Using Genetic Transformation to Produce Enhanced Levels of Erucic Acid and Novel Wax Esters in Crambe Abyssinica

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Using Genetic Transformation to Produce Enhanced Levels of Erucic Acid and Novel Wax Esters in Crambe abyssinica

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Content Chapter 1: General introduction 7 Chapter 2: Crambe abyssinica in vitro regeneration and transformation 17 Chapter 3: The generation of marker-free, genetically modified plants in Crambe abyssinica 37 Chapter 4: Increasing erucic content of Crambe abyssinica seed-oil by genetic modification 53 Chapter 5: Producing wax esters in Crambe abyssinica 71 Chapter 6: General discussion 89 References 99 Summary 107 Samenvatting (Summary in Dutch) 109 Acknowledgements 113 Curriculum vitae 11͸ Education statement of the graduate school experimental plant sciences 11͹

Chapter 1: General introduction

1.1 Crambe abyssinica (crambe), general characteristics

The oil (mostly as triacylglycerol (TAG)) stored in plant seeds is not only a very crucial resource for seedling growth (Gurr et al., 1974; Carlsson, 2009; Chapman and Ohlrogge, 2012), but also an essential renewable resource for humans. Oil seed crop cultivation is an important part of global agriculture. The vegetable oil produced by oil seed crops, is being used in diets (as nutrition, flavor, additive and for frying), as well as feedstock for modern chemical industrial products like lubricants, detergents, cosmetics and pharmacology. To meet the growing demand of vegetable oil, it is pivotal to enhance the yield and quality of oil seed crops. For this reason, breeders keep making efforts to improve the traditional oil seed crops, such as soybean, rapeseed, oil palm and sunflower. On the other hand, there is also a lot of breeding research conducted on new oil seed crops, trying to use novel interesting plants for the production of specific types of oil.

A B C

Figure 1.1 Crambe morphology: A, Morphological description of a crambe plant (a/stem and branch; b/leaf; c/flower; d/petal & calyx; e/pistil & calyx; f/petal; g/stamens & pistil; h/stamens; i/pistil & ovary; k/developing seedpod; l/pod containing a seed; m/seed); B, Flowers and developing seedpods of crambe; C: A field of crambe plants about to be harvested.

The oil seed plant crambe, which is also known as Abyssinian mustard, Abyssinian kale, colewort or datran, is a new crop cultivated usually to produce high erucic acid plant oil desired by the chemical industry (Bruun and Matchett, 1963; White and Higgins, 1966). This crop is an erect, annual herb with large pinnately lobed leaves similar to mustard leaves (Figure 1.1 A and B). The plant height normally varies between one to two meters, depending on the season and the plant density. It shows an indeterminate flowering habit and may continue to set seeds even late in the season. Although crambe is mainly self-pollinated, still outcrossing was reported to occur at a rate of 4 to 7% (Meier and Lessman, 1973). Typically, the flowering period is approximately three weeks (White and Higgins, 1966). The flowers are numerous, small and produce small brownish and round seedpods of 5 mm in diameter. The seedpod consists of two parts: a terminal spherical capsule and a much smaller cylindrical lower segment (Figure 1.1 A, B and C). Each capsule-like seedpod contains a single spherical seed, which are greenish brown and 0.8-2.6 mm in diameter. As a plant preferring moderate rainfall, crambe can be field-cultivated in warm-temperate regions and does not possess a high level of frost resistance. Its center of origin is the Mediterranean region (White, 1975), and it is also prevalent across Asia and Western Europe, having been grown experimentally and commercially in a wide climate range (Wang et al., 2000).

Crambe abyssinica is an allohexaploid (2n=6x=90), which is a member of the genus Crambe (Brassicaceae family) (Rudloff and Wang, 2011). Genus Crambe has a disjunctive distribution among four major geographical regions: Macronesian (12 species), Mediterranean (four species)

ͺ that C. abyssinica belongs to, east African (three species), and Euro-Siberian-southwest Asian (ca 20 species) (Francisco-Ortega et al., 1999). Crambe abyssinica is morphologically similar to the other two species C. hispanica (n=30) and C. glabrata (n=15). A study based on morphological as well as molecular data sets including chloroplast DNA restriction sites and internal transcribed spacer regions analyzes, showed that C. abyssinica taxonomically should be organized as a subdivision under C. hispanica and with C. glabrata as a distinct species (Rudloff and Wang, 2011). So the full name of C. abyssinica is Crambe hispanica subsp. abyssinica Hochst. ex R.E.Fr. In fact, it is the only one cultivated within the Crambe genus because of the high erucic acid content in its seed oil. The oil content of crambe seed is about 35-40% per seed with pod (up to 60% when expressed per hulled seed) and the protein content is about 20-40% (Bruun and Matchett, 1963). The fatty acid composition is shown in Table 1.1. About 55% to more than 60% of total fatty acid is erucic acid (Lazzeri et al., 1994), which is 10-15% higher than in high erucic acid rapeseed (HEAR). A cloned fatty acid elongase 1 (FAE1) gene from the genome of C. abyssinica (cv. ‘Prophet’) transformed and expressed in Arabidopsis could significantly elevate erucic acid content in the offspring seed (Mietkiewska et al., 2007). Asymmetric somatic hybridization between Brassica napus and C. abyssinica indicated that the seeds from the hybrids accumulated more erucic acid in their seed oil compared to the original B. napus (Don Palmer and Keller, 2011).

Table 1.1 Proportion of fatty acids in crambe oil

Fatty Acid % of Total Palmitic Acid C16:0 1.8 Stearic Acid C18:0 0.7 Oleic Acid C18:1 17.2 Linoleic Acid C18:2 8.7 Linolenic Acid C18:3 5.2 Eicosanoic Acid C20:1 3.4 Erucic Acid (cis-13-docosenoic) C22:1 58-66 Brassidic Acid (trans-13-docosenoic) C22:1 0.7 Tetracosanoic C24:0 0.7 Nervonic C24:1 1.6 Others 2.5

Crambe seed oil, due to its fatty acid composition, is not suited for making food or food additives. However, for the same reason crambe is an interesting crop for feedstock production for the chemical industry. The possible uses of crambe oil are listed in Table 1.2. Because of the high erucic acid content in crambe seed oil, its most significant value is for producing erucamide, which is in wide and permanent request all around the world. Erucamide is a derivative of erucic acid and added to plastics. The main purpose thereof is acting as lubricant to ease the production of plastic parts and to provide a thin layer of lubrication on the extruded plastic film surface, hereby reducing the coefficient of friction, acting as a ‘slip-agent’. Some of the other derivatives of erucic acid like erucyl alcohol, behenic acid and behenyl alcohol are also widely used for producing wax esters, nylon, surfactants and pharmaceutical products.

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Table 1.2 Oil product uses of crambe

Oil Product Uses Triglyceride (refined oil) Pharmaceuticals, lubricants, heat transfer fluids, dielectric fluids, waxes and fish food coating agent. Erucic acid/Erucamide Antifriction coatings, plasticizers, anti-stats and corrosion inhibitors. Behenic acid/Behenamide Antifriction coatings, mold release agents, processing aids flow improvers and cosmetics. Erucyl alcohol Surfactants slip and coating agents. Behenyl alcohol Surfactants slip, coating agents and pharmaceutical products. Wax ester Lubricants cosmetics Brassylic acid Nylon, perfumes, plasticizers, synthetic lubricants and coatings, flavors. Pelargonic acid Perfumes, plasticizers, synthetic lubricants, coatings and flavors. Fatty acids Existing markets for C18-C22 acids and their amides.

1.2 The history of crambe breeding

Cultivation of crambe started in Europe, first in Russia in the 1930s (Mastebroek et al., 1994). After that, the cultivation extended from Russia to Sweden and Poland in the period leading up to the Second World War. Due to the war breeding in the crop almost ceased.

1.2.1 Breeding in USA

After the war in the 1960s, testing of 11 lines introduced into USA and Canada from Sweden and Russia did not show adequate crop yields. Breeding was therefore initiated at the agricultural experimental station of Purdue University (Maryland, USA) and three varieties were released in the 1970s, namely ‘Prophet’, ‘Indy’ and ‘Meyer’. Through the continuation of the breeding work during the 1980s at the United States Department of Agriculture, Agricultural Research Service (USDA ARS) in Beltsville (Maryland, USA), ‘BelEnzian’, ‘BelAnn’ and C-22, C-29 and C-7 were released around 1985. Their yields and oil contents had improved compared to the earlier released cultivars (Carlsson, 2009).

1.2.2 Breeding in Europe

In the beginning of the 1990s crambe breeding in Europe was resumed at Plant Research International (PRI), Wageningen University & Research Centre the Netherlands (Mastebroek et al., 1994). About 20 accessions of old European landraces and American cultivars from the 70s and 80s were evaluated for agronomical characteristics. This study showed that the breeding effort in the USA had resulted in a 15% oil yield increase per hectare. The breeding targets of PRI aimed at improving seed and oil yield, enhancing the erucic acid content in the oil, improving the disease resistance and decreasing the glucosinolate content in the seeds. The materials selected for further breeding at PRI were one late flowering old European landrace and two early flowering newer American lines. The three crambe-lines were intercrossed and, from F3 generation onwards, selection was performed for agronomical characteristics. From that research, several cultivars have been released during the 1990s. Independently, there was another genotype (Mario) released in 1995 by the Institute Sperimentale per le Colture Industriali in Bologna, Italy (Fontana et al., 1998).

