means total oil proportion that can be extracted in plant harvested organ (for crambe and camelina is in their seed part). Oil

9 content can be scaled by calculating oil weight percentage in total seed weight of crambe and camelina. The scale will be in %. If the plant can produce high amount of seed per hectare, then the higher their oil percentage in the seed means the more valuable the crops as oil producer plant. There are several methods available to gain the information about oil content and oil composition in crambe and camelina seed. First method for measuring oil content in a seed is destructive method by using chemical (solvent) extraction. This method requires seed destruction to remove oil material from solid material of the seed by using a liquid solvent. It is also commonly known as leaching. To optimize oil extraction by this method, the seed needs to grind in small particle so the interfacial area between seed and solvent increase (Lalas, 2012). Grinding the seed also useful to break the cell wall which usually trapping oil in a seed. Sufficient level of temperature and agitation can be useful to increase oil extraction efficiency of this method. n-hexane or hot water can be used as the liquid solvent for this oil extraction process. The last step is to distinguish the oil from the liquid solvent so the oil content of the seed can be calculated. However, in the present situation, solvent extraction can only be done in a largescale oil plant production. Fifty tonnes bio-oil per day are the minimum scale for extraction by using solvent method (ibid.). Second method for oil content measurement is using non-destructive instrument which able to distinguish the seed composition (water, solid, gas, and oil) without destroying the seed. Nuclear magnetic resonance (NMR) is a quite common instrument for measuring oil content by this method as used in Warwick and Gugel (2001) and Mastebroek et. al., (1994) to measure oil content in crambe and rapeseed seed. NMR instrument works based on magnetization field pulses to the seed. Magnetic wave from the instrument can cause proton particle (Hydrogen atom) in the sample become spinning. The spin pattern of proton in the sample is then received by the machine as pulses. Proton has different pulses signal when it is in water, solid, gas or oil compound. Based on different pulses signal received, the instrument can distinguish the solid composition, the water composition, the gas composition, and the oil composition of the sample (van As, 2016, personal communication). NMR test is one of favourite instrument in measuring oil content in the seed(s) because it can give the oil content estimation very fast (up to intact seeds per hour) in non-

10 destructive way and without harmful side effect. As the consequence, the seed can still be planted after the oil content is determined (Colnago, 2007). To determine fatty acid composition from oil plant sample, gas chromatography (GC) test is a common method as used in Lalas (2012), Warwick and Gugel (2001) and Yaniv (1998) experiments as well. GC experiment works based on different fatty acid compound weight separation. By adding compound (such as KOH) that can methylate fatty acids in the oil and manipulate the temperature of the solution, fatty acids composition in oil solution can be distinguished. GC machine from Agilent Technologies series 7890A GC System can calculate the fatty acids composition of oil plant (van Loo, 2016, personal communication). 1.6 Genetic x Environment interaction in crambe and camelina In order to develop a better crambe and camelina cultivar for industrial sector, genetic x environment interaction in crambe and camelina cultivation need to taking in to account. According Allard and Bradshaw (1964), plant breeders community have common agreement that genetic x environment interaction is an important factor for developing a better crop varieties. Additionally, Cooper and DeLacy stated that G X E interaction is a part of plant adaptation process. The heterogeneity of G X interaction can be used to investigate and to select desire genotypes for the desire trait(s) (ibid.). By having a better understanding G X E interaction of oil composition in crambe and camelina, the opportunity to breed a better cultivar for industrial sector become higher. 1.7 Research objectives As already mentioned earlier, oil production from crambe and camelina are the main interest of this research (part of COSMOS project). Another target is developing new type of cultivars which able to produce oil with better fatty acids composition for industrial sector. Taking as an important consideration, crambe and camelina cultivars should be able to cultivate in diverse environment background in Europe and have stable results in terms of oil production per hectare, oil content in the seed, and good oil composition (good fatty acid composition in the oil). The study focuses on different oil contents and their fatty acids composition from crambe and camelina that grow in different Europe region and in different years from various crambe and camelina cultivars. Specifically, this research attempts to

11 understand the factors which may influence production of oil in crambe and camelina and its industrial fatty acids composition to improve the production of those traits. Field test in various years and various locations throughout Europe as lab experiment (GC experiment, NMR experiment, solvent experiment) were used as methods to answer research questions. In brief, the main questions of this research are: I. Does the genetic x environment correlation strongly occur for crambe and camelina plantation in different location and different years? The research mainly focused on analysing the differences of oil content and its fatty acids composition traits from various crambe and camelina seed samples (having different years, different locations, and different cultivars). As the hypothesis, one crambe elite cultivar and one camelina elite cultivar can produce stable level of oil content and stable level of oil composition when planted in different year and different location. II. Does the proportion of biomass in crambe and camelina elite cultivar will be same? The research was focused more on comparing biomass proportion from crambe and camelina elite cultivars (three cultivars per each species) that were planted in Wageningen University field trial in The observed parameter for this research are stem dry weight, leaf dry weight, and grain dry weight. As the hypothesis, different camelina and crambe elite cultivars were expected to have different proportion of biomass. III. What can be the weaknesses of crambe and camelina elite cultivar when in the open field? The challenge from cultivating crambe and camelina current elite cultivars (three cultivars per each species) in the open field that might influence yield production, oil content, and fatty acids composition was known by cultivating it in Wageningen University field trial during spring 2016 cultivation. The information was described in this report as guidance to improve crambe and camelina elite cultivars in next season cultivation.

12 2. MATERIAL AND METHOD 2.1 Seed material Crambe and camelina seed material from Wageningen, The Netherlands, field trial in 2015 and 2016, Poland field trial 2016, and Italy field trial 2016 were used in this research. The seed material was used for GC test, NMR test, solvent experiment to calculate the difference of oil content and oil composition in crambe and camelina from different cultivar, different cultivation regions in Europe, and different plantation year. In addition, selected elite cultivars from 2015 Wageningen field trial were replanted in 2016 to find stable yield pattern over the year. 2.2 Field trial Field trial was held in Droevendalsesteeg field trial, Wageningen, The Netherlands, to see growing pattern of crambe and camelina when planted in an open field which represents The Netherlands environment background. The split plot design was used for the field trials, whereas different harvest time was considered as main plot and different cultivar factor as the sub plot. Three elite cultivars of crambe and three elite cultivars camelina were cultivated in Droevendalsesteeg field trial. Three crambe elite cultivars for Wageningen field trial were: Crambe_PRI _48, Crambe_Galactica, Crambe_PRI _59. On the other hand, three camelina elite cultivars for Wageningen field trial were: Camelina_Midas, Camelina_WUR _7, Camelina_13CS _31. Seed material from field experiment were also collected to supply for GC test, NMR test, solvent experiment, and for next season plantation. Field trial was held to collect the information on growing pattern, pest and disease attack, lodging level, leaf dry mass, flowering time, stem dry mass, seed and pod dry mass, bulk dry weight, specific leaves area index, and biomass distribution among crambe and camelina elite cultivars. The difficulties (obstacles) in cultivating crambe and camelina current elite cultivars (three cultivars per each species) in the open field that might influence yield production, oil content, and fatty acids composition were known by cultivating it in Wageningen University, The Netherlands, field trial during spring 2016 cultivation. The information was described in this report as the guidance to improve crambe and camelina elite cultivars in next season cultivation.

