Biology Reference
In-Depth Information
In vitro assays were typically performed by completely coating stigmas of cut
pistils with pollen (>100 grains per stigma); in contrast, for the repulsion assays
only 20-30 pollen grains were deposited per stigma, making it possible to clearly
observe individual tube behaviour. Based on experiments with limited amounts
of pollen, we typically observed 50-80% of the pollen grains produced tubes that
emerged from the style.
LAT52:GFP Transgenic Plants
A HindIII fragment encoding GFP expressed from a post-meiotic, pollen-specific
LAT52 promoter [25], was cloned into PBI121 (Clonetech, CA) and introduced
into A. thaliana (Columbia) plants by Agrobacterium-mediated transformation.
Kanamycin-resistant transgenic plants were selected, and a line containing a single
transgene insertion, based on segregation of kanamycin resistance and GFP, was
chosen for this study. This line had no detectable reproductive defects.
Microscopy
Ovules targeted by pollen tubes in the in vitro assay were counted under a Zeiss
fluorescent dissecting stereoscope. For calculating targeting efficiencies, we in-
cluded only ovule micropyles within 100
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m of a growing pollen tube; within
this range pollen tubes exhibited responses that are typical for cells undergoing
attraction: significant reorientation of growth towards the signal source, followed
by a steady advance towards the target. For time-lapse fluorescent microscopy,
GFP-labelled pollen tubes were observed using a Zeiss Axiovert 100 fitted with
an automated shutter, motorized stage and CCD camera (CoolSNAP fxHQ,
Roper Scientific, Inc Tucson, AZ). Images were captured at 10-minute intervals,
converted to a TIFF format per the manufacturer's instructions using Slidebook
(Intelligent Innovations Imaging, Santa Monica, CA). Pollen tube behaviours
(growth rate, angle of turning, distance from micropyle) were measured and im-
ages were assembled into movies using ImageJ image analysis software (http://rsb.
info.nih.gov/ij/download.html).
Diffusion Rates
To estimate the size of pollen tube signalling molecules, we performed time-lapse
imaging of diffusion of a series of fluorescein-conjugated dextrans (Invitrogen,
Carlsbad, CA) ranging in molecular weight from 3 to 70 kD on the pollen growth
medium used for performing the in vitro pollen tube guidance assay. We dissolved
each dextran compound in pollen growth medium, and spotted 2 ul each of 10
