Bx 60 of confocal laser scanning microscope

FITC (fluorescein isothiocyanate) and TRITC (tetramethylrho damine) filters were used to assess green fluorescence for GFP and red fluorescence for chlorophyll autofluorescence from chloroplasts. The fluorescence intensity of merged images with orange color revealed the expression of GFP. We selected young leaves of single copy positive plants at 30 days after germination (DAG) to analyze the expression of GFP in transgenic rice with TaAFP-Ba-GFP and TaAFP-Bb-GFP constructs mounted on covered glass slides by an Olympus BX-60 of Confocal Laser Scanning Microscope. We used excitation lasers at 488 and 568 nm to excitate GFP and chlorophyll, respectively; images were collected by FITC and TRITC filters, and single-channel images were superimposed to observe the expression of GFP in leaves. At the same time, Anti-GFP (TransGen Biotech) was selected as a probe to detect the TaAFPB fusion protein in transgenic rice.

When the leaves of wheat grew to the period of one leaf and one terminal bud, the first leaf was cut off about 4 cm from the tip of the leaf, and soaked in 70% ethanol for 3 min. Then, the leaves were washed with distilled water 3 times, the leaf surface was cleaned, and the leaves were attached to a piece of glass. Finally, plasmid DNA (1 μg/μl) were transferred into leaves by biolistic bombardment (PDS-1000/He system of BioRad) transformation. He pressure of the rupture disc was 1100 psi and vacuum degree was 28 inHg. The bombardment distance was 9 cm [50 (link), 51 ]. Every experiment had three biological replicates.
The tdTomatoER expression was viewed in whole leaves mounted on glass slides using the Olympus BX-60 of Confocal Laser Scanning Microscope (excitation light 540 nm, emitted light 580 nm). Images were processed with Adobe Photoshop (Mountain View, CA). Then, for observation of GUS expression, the samples were incubated overnight in a solution of 1 mM X-Gluc in 50 mM phosphate buffer (pH 7.0) at 37 °C. After that, tissues were cleared of chlorophyll in 70% ethanol and photographs of whole-mounted tissues were taken using an optical microscope. Finally, all transgenic plants for each construct were analyzed.