13 Field trial was started in 14 th of April 2016 by sowing crambe and camelina elite cultivars from Wageningen 2015 field trials. Each cultivar was harvested five times to measure growing pattern of their vegetative and generative stage. Each plot of experiment has 1.5mx1.5m dimension. Plants from sub plot that grown in 0.5mx0.5m area in the middle of each plot were taken as the harvest sample while the rest were used as border plant. Various methods were used to collect information from field trial. Ten per cent of samples were taken as subsample to separate their leaves, stem, and grain while the rest 90% was used to measure bulk fresh weight and bulk dry weight. The fresh and dry weight of their leaves, stem, and grain were measured separately by using analytical weight scale. After the fresh weight was measured, the sample was put in an oven with 105 C temperature for 25 hours to gain fresh weight. Data from dry weight and fresh weight were analysed to determine the biomass composition of sample. Biomass composition data from the sample can be used to determine the growing pattern of crambe and camelina during their field trial. Pest and disease attack pattern from this field trial were known by using visual observation method. Flowering time and lodging level were measured by using visual scoring. Flowering time was measured in every harvest time while lodging level was measured a week after harvest two. The last, specific leaves area index was measured by using Licor machine (to measure their leaves area meter) in Unifarm Wageningen and compared to leaves weight. The details harvest date order of this field trial were May 12 th (1 st harvest), June 13 th (2 nd harvest), July 5 th (3 rd harvest), August 1 st (camelina final harvest) and August 16 th (crambe final harvest). Based on Julian calendar, sowing date started in day 107 while harvest one until harvest three was done in day 133, day 165 and day 187 respectively. For final harvest (harvest four), it was split as day 214 for camelina and day 229 Julian date for crambe. In between plantation period weeding (in June 9 th ) and additional chemical fertilizer (in May 10 th ) were also done.

14 2.3 NMR test The NMR test was done in this research as a non-destructive method to measure percentage oil content (w/w) in crambe and camelina seed sample. By using NMR measurement, the seed that already used in this experiment is still expected to cultivate again next season without any side effect. This measurement was held by using NMR machine in NMR centre, Biophysics department, Wageningen University. The result of this test was expected to confirm oil content data that gained from solvent extraction method. Seeds were randomly taken as samples to measure in the NMR machine. To run the NMR test, the sample only required gr or equal ml in volume (depends on the species and the cultivars) of seed sample. NMR measurement protocol for small seed content from NMR centre, Biophysics department, Wageningen University was used. A minor adjustment was set so the machine can suit better to measure oil content specifically in crambe and camelina seed. The sample was tested by CPMG (Carr-Purcell-Meiboom-Gill) experiment in the NMR machine to measure T2 relaxation signal. It started with a 90 rf pulse nand continued by a series of 180 pulses. The time between the 90 and first 180 rf pulses was 520 microseconds (us). The time between the 180 pulses was 2x520. In between each 180 pulse an echo was detected at nx1040 us, where n is the echo number. NMR settings for the test were: 90 rf pulse: 3.5 us, 180 rf pulse: 6.8 us; number of data points per echo: 5, time between these data points 20us (also called oversampling per echo). It used rf pulses, so we generated 1500 echoes (although at longer time they disappeared in the noise). The time between each CPMG measurement (TR or repetition time) was 4 s. Each seed sample had eight measurements in average. All measurements were done in the same way. The decay curves were analysed by CONTIN software program, resulting in T2 spectra. The experiment integrated the T2 spectra between certain T2 values were only oil observed. The integration intervals results were served in Microsoft xl files.

15 2.4 Solvent oil extraction This research was used solvent oil extraction to gain more information on oil content percentage (w/w) of available crambe and camelina seed sample. Oil seed extraction protocol method from biochemical lab, plant breeding department, Wageningen university was used to extract the oil from the seed. All the crambe and camelina seed sample in this research was expected to have oil content in range of 30-50%. The fatty acid (FA) fraction was extracted from several crambe and camelina seeds (approximately 1.5 g seeds). Initially the seed was grinded using coffee mortar. Five hundred mg grinded material from every sample seed was randomly taken for oil extraction from total 1.5 g seed material in the coffee mortar. 1ml n-hexane per 100mg of the ground seed material was added in a test tube. The samples then were shaken for 30 minutes at 40 C before it went to centrifuge machine for separating the oil from grinded seed. Next, the supernatant that contains oil was taken to the new test tube. The extraction process was repeated three times and producing 12ml supernatant in total from 500mg grinded seed material. The hexane in new tube test was evaporated by using RapidVap machine or Nitrogen gas flow until only the pure oil left in the tube. 2.5 Gas Chromatography (GC) test GC test was done to measure fatty acids composition (%) of oil that produced from specific sample of crambe and camelina seed. Oil from crambe seed is expected to contain high level of erucic acid, while oil from camelina seed is expected to have high composition of gondoic acid, linoleic acid, and linolenic acid. Sample preparation for fatty acids composition of crambe and camelina oil seed measurement were prepared using isolation and methylation of fatty acids compounds standard protocol which is provided by biochemical lab, plant breeding department, Wageningen university. The protocol was slightly adjusted to maximize the fatty acids profiling in specific crambe and camelina seed. The details protocol for GC sample material preparation is attached. Furthermore, the detail setting of GC machine in this experiment followed the same setting as used by Cheng (2014). The information from fatty acids composition can be used to determine genetic x environment interaction in crambe and camelina plantation.

16 2.6 Genotype x Environments interaction in crambe and camelina Several cultivars seed samples from multiple field test locations and multiple years had been collected to gain more information to understand genotype x environment interaction of crambe and camelina. The difference of oil content and oil composition from seeds sample will be the main focus to understand genotype x environment interaction in crambe and camelina in this research. The data distribution was served in Microsoft xl file and analysed using ANNOVA in Genstat statistical software to see the correlation of genotype and environment factors of oil content and oil composition traits in crambe and camelina. 2.7 Statistical analysis The variability data from field trial, NMR test, Solvent oil extraction test, and the GC test was analysed by one-way analysis of variance (ANOVA) using GenStat statistical software (the split plot field trial data) and using R studio statistical software for other data to test environment treatment effect of crambe and camelina oil content and oil composition. If the treatment value were significant (confidence level was 5%), the tuckey statistical post-test was used to determine the difference among the means. Tabel 3. Example of Analysis of variance result in crambe and camelina fatty acids content data. Analysis of variance ==================== Variate: C18_1 Source of variation d.f. s.s. m.s. v.r. F pr. Country <.001 Species <.001 Country.Species <.001 Species.CV_name <.001 Country.Species.CV_name <.001 Residual Total

