Thesis report PRI_Martin Beers_final - The Lilium information page

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M. Beers / Thesis Report, May 2006, Plant Research International medium consisted of 2.2 g/l MS salts (+MS vitamins), 50 g/l sucrose and 4 g/l gelrite. The pH was adjusted to 5.8. The plants which were already in tissue culture were simply transferred to new propagation medium. Hereby every individual bulb scale was planted out in a tube. The medium used for this propagation is the same as described above. Both methods were used in order to grow root tips needed for the GISH analysis. 3.3 Mitotic chromosome preparation Root tips were collected and incubated in 0.7 mM cyclohexamide for 5 to 6 h and then fixed in ethanol acetic acid solution (3:1) and stored at 4°C until further use. The root tips were then incubated in a pectolytic enzyme mixture containing 1% (w/v) cellulose RS and pectolyase Y23 in 2 mM citrate buffer (pH 4.5) at 37°C for about 1 – 1.5 h depending on the size of the root tip. Squash preparations were made in a drop of 45% acetic acid and frozen in liquid nitrogen; the cover slips were removed by using a razor blade. Slides were dehydrated in absolute ethanol and airdried. 3.4 DNA probe preparation Genomic DNA from the Oriental cultivar “Sorbonne” and the Asiatic cultivar “Connecticut King” was isolated and respectively used as probe and block. For the GISH analysis the probe was boiled for 10 min to obtain a DNA size of 1 – 10 kb and the block DNA was boiled for 30 min. (100 bp – 500 bp). The probe was then labelled with fluorescein high prime by heating for 10 min at 70 °C and immediately chilled on ice for 5 min. Labelling was done according to the instructions of the manufacturer (Roche, Germany). 3.5 In situ hybridization Slides were incubated at 37°C in RNase A with a concentration of 100 g/ml for 1 h, and incubated in pepsin (5 g/ml) for 10 min, followed by 4% paraformaldehyde for 10 min at room temperature. Between every step the slides were briefly washed in 2X SSC and finally dehydrated with 70%, 90% and absolute ethanol for 3 min in each and airdried. Hybridization followed using a hybridization mixture consisting of 2X SSC, 50% 18
M. Beers / Thesis Report, May 2006, Plant Research International formamide, 10% dextransulphate, 0.25% SDS, 1.25 ng of probe and 0.5 g of block DNA per slide. The hybridization mixture was heated at 70°C for 10 min and then placed on ice for at least 5 min. For each slide 40 l hybridization mixture was used. The preparations were denatured at 80°C for 5 min and incubated overnight at 37°C in a humid chamber. Slides were washed 3X at room temperature in 2X SSC for 5 min and then washed in 0.1X SSC for 30 min at 42°C. Here after the slides were again washed for 3X in 2X SSC for 5 min each. Chromosomes were counterstained with 1 mg/ml DAPI (4,6diamidino2 phenylindole) and a drop of Vectashield antifade (Vector laboratories, Burlingame, CA) was added for its examination under a Zeiss Axioplan 2 Photomicroscope equipped with epifluorescent illumination, filter sets of DAPI and FITC. Images were captured by a Photometrics Sensys 1,305 x 1,024 pixel CCD camera and processed with Genus Image Analysis Workstation software (Applied Imaging Corporation). The DAPI images were sharpened with a 7x7 High Gauss spatial filter and pseudocoloured in blue. The probe fluorescent was green pseudocoloured. Optimal brightness and contrast were achieved by Adobe Photoshop image processing. 19
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