2 Material and Methods
2.10 Cultivation conditions
2.10.1 Algae
O. lucimarinus cultures were grown as batch cultures in 200 ml sterile liquid L1 me-dium, T. pseudonana cultures were grown as batch cultures in 200 ml sterile liquid 1/2 SWES medium with 1 % silicate. All cultures were cultivated at 20 °C, 110 µmol pho-tons*m-2*s-1 and a day length of 14 hours in a Percival climate chamber (CLF Plant Climatics).
L1 medium (Guillard, 1993) 75 mg/l NaNO3
5 mg/l NaH2PO4*H2O 30 mg/l
1 ml/l
Na2SiO3*9 H2O
L1 trace element solution 0.5 ml/l f/2 vitamin solution
The medium was set up in filtered seawater (Biologische Anstalt Helgoland, Germany) and autoclaved at 120 °C for 20 min. The vitamin solution was added afterwards, the medium was stored at 4 °C.
Material and Methods
L1 trace element solution
Stock solution (g/l) Quantity
Na2EDTA*2 H2O 4.36 g
FeCl3*6 H2O 3.15 g
MnCl2*4 H2O 178.1 1 ml
ZnSO4*7 H2O 23 1 ml
CoCl2*6 H2O 11.9 1 ml
CuSO4*5 H2O 2.5 1 ml
Na2MoO4*2 H2O 19.9 1 ml
H2SeO3 1.29 1 ml
NiSO4* 6H2O 2.63 1 ml
Na3VO4 1.84 1 ml
K2CrO4 1.94 1 ml
The solution was set up in ddH2O, autoclaved at 120 °C for 20 min and stored at 4 °C.
f/2 vitamin solution (Guillard and Ryther, 1962) 1 mg/l Cyanocobalamine (vitamin B12) 1 mg/l Biotin (vitamin H)
100 mg/l Thiamine chloride (vitamin B1)
The solution was set up in ddH2O, filter sterilized and stored at 4 °C.
1/2 SWES medium with 1 % silicate 0.2 g/l KNO3
20 mg/l MgSO4*7 H2O 20 mg/l KH2PO4
30 ml/l Soil extract#
5 ml/l Micronutrient solution 450 ml/l ddH2O
455 ml/l 50 ml/l
Filtered seawater (Biological Institute Helgoland, Germany) Saturated solution of Na2SiO3*9 H2O
#Soil extract was produced from garden soil. The soil was mixed with water in the ratio 1:2 and autoclaved three times at 120 °C for 20 min. The supernatant was filtered through a round filter (Whatman GmbH, Dassel, Germany) and the solution was stored at 4 °C.
The medium was autoclaved at 120 °C for 20 min and cooled down. Then sterile fil-trated vitamin B12 (5 µg/l) was added. The solution was stored at 4 °C.
Micronutrient solution
I Stock solution (g/100 ml) Used volume (ml)
ZnSO4*7 H2O 0.1 1
MnSO4*4 H2O 0.1 2
H3BO3 0.2 5
Co(NO3)2*6 H2O 0.02 5
Na2MoO4*2 H2O 0.02 5
CuSO4*5 H2O 0.0005 1
ddH2O 881
II
FeSO4*7 H2O 0.7
EDTA 0.8
ddH2O 100
Both solutions were set up separately in ddH2O to avoid precipitations. They were autoclaved at 120 °C for 20 min, then combined and stored at 4 °C.
2.10.2 Bacteria
E. coli XL1blue cells were grown at 37 °C in liquid Luria and Bertani (LB) medium while shaking at 200 rpm or on solid LB medium. A. tumefaciens cells were grown at 28 °C in liquid YEB medium while shaking at 200 rpm or on solid YEB medium. For the produc-tion of solid medium 1.5 % agar (w/v) (Duchefa Biochemistry, Haarlem, Netherlands) was added to liquid medium. All media were set up in ddH2O and autoclaved at 120 °C for 20 min. For selection purposes different media additives were added.