17 3. RESULTS 3.1 Oil content and oil composition in camelina and crambe seed from different cultivar, different year and different plantation location. To gain more information regarding consistency of oil content and oil composition from various crambe and camelina seed sample, solvent oil extraction, NMR experiment and GC experiment were done. Seed collection of crambe and camelina from various cultivars that planted in multiple plantation location and in multiple year were tested with those test. NMR test was chosen to measure oil content in the seed mainly because it is a nondestructive experiment. Data from solvent oil extraction was expected to confirm data from NMR experiment. The GC test was also done in order to measure oil composition (fatty acids composition) of all samples. The data of percentage oil content in crambe and camelina seed gained from solvent extraction and NMR measurement were slightly difference. The differences are described in figure y = x R² = Figure 1. Scatter correlation between solvent extraction and NMR measurement method in percentage oil content measurement of crambe and camelina seed sample Fatty acids composition from GC test GC test in this research project mainly focused on analysing the differences of fatty acids composition from crambe and camelina seed samples. The experiment can compare the diversity of fatty acids composition in camelina and crambe seed sample. the main interest industrial fatty acids in camelina is gondoic acids (C20:1). On the other hand, the main interest industrial fatty acids in crambe is erucic acids (C22:1). From GC test, fatty acid

18 composition is displayed in peaks. The fatty acids composition can be quantify by comparing the area under the peak (figure 2 and figure 3). Figure 2. Interface of the fatty acids quantification in camelina oil from GC machine. Figure 3. Interface of the fatty acids quantification in crambe oil from GC machine. Erucic acid has the highest peak among all the fatty acids in crambe oil. The composition of fatty acids in crambe and camelina oil is showed in figure 4 and figure 5. From figure 3 and figure 4, it is clearly seen that crambe and camelina have different fatty acids composition in their seed. Majority of fatty acids in crambe seed in this research is erucic acids which already consisting two per third of total fatty acids in crambe while gondoic acids content in camelina is only 17,9%. Poly Unsaturated Fatty acid (PUFA), combination from linoleic acids (C18:2) and linolenic acids (C18:3), is dominating fatty acids composition in camelina with total for almost a half of total fatty acids. With high content of erucic acids, Crambe seeds in this project already shows promising fatty composition for industrial interest. Meanwhile, fatty acids composition in camelina seed can be improved further by increasing the content of gondoic acids and decreasing the PUFA content.

19 Figure 4. Fatty acids composition from GC test in crambe seed samples. The percentage of erucic acids (C22:1) is dominating by two per third of total fatty acids content of crambe seeds, followed by C18:1 (13,5%), PUFA (10,6%), SAT (4,2%) and C20:1 (3.3%).. Figure 5. Fatty acids composition in oil from GC test in camelina seed sample. The percentage of PUFA (18:2+3) is dominating fatty acids composition in almost a half of the total oil. The rest of fatty acids composition is C18:1 (18,2%), C20:1 (17,9%), SAT (4,2%) and C22:1 (4,1%).

20 The differences level of specific fatty acid content of all seed sample was analysed by using annova table (attached). Gondoic acid, oleic acid, erucic acid, and saturated fatty acids (C16:0, C18:0, C20:0, C22:0, and C24:0) are the specific fatty acids that analysed in statistic software. The result is shown in figure 6 (gondoic acid), figure 7 (oleic acid), figure 8 (erucic acids), figure 9 PUFA, figure 10 (saturated fatty acid), figure 11 (erucic acid content in crambe sample only), figure 12 (gondoic acid in camelina sample only). The treatment factors are plantation site (country), plantation year, species (crambe and camelina), and cultivars. In this research, each fatty acid was influenced by different combination factors. From the statistical analysis, gondoic acid content was significantly influenced by country and species factor and interaction between country x species factor. Oleic acid content was influenced by country and species factor and interaction between country x species factor. Erucic acids was influenced by country and species factor. PUFA content was influenced by country and species factor and interaction between country x species factor. Saturated fatty acid content was significantly influenced by country and species factor. In addition, cultivar factors did not show significantly differences of erucic acid in crambe species and gondoic acid in camelina. More information of statistical analysis in fatty acids content is attached. Figure 6. Percentage of gondoic acid (C20:1) in crambe and camelina oil from GC test. When planted in Italy, Poland and Wageningen, gondoic acid content in camelina oil is significantly higher compare than in crambe oil.

21 Figure 7. Percentage Oleic acid (C18:1) in crambe and camelina oil from GC test. When planted in Italy and Wageningen, Oleic acid content in camelina oil is higher compare than in crambe oil. Figure 8. Percentage Erucic acid (C22:1) in crambe and camelina oil from GC test. When planted in Italy, Poland and Wageningen, Erucic acid content in crambe oil is always significantly higher compare than in camelina oil. Erucic acids in crambe comprise for about two per third of total fatty acids, while in camelina erucic acids is only comprise for about 4% of total fatty acids.

22 Figure 9. Percentage Poly Unsaturated Fatty acid (PUFA; C18:2+3) in crambe and camelina oil from GC test. PUFA content in camelina crambe oil is always higher compare than in crambe oil. Erucic acids in camelina is always comprise for about 40% until 50% total fatty acids, while in crambe erucic acids is only comprise for about 10% of total fatty acids. Figure 10. Percentage Saturated Fatty acid in crambe and camelina oil from GC test. Saturated Fatty acid composition in crambe and camelina are ranged between 3,5% - 4,5%.

23 Figure 11. Percentage Erucic acid (C22:1) content in various crambe elite cultivars. Erucic acid content in crambe seed are always in between 60% until 67%. Figure 12. Percentage Gondoic acid (C22:1) content in various camelina elite cultivars. Erucic acid content in camelina elite cultivars shows in between 16% until 19,5%.

24 3.1.2 Percentage oil content from NMR measurement. NMR measurement in this research project mainly is focused to see the percentage oil content from various crambe and camelina seed samples (different cultivation years, different locations and different cultivars) in a non-destructive method. The experiment was held to see the diversity of percentage of oil content in every seed sample in this research. Figure 13. Percentage oil content from crambe and camelina in different plantation locations in For Wageningen location, the plantation was done twice, in 2015 and in Generally, oil content in crambe crop 2015 treatment is higher than oil content in the rest of crambe and camelina seed sample. Figure 14. Percentage oil content from crambe and camelina elite cultivars in different plantation locations field trial. The oil content in seed sample was measured by using NMR machine. Both in crambe and camelina seed sample, Wageningen field trial tends to have higher oil content but not significant compared than Poland and Italy field trial.

25 Figure 15. Mean of oil content of crambe and camelina, planted in Wageningen 2016 which measured using NMR. Generally, camelina elite cultivars produced higher oil content compared than crambe elite cultivars but not significantly different compared than crambe oil content Percentage oil content from solvent extraction. Solvent extraction was held to measure the percentage oil content of every crambe and camelina seed sample. The oil from grinded seed material was extracted using hexane to separate the oil from other seed material. In the last part, hexane was evaporated and leaving pure oil material in the tube. By comparing initial seed material used and the oil weight in the tube, percentage oil content of the seed can be known. Percentage oil content of crambe and camelina seed from different plantation site was compared in statistical analysis. As the result crambe and camelina seeds have a significantly difference percentage oil content. In addition, different cultivation site gave different percentage oil content in crambe and camelina seed. However, interaction between two factors were not significant in NMR measurement.