1.3 Potential use of crambe as oil seed crop

The main argument for developing crambe into a cultivated oil seed crop is the demand for high erucic acid-containing plant oil (Vargas-Lopez et al., 1999). Erucic acid that constitutes up to 60% of crambe oil is an expensive chemical compound compared with other fatty acids, due to its

ͳͲ various utilities in the chemical industry. It belongs to the very long chain fatty acid (VLCFAs; very long chain fatty acids are fatty acids with aliphatic tails longer than 22 carbons) class. Although erucic acid is a fatty acid compound, which is not considered to be suitable for the human diet, there is certainly a global demand for specific and significant quantities as an important industrial feedstock. At present, the high erucic acid plant oil used in industry is mainly produced by HEAR. However, HEAR cultivation poses some problems, because of the risk of cross-contamination of seeds during processing and of genetic crossing with food quality rapeseed (canola). The canola used primarily in foods belongs to the kind of low erucic acid rapeseed (LEAR). The occurrences of crossing or cross-contamination between HEAR and LEAR will have a severe negative impact on the commercial applications and value of both crops. Currently, the upper limit of erucic acid in edible oil has been set to 5% in most countries. For LEAR, the increase in erucic acid content resulting from crosses with HEAR can badly influence its edible oil quality. On the other hand, for HEAR, the decrease of erucic acid content will also make its oil less valuable. An additional problem will be caused by the changes in omega 3 (linoleic acid) and omega 6 (alpha linoleic acid) unsaturated fatty acids which make up to 37% in LEAR oil but only a mere 20% in HEAR (Renner et al., 1979). Because these fatty acids belong to the class of polyunsaturated fatty acids (PUFAs, having two or more double bonds in their carbon chain), they are believed to be valuable components with health promoting effects in the human diet (Roche, 1999). On the other hand for erucamide production, a higher level of PUFAs just means more costs for their elimination in the downstream processing phase.

If crambe would be used to produce high erucic acid plant oil, there will be no such consequences, because it is very difficult for crambe to cross-pollinate with Brassica species including oilseed rape (Youping and Peng, 1998). Along with this fact, crambe has several other advantages over HEAR for producing erucic acid: 1) It already has a higher amount of erucic acid in its seed oil than HEAR; 2) it is useful in crop rotations for alleviating weed, pest and disease build-up in areas where rapeseed was planted; 3) crambe is a moderate input crop, requiring fewer pesticides. Pests are easily dealt with by tillage and/or pesticides; 4) the oil profile is of interest to the chemical industry as it contains fewer PUFAs than HEAR; 5) It grows well on all soil types from heavy clays to light sands, and is more tolerant to late-season drought (White and Higgins, 1966; Stymne and Dyer, 2007; Falasca et al., 2010). Although crambe apparently has so many superior characteristics, there is still a major disadvantage that obstructs large-scale, global cultivation of crambe instead of HEAR. This is the fact that crambe is not yet competitive with HEAR with respect to its lower seed yield and less value for the seed cake. Therefore, to make crambe competitive with HEAR, its economic value needs to be improved (Stymne and Dyer, 2007).

1.3.1 Use of crambe instead of rapeseed for current erucic acid production

One strategy for the improvement of crambe’s economic value is to increase the erucic acid content and decrease the amounts of PUFA further in crambe seed oil. This will mean more erucic acid yield per hectare, and less input required for the following downstream chemical modifications. With increasing erucic acid content in crambe to more than 66%, e.g. by genetic engineering, this crop can be an economically attractive alternative to HEAR. As a result of the research performed on the lipid biosynthesis pathway in plant seeds, several important genes have been identified and proposed critical to control the level and composition of erucic acid and PUFA, accumulating in seeds of Brassicaceae species.

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1.3.2 Genes positively modulating erucic acid accumulation

Erucic acid is synthesized in the cytosol by extension of oleic acids. After the C18:1 CoA is synthesized in the plastid and released into the cytosolic pool, the fatty acid elongase complex catalyzes its elongation reaction to produce C20:1 CoA and then C22:1 CoA. Every cycle of fatty acid elongation attaches two carbons to the acyl moiety (Ohlrogge and Browse, 1995). Within each cycle of reaction there are four steps, firstly condensation of malonyl-CoA with an acyl primer, followed by a reduction, then a dehydration, and finally a second reduction. The four enzymes catalyzing this biosynthesis are known as the fatty acid elongase complex (Whitfield et al., 1993). The 3-ketoacyl-CoA synthase (KCS) (EC 2.3.1.199) catalyzing the condensation reaction plays a key role in determining the chain length of fatty acid products found in seed oils (Katavic et al., 2000). It has been reported that crambe’s own KCS1 is most likely responsible for its high seed-oil erucic acid content compared to the other members of the Brassicaceae family. This was also supported by the introduction and expression of crambe KCS1 into Arabidopsis by genetic modification, which resulted in an elevation of the erucic acid content (Mietkiewska et al., 2007). Therefore, a stronger expression of its own KCS1 (also known as fatty acid elongase FAE, CaFAE; in this thesis, KCS will always be referred to as FAE) might be helpful for increasing the erucic acid content in crambe seed. However, this approach was not followed in the research for this thesis.

Figure 1.2 Difference between triacylglycerol and WE synthesis: Normally, oil seed plants accumulate TAG as main storage of lipids for use in seed germination. TAG is synthesized by the Kennedy pathway, in which the glycerol backbone is acylated at the positions sn-1, sn-2 and sn-3 (A). In jojoba seed, WE make up > 90 % of the seed oil (B). A fatty acid reductase or fatty acyl reductase (FAR) converts acyl-moieties from fatty acyl-CoAs to fatty alcohols: fatty alcohol acyltransferase (wax synthase, WS) catalyzes the esterification of the fatty alcohols with fatty acyl-CoAs.

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Another bottleneck for raising the erucic acid content in the seed oil of crambe or even in any other Brassicaceae species is the endogenous lysophosphatidate acyltransferase 2 (LPAT2) (EC 2.3.51). This enzyme catalyzes acyl-CoA attachment to the sn-2 position (Figure 1.2 A) of the glycerol backbone for TAG, by an esterification bond. In vitro experiments indicated that the LPAT2 from Brassicaceae species, all have extremely low affinities to erucoyl-CoA (Kuo and Gardner, 2002), while the enzymes (glycerol phosphate acyl-transferase and diacylglycerol acyltransferase) (Kennedy, 1961) catalyzing the sn-1 and sn-3 positions are without such kind of limitation (Kuo and Gardner, 2002). It means that the highest percentage of erucic acid within seed oil will be around 66%. Interestingly, Cao et al. (1990) found in the seed of Limnanthes douglasii a LPAT2 (LdLPAT2) that has a high affinity to erucoyl-CoA (Cao et al., 1990). The insertion of a gene encoding a L. douglasii LPAT2, capable of acylating erucic acid at the sn-2 position, led to a redistribution of erucic acid esterified at sn-2 in the oil of the transgenic seeds, and a slight increase of the total amount of erucic acid even in combination with an extra copy of FAE1 from rapeseed (Lassner et al., 1995; Han et al., 2001; Jadhav et al., 2005). To produce a GM crambe with high erucic acid content (more than 66%) in its seed oil, the LdLPAT2 is likely to be an essential factor that should be incorporated.

1.3.3 Genes negatively modulating erucic acid accumulation

The fact that if the erucic acid content can be as high as 66% means a sufficient supply of erucoyl-CoA, and hardly any other fatty acid moieties than erucoyl are built in on the sn-1 and sn- 3 positions in crambe. When LPAT2 from L. douglasii is introduced, theoretically it becomes possible to be able to achieve 100% C22:1 on the sn-2 position, and then plant oil with 100% erucic acid could be obtained. However, in practice, L. douglasii LPAT2 transformation of Brassica napus L. has so far never led to an erucic acid level higher than 80% of the seed oil (Brough et al., 1996; Wilmer et al., 2000; Han et al., 2001; Nath et al., 2009). The reason why this is not 100% might be that the endogenous CaLPAT acts as a competitor to the introduced exogenous LdLPAT. It is proposed to test this hypothesis by using RNA interference to down- regulate specifically the endogenous LPAT2; if competition for the sn-2 position plays a role, the down regulation of the crambe endogenous LPAT2 (CaLPAT2) should lead to higher erucic-acid content at the sn-2 position because of the action of LdLPAT2 and to a higher overall percentage of erucic acid in the seed oil. An endogenous LPAT2 cDNA of crambe has been cloned and sequenced and to down regulate this gene an RNA-interfering DNA fragment (CaLPAT2-RNAi) will be designed according to this sequence. A suitable conserved region has been identified consisting of the NHXXXD box, FP/VEGTR box and the sequence between them (Murphy, 2009).

In addition to the endogenous LPAT, endogenous Fatty acid desaturase 2 (FAD2) (EC 1.14.19.1) has also been found to play a limiting role in erucic acid biosynthesis. Fatty acid desaturase 2 is an enzyme that catalyzes oleic acid (C18:1) conversion into linoleic acid (C18:2) (Okuley et al., 1994; Dyer and Mullen, 2001; Yang et al., 2012). After that, the produced linoleic acid will enter the PUFA pool followed by several steps of modification (further desaturation and/or elongation), which means that once the C18:1 is transformed into C18:2, those carbon chains will no longer be available for C22:1 biosynthesis. This competition between FAD2 and erucic acid biosynthesis for C18:1 oleic acid as substrate has been proven to occur indeed by the transgene Brassica carinata with native FAD2 silenced, which allowed it accumulated more erucic acid in its seeds than wild type (Jadhav et al., 2005). The erucic acid content also further increased (to 70 %) by crossing the mutant line defective in FAD2 with GM rapeseed lines carrying the L. douglasii LPAT and FAE (Nath et al., 2009). So, another alternative for engineering increased erucic acid

ͳ͵ ͳ accumulation in crambe seed oil would be by inhibiting the desaturation of oleic acid by knocking down FAD2 (CaFAD2-RNAi) when the L. douglasii LPAT or/and a strong expression of native KCS are being introduced. Simultaneously with such increases in erucic acid, a decrease in the PUFA content will be expected.