LB medium (Silhavy et al., 1984) 10 g/l Peptone
5 g/l Yeast Extract 10 g/l NaCl
Material and Methods
Solvent Stock solution End concentration
Carbenicillin ddH2O 100 mg/ml 100 mg/l
Kanamycin ddH2O 50 mg/ml 25 mg/l
Rifampicin Dimethylsulfoxide (DMSO) 50 mg/ml 50 mg/l IPTG
Tetracycline Ethanol 10 mg/ml 10 mg/l
2.10.3 Yeast
All S. cerevisiae strains were grown in liquid or on solid YPD or SD medium at tem-peratures between 16 °C and 30 °C as indicated for each experiment. Liquid cultures were shaken at 150-200 rpm. For production of solid medium 2 % (w/v) agar (Duchefa Biochemistry, Haarlem, Netherlands) were added to liquid medium. All media were set up in ddH2O and autoclaved at 120 °C for 15 min.
YPD medium (complete medium) 10 g/l Yeast Extract
20 g/l Peptone
20 g/l Glucose Monohydrate
For selection purposes, hygromycin B was added to a final concentration of 300 µg/ml after autoclaving the medium.
SD medium (selection medium)
1.7 g/l Yeast Nitrogen Base (MP Biomedicals, Heidelberg, Germany) 2.5 g/l (NH4)2SO4
After autoclaving and before use, 2 % (w/v) sugar (sterile filtrated glucose, raffinose or galactose) as well as synthetic complete drop-out medium mix to a final concentration of 1 x were added.
50 x Synthetic complete drop-out medium mix 2 g Adenine hemisulfate
2 g L-Arginine HCl 2 g L-Histidine HCl 2 g L-Isoleucine 4 g L-Leucine 2 g L-Lysine HCl 2 g L-Methionine 3 g L-Phenylalanine 2 g L-Serine
2 g L-Threonine 3 g L-Tryptophan 2 g L-Tyrosine 1.2 g Uracil 9 g L-Valine
Substances were mixed, leaving out the respective component for selection, and ground. They were resuspended to a concentration of 100 g/l in sterile ddH2O and stored at 4 °C.
2.10.4 Plants
2.10.4.1 Surface sterilization of A. thaliana seeds
Prior to sterile cultivation of A. thaliana plants on plates, seeds were sterilized. For a 150 mm plate, 40 mg of seeds were mixed with 1 ml 6 % (w/v) NaOCl containing 0.1 % (v/v) Triton X-100 in sterile tubes and were incubated on a rocking table for 15 min.
Supernatants were discarded and seeds were washed four times with 1 ml of sterile ddH2O. Afterwards, seeds were resuspended in 6-8 ml 0.1 % (w/v) agar.
2.10.4.2 Cultivation on plates
Surface-sterilized seeds were sown onto plates containing 1/2 Murashige Skoog (MS) medium. For selection of transgenic plants, kanamycin was added to a final concentra-tion of 40 µg/ml after autoclaving. For freshly transformed seeds, also cefotaxime was added to a final concentration of 100 µg/ml to prevent agrobacteria from growing on the plates. Plates were wrapped with 3M micropore tape and then incubated at 23 °C and under constant illumination of 120 µE.
Material and Methods
1/2 MS medium
2.2 g/l Murashige Skoog powder 1 % (w/v) Sucrose
7 g/l Microagar
The medium was set up in ddH2O, adjusted to pH 5.9 with KOH and then autoclaved for 20 min at 120 °C.
2.10.4.3 Cultivation on soil
Prior to usage, soil was incubated for 8 hours at 180 °C to reduce fungal contamina-tions. A. thaliana seeds were sown non-sterile on soil (Frühstorfer Erde Typ EP Nr.
340, Industrie Erdwerk Archut, Lauterbach-Wallenrod, Germany) and incubated for stratification 2-4 days at 4 °C. Afterwards, plants were either cultivated in the green-house at a day length of 16 hours and temperatures between 16-22 °C or in the climate chamber at a day length of 16 hours (120 µmol/m2s), air humidity of 60 % and a tem-perature of 22 °C. C. sativa plants were grown non-sterile on soil (Frühstorfer Erde Typ T 25, Industrie Erdwerk Archut, Lauterbach-Wallenrod, Germany) in the greenhouse at a day length of 16 hours and temperatures between 16-22 °C. The herbicide Basta (Bayer CropScience, Monheim, Germany) containing glufosinate as active ingredient was used for selection of transgenic plants and sprayed one and two weeks after sowing onto the plants.