26 Figure 16. The percentage oil content comparison among crambe and camelina which planted in Italy, Poland and Wageningen (2015 and 2016) from solvent extraction. The differences of oil content percentage crambe and camelina Italy and Poland plantation site are hardly seen. However, Wageningen field trial showed significance higher oil content compare than crambe and camelina oil content from Italy and Poland plantation site. Figure 17. Percentage oil content from crambe and camelina in different plantation locations. The oil content percentage was measured by using solvent extraction. Oil content in Wageningen was significantly higher compare than Italy and Poland field trial. 3.2 The proportion of biomass in crambe and camelina elite cultivar during their growth stage. Field trial experiment of crambe and camelina was done in Wageningen University field trial in the early spring of 2016 to gain information on biomass proportion of crambe and camelina during their growth. The research was focused more on comparing biomass proportion from crambe and camelina elite cultivars (three cultivars per each species) that were planted in Wageningen University field trial in The observed parameter for this research are leaf area meter, stem dry weight, leaf dry weight and grain dry weight. By

27 comparing those data, the proportion of crambe and camelina biomass composition can be described better. As the hypothesis, different camelina and crambe elite cultivars were expected to have different proportion of biomass. Based on Julian calendar, sowing date in this field trial was started in day 107 while harvest one until harvest three was done in day 133, day 165 and day 187 respectively. For final harvest (harvest four), it was split as day 214 for camelina and day 229 Julian date for crambe. Statistical analysis had been done in crambe and camelina 2016 field trial data. Harvest time and cultivar were used as the treatment factor. From the data analysis, only stem dry weight parameter is significantly influenced by harvest time factor and cultivar factor and interaction between harvest time and cultivar. On the other hand, leaves dry weight, pod, grain and harvest index of crambe and camelina are only significantly influenced by their cultivar factor while total biomass of the crop is only influenced by their harvest time. More info of the 2016 biomass statistical analysis is attached. In the harvest one, two and three, biomass in camelina only contained with leaves and stem. Camelina pod was having the maximum weight in harvest four and ready to be harvested. In contrast with pod content, leaves content in camelina starting to decrease in harvest three and the leaves was totally disappear in harvest four. Camelina was producing the highest stem mass content in harvest three and decrease in harvest four. Furthermore, Camelina_13CS _31 cultivar showed the best seed production among all the species planted. In the final harvest (harvest four), average harvest index for camelina was Different growing stage in a crop will requiring different nutrient composition. From the growing curve and biomass proportion graph that showed in figure 16 and 17. A more efficient fertilizer application for crambe and camelina crops cultivation can be predicted in order to maximize harvest yield in crambe and camelina.

28 Kg/Ha Camelina day 133 day 165 day 187 day 214 Julian Date seed pod Leaf Stem Total Figure 18. Growth curve of camelina in Wageningen field trial. Total biomass of camelina is constantly increase over the time. Camelina leaves only remains until harvest three. In harvest four camelina leaves organ was already fully disappeared and replaced by pod and seed organ. Crambe Kg/Ha day 133 day 165 day 187 day 229 Julian Date Figure 19. Growth curve of crambe in Wageningen field trial. Total biomass of crambe is constantly increase over the time and reach the peak in harvest four. In harvest four, all the leaves in crambe was already disappeared and replaced by pod organ. seed pod Leaf Stem Total 3.3 The possible obstacle of crambe and camelina elite cultivar plantation in the field. Described by Mastebroek (1994), environment and growing season are influencing the yield of crambe. The pattern of environment effect for growing crambe and camelina and the effect of the environment to the crambe and camelina elite cultivar in The Netherlands is not much known, especially for the six crambe and camelina elite cultivar (Ca_PRI , Ca_Galactica, Ca_PRI , Cs_Midas, Cs_WUR , Cs_13CS ). In order to gain better understanding of environment negative effect and agronomical weaknesses of

29 crambe and camelina elite cultivars in the field, crambe and camelina cultivation was held in Droevendalseesteg, Wageningen University field trials, in spring 2016 plantation season. Several information of possible obstacles were collected from the 2016 Wageningen field trial. There are pest and disease attack level, cultivar lodging level and agricultural general information. Having insight of these information can help farmers or researcher to design a better farming system that might useful in order to maximize crambe and camelina productivity level in the next growing season. This information can also be used to select some essential traits for developing a better crambe and camelina elite cultivars compare than the current cultivars. Crambe and camelina have different problem when planted in 2016 Wageningen field trial. The most clear problem from cultivating camelina is lodging. Camelina cultivar Cs_WUR was having significantly higher lodging level compared than other cultivars in 2016 field trial. On the other hands, the major problem in crambe 2016 field trial was pest attack. Camelina relatively resistant against pest attack during the experiment while no major lodging problem was observed in crambe Figure 20. Lodging level in crambe and camelina selected cultivar when planted in Wageningen field trial (higher score is more lodging). Camelina cultivar WUR has the worst lodging level and significantly higher compare than other cultivar.

30 Figure 21. Camelina cultivar with high lodging level in field trial. Figure 22. Leaves of crambe cultivar were eaten by plutella worm (diamondback moth larvae) in Wageningen field trial and zoom in photo of plutella worm. 3.4 Genetic x Environment interaction in crambe and camelina Genetic and environment factors have major role to influence yield result in cultivated crops. Stated by Allard and Bradshaw (1964), diversity of genotype x environment interaction can be used in a breeding program to develop a better cultivar. This breeding program can be set on crambe and camelina for industrial oil purpose. By combining several beneficial traits of crambe and camelina, a better cultivar can be produced in the near future. This elite cultivar is expected to produce high oil content with good fatty acids composition for industrial sector and can produce stable yield when planted in various European environmental background.

31 Oil content and oil composition traits are the major genotypes in this research, especially oleic acid, gondoic acid, erucic acid, PUFA and percentage oil content traits. By comparing oil result from several cultivars when planted in Italy, Poland and Netherlands, information G X E interaction of crambe and camelina was gained. G X E interactions in crambe and camelina are showed in figure 23 (PUFA traits), figure 24 (oleic acid traits), figure 25 (gondoic acid traits), figure 26 (erucic acid traits) and figure 27 (percentage oil content traits). Two major G X E interactions pattern were observed in this research. Potential G X E interaction on crambe and camelina were not much obtained in PUFA content and percentage oil content traits. In contrast, G X E interaction in oleic acid and in erucic acids traits seem more higher compare than in PUFA and oil content traits. Figure 23. Genetic x environment interaction curve from camelina and crambe oil in PUFA content traits when planted in Italy, Poland and Netherlands (Wageningen). Cultivar and in crambe variety show potential genetic x environment interaction. In camelina, cultivar , 887, and omega also show potential genetic x environment interaction.