1.3.4 Producing Simmondsia chinensis (jojoba) like wax esters in crambe

Another option for increasing the economic value of crambe is to have some other precious chemical compounds produced in its seed. Jojoba-like wax esters (WE; Figure 1.2B) are good candidates. Such long-chained wax esters have important applications in cosmetics and lubrication with high added value (Stymne and Dyer, 2006). An amazing characteristic jojoba, a desert plant from North America, is that it has WE as the main storage lipid in its seeds (Gentry, 1958; Miwa, 1971), instead of having TAG (Figure 1.2A) that accumulates in seeds of most other plants. Compared to conventional vegetable oils (TAGs), jojoba oil has many superior properties and can be extensively used in lubrication and transmission fluids. Jojoba WEs are ester molecules between fatty alcohols and fatty acids with long carbon chains both (C20:1, C22:1 and C24:1). The key steps of WE assembly in jojoba seeds are: first oleic acid is converted into eicosanoic acid, erucic acid or even nervonic acid (24:1) CoA by jojoba fatty acid elongase (ScFAE) (Lassner et al., 1996); and then fatty acid reductase (ScFAR) catalyzes those fatty acids to become fatty alcohols; finally, fatty acids and fatty alcohols are joined together by an esterification bond (Figure 1.2B), catalyzed by jojoba wax synthase (ScWS). The genes encoding the enzymes for each step have been cloned from the mRNAs isolated from developing jojoba seed (Lassner et al., 1999; Metz et al., 2000).

Presently, the amount of jojoba WEs that is used worldwide is very small due to the high price and low production. The price of the jojoba oil is about 5000 €/ ton. The global production of jojoba oil is about 3500 tons per year. The biggest markets for the use of jojoba oil are cosmetics (2000 tons per year) and the pharmaceutical sector (jojoba oil is said to heal acne, pimples and warts.). The jojoba oil consumption for lubrication is only around 100 tons annually. Although WE compounds comparable to jojoba oil can also be produced enzymatically from alcohols and fatty acids (bio-based or mineral-based) by artificial chemical engineering, this production system is rather expensive. To find another, cheaper alternative for production is very lucrative and necessary, if people want to extend the range of its uses, especially to the global lubricant market (polyalphaolefins 0.5 million tons and organic esters 0.05 million tons annually). One way of achieving this could be to transform ScFAE, ScFAR and ScWS into another plant species and use it as bio-platform to produce large quantities of jojoba WE (Lassner et al., 1999). A preliminary study has shown the encouraging result that insertion of ScFAR and ScWS from jojoba and FAE1 from Lunaria annua in Arabidopsis resulted in transgenic plants producing seed oil with up to 70% of WE instead of the common TAGs (Lardizabal et al., 2000). An alternative approach that would bypass the need to use an elongase gene like FAE1 is to choose plants that naturally contain high amounts of erucic acid (C22:1). Crambe, which has 60% of erucic acid in its seed oil, thus has an ideal fatty acid composition for production of jojoba type WE and becoming the desired bio-production platform (Mietkiewska et al., 2007).

1.4 European Union 7th Frame Work Project ICON and the scope of this thesis

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Crambe is one of the target crops within EU 7th Frame Work Project ‘Industrial Crops producing added value Oil for Novel chemicals’ (ICON, website: http://icon.slu.se/ICON/). ICON was a 4- year international cooperative research project in which multiple partners from different countries were involved. The main goal for the ICON researchers was to find possibilities to replace non- renewable fossil oil as industrial chemistry feedstock with renewable plant oils. The main targets for the new crop that ICON proposed, i.e. crambe, were to increase the erucic acid content and produce jojoba-type WE in its seeds by genetic modification. All the work described in this thesis belonged to three work-packages (WP) of the ICON project.

Chapter 2: Crambe abyssinica in vitro regeneration and transformation

The work described in this chapter was part of the ICON WP1.1 ‘Increase efficiency of Crambe transformation method and determine optimal selectable marker gene’. A reliable protocol for transformation of crambe is crucial for further genetic engineering. By testing different tissues of crambe seedlings with various combinations of hormones and Agrobacterium inoculating procedures, we have developed a protocol of Agrobacterium-mediated transformation using cotyledonary nodes as explants, based on a protocol developed by Krens and coworkers for sugar beet (Krens et al. 1996). In addition to this, an efficient amplification method for in vitro crambe material was also set up. Both protocols were applied to the work described in the following chapters.

Chapter 3: Test the Marker-free system in the transformation of Crambe abyssinica

The work described in this chapter was part of the ICON WP1.2 ‘Test clean vector technology of PRI in Crambe’. To explore the possibilities to make GM crambe more consumer-friendly, the marker-free technology of PRI, WUR (Schaart et al., 2004) was tested in crambe transformation. In this part of the research, we first made GM plants using a pMF1-based vector (marker-free system). Then, two independent lines were selected from them, and after multiplication by in vitro propagation they were treated with DEX to trigger marker elimination by recombination. After applying 5-FC as the selection agent, marker-free plant material was obtained.

Chapter 4: Improve the erucic acid content of Crambe abyssinica seed-oil with genetic modification

Table 1.3 Gene combinations for improvement of the erucic acid content in crambe seeds

Goals Gene combinations Increase erucic acid content in seed oil LdLPAT + BnFAE1 Increase erucic acid and decrease PUFA content LdLPAT + BnFAE1 + CaFAD2-RNAi Identify the proper domain in the mRNA of CaLPAT2 as target for CaLPAT2-RNAi RNAi Increase erucic acid and decrease PUFA content LdLPAT + BnFAE1 + CaFAD2-RNAi+CaLPAT2- RNAi

The work described in this chapter was partially of the ICON WP2.2 ‘Production of ultra-high amounts of erucic acid in Crambe and B. carinata’. In this chapter, by using the protocol developed in Chapter 2, we introduced L. douglasii LPAT, and B. napus FAE and crambe FAD2- RNAi genes (Table 1.3) all driven by a napin promoter (Rask et al., 1998) into crambe. The T1 offspring seeds were analyzed by gas chromatography for their fatty acid composition. In this chapter we also report on the work of selecting specific sequences of crambe LPAT2 for RNAi targeted silencing. The target sequence of the RNAi approach was from a conserved region of

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Brassicaceae LPAT2 genes. It contained the conserved region consisting of the NHXXXD box, FP/VEGTR box and sequences between them (Murphy, 2009). The RNAi vector (Table 1.3) was used for transformation of crambe and Arabidopsis. The efficiency of RNAi was indicated by the knockdown of the endogenous LPAT2 (CaLPAT2) in transformed crambe and Arabidopsis. Finally, this CaLPAT2-RNAi was assembled together with LdLPAT, BnFAE1 and CaFAD2-RNAi into one construct for transforming crambe. Chapter 5: Production of Wax ester in Crambe abyssinica

The work described in this chapter were for ICON WP4.2 ‘Production of long-chain wax esters in Crambe and B. carinata; Expression of ScWS+ScFAR in Crambe’, and WP5.4 ‘Expression of [Jojoba WS]+[Jojoba FAR]+[FAD2-RNAi] in Crambe’. The genes needed to enable WE production are the jojoba WS and FAR either with or without the jojoba FAE (Table 1.4). Two constructs with these genes were available from the ICON project, provided by Dr. Edgar Cahoon (University of Nebraska-Lincoln, USA) with kanamycin as selectable marker (in one case with DsRed as additional visual marker for selection) and with a glycinin promoter from soybean (Iida et al., 1995) to drive the functional genes. After the establishment of GM plants carrying either ScWS+ScFAR or ScWs+ScFAR+ScFAE, they were maintained in the greenhouse for further seed ripening. The offspring seeds from T1 to T3 were analyzed and selected for increased WE content. The influence of WE content on seed germination and seedling vigor and growth was studied. In addition to the introduction of the jojoba WE genes (FAR and WS) and the introduction of the jojoba FAE to increase the conversion of C18:1 to C22:1, it was tested whether suppression of the native FAD2 using RNAi can increase the substrate, C18:1, availability for conversion into C22:1 for WE yield in developing seeds.

Table 1.4 Producing jojoba-type WE in crambe

Goals Gene combinations

WE accumulation in seeds of crambe ScWS + ScFAR

Production of very long chain WE of the jojoba type to at least 20% in the seed ScWS + ScFAR + ScFAE oil of crambe Suppression of the native FAD2 to increase the supply of C18:1 for C22:1 ScWS + ScFAR+ CaFAD2-RNAi conversion and uptake in WE

In summary, we describe a series of studies on transgene breeding of crambe, from the establishment of the protocol to the actual transformations with functional genes. These experiments demonstrated the potential value of GM crambe as oil seed platform crop and opened up the way for continuation of this work in the future.

