Biology Reference
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(CS22562), C. rubella (CS22561) and S. irio (CS22653), deposited in the A.
thaliana Biological Resources Center, Ohio State University. Female structures
are unaffected by ms1, making it a convenient source of virgin pistils without
the need for emasculation. No difference was detected between assays performed
with materials derived from the Landsberg or Columbia ecotypes (not shown).
Seeds were sown in soil and stratified at 4°C for 2 days, and plants were grown
under fluorescent light (100
µ
E) for 16 or 24 hrs/day at 40% humidity. To con-
sistently isolate pistils of varying developmental stages, we correlated the initial
day of flowering of our plant population with previously defined floral develop-
ment stages [26]. First, we confirmed that the youngest open flower is similar to
stage 14 [26]. In A. thaliana, flowers continuously arise at the floral apex and are
arranged in a spiral, with the younger buds on the inside. This predictable pattern
allowed us to select stage 14 flowers as a starting point and identify older flowers
(up to stage 18) and younger buds (up to 12a).
In Vitro Pollen Tube Guidance Assay
Growth medium for in vitro manipulations of pollen tubes [10] was determined
to be optimal for also growing pollen tubes through a cut pistil. For the in vitro
assays described here, pollen growth medium (3 ml) was poured into a 35 mm
petri dish (Fisher Scientific, Hampton, USA). This volume of medium was ideal
both for pollen tube growth and for microscopically viewing the interactions be-
tween pollen tubes and ovules. Excised pistils were pollinated under a dissection
microscope (Zeiss Stemi 2000), cut with surgical scissors at the junction between
the style and ovary (World Precision Instruments, Sarasota, USA), and placed
horizontally on pollen growth medium. Pollen tubes emerged from the pistil ~3
hours after pollination and dispersed along the agarose surface for up to ~3 mm
from the pistil.
Unlike previous reports [12,18], ovules were excised dry under a dissection
microscope with a 27.5 gauge needle, from pistils that were held horizontally on
double-sided tape (Scotch brand, 3M, St. Paul, USA). Excised ovules were im-
mediately placed on the pollen growth medium, ~2 mm from the pistil, a distance
that was typically accessible by the emerging pollen tubes. To maximize pollen
tube-ovule interactions, 8-10 ovules were placed at the base of a pistil as shown
in Fig. 1d. Because pollen tubes tend to disburse and grow randomly after leaving
the style, not all ovules, particularly those placed near the cut pistil, are visited by
a pollen tube (Fig. 1e).
For time-lapse imaging, ovules were placed with their micropylar end clos-
est to the pistil excision site. Although not essential for targeting, ovules were
oriented in this manner to reduce the time elapsed before targeting was achieved.