32 Figure 24. Genetic x environment interaction curve from camelina and crambe oil in C18:1 fatty acids (oleic acid) content traits when planted in Italy, Poland and Netherlands (Wageningen). Cultivar , , , , Nebula, , Galactica, , in crambe variety show potential genetic x environment interaction. In camelina, cultivar , , , , , Omega, 887 also show potential genetic x environment interaction.. Figure 25. Genetic x environment interaction curve from crambe and camelina oil in C20:1 fatty acids (gondoic acids) content traits when planted in Italy, Poland and Netherlands (Wageningen). Cultivar , , Galactica, in crambe variety show potential genetic x environment interaction. In camelina, cultivar , , 887 also show potential genetic x environment interaction.

33 Figure 26. Genetic x environment interaction curve from camelina and crambe oil in C22:1 fatty acids (erucic acids) content traits when planted in Italy, Poland and Netherlands (Wageningen). Cultivar , , , in crambe variety show potential genetic x environment interaction. In camelina, cultivar WUR, , , Midas, 887 also shows potential genetic x environment interaction. Figure 27. Genetic x environment interaction curve from camelina and crambe oil percentage in their seed. Genetic x environment interaction is hardly seen and environment influence percentage oil content in crambe and camelina seed.

34 4. DISCUSSION This research mainly focused on exploring three aspect; (1) knowing crambe and camelina oil production level and their fatty acids composition as the alternatives oil crop for industrial sector, (2) gaining more understanding about crambe and camelina agronomical information and (3) gathering more information regarding possible obstacle on current cultivars of crambe and camelina when cultivated in an open field. To achieve those purposes, several experiment was already held, in laboratory and in field trial. NMR measurement, solvent extraction and GC test were the lab experiment in this research project. NMR measurement and solvent experiment were done in order to measure oil content percentage in every seed sample in this research. Additionally, GC test was done to see fatty acids composition of the crambe and camelina oil seed sample. Lastly, cultivation of six crambe and camelina current cultivar was held in Droevendalsesteeg field trial. All the research was done in Wageningen university, Netherlands. Information from oil and fatty acids production in crambe and camelina was used to determine genetic and environment interaction regarding oil production in crambe and camelina cultivation. Further, oil production result was measured through characterization and differentiation percentage oil content and oil composition (fatty acids composition in the oil) among crambe and camelina seed from several cultivation site in Europe. The cultivation sites for this research were; The Netherlands, Italy and Poland. Several crambe and camelina cultivars seed sample had been used in this research to compare their oil content and oil composition. Additionally, to gain more information on crambe and camelina elite cultivars agronomical traits, multiple years plantation had been tested in The Netherlands cultivation site, in Wageningen university field trial. 4.1 Genetic X environment interaction of crambe and camelina from different location and different cultivation year. The genetic x environment interaction oil traits of crambe and camelina modern cultivars is not much known yet. Specific of industrial sector purpose in the scope of this research project, percentage oil content in the seed and fatty acids composition of the oil are two most interesting traits to develop crambe and camelina crops.

35 Fatty acids composition in crambe and camelina seeds, percentage oil content from NMR measurement and percentage oil content from solvent experiment were used to understand G X E interaction regarding oil production in crambe and camelina. Different oil content and different oil composition from crambe and camelina that planted in different cultivation sites showed that genetic x environment interaction does exist clearly in crambe and camelina seeds oil content traits. The data from oil content percentage and the fatty acids composition in crambe and camelina seeds can be used to analyse which cultivar are possibly the most promising cultivar as an alternative oil crop for industrial sector. The information from this research also can be used as a guidance selection material to develop a better crambe and camelina cultivars for industrial purpose. This potential is similar with Cooper and DeLacy (1994) statement that the heterogeneity genotypes among environments factor can be used to improve crop adaptation level in two environment (or more). By combining the good genotypes where G X E interaction is seen a new cultivar with higher and more stable yield result can be developed. The genetic x environment interaction information from this research can be used to help designing a breeding program to upgrade current crambe and camelina become more beneficial for industrial sector green oil production Fatty acids composition in crambe and camelina seeds, Percentage oil content from NMR measurement. Percentage oil content from solvent experiment. Erucic acids (C22:1) is the most favourable industrial fatty acids in crambe seeds, while gondoic acid (C20:0) is the most desired industrial fatty acids in camelina seed. The composition of erucic acids in crambe seed and the composition of gondoic acids in camelina seed on this research was measured by using GC analysis. Oil seed sample was used to analyse their fatty acids composition in GC machine. From the GC experiment, erucic acids content is very dominating fatty acids composition in crambe oil seed. This data is expected since erucic acids is the favourable fatty acids from crambe oil. Moreover, crambe cultivars in this research was the elite cultivars which resulted through several breeding programs. Thus, crambe seed with high erucic acids content was a desirable result in this project.

36 Aside of erucic acids and gondoic acids composition in crambe and camelina oil sample, various fatty acids composition in the sample was also identified. They are oleic acid (C18:1), saturated fatty acids (C16:0, C18:0, C20:0, C22:0, and C24:0) and Poly Unsaturated Fatty acid (PUFA), combination from linoleic acids (C18:2) and linolenic acids (C18:3). Statistical analysis in this research showed that fatty acids composition in crambe and camelina oil was influenced by different combination factor. Gondoic acid content was significantly influenced by country and species factor and interaction between country x species factor. Oleic acid content was influenced by country and species factor and interaction between country x species factor. Erucic acids was influenced by country and species factor. PUFA content was influenced by country and species factor and interaction between country x species factor. Saturated fatty acid content was significantly influenced by country and species factor. In addition, cultivar factors did not show significantly differences of erucic acid in crambe species and gondoic acid in camelina Percentage oil content from NMR measurement. High percentage oil content in the crambe and camelina seed is an beneficial trait for industrial sector. The higher oil content in a seed the more economical the cultivar. Additionally, in order to produce high amount oil for industry, crambe and camelina should be able to have stable oil content performance when planted in different cultivation site in Europe and in multiple plantation years. To confirm the oil content in crambe and camelina seed samples, measurement using NMR machine was done. NMR machine has capability to distinguish oil, water, solid and air component in a seed. This research only focused in oil content in the seed. NMR was chosen because it is a non-destructive oil seed content measurement and can measure many seed samples in a relatively short time. From NMR measurement, oil content crambe in Poland and Italy field trial showed higher level compare than camelina oil content and camelina showed higher oil content when cultivated in Wageningen field trial. However, statistical analysis shows that percentage oil content in NMR measurement is only significance in plantation site. Compared than In Italy,

