Slutrapport till Partnerskap Alnarp ( ) för projektet: Omega 3 havre Havre med förbättrad fettbalans och förhöjt näringsvärde.

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1 Slutrapport till Partnerskap Alnarp ( ) för projektet: Omega 3 havre Havre med förbättrad fettbalans och förhöjt näringsvärde. Projekttid ( ) Projektansvarig: Anders S. Carlsson, SLU Medsökande: Rickard Jonsson, SW och Olof Olsson, GU, Sammanfattning på svenska (för ytterligare detaljerad information se engelska versionen Bilaga 1.) Bakgrund Projektet har varit ett sammarbete mellan undertecknad, Rickard Jonsson, Svalöf Weilbul och Olof Olsson, Göteborgs Universitet. Det är en del av ett större forskningsområde där fokus ligger på att med bioteknik modifiera kvaliten av havreoljan med avseende på omega 3 fetter. Havrelinjer med en nutritionellt förbättrad havreolja kommer att studeras utifrån vilka förändringar som har skett med oljan och hur en sådan olja påverkar olika näringsrelaterade parametrar. Målsättningen med detta projektet var att testa metoder och teknik framtagna av Olof Olsson för gentransformering av havre samt kloning av gener nödvändiga för modifieringen av havreoljan. Resultat Ett flertal metoder utvärderades under hösten 2005 och våren 2006 vid laboratoriet i Alnarp: Framtagning av materialet dvs havrehypokotyler som erhölls genom groning av havrefrön efter ytsterilisering. Hypokotylmaterialet behandlades med olika hormonblandningar och utvecklade kallus. Från kallus regenererades nya skott efter ytterligare hormonbehandling. Alla metoder fungerade tillfredställande efter ett antal försök. Under senare delen av projekt tiden introducerades transformerings protokoll och olika agrobakterium sorter och två nya selektionsmarkörer, hygromycin och basta prövades. På grund av tidsbrist i slutet av projekttiden kunde inte tillräckligt många tester göras för att dessa parametrar (sorter och markörer) på ett tillfredställande sätt kunder utvärderas. Vid ansökningstiden till forskningsråden i April 2006 fanns resultaten av havreregenerering från kallus samt en del preliminära resultat av tester av transformerings protokol tillgängliga och kunde användas i ansökningar. Den stora ansökan om Omega 3 Havre blev sedan beviljad med ett i första hand 3- årigt forskningsanslag från FORMAS.

2 Vi anser att medlen från Partnerskap Alnarp har varit av stor vikt för detta gynnsamma utslag, genom att det gav oss möjlighet att generera resultat från utprovingen av metoderrna som sedan användes i ansökan. Ekonomisk redovisning Ekonomisk redovisning för SLU lämnas i Bilaga 2. Ekonomisk redovisning för Svalöf Weibull och Göteborgs Universitet återfinns i bilagorna 3 och 4. Anders Carlsson Alnarp Bilagor 1. Resultatredovisning (engelska) 2. Ekonomisk redovisning (SLU) 3. Ekonomisk redovisning (GU) 4. Ekonomisk redovisning (SW)

3 Report from the pilot-study testing techniques and methods in a Agrobacterium-Mediated Transformation of Oat Background Many reports support the idea that an ideally balanced ratio of the omega 6 and omega 3 fatty acids in the grains of oat significantly can improve its nutritional qualities. This suggestion is explored in research collaboration between me, Olof Olsson, GU and Rickard Johnsson, Swalöf Weibul. Our project has an overall goal to investigate the feasibility in developing a concept oat, Omega 3 Oat, with improved fat quality and nutritional value. We combine unique biotechnology and solid experience in lipid biochemistry in order to achieve a raise of the exceedingly low levels of important omega-3 fatty acids in the oat grain. Such a change will radically improve the unfavourable fat quality presently existing in oat. This model oat, with its novel fat quality, will be a unique tool in investigations on how changes in fat composition influence other nutrients in the grain. The Omega 3 Oat with its modified fat quality will constitute an invaluable platform in research aimed at exploring the nutritional beneficial of omega 3 fatty acids. In addition, this utility oat will ultimately represent a positive example of an agricultural application made by gene technology. Results The omega 3 fatty acid qualities will be produced with an Agrobacteriummediated transformation protocol developed by Olof Olsson and Gokarna Gharti- Chetri, Gothenburg University. The different methods included in the transformation protocol have been tested in a pilot project at the Department of Plant Breeding and Biotechnology with the support from the Partnerskap Alnarp. The following report describes the results from the pilot-project. Svetlana Leonova, a guest student from the Vavilov institute in St Petersburg has been attached to the pilot project and done most of the work. She spent time at the Plant Molecular Biology lab, Gothenburg University and was trained by Olof Olsson and Gokarna Gharti-Chetri in basic tissue culture techniques such as seed surface sterilization, preparation of media for seed germinating, callus induction, shoot and root regeneration, handling of the special kind of explants from oat (mesocotyle joints from seedlings) needed to get callus induction and finally the specific transformation procedures. During the one year pilot project Svetlana worked with testing regeneration methods and techniques for Agrobacterium-mediated oat transformation at the Department of Plant Breeding and Biotechnology in Alnarp. Protocols regarding seed germination, sampling of plant material (hypcotyles), callus induction and shoot regeneration have been established and are working well. At the later part of the project protocol for transformation was introduced but there was not enough time available at the end to fully evaluate this part of the work.