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Chapter 2: Crambe abyssinica in vitro regeneration and transformation

Weicong Qi1, 2, , Iris E M Tinnenbroek-Capel1, Jan G Schaart 1, Bangquan Huang3, Jihua Cheng 1,4, Richard G F Visser 1, Eibertus N Van Loo1, Frans A Krens1§

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Chapter 2

Abstract:

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18 Crambe abyssinica in vitro Regeneration and Transformation

2.1 Introduction

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19 Chapter 2

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2.2.3 Regeneration capacity of in vitro plants

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20 Crambe abyssinica in vitro Regeneration and Transformation

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21 Chapter 2

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Table 2.1 Experimental design for setting up Agrobacterium-mediated transformation of seedling explants

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22 Crambe abyssinica in vitro Regeneration and Transformation

2.2.7 Transformation frequency using different vectors

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23 Chapter 2

'LVFRYHU\9 HTXLSSHG ZLWK 3HQWD)OXDU 6 IOXRUHVFHQFH HTXLSPHQW DQG 352,5 /XPHQ LOOXPLQDWLRQV\VWHP 2.3 Results

2.3.1 Shoot regeneration from seedling parts

Table 2.2 Cotyledon regeneration result from media with Microagar and Phytoblend

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24 Crambe abyssinica in vitro Regeneration and Transformation

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Table 2.4 Hypocotyl regeneration result from media with Microagar and Phytoblend

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25 Chapter 2

2.3.2 Regeneration capacity of in vitro plant parts

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Figure 2.2 Regeneration capacity of explant types from in vitro plants: $IWHUEHLQJVXEMHFWHGWRUHJHQHUDWLRQPHGLXPIRUWZR ZHHNVWKHD[LOODU\EXGVEHJDQWRJLYHVKRRWUHJHQHUDWLRQ GLUHFWDQGLQGLUHFW EXWWKHRWKHUNLQGVRIH[SODQWVMXVWVWDUWHGWKHLUVKRRW UHJHQHUDWLRQDIWHUWKUHHZHHNV$IWHUILYHZHHNVWKHVWHPH[SODQWVDOVRKDYHVLJQLILFDQWO\KLJKHUVKRRWUHJHQHUDWLRQIUHTXHQF\WKDQ WKHSHWLROHVOLFHVEXWORZHUWKDQWKHD[LOODU\EXGV5HJHQHUDWLRQZDVVFRUHGDWGLIIHUHQWWLPHLQWHUYDOVIRUIRXUGLIIHUHQWH[SODQWW\SHV 7KHOHWWHUVDERYHWKHHUURUEDUVVKRZHGWKHUHVXOWRIRQHZD\$129$DQDO\VLV

2.3.3 Selectable agent testing

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26 Crambe abyssinica in vitro Regeneration and Transformation

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Table 2.5 Effect of different kanamycin concentrations on the explants of hypocotyl, cotyledonary node and cotyledon

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Table 2.6 Effect of different hygromycin concentrations on regeneration of cotyledonary node explants of crambe

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Table 2.7 Effect of different PPT and bialaphos concentrations on regeneration of cotyledonary node explants of crambe

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2.3.4 Transformation

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27 Chapter 2

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Figure 2.3 GUS staining and PCR test of putative transgenic shoots $ *86 VWDLQLQJ RI OHDI VDPSOHV IURP D WUDQVIRUPDWLRQ FDQGLGDWHREWDLQHGIURPFRW\OHGRQDU\QRGHVZLWKDVKLIWLQJVHOHFWLRQVFKHPH%*86VWDLQLQJRIDOHDIVDPSOHIURPWKH:7FRQWURO &3&5XVLQJHLWKHUSULPHUVIRU137,, XSSHUSDQHO RUYLU* ORZHUSDQHO '1$VDPSOHVIURPIRXUUDQGRPO\FKRVHQWUDQVIRUPDWLRQ FDQGLGDWHVREWDLQHGIURPFRW\OHGRQDU\QRGHVRQPHGLXP$ZHUHWHVWHG'1$IURP:7FUDPEHZDVXVHGDVQHJDWLYHFRQWURODQG $JUREDFWHULXPDVSRVLWLYHFRQWURO

Table 2.8 Effect of different explant types and selection methods on transformation frequencies

No. of No. of explants No. of explants with Transformation Explant type Medium Selection with GUS+ Explants regenerating Frequency regenerating shoots shoots

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2.3.5 Transformation of axillary bud explants

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Figure 2.4 DsRed fluorescence in a transformation candidate after transformation by pBinGlyred (ASCI) (A) and a WT control (B): 7KH IOXRUHVFHQW VKRRW LQ FKDUW $ ZDV REWDLQHG IURP WKH WUDQVIRUPDWLRQ RI S%LQ*O\UHG $6&, XVLQJ WKH D[LOODU\ EXG H[SODQWIURPLQYLWURSODQWIRULQRFXODWLRQ

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Table 2.9 Transformation frequencies using axillary buds from in vitro grown crambe plants

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29 Chapter 2

2.3.6 Differences in frequencies of cotyledonary node explant based transformation with different binary vectors

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Table 2.10 Using different vectors in two Agrobacterium strains

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32 Crambe abyssinica in vitro Regeneration and Transformation

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33 Chapter 2

Supplementary Table

Supplementary table 1: Media components and observed result of regeneration experiments

No. of Direct shoot Regeneration from callus Microagar explants Callus induction regeneration cultures evaluated

Explant type No. % No. % No. % Hormones (μM) Cotyledons 40 40 100 0 0 0 0 BAP 0.44 petcot 40 10 25 36 90 0 0 hypocotyl 60 60 100 1 2 0 0 Cotyledons 35 35 100 0 0 0 0 BAP 2.2 petcot 40 19 48 39 98 0 0 NAA hypocotyl 40 40 100 0 0 0 0 0.5 Cotyledons 40 40 100 0 0 0 0 BAP 4.4 petcot 40 12 30 34 85 1 3 hypocotyl 60 56 93 0 0 1 2 Cotyledons 40 40 100 0 0 0 0 BAP 22 petcot 30 2 7 27 90 0 0 hypocotyl 40 40 100 0 0 0 0 Cotyledons 40 40 100 0 0 0 0 BAP 0.44 petcot 40 31 78 21 53 4 10 hypocotyl 60 35 58 0 0 0 0 Cotyledons 40 38 95 0 0 0 0 BAP 2.2 petcot 40 34 85 9 23 2 5 hypocotyl 60 52 87 0 0 1 2 NAA 5 Cotyledons 40 40 100 0 0 3 8 BAP 4.4 petcot 30 17 57 22 73 2 7 hypocotyl 60 43 72 0 0 0 0 Cotyledons 40 40 100 0 0 0 0 BAP 22 petcot 40 33 83 13 33 2 5 hypocotyl 60 17 28 0 0 0 0 Cotyledons 40 0 0.0 0 0 0 0 NAA 0 BAP 0 petcot 40 0 0.0 0 0 0 0 hypocotyl 60 0 0.0 0 0 0 0

34 Crambe abyssinica in vitro Regeneration and Transformation

No. Of explants Callus Direct shoot Regeneration from callus Phytoblend evaluated induction regeneration cultures

Hormones (μM) Explant type No. % No. % No. % Cotyledons 40 22 55 1 3 20 50 BAP petcot 40 14 35 40 100 6 15 2.2 Hypocotyl 40 35 88 0 0 0 0 Cotyledons 40 38 95 0 0 14 35 NAA BAP petcot 40 14 35 39 98 3 8 0.5 4.4 Hypocotyl 40 38 95 0 0 1 3 Cotyledons 40 12 30 0 0 2 5 BAP petcot 40 6 15 40 100 1 3 22 Hypocotyl 40 35 88 0 0 0 0 Cotyledons 40 39 98 0 0 19 48 BAP petcot 40 20 50 40 100 1 3 2.2 Hypocotyl 40 40 100 0 0 1 3 Cotyledons 40 40 100 0 0 18 45 NAA BAP petcot 40 21 53 38 95 0 0 2.5 4.4 hypocotyl 40 40 100 0 0 1 3 Cotyledons 40 32 80 0 0 5 13 BAP petcot 40 14 35 40 100 0 0 22 hypocotyl 40 40 100 1 3 0 0 Cotyledons 40 40 100 0 0 21 53 BAP petcot 40 18 45 40 100 1 3 2.2 hypocotyl 40 40 100 0 0 0 0 Cotyledons 40 40 100 0 0 23 8 BAP NAA 5 petcot 40 23 58 40 100 0 0 4.4 hypocotyl 40 40 100 0 0 0 0 Cotyledons 40 19 48 0 0 8 20 BAP petcot 40 3 8 20 50 0 0 22 hypocotyl 40 40 100 0 0 0 0.