37 in Poland and in Wageningen 2016, crambe cultivation in Wageningen 2015 shows significantly higher oil content. Environment background might play major role to determine percentage oil content in crambe seed. During early stage cultivation in Wageningen 2016, rain was rarely occur while starting in harvest three until final harvest, rain fall pretty heavy. In this case, climate might influence yield production of the crops. This finding is linier with Falasca et. al., (2010) findings that crambe can produce varies yield result depends on their cultivating regions. further, good water supply during blooming is essential to make crambe able to produce good yield. Fontana et. al., (1998) state that good weather condition in seed filling, flowering and emergence stage is essential phases for camelina production. Less favourable climate and environmental background might causing crambe and camelina in Italy, Poland and Wageningen 2015 were unable to achieve optimum oil content production in their seed. This findings might indicate that Wageningen environment might more suitable for growing crambe crop with higher oil content compare than Italy and Poland region Percentage oil content from solvent experiment. Solvent extraction is one of the most common method to extract oil from plant material. Because this method already commonly used to extract oil from plant, the protocol and the material for this method already good/settle and can be easily to found. The weaknesses of this method are destructive measurement, laborious and time consuming. The solvent experiment showed that crambe and camelina in Poland and Italy field trial is having equal oil content level. In Wageningen field trial the oil content level in crambe and camelina is relatively not stable, depends on cultivation years. The data is different from oil content data in NMR measurement. From the statistical analysis in solvent extraction data, species and cultivation site (country) were significantly influencing crambe and camelina oil content. It is possible that crambe and camelina need specific environment background, especially climate factor in order to be able to produce maximum oil content, or pest attack in crambe case. This data similar with Falasca et. al., (2010) and Fontana et. al., (1998) statement that wrote environmental background have a power to influence oil content in crambe and camelina crops.

38 In this research, data of oil content level from NMR measurement might more reliable than from solvent reaction. During the solvent experiment, rapidvap machine for evaporating hexane from oil solution was broke. As an alternative, this research was using nitrogen flow that combined with thermo-shaker machine to collect crambe and camelina pure oil from hexane solution. However, for this specific experiment, using nitrogen flow and thermoshaker machine is a time consuming method and relatively less reliable method compare than using RapidVap machine. This information can be seen from final oil content of crambe and camelina that can reach 65% in the end of the experiment. Normally, oil content in crambe and camelina is not more than 50%. This result appear Even though the time allocation for evaporating hexane from oil was already more than twice if using RapidVap machine. this condition might influence the data quality of oil content from solvent experient. As the implication, we are unable to make any reliable conclusion from solvent experiment in this research. for the additional mark, it is important to have a good back up machine or a back up protocol to evaporate hexane from oil solution in case in the middle of experiment using rapidvap machine is no longer possible to do. 4.2 The proportion of biomass in crambe and camelina elite cultivars during their growth stage Crambe and camelina have different biomass proportion during their vegetative and generative stage. To gain more information regarding biomass composition and their harvest index, three crambe elite cultivar and three camelina elite cultivar were cultivated in Droevendalsesteeg field trial, Wageningen university, The Netherlands. Biomass data, especially the dry weight of stem, leaves and grain were collected. In the earlier stage, biomass composition is dominated in stem and leaves part. However, in the late stage, all the leaves in crambe and camelina was fall. Thus, only grain, pod and stem part biomass that still available in the harvest time. Crambe and camelina are tend to accumulate their energy and nutrient for their stem and leaves in their early growth stage (the vegetative phase). Reaching the vegetative stage, crambe and camelina gradually decrease investing their energy and their nutrient in their leaves and their stem because they have to start growing their generative organ (flower and

39 grain). It is essential to distinguish different fertilizer type depends on their vegetative or generative stage. furthermore, crambe and camelina are also requiring good weather condition during their blooming and their seed feeling phase in order to produce stable and high yield. Cultivar factor was significantly influencing biomass production in tem, leaves, pod, grain and the harvest index of crambe and camelina crop while harvest time significantly was influencing stem and total biomass production. Camelina_13CS _31 cultivar gives the best seed production among all the species planted. This result also supported by the fact that this cultivar also producing the highest stem and leaves dry weight. It probably that among all the cultivars were tested, Camelina_13CS _31 cultivar is the most suitable cultivar to grow in Wageningen environment. This is similar with Moser (2010) statement that the variety in yield might attribute to climate, the quality of the growing location (such as soil type, water supply, pest level) and the agricultural input of the crop. From this findings, Camelina_13CS _31 cultivar can be used as a breeding material to develop a higher and more stable yield for industrial green oil production. In opposite for 2016 Wageningen field trial crambe, no specific cultivar from this experiment can be determined clearly as the most promising cultivar for further crambe breeding material. 4.3 The possible obstacle of crambe and camelina elite cultivar plantation in the field. In order to gain more insight of major obstacle in crambe and camelina cultivation, observation in crambe and camelina growth in Wageningen field trial was held. Several factor that might give major impact in crambe and camelina yield were identified. Major factor might influence crambe and camelina yield. For camelina, major obstacle was lodging level, while in crambe major obstacle was pest attack from plutella worm (diamondback moth larvae). As like wrote by Ehrensing and Guy (2008), camelina seed and pod relatively strong and not easy to be scattered. As the consequence, even though lodging cultivar is occur in camelina, the seed production relatively will not decrease significantly. This might be the reason why camelina_13cs _31 cultivar yield is significantly higher than crambe yield. This result

40 might also happen because camelina crops suffer less severe pest attack compare than crambe crop. During the cultivation until harvest time, camelina leaves organ relatively remains in good condition and almost without major pest attack. This findings is supported by Brown et. al., (1999) that insect pest attack in Brassica crop can decrease the crop yield up to 37%. The expected crambe yield in field trial can be higher than the actual 2016 Wageningen field trial if less insect attack occur in field trial. On the other hand, breeding program to develop a better pest resistant crambe cultivar might be essential for the future plan to develop a better crambe cultivar. Although 2016 Wageningen field trial is already giving some insight for improving crambe and camelina current cultivar, the breeding program should also be tested in more diverse environment background in Europe and in more experiment year. This need to be done so the future cultivar can overcome lodging and pest susceptibility traits. A new crambe and camelina cultivar which able to overcome these weaknesses and able to show stable result throughout Europe landscape can be rally beneficial for promoting crambe and camelina cultivation in industrial scale. 5. CONCLUSION This research showing that crambe and camelina have a big potential to develop as the alternative industrial oil crop. Crambe is able to produce high content of erucic acids while camelina has a potential to develop to produce high content of gondoic acids. Both of fatty acids has a promising market as a material to produce plasticizers, bio-lubricants and high quality nylon which has high economical value. Cultivation site, crop species and interaction of those factors can significantly influence oil content and desire fatty acids composition in crambe and camelina oil. Two possible obstacle in crambe and camelina plantation were identified. Lodging level in camelina and pest susceptibility in crambe are the weaknesses that might be essential to improve in crambe and camelina cultivars. Overcoming those traits might be useful in order to make crambe and camelina become more able to produce a stable high yield level through various European environmental background. Especially in camelina, the fatty acids composition can be upgraded further by increasing gondoic acids content and decreasing the PUFA content. From