4 Below is a description of the methods drawn out. Callus induction (See Figure 1.) Tissue culture is a technique which allows the generation of entire plants from small pieces of tissue by supplying essential nutrients and hormones. One of the key aspects for a successful accomplishment of this method is sterility that provides the plant cells with an environment in which they are protected from for example attacks of fungus. To provide sterile conditions, all the steps of the procedure are done in a sterile fume-hood, autoclaved solutions and tools must be used, seeds (preliminary dehulled) have to be surface sterilized by exposing them to 95% of ethanol, washing in detergent and a disinfecting solution followed by subsequent rinsing in autoclaved water. Callus, is a thickened surface of a plant tissue (leaf, stem etc.). It consists of bulk of cells which at certain conditions (depending on the hormones provided in the media) can regenerate any type of tissue and grow into a whole plant. Thus, callus, infected with Agrobacteria which carries a gene of interest, therefore has an ability to produce transformed plants. After surface sterilization of seeds, they are placed on Petri dishes containing agar with nutrient solution. When seeds have germinated the joints on the hypocotyls are sampled on the third day of germination and placed on new Petri dish containing callus induction media (containing different nutrients) for approximately two weeks in order for the callus to develop. The joints are located on the border between embryonic shoot (hypocotyl) and embryonic root and are comprised by non-specific cells which are able to proliferate intensively. Therefore this tissue from young seedlings was chosen for callus production. When joint has been collected, it is not possible to dissect completely the area of non-specific cells on the seedling from the hypocotyle and embryonic root area. Therefore, during callus induction period, not only callus cells are produced but also those cells that continue to form shoots and roots. Those cells have to be removed for more rapid and efficient callus formation. Figure 1. Oat callus at different developmental stages. A. 1 week old, B. 4 weeks old and C. 8 weeks old.

5 Transformation (See Figure 2.) The calli are then co-cultivated with Agrobacterium (containing the specific transgene) and again placed on the callus induction (CI) media for 48h. To make Agro infection more efficient, a special matter (acetosyringone) is added to the Agrobacterium solution before infection procedure. This substance is normally produced by wounded plant tissue and recognized by the Agrobacterium as a sign that this tissue has lost its integrity and is easy to infect. By adding this substance to the solution with Agrobacterium just before co-cultivation with the plant callus, the natural conditions are mimicked and the bacteria are stimulated. Since infection of the callus is performed during shaking, this procedure usually results in some of the callus tissue to wear off. Therefore, after being rinsed in Cefotaxime (to kill the Agrobacterium after transformation) containing water (300mg per 1L) the callus pieces are placed on Cefotaxime containing CI media (300mg per 1L) for one-two weeks to develop more calli tissue. During the testing of transformation protocol several Agrobacterium strains (GV3101, EHA105 and GV2260) were included in the test. Figure 2. Callus 8 weeks after transformation with Agrobacterium. Shoot regeneration (see Figure 3.) When calli have grown back, they are transferred on to shoot regeneration media containing selection antibiotic (hygromycin or basta). At this step calli are exposed to light (all the previous processes are performed in the darkness). Shoot regeneration is stimulated by adding growth hormones (auxins and cytokinins at specific ratios) into the media. After shoot have emerged in mass they are transplanted onto new media for root development and later onto soil.

6 Figure 3. Figures showing the efficient regeneration of shoots from non-transformed oat callus. Cloning of delta 15 desaturase gene from Arabidopsis The Arabidopsis delta 15 desaturase gene (FAD3) was amplified from an ESTclone and inserted downstream the 35S promoter in the vector part7. After sequencing the inserted gene the expression cartridge of the part7 vector was cut at the Not1 sites and inserted into the binary vector system pgii0179 containing hygromycin as plant selection marker. The resulting plasmid pgii0179::atfad3/35s (see figure 4.) was transformed into Agrobacterium strain GV3101 that is used in the transformation protocol for oat. SpeI (8 446) NotI (1 ) PstI (8 428) XbaI (8 ) Ocs-R2 Ocs-R3 XbaI (770 4) SpeI (1 4) BamHI (20) XmaI (26) AtFAD3-2r-XbaI AtFAD3/6r SalI (7456) SmaI (28) PstI (36) EcoRI (38 ) AtFAD3-4s EcoRV (46) HindIII (7132) HindIII (50) CDS 1 (AtFAD3) AtFAD3-3s ClaI (57) SalI (65) AtFAD3-1s-XhoI XhoI (71) XhoI (6492) KpnI (90) pgii0179::part735s::atfad3 P735S-3S PvuI (242) 8482 bp promoter (35S) PvuI (1 08 9) P1 P hygro NotI (5139) ClaI (2135) SacII (5133) Left border SacI (5126) Rep Origin (psa) Right border CDS (NPTI) 2 PvuI (3458) Figure 4. Map of the plasmid construct pgii0179::atfad3/35s that contain the FAD3 ddna sequence from Arabidopsis under the control of the 35S promoter. 15 desaturase gene from Soybean We wanted to investigate other delta 15 desaturase as well as other promoters (other than 35S) and therefore a delta 15 desaturase from soybean was kindly provided from Dr. Anai at the SAGA university in Japan. This gene sits in the

7 construct phts-ubi-gmfad3 were it is driven by the ubiquitin promoter from maize. This promoter has proven to have high activity when used in Rice. This construct has been transformed into Agrobacterium and was used in the transformation tests of oat hypocotyl tissue. Alnarp Anders Carlsson