6XSSOHPHQWDU\WDEOHT-DNA insertion copy-number of the T0 Crambe

Copy number of T0 Plant

Vector 1 2 3 4 5 pMF1-gus -gfp 2 2 2 0 0 1 Number of T0 Plant with Phan2 Certain T-DNA insertion Copy- 0 1 1 0 0 1 number pBinGlyRed ScFAR+ScWS (Asc I) 14 1 2 2 1 1 pBinGlyRed ScFAR+ScWS+ScFAE (Kpn I) 2 0 1 1 1 0

Chapter 3: The generation of marker-free, genetically modified plants in Crambe abyssinica

Weicong Qi1, 2, , Iris E M Tinnenbroek-Capel1, Jan G Schaart 1, Zhao Zhang 3, Bangquan Huang4, Jihua Cheng 1,5, Richard G F Visser 1, Eibertus N Van Loo1, Frans A Krens1§

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Chapter 3

Abstract

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38 The eneration of Marker-free, Genetically Modified Plants in Crambe abyssinica

3.1 Introduction

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39 Chapter 3

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3.2.1 Plant materials, vector and transformation

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3.2.2 Determination of the effect of dexamethasone on regeneration

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40 The eneration of Marker-free, Genetically Modified Plants in Crambe abyssinica

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3.2.3 Determination of the effect of 5-FC and 5-FU on regeneration

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3.2.4 Transformation of crambe with the model construct pJS-M14

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3.2.5 Verification of transformation

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41 Chapter 3

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3.2.6 qRT-PCR

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3.2.6 Application of DEX and subsequent selection steps

Table 3.1: The scheme of DEX treatments and 5-FC selection

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42 The eneration of Marker-free, Genetically Modified Plants in Crambe abyssinica

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DEX treatment to induce recombination (Step 1, 2 and 3)

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Table 3.2 The DEX treatments on the chosen T0 lines

T0 line DEX Con. ȝ0 Rooting Plants Axillary buds 1 Total 2 Total

Selection for marker-free shoots (Step 4 to step 8)

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43 Chapter 3

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3.3.1 Determination of the effect of dexamethasone on regeneration

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3.3.2 Determination of the proper concentration of 5-FC for selection

Regeneration of WT axillary bud explants on medium with 5-FC

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Regeneration of WT axillary bud explants on medium with 5-FU

Table 3.3 The 5-FU treatment on WT crambe axillary bud explant regeneration

Treatment (mg·L-1)\ status All Green Half bleached All bleached Total

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44 The eneration of Marker-free, Genetically Modified Plants in Crambe abyssinica

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3.3.3 Transformation of crambe with the marker-free vector pJS-M14

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Figure 3.2 T-DNA insertion number verification by Southern blotting: 7KHSUREHXVHGZDVDIUDJPHQWRIWKHJISJHQHDQG(FR5, ZDVFKRVHQIRUWKHUHVWULFWLRQEHFDXVHWKLVFRPELQDWLRQDOORZHGFRS\QXPEHUGHWHUPLQDWLRQ/LQHDQGOLQHUHSUHVHQWWZRVHSDUDWH 7HYHQWVZLWKDVLQJOH7'1$LQVHUWLRQDQGGRXEOHFRS\LQVHUWLRQUHVSHFWLYHO\:7UHSUHVHQWWKHZLOWW\SHQRQ*0FUDPEHFY µ*DODFWLFD¶FRQWUROWKHRXWHUOHIWODQHVKRZVDPROHFXODUZHLJKWPDUNHU+\EULGL]LQJIUDJPHQWVVKRXOGKDYHDPLQLPDOVL]HRINE 7KHZLGHDUURZVLQWKHFKDUWPDUNHGWKHSRVLWLRQRIWKHEDQGV

Figure 3.3 qRT-PCR analysis on in vitro vegetative material of line 1 and line 2: 7KHFKDUWSURYLGHWKHT573&5GDWDRQWKH H[SUHVVLRQRIWKHQSW,,JHQHDQGWKHFRG$JHQHLQWKHLQYLWUROHDIPDWHULDORIWZRVHOHFWHGRULJLQDOOLQHVZLWKRXWDQ\WUHDWPHQW7KH DYHUDJHUHODWLYHH[SUHVVLRQOHYHORIWKHKLJKHVWSHUIRUPLQJOLQH /LQHIRUERWKJHQHV ZDVVHWDW6WDWLVWLFDODQDO\VLVGRLQJD7 WHVW VWXGHQWWWHVW VKRZHGWKDWWKHGLIIHUHQFHLQH[SUHVVLRQEHWZHHQERWKOLQHVZDVVLJQLILFDQW S IRUERWKJHQHV

45 Chapter 3

3.3.4 The effect of the DEX treatment on rooting of in vitro shoots

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Figure 3.4 The effect of a 6-week DEX treatment on the rooting of shoots: 7KHSHUFHQWDJHVRIVKRRWVJLYLQJURRWVRQPHGLDZLWK GLIIHUHQW'(;FRQFHQWUDWLRQVDUHJLYHQWRJHWKHUZLWKWKHVWDQGDUGHUURURIPHDQVDVEDUVRQWKHFROXPQV7KHSHUFHQWDJHRIURRWLQJ VKRRWVZDVVLJQLILFDQWO\FRUUHODWHG DWOHYHO ZLWKWKH'(;FRQFHQWUDWLRQDFFRUGLQJWR3HDUVRQFRUUHODWLRQDQDO\VLV WDLOHG LQ 6366

Figure 3.5 The effect on survival of regenerating shoot clusters of 5-FC and Km at subcycle 1: &KDUW$GHPRQVWUDWHVWKHHIIHFWRI )&VHOHFWLRQIRUERWKOLQHVDIWHUGLIIHUHQWWUHDWPHQWZLWK'(;&KDUW%JLYHVWKHHIIHFWRI.PVHOHFWLRQ7KHEDUVRQWKHFROXPQ UHSUHVHQWWKHVWDQGDUGHUURU 6(

3.3.5 The efficiency of marker-free plant generation as monitored by the treatments with 5- FC and Km at subcycle 1

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46 Ǧǡ Crambe abyssinica individual shoots kept regenerating and became regeneration clusters at the end of the term. The regeneration clusters consisted of green shoots, white shoots or a mixture. The survival rate is defined as the number of clusters that still had green shoots left. The survival rates after the 5-FC treatments for 3 weeks are presented in Figure 3.5A. According to a Chi-square test, the survival rates of DEX (5, 15, 25 ȝM) treated material of both lines were significantly higher than the ones without DEX. Comparing the two GM lines, their 5-FC survival rates were significantly different from each other, with the fraction of 5-FC survival being generally lower for line 2 than for line 1. Moreover, the Km treatments also showed differences related to the various DEX treatments. Figure 3.5B displays the rates of subjected explants giving no bleaching of shoots. Surprisingly, the explants of line 1 without DEX showed bleaching of regenerating shoots at 12.8%, while the materials without DEX from line 2 showed no bleached shoot at all, as expected. Chi-square tests also showed that on Km selection, the shoot-clusters on any treatment with DEX (5, 15, 25 ȝM) gave significantly lower percentages of survival shoots than the explants on 0 DEX; no significant differences were found between the various concentrations tested. The material of line 1 gave more serious bleaching than line 2.

3.3.6 Survival of regenerating shoot-clusters after each subcycle of 5-FC selection

Figure 3.6 The efficiency of 5-FC selection: The efficiency of 5-FC selection is demonstrated by plotting survival at different subcycles, 1 to 4 against the theoretical multiplication rate that can be achieved without any selection. The dotted and dashed lines (with Y axis on the left) display the dynamics of shoot survival for respectively line 1 and line 2. For the X-axis, ‘start’ means the beginning of subcycle 1, and the rest means the ends of each subcycle. The start value was calculated as the total number of shoots starting 5-FC selection, from the axillary bud regeneration, divided by the number of initial rooting plants times three as the number of axillary buds isolated from them. The start number for line 1 is nine, and for line 2 is six. As start value for the ideal curve 8 was taken which is close to the average start-value of line1 and line 2. The full line (with Y axis on the right) shows the ideal shoot multiplication curve starting from eight shoot in the beginning, assuming that one shoot in regeneration medium for 3 weeks without selection will produce 3 shoot clusters on average. Then, after 4 rounds of shoot to shoot amplification, there will be 648 shoots in the end.

The regeneration medium was used as the basic medium for the selection in each subcycle. So, there was always regeneration in parallel with selection. According to the results of preliminary experiments, without selection, each shoot subjected to regeneration medium for 3 weeks would give rise to on average 3 newly regenerating shoots. Hence, the theoretical multiplication rate for a plant put on rooting medium, from which three axillary buds can be isolated which are put through four rounds of multiplication subsequently would lead to 243 shoots. The theoretical curve for this experiment based on the average number of axillary buds per rooting plant of both lines taken together is shown in Figure 3.6 and compared there with the actual number of regenerating shoots as obtained after treatment with DEX and selection on 5-FC visualizing the effect of selection in this stage. It is clear that in this experiment, because of the 5-FC selection, the number of surviving shoots was constantly decreasing step-wisely. In Figure 3.6, the dotted and dashed lines demonstrate the declining numbers of shoots on 5-FC for the two lines. The actual numbers of surviving shoot-clusters at the end of each subcycle are shown in the Table 3.4.

Ͷ͹ ͵

At the end of subcycle 4, there were 18 surviving shoots for line 1, and those were obtained from 8 regenerating axillary buds of step 2, and from 5 originally rooting plants in step 1; for line 2, there were 15 survivors, which were derived from 4 axillary buds of step 2, and from 4 rooting- plants of step 1(Table 3.1).

Table 3.4 The numbers of surviving shoot-clusters at the end of each subcycle

# # # # Number Number Number Number of of of of plants axillary axillary Line axillary Sub 1 Sub 2 Sub 3 Sub 4* put on buds buds at buds rooting put on the isolated medium 5-FC start of (step2) (step 1) (step 3) Sub 1 1 66 212 212 201 346 57 32 18 (4;5) 2 46 108 108 101 141 12 26 15 (4;4) Note: As shown in the table, from the step 3 to the start of Sub 1, a few axillary bud explants were discarded because that they gave no green regeneration shoots. Between brackets are the number of axillary buds at step 2 from which the surviving shoot cluster are derived and the number of original rooting plants at step 1,from which they are derived respectively.