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43 APPENDIX Appendix 1. Mean of fatty acids composition and percentage oil content of crambe and camelina seed. C18:1 COMPOSITION IN CRAMBE AND CAMELINA CULTIVARS CV_name Names Italy Poland Netherlands Mean Ca_PRI_Elst_ ,44 12,74 11,94 12,7 Ca_PRI_Elst_ ,93 12,84 13,65 13,1 Ca_PRI_Elst_ ,18 12,86 12,4 13,1 Ca_PRI_ ,63 12,46 14,5 13,2 Ca_PRI_ ,36 13,19 13,08 13,2 Ca_Nebula Nebula 14,71 12,92 12,67 13,4 Ca_Galactica Galactica 14,03 12,69 14,09 13,6 Ca_PRI_Elst_ ,59 13,37 14,38 13,8 Ca_PRI_Elst_ ,9 13,47 13,02 13,8 Ca_PRI_Elst_ ,26 14,82 14,08 14,1 Cs_13CS ,96 12,23 17,5 15,6 Cs_Omega Omega 19,57 13,12 16,3 Cs_Midas Midas 17,86 15,13 17,69 16,9 Cs_13CS WUR 16,57 15,71 18,68 17,0 Cs_13CS ,78 13,53 17,88 17,1 Cs_WUR ,63 13,75 19,89 17,1 Cs_14CS ,64 13,68 17,04 19,1 Cs_14CS ,93 13,48 26,03 19,1 Cs_13CS ,92 13,89 18,46 19,4 Cs_14CS ,34 21,9 17,67 19,6 Cs_13CS ,93 18,77 23,43 20,0 Mean 16,82 14,12 17,22 LSD Country (p=0.05) 0.65 LSD Species 0.49 LSD Species.Cv 2.07 Fprob Country <0.001 Fprob Species <0.001 Fprob Species/Cultivar <0.001 Fprob C*S/C <0.001

44 PUFA (C18:2+3) COMPOSITION IN CRAMBE AND CAMELINA CULTIVARS CV_name Names Italy Poland Netherlands Mean Ca_Nebula Nebula 8,97 10,49 9,61 9,7 Ca_PRI_Elst_ ,43 10,18 9,83 9,8 Ca_PRI_Elst_ ,15 9,52 10,95 9,9 Ca_PRI_Elst_ ,21 10,12 10,83 10,1 Ca_PRI_Elst_ ,47 10,18 10,67 10,1 Ca_Galactica Galactica 8,7 10,72 11,15 10,2 Ca_PRI_ ,63 10,7 10,78 10,4 Ca_PRI_Elst_ ,27 13,61 9,45 10,4 Ca_PRI_Elst_ ,96 12,38 10,7 10,7 Ca_PRI_ ,38 10,5 11,71 10,9 Cs_14CS ,75 45,25 44,56 45,9 Cs_14CS ,53 53,82 44,92 46,1 Cs_13CS WUR 46,26 48,46 45,12 46,6 Cs_14CS ,9 53,27 40,91 46,7 Cs_13CS ,32 51,09 44,71 47,4 Cs_13CS ,33 53,93 45,98 47,4 Cs_13CS ,01 53,24 45,8 48,0 Cs_WUR ,37 53,2 44,5 48,0 Cs_Midas Midas 46,24 51,77 46,88 48,3 Cs_Omega Omega 44,03 53,51 48,8 Cs_13CS ,82 53,39 46,46 48,9 2 LSD Country (p=0.05) 0.65 LSD Species 0.49 LSD Species.Cv 2.07 Fprob Country <0.001 Fprob Species <0.001 Fprob Species/Cultivar <0.001 Fprob C*S/C <0.001

45 C20:1 COMPOSITION IN CRAMBE AND CAMELINA CULTIVARS CV_name Names Italy Poland Netherlands Mean Ca_PRI_ ,55 3,79 2,58 3,0 Ca_PRI_Elst_ ,09 3,34 2,87 3,1 Ca_Galactica Galactica 4,7 2,32 2,55 3,2 Ca_PRI_ ,12 3,4 3,81 3,4 Ca_PRI_Elst_ ,23 2,94 3,28 3,5 Ca_PRI_Elst_ ,51 3,59 3,44 3,5 Ca_PRI_Elst_ ,24 4,22 3,08 3,5 Ca_PRI_Elst_ ,1 3,74 3,7 3,5 Ca_Nebula Nebula 3,55 3,59 3,42 3,5 Ca_PRI_Elst_ ,57 3,76 3,71 4,0 Cs_14CS ,51 16,31 18,27 17,0 Cs_13CS ,47 16,14 18,29 17,3 Cs_14CS ,37 16,23 17,54 17,4 Cs_Omega Omega 18,88 16,31 17,6 Cs_13CS WUR 17,99 16,54 18,4 17,6 Cs_13CS ,47 16,39 18,47 17,8 Cs_Midas Midas 18,33 17,06 17,98 17,8 Cs_WUR ,36 16,07 19,08 17,8 Cs_13CS ,87 16, ,1 Cs_13CS ,56 20,53 18,17 18,8 Cs_14CS ,94 18,91 19,0 LSD Country (p=0.05) 0,31 LSD Species 0,24 LSD Species.Cv 1,00 Fprob Country <0.001 Fprob Species <0.001 Fprob Species/Cultivar 0,006 Fprob C*S/C 0,002

46 C22:1 COMPOSITION IN CRAMBE AND CAMELINA CULTIVARS CV_name Names Italy Poland Netherlands Mean Ca_PRI_Elst_ ,56 60,86 65,93 64,8 Ca_PRI_Elst_ ,91 62,4 68,07 65,5 Ca_PRI_ ,03 66,54 65,8 66,1 Ca_PRI_Elst_ ,78 66,64 67,16 66,2 Ca_PRI_ ,01 66,66 65,3 66,3 Ca_Nebula Nebula 67,05 66,81 65,51 66,5 Ca_PRI_Elst_ ,28 67,9 64,61 66,6 Ca_Galactica Galactica 67,44 67,53 64,88 66,6 Ca_PRI_Elst_ ,13 68,38 67,46 67,0 Ca_PRI_Elst_ ,93 66,24 68,59 67,3 Cs_13CS WUR 4,53 2,32 3,14 3,3 Cs_WUR ,32 3,27 3,12 3,6 Cs_13CS ,89 3,62 4,29 3,6 Cs_13CS ,14 3,59 4,85 3,9 Cs_13CS ,32 3,55 4,02 4,0 Cs_14CS ,27 3,61 4,16 4,0 Cs_14CS ,48 3,42 4,16 4,0 Cs_13CS ,59 3,67 4,1 Cs_Omega Omega 4,81 4,05 4,36 4,4 Cs_Midas Midas 4,3 3,46 6,14 4,6 Cs_14CS ,32 4,73 3,96 4,7 LSD Country (p=0.05) 0.77 LSD Species 0.59 LSD Species.Cv 2.47 Fprob Country <0.001 Fprob Species <0.001 Fprob Country*Species n.s. Fprob Species/Cultivar n.s. Fprob C*S/C n.s.