3.3.7 GUS staining of surviving shoot-clusters after prolonged 5-FC selection

GUS staining was done on the surviving shoots at the end of subcycle 3 and subcycle 4. Among those survivors, GUS negative shoots (as white as WT material) were found in both lines, which implied that the starting material was chimeric, containing both transformed and untransformed cells in both cases. For line 1, 14 out of 18 total green shoot clusters showed no GUS staining (without any blue color) coming from three of the five original rooted shoots. For line 2, only one of the shoot clusters proved to be GUS negative derived from one original rooted plant, while the other 14 clusters stained positive. All in all, the overall percentage of GUS positive shoots from both lines, indicating putative transgenic, marker-free material was 54.5% of the remaining shoot cultures after prolonged selection on 5-FC. The GUS positive survivors of line1 originate from two Step-1 rooting plants of the 15 ȝM DEX treatment; those of line 2 were from three Step-1 rooting plants, one of the 0 ȝM DEX (spontaneous recombination) and two of the 15 ȝM DEX treatment.

3.3.8 Identification of true marker-free plant material

PCR analysis was performed on two separate leaves from each shoot culture at the end of subcycle 3 and at the end of subcycle 4 in parallel with the GUS staining. The results showed that the surviving shoot clusters consisted of marker-free, non-marker-free or WT material or a mixture of any of the three types. Marker-free shoots testified by PCR only presented a small fraction of the total number of plants. The PCR results at the end of subcycle 4 showed that from the GUS positive regenerating shoot clusters, 37.5% were marker-free and 62.5% were non- marker-free. All of those marker-free shoots were derived from line 2, and amounted up to 15 in total. Among them, one was from obtained from the 0 ȝM DEX treatment, the rest from 15 ȝM DEX treatment.

The putative marker-free shoots were evaluated again by taking another two leaves from the cluster for a new DNA isolation and PCR run, as well as for a GUS assay after subcycle 4. The GUS staining result was positive for all and proved the transgenic nature of this material to be consistent. However, the second PCR test showed that this time among the shoot clusters, previously found to be QSW,, negative for both leaves, only 30% could be reconfirmed as marker- free, which implied that among the surviving shoots, many of them were still chimeras of marker- free and non marker-free cells. All of the double confirmed marker-free shoots were from line 2, 15 ȝM DEX treatments. Figure 3.7 provides a flow-diagram demonstrating how a batch of marker-free shoots was finally acquired at the end of subcycle 4 starting from one rooting plant as

Ͷͺ The generation of Marker-free, Genetically Modified Plants in Crambe abyssinica

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49 Chapter 3

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Table 3.5 GUS staining and PCR tests on T1 seedling obtained from the marker-free shoots identified earlier

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50 The eneration of Marker-free, Genetically Modified Plants in Crambe abyssinica

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51 Chapter 3

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Chapter 4: Increasing erucic content of Crambe abyssinica seed-oil by genetic modification

Weicong Qi1, 2, Iris E M Tinnenbroek-Capel1, Elma M J Salentijn1, Zhao Zhang 3, Dongli Gao1, Bangquan Huang4, Jihua Cheng 1,5, Richard G F Visser 1, Frans A Krens1, Eibertus N Van Loo1§

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Chapter 4

Abstract

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54 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

4.1 Introduction

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55 Chapter 4

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A. pHellsgate CaLPAT2-RNAi

B. pWatergate-4G

C. pHan2

56 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

D. pWatergate-3G

Figure 4.1 the functional genes contained by the 4 vectors used in the present research: 7KHUHDUHYHFWRUVZHUHXVHGLQSUHVHQW VWXG\$QGDOORIWKHPKDYH137,,DVWKHVHOHFWDEOHPDUNHUIRUSODQWWUDQVIRUPDWLRQ,QS+HOOVJDWH&D/3$751$L A WKH&D/3$7 51$LZDVFRQWUROHGE\S&D09VSURPRWHU,QWKH UHVWYHFWRUVJHQHVDGMXVWLQJSODQWVHHGRLOFRPSRVLWRQLQS:DWHUJDWH* B S+DQ C DQGS:DWHUJDWH* D ZHUHUXOHGE\%UDVVLFDQDSXV1DSLQSURPRWHU S1DSLQ DEEUHYLDWLRQV/%OHIWERUGHU5%± ULJKWERUGHUWQRV126WHUPLQDWRUW2&62&6WHUPLQDWRUW.&6±.&6WHUPLQDWRU .&6 )$( $V VKRZQ LQ )LJXUH IRXU ELQDU\ YHFWRUV ZHUH XVHG LQ WKLV VWXG\ 7KH YHFWRU S+HOOVJDWH &D/3$751$L ZDV FRPSRVHG RI WKH 51$ LQWHUIHUHQFH JHQH DJDLQVW &D/3$7 &D/3$7 51$L ZKLFKZDVFRQWUROOHGE\WKH6SURPRWHUDQGWHUPLQDWRU7KHS+DQDQGS:DWHUJDWH *YHFWRUVZHUHDFTXLUHGIURPWKH8QLWRI%RWDQ\$DFKHQ8QLYHUVLW\RI7HFKQRORJ\7KHS+DQ YHFWRU LV FDUU\LQJ WZR JHQHV ZKLFK DUH /G/3$7 DQG %Q)$( DQG S:DWHUJDWH* FRQWDLQV /G/3$7%Q)$(DQG&D)$'51$L7KHS:DWHUJDWH*FRQVWUXFWFRQWDLQV/G/3$7%Q)$( &D)$'51$L DQG &D/3$751$L ZDV DFTXLUHG DIWHU WKH PROHFXODU PRGLILFDWLRQ RI S:DWHUJDWH*)XQFWLRQDOJHQHVLQS+DQS:DWHUJDWH*DQGS:DWHUJDWH*ZHUHFRQWUROOHG E\WKH1DSLQSURPRWHU$OORIWKRVHYHFWRUVFRQWDLQHGWKH137,,JHQHDVVHOHFWDEOHPDUNHU)RU LQRFXODWLQJ SODQWV S+HOOVJDWH &D/3$751$L S:DWHUJDWH* DQG S:DWHUJDWH* ZHUH WUDQVIRUPHGLQWR$JUREDFWHULXPVWUDLQ$*/ZKLOHS+DQZDVLQWURGXFHGLQ$*/ /D]RHWDO

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Modifying pWatergate-3G into pWatergate-4G

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57 Chapter 4

4.2.1 Plant transformation

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58 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

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Table 4.1 The primers of Q-PCR for testing LPAT2 expression level in Arabidopsis leaf, crambe leaf and crambe developing seed

Target Gene Primers name Sequence $W/3$7B) **7**77*&$*$$$&&77*7 $UDELGRSVLV/3$7 $W/3$7B5 $*&$7*77&777*&&&$77& *, $W/3$7B) **7**77*&$*$$$&&77*7 $W/3$7B5 *$*&$7*77&777*&&&$77

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4.2.4 Gas chromatography analysis

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59 Chapter 4

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Table 4.2 The amount of seeds used for FAME analysis

Generation Vector Seed Family No. Seed No.

:LOGW\SH

7 S+HOOVDJDWH&D/3$751$L

7 S+DQ

7 S:DWHUJDWH*

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Figure 4.2 the target sequence picked up for C. abyssinica endogenous LPAT2 RNAi: $ SDUW RI P51$ RI & DE\VVLQLFD/3$7DQGWKHFRUUHVSRQGLQJSHSWLGHZHUHGLVSOD\HGLQWKHFKDUW7KHORQJEDUPDUNHGWKHZKROHVHTXHQFH ES LQFRUSRUDWHGLQWRWKH51$LYHFWRUODWHUDQGWZRVKRUWEDUVVKRZHGWZRFRQVHUYHGUHJLRQVWKH1+;;;'ER[ DQG)39(*75ER[

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60 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

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Figure 4.3 qRT-PCR tests on pHellsgate-CaLPAT2-RNAi GM plant materials of Arabidopsis and crambe: (QGRJHQRXV/3$7 H[SUHVVLRQOHYHOVRIOHDIPDWHULDOIURP7$UDELGRSVLVDQG7FUDPEHSODQWVDQGRIGHYHORSLQJ7FUDPEHVHHGVZHUHDQDO\]HGE\ T573&57KH*0OLQHVWKDWVKRZHGWKHODUJHVWGHFUHDVHVDUHVKRZQLQWKHFKDUWV7KHFROXPQLVWKHDYHUDJHRIUHSHDWVDQGWKHEDULV WKHVWDQGDUGGHYLDWLRQ,QFRPSDULVRQZLWKWKHZLOGW\SHWKH/3$7H[SUHVVLRQLQWUDQVJHQLF$UDELGRSVLVDQGFUDPEHZDVVXSSUHVVHG VLJQLILFDQWO\LQGLIIHUHQWWLVVXHV DFFRUGLQJWRWKHVWXGHQW7WHVW

Figure 4.4 The phenotype of LPAT2 knockdown in Arabidopsis by RNAi: 7KH 7 SODQW RI $UDELGRSVLV WUDQVIRUPHG ZLWK S+HOOVJDWH&D/3$751$LVKRZHGVKRUWHUVLOLTXHV SDQHO$ WKDQWKRVHRIWKHZLOGW\SH SDQHO% ,QVLGHGHYHORSLQJVLOLTXHVVRPH GHYHORSLQJVHHGVZHUHDERUWHG SDQHO&PDUNHGE\DUURZV 7KHH[SUHVVLRQRIHQGRJHQRXV/3$7LQWKHWUDQVJHQLFSODQWVZDVGRZQUHJXODWHGEHFDXVHRIWKH 51$LZKLFKZDVFRQILUPHGE\WKHT573&5$PRQJWKH7OLQHVRI$UDELGRSVLVWKHPRVW HIILFLHQW51$LHIIHFWLVVKRZQLQ)LJXUH SDQHO$ 7KH/3$7H[SUHVVLRQOHYHOLQWKH*0 SODQWOHDIWLVVXHZDVDERXWIROGORZHUWKDQWKHZLOGW\SH VLJQLILFDQWS E\VWXGHQW7

61 Chapter 4

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Figure 4.5 Southern blotting analysis on T0 crambe plant of pHan2 transformation: 7KHSUREHLVGHVLJQHGWDUJHWLQJWKH137,, JHQHDQGWKUHHUHVWULFWLRQHQ]\PHV 'UD,;ED,DQG(FR5, ZHUHXVHGIRU'1$GLJHVWLRQ7KH6RXWKHUQEORWWLQJGHPRQVWUDWHGWKDW RQHOLQH ,' FDUULHGPRUHWKDQ7'1$LQVHUWLRQVOLQHFRQWDLQHG7'1$LQVHUWLRQVDQGWKH7'1$LQVHUWLRQQXPEHURI ZDV RU 'LVWLQJXLVKDEOH EDQGV RQ WKH EORW DUH PDUNHG ZLWK DQ DVWHULVN WKH DUURZV LQGLFDWH WKH GLUHFWLRQ RI WKH HOHFWURSKRUHVLV

Figure 4.6 qRT-PCR analysis on the developing GM seeds of pWatergate-3G and pWatergate-4G transformations: 7 GHYHORSLQJVHHGVRIGD\VDIWHUSROOLQDWLRQZHUHXVHGIRUWKLVDVVD\7ZR7S:DWHUJDWH*SODQWVDQGRQH7S:DWHUJDWH*SODQW ZHUHVHOHFWHG7KHZLOGW\SHVHHGVZHUHDFTXLUHGIURPFRQWUROSODQWVDIWHUDWLVVXHFXOWXUHSKDVHDVZHUHWKHWUDQVIRUPHGRQHV3DQHO$ VKRZVWKHH[SUHVVLRQOHYHORI&D)$'SDQHO%VKRZVWKHH[SUHVVLRQOHYHORI&D/3$7

62 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

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Table 4.3 Fatty acids content variation in the seeds of T1 generation transgene and wild type control

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Variation of polyunsaturated fatty acid (PUFA) content of the T1 seeds’ oil

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63 Chapter 4

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The variation of erucic acid and other fatty acids in the T1 seeds’ oil of transformants pHellsgate CaLPAT2-RNAi

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64 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification pWatergate-4G

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In summary

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The fatty acid content variation because of genetic modification

Figure 4.8 Correlation analysis between different fatty acid compounds in wild type and GM C. abyssinica seeds: 7KH*& DQDO\VLVGDWDRIDOO*0VHHGVDQGWKHZLOGW\SHFRQWUROZHUHDVVHPEOHGWRDQDO\]HWKHFRUUHODWLRQHIIHFWEHWZHHQGLIIHUHQWIDWW\DFLGV IROORZLQJWKHPHWKRGRI3HDUVRQFRUUHODWLRQ7KHQXPEHULQHDFKFHOOLVWKHYDOXHRI3HDUVRQ¶V5)URPWRWKHQWRWKHFRORU FKDQJHGIURPEOXHWRZKLWHDQGWKHQWRUHG([FHSWWKHFRUUHODWLRQVRI&)$&DQG&)$&WKHRWKHUFRUUHODWLRQV UHGLVSRVLWLYHRUEOXHLVQHJDWLYH ZHUHDOOVLJQLILFDQW SRU

65 Chapter 4

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Trierucin content of the transgenic seeds

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Figure 4.9 the trierucin content of pHan2 and pWatergate-3G transgene seeds: $IUDFWLRQRIVHHGVIRUIDWW\DFLGFRPSRVLWLRQ DQDO\VLVZDVDOVRDQDO\]HGIRUWKHLUWULHUXFLQFRQWHQW7KH7VHHGVRIS+DQDQGS:DWHUJDWH*WUDQVIRUPDWLRQDUHVKRZQLQWKH FKDUW$FFRUGLQJWRD3HDUVRQFRUUHODWLRQDQDO\VLVIRUVLQJOHVHHGVWKHFRQWHQWRIWULHUXFLQFRUUHODWHGVLJQLILFDQWO\DWS ZLWKWKH HUXFLFDFLGFRQWHQW

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66 Increasing rucic ontent of Crambe abyssinica eed-oil by enetic odification

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68 Increasing erucic content of Crambe abyssinica seed-oil by genetic modification

Supplementary Figure

Supplementary Figure 4.1 the identification of the successful recombination for constructing pHellsgate-CaLPAT2-RNAi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

Chapter 5: Producing wax esters in Crambe abyssinica

Weicong Qi1, 2, Iris E M Tinnenbroek-Capel1, Elma M J Salentijn1, Edgar B Cahoon3, Ivo Feussner4, Bangquan Huang5, Jihua Cheng 1,6, Richard G F Visser 1, Frans A Krens1, Eibertus N Van Loo1§

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Chapter 5

Abstract

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72 Producing Wax Esters in Crambe abyssinica

5.1 Introduction

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73 Chapter 5

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5.2 Materials and Methods

5.2.1 Plant materials and transformation vectors

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A: pBinGlyRed ScFAR+ScWS

B: pBinGlyRed ScFAE+ScFAR+ScWS

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Transformation and selection

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74 Producing Wax Esters in Crambe abyssinica

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5.2.2 Identification of potential transformants

Screening for fluorescence

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Southern blotting

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5.2.3 Crossing

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75 Chapter 5

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5.2.4 Analysis of fatty acid and fatty alcohol composition of the WE in GM crambe seeds

Gas chromatography

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Electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS)

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5.2.5 Half-seed analysis and seedling growth evaluation

76 Producing Wax Esters in Crambe abyssinica

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Figure 5.2 Crambe seed image under binoculars: 7KHSLFWXUHVKRZVWKHFRW\OHGRQVDWWKHRXWHUVLGHRIWKHVHHGDQGEHWZHHQWKH FRW\OHGRQVWKHUDGLFOHLVYLVLEOH)RUFKLSSLQJRQHFRW\OHGRQLVFXWRIIDORQJWKHYHUWLFOHOLQHDVLQGLFDWHG$Q\GDPDJHWRWKHUDGLFOH VKRXOGEHDYRLGHG

5.2.6 Statistical analysis

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5.3 Results

5.3.1 Identification of transformants

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77 Chapter 5

Figure 5.4 DsRed Fluorescence: &RQVWUXFW)$5:6KDVDIXQFWLRQDO'V5HGJHQHDVDUHSRUWHUJHQHSLFWXUH$DQG%VKRZD7 GHYHORSLQJVHHGLQ89DQGEULJKWILHOGPLFURVFRS\UHVSHFWLYHO\FKHFNLQJIOXRUHVFHQFHRIWKHSRG&DQG'VKRZWKH:7(DQG) VKRZVGHYHORSLQJ7HPEU\R*DQG+VKRZWKHGHYHORSLQJHPEU\RRI:7

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5.3.2 Determination of WE content in the seed oil of different transgenic lines

78 Producing Wax Esters in Crambe abyssinica

Figure 5.5 Relation between T-DNA insert number of FAR+WS and FAE+FAR+WS and WE content in GM crambe lines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

5.3.2. Modulating the WE content by targeted selection in subsequent generations

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Figure 5.6 Effect of selection between and within different GM lines of crambe with the FAR+WS construct:$7 ,QGLYLGXDO 7VHHGVRIDVLQJOH7SODQWZLWKD7'1$LQVHUWLRQQXPEHURIWKUHH7 ,QGLYLGXDO7VHHGVIURPDOO7VHHGVWKDWJHUPLQDWHG IRXU7SODQWV 7 LQGLYLGXDO7VHHGVRIWKHEHVWWZR7IDPLOLHV)RU7VHHGWKHVHOHFWLRQRQWKHLUIOXRUHVFHQFHQDWXUHZDV DSSOLHG7KH\D[LVLQGLFDWHVWKHUHODWLYHIUHTXHQF\RIWKHDSSHDUDQFHRIWKHVHHGZLWKLWV:(FRQWHQWLQFHUWDLQUDQJHPDUNHGLQ[D[LV GXULQJWKHDVVD\7KHJHUPLQDWHGVHHGVZLWKRXWIOXRUHVFHQFHZHUHGLVFDUGHG)URP7WR7WKH:(FRQWHQWLVWKHFULWHULRQWKHWZR 7OLQHVZLWKKLJKHVW:(ZHUHFKRVHQWRJHQHUDWHWKH7IDPLOLHV%$IWHUVHOHFWLRQWKH7VHHG¶VUHODWLYH:(FRQWHQWLVVLJQLILFDQW KLJKHUWKDQWKH7DQG7GLIIHUHQWOHWWHUVRQWKHWRSRIHUURUEDUVLQGLFDWHGDVLJQLILFDQWGLIIHUHQFHDWWKHS OHYHODFFRUGLQJWRD RQHZD\$129$DQDO\VLV

79 Chapter 5

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Figure 5.7 Effect of selection between and within different GM lines of crambe with the FAE+FAR+WS construct: ,QWRWDOWKHUH ZHUHVHHGVIURP7IDPLOLHVZLWKRQHWRILYH7'1$LQVHUWLRQVXVHGIRUWKLVUHVHDUFK7KH7VHHGVLQGLFDWHG:(FRQWHQWPRUH WKDQZHUHDOOIURPOLQHVZLWKILYH7'1$LQVHUWLRQV7KHDYHUDJHRI7VHHG:(FRQWHQWZDVEXWWKH7¶VIHOOLQWR GXHWRWKHORVVRIJHUPLQDWLRQDQGVHOHFWLRQ