47 Appendix 2. Statistical analysis. STATISTICAL ANALYSIS Saturated Fatty Acid ## Analysis of Variance Table ## ## Response: SFA ## Df Sum Sq Mean Sq F value Pr(>F) ## Country e-07 *** ## Species ## Country:Species ** ## Residuals ## --- ## Signif. codes: 0 '***' '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 ## LSD t Test for SFA ## Groups, Treatments and means ## a Italy:Crambe ## a Wageningen:Camelina ## ab Wageningen:Crambe ## ab Italy:Camelina ## b Poland:Crambe ## c Poland:Camelina Analysis of variance ==================== Variate: C18_1 Source of variation d.f. s.s. m.s. v.r. F pr. Country <.001 Species <.001 Country.Species <.001 Species.CV_name <.001 Country.Species.CV_name <.001 Residual Total Least significant differences of means (5% level) Table Country Species Species CV_name rep. unequal unequal unequal d.f l.s.d X min.rep max-min 0.418X 1.248X max.rep Except when comparing means with the same level(s) of Species 2.646X min.rep max-min 1.247X max.rep

48 Analysis of variance ==================== Variate: PUFA_C18_2_3 Source of variation d.f. s.s. m.s. v.r. F pr. Country <.001 Species <.001 Country.Species <.001 Species.CV_name <.001 Country.Species.CV_name <.001 Residual Total Least significant differences of means (5% level) Table Country Species Species CV_name rep. unequal unequal unequal d.f l.s.d X min.rep max-min 0.418X 1.247X max.rep Except when comparing means with the same level(s) of Species 2.642X min.rep max-min 1.246X max.rep (No comparisons in categories where l.s.d. marked with an X) ==================== Variate: C20_1 Source of variation d.f. s.s. m.s. v.r. F pr. Country <.001 Species <.001 Country.Species <.001 Species.Cultivar Country.Species.Cultivar Residual Total Least significant differences of means (5% level) Table Country Species Species Cultivar rep. unequal unequal unequal d.f l.s.d X min.rep max-min X X max.rep Except when comparing means with the same level(s) of Species X min.rep max-min X max.rep (No comparisons in categories where l.s.d. marked with an X)

49 Analysis of variance ==================== Variate: C22_1 Source of variation d.f. s.s. m.s. v.r. F pr. Country <.001 Species <.001 Country.Species Species.Cultivar Country.Species.Cultivar Residual Total Least significant differences of means (5% level) Table Country Species Species Cultivar rep. unequal unequal unequal d.f l.s.d X min.rep max-min 0.499X 1.489X max.rep Except when comparing means with the same level(s) of Species 3.156X min.rep max-min 1.488X max.rep (No comparisons in categories where l.s.d. marked with an X) Oil (Solvent Extraction) ## Analysis of Variance Table ## ## Response: Oil_ME ## Df Sum Sq Mean Sq F value Pr(>F) ## Country < 2.2e-16 *** ## Species *** ## Country:Species ## Residuals ## --- ## Signif. codes: 0 '***' '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1 ## LSD t Test for Oil_ME ## Groups, Treatments and means ## a Wageningen ## b Italy ## c Poland ## Groups, Treatments and means ## a Camelina ## b Crambe

50 Oil (NMR) ## Analysis of Variance Table ## ## Response: Oil_NMR ## Df Sum Sq Mean Sq F value Pr(>F) ## Country e-08 *** ## Species ## Country:Species ## Residuals ## LSD t Test for Oil_NMR ## Groups, Treatments and means ## a Wageningen ## b Poland ## b Italy Crambe: Erucic acid ## Analysis of Variance Table ## ## Response: Erucic.acid ## Df Sum Sq Mean Sq F value Pr(>F) ## Cultivar ## Residuals Camelina:Gondoic acid ## Analysis of Variance Table ## ## Response: Gondoic.acid ## Df Sum Sq Mean Sq F value Pr(>F) ## Cultivar ## Residuals Lodging Level Variate: Lodging level Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum Rep.Main_plot stratum CropVar_name Residual Rep.Main_plot.Plot_within_main_plot stratum H_time_no CropVar_name.H_time_no Residual Total

51 Fisher's protected least significant difference test CropVar_name Mean Crambe_Galactica_10 Camelina_Midas_33 Camelina_13CS _31 Crambe_PRI _48 Crambe_PRI _59 Camelina_WUR _ a a a a a b Stem Analysis of variance Table Variate: Stem Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum Rep.Plot_nr stratum HTIME <.001 Spec_Cult <.001 HTIME.Spec_Cult Residual Total Fisher's protected least significant difference test HTIME Mean HARVEST 1 HARVEST 2 HARVEST 5 HARVEST a 2537 b 3306 c Leaf Analysis of variance Variate: Leaf Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Rep stratum Rep.Plot_nr stratum HTIME 2 (1) Spec_Cult HTIME.Spec_Cult 10 (5) Residual 51 (18) Total 71 (24)

52 POD Analysis of variance Variate: pod Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Rep stratum Rep.Plot_nr stratum HTIME 0 (3) Spec_Cult HTIME.Spec_Cult 0 (15) 3. Residual 15 (54) Total 23 (72) Seed Analysis of variance Variate: seed Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Rep stratum Rep.Plot_nr stratum HTIME 0 (3) Spec_Cult 2 (3) HTIME.Spec_Cult 0 (15) Residual 6 (63) Total 11 (84) Fisher's protected least significant difference test Spec_Cult Mean Camelina_WUR _7 Camelina_Midas_33 Crambe_Galactica_10 Crambe_PRI _48 Crambe_PRI _59 Camelina_13CS _ a 2027 a 2132 a 2132 a 2132 a 2570 b Total Biomass Analysis of variance Variate: Total_Biomass Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum Rep.Main_plot stratum

53 CropVar_name Residual Rep.Main_plot.Plot_within_main_plot stratum H_time_no <.001*** CropVar_name.H_time_no Residual Total H_time_no Mean a b b b Harvest Index Analysis of variance Variate: %_grain_harvest_index Source of variation d.f. (m.v.) s.s. m.s. v.r. F pr. Rep stratum Rep.Plot_nr stratum HTIME 0 (3) Spec_Cult <.001 HTIME.Spec_Cult 0 (15) 0.00 Residual 15 (54) Total 23 (72) Fisher's protected least significant difference test Spec_Cult Mean Crambe_PRI _59 Camelina_WUR _7 Camelina_Midas_33 Camelina_13CS _31 Crambe_PRI _48 Crambe_Galactica_ a a a a b b

54 Appendix 3. Documentation of research activities. PHOTOS Photo 1. Seed sample collection, Photo 2. Field trial two weeks after sowing. Photo 3, 4 and 5. Crambe and camelina crops in field trial. Photo 6, 7 and 8. Discrimination of crambe and camelina organ after harvested.

55 Photo 9. NMR machine in biophysics department, Wageningen University. Photo 10. GC machine from Agilent Technologies series 7890A GC System in biochemical lab, Plant Breeding department, Wageningen University.