5.3.3 Characterization of WE species in the GM crambe lines by ESI-MS/MS

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80 Producing Wax Esters in Crambe abyssinica

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Figure 5.8 Analyzes of WE composition in GM crambe seeds by ESI-MS/MS: ,QGLYLGXDO VHHGV IURP IDPLOLHV :7% :7%DQG:7%UHVSHFWLYHO\ ZLWKWKHKLJKHVW:(FRQWHQWDIWHUVHOHFWLRQZHUHWDNHQ $LV WKHDEVROXWHVHHG:(FRQWHQWRIWKH IDPLOLHVRXWRIWKHVHOHFWLRQ:7%DQG:7%DUH7IDPLOLHVRI)$5:6DQG:7%LV7IDPLO\ RI)$()$5:6)LYH VHHGVZHUHUDQGRPO\VHOHFWHGIURPHDFKIDPLO\%VKRZVGLIIHUHQW:(VSHFLHV¶UHODWLYHFRQWHQWLQWKHWRWDO:(RIWKH*0FUDPEH VHHGVGHPRQVWUDWHGE\FKDUW$WKHFROXPQVVKRZWKH:(VSHFLHVRIWRSFRQFHQWUDWLRQWKHOHIWSDUWLVIDWW\DOFRKROPRLHW\DQG ULJKWIDWW\DFLG7KHOHWWHUVEHWZHHQDQGPHDQVWKDWIROORZWKHRQHZD\$129$WHVWWKHFRQFHQWUDWLRQVRIWKHVH WZR:(VLQ)$()$5:6DUHVLJQLILFDQWO\GLIIHUHQWIURPWKHRWKHUWZR)$5:6IDPLOLHVUHVSHFWLYHO\ZLWKS &GLVSOD\VWKH IDWW\DOFRKROVDQGIDWW\DFLGVEXLOWLQWRWKH:(VDQGWKHLUFRQFHQWUDWLRQVFRQWDLQHGE\WKHVHHGVRIWKRVHWKUHHIDPLOLHV

81 Chapter 5

5.3.4 Tailoring WE content after crossing FAR+WS GM plants with FAD2-RNAi GM plants

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Table 5.1 Crossing combinations and numbers of F1 plants used for further research

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82 Producing Wax Esters in Crambe abyssinica

Figure 5.9 WE content in seed oil of WT, FAR+WS line, CaFAD2-RNAi line and of the cross combination ScFAR+ScWS+CaFAD2-RNAi: :W :LOG W\SH FY *DODFWLFD )$'51$L WUDQVJHQLF OLQH RI &D)$'51$L :6)$5 WUDQVJHQLFOLQHRI)$5:6; :6)$5 [ )$'51$L FURVVEHWZHHQ)$5:6WUDQVJHQLFOLQHDQG&D)$'51$LWUDQVJHQLF OLQH

Figure 5.10 PUFA-content (C18:2+C18:3) in seed oil of WT, FAR+WS line, CaFAD2-RNAi line and of the cross combinating ScFAR+ScWS+CaFAD2-RNAi: WKHODEHOVDUHDVVDPHDV)LJXUH

5.3.6 Seed germination and seedling growth of the GM seeds containing WE

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83 Chapter 5

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85 Chapter 5

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Table 5.2 The comparison of fatty acids and fatty alcohols in Jojoba WE and GM crambe WE

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86 Producing Wax Esters in Crambe abyssinica

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87 Chapter 5

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Chapter 6: General discussion

Chapter 6

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6.1 Observations and remarks

6.1.1 Avoiding chimerism

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90 General Discussion

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6.1.2 Optimizing the copy-number of T-DNA insertion for a desired trait

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91 Chapter 6

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6.1.3 How to get seeds with high WE content still able to germinate

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92 General Discussion

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6.2 The technical route in engineering seed oil metabolism

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6.2.1The biosynthesis pathway of seed oil: from sucrose to TAG

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93 Chapter 6

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94 General Discussion

6.2.2 Use of metabolic engineering to reformulate the lipid biosynthesis system in developing seed

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95 Chapter 6

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6.3 Potential beneficial genes for crambe GM breeding

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96 General Discussion

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Table 6.1 A list of genes possibly interesting for crambe transformation to alter/modify oil content and/or composition

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6.4 Conclusions and prospective

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97 Chapter 6

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References

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115

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11͸ 115

Education Statement of the Graduate School Experimental Plant Sciences ,VVXHG WR :HLFRQJ 4L 'DWH $XJXVW *URXS 3ODQW%UHHGLQJ :DJHQLQJHQ 8QLYHUVLW\ 5HVHDUFK &HQWUH

6WDUWXS SKDVH GDWH Ź )LUVWSUHVHQWDWLRQRI\RXUSURMHFW Using Genetic Transformation to produce Erucic Acid and Novel Wax Esters in &UDPEHDE\VVLQLFD Jan 05, 2009 Ź :ULWLQJRUUHZULWLQJDSURMHFWSURSRVDO Production and modification of Erucic Acid and Novel Wax Esters in &UDPEHDE\VVLQLFD Oct 2008 Ź :ULWLQJDUHYLHZRUERRNFKDSWHU Ź 06FFRXUVHV Ź /DERUDWRU\XVHRILVRWRSHV 6XEWRWDO6WDUWXS3KDVH FUHGLWV

6FLHQWLILF ([SRVXUH GDWH Ź (363K'VWXGHQWGD\V EPS PhD student day, Utrecht University Jun 01, 2010 EPS PhD student day, Wageningen University May 20, 2011 Ź (36WKHPHV\PSRVLD EPS theme 3 'Metabolism and Adaptation', Leiden University Feb 19, 2010 EPS theme 3 'Metabolism and Adaptation', Wageningen University Feb 10, 2011 Ź 1:2/XQWHUHQGD\VDQGRWKHU1DWLRQDO3ODWIRUPV ALW meeting "Experimental Plant Sciences", Lunteren Apr 19-20, 2010 ALW meeting "Experimental Plant Sciences", Lunteren Apr 04-05, 2011 ALW meeting "Experimental Plant Sciences", Lunteren Apr 02-03, 2012 Ź 6HPLQDUV VHULHV ZRUNVKRSVDQGV\PSRVLD Symposium 'Photosynthesis: from femto to Peta and from nano to Global', Wageningen University Nov 05, 2009 Plant Breeding Seminar Series Feb 2009-2013 Workshop: Plant facility Sep 07, 2012 Minisymposium" How to write a world class paper" Apr 19, 2011 Minisymposium" Plant Breeding in the Genomics Era" Nov 25, 2011 Plant Breeding Research Day (Arnhem) Feb 28, 2012 Conference Next Generation Plant Breeding (Ede, The Netherlands) Nov 12-13, 2012 Invited seminar by Anne Endler 'Dissecting Cellulose Production in Arabidopsis' Jun 15, 2010 Invited seminar by BGI: Genomics in China Apr 01, 2010 Plant Sciences seminars by Andries Koops: 'Bioscience strategy on plant-based raw materials for a biobased economy' and Luisa Trindade: 'Targeted breeding for a Biobased Economy' Dec 14, 2010 Ź 6HPLQDUSOXV Ź ,QWHUQDWLRQDOV\PSRVLDDQGFRQJUHVVHV 5th European Symposium on Plant Lipids, Gdansk Jul 08-13, 2011 Ź 3UHVHQWDWLRQV Poster in ALW Lunteren meeting Apr 04-05, 2011 Poster in 5th European Symposium on Plant Lipids, Gdansk Jul 08-13, 2011 Ź ,$%LQWHUYLHZ Meeting with a member of the International Advisory Board Feb 18, 2011 Ź ([FXUVLRQV 6XEWRWDO6FLHQWLILF([SRVXUH FUHGLWV

,Q'HSWK 6WXGLHV GDWH Ź (36FRXUVHVRURWKHU3K'FRXUVHV Spring School 'RNAi' Apr 14-16, 2010 Postgraduate course 'Basic statistics' Jun 22-23, 27-29, 2011 Postgraduate course 'Bio-energy Production from Crop Plant and Algea' Nov 21-23, 2012 Ź -RXUQDOFOXE member of literature discussion group at PBR 2009-2012 Ź ,QGLYLGXDOUHVHDUFKWUDLQLQJ Training in Bangquan's laboratory, Life science college, Huibei University, China Oct-Nov 2009 6XEWRWDO,Q'HSWK6WXGLHV FUHGLWV

3HUVRQDO GHYHORSPHQW GDWH Ź 6NLOOWUDLQLQJFRXUVHV Moral dilemmas in Daily Scientific Practices Feb 15-18, 2011 Project and Time management Oct-Dec 2011 Mobilising your Scientific Network Sep 18 & 25, 2012 Ź 2UJDQLVDWLRQRI3K'VWXGHQWVGD\FRXUVHRUFRQIHUHQFH Ź 0HPEHUVKLSRI%RDUG&RPPLWWHHRU3K'FRXQFLO 6XEWRWDO3HUVRQDO'HYHORSPHQW FUHGLWV

727$/ 180%(5 2)&5(',7 32,176 Herewith the Graduate School declares that the PhD candidate has complied with the educational requirements set by the Educational Committee of EPS which comprises of a minimum total of 30 ECTS credits $FUHGLWUHSUHVHQWVDQRUPDWLYHVWXG\ORDGRIKRXUVRIVWXG\

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