Biology Reference
In-Depth Information
Materials and Methods
Plant Lines and Growth Conditions
The wild-type control lines C24 and Col were supplied by the ABRC stock center.
The line met1a/s (C24) was supplied by J. Finnegan [6]. The line met1-3 (Col)
was supplied by J. Paszkowsky and contains a TDNA insert conferring resistance
to BASTA [17]. The met1-3 line was maintained as heterozygous by repeated
backcrosses to wild-type plants in order to avoid accumulation of epigenetic de-
fects. Once allowed to self, the resulting segregating homozygous plants were used
for emasculation for crosses to wild-type plants and for observation of autono-
mous development.
Plants were grown at 22 C and 60% hygrometry in short days (16 h
night) for three weeks followed by long days (8 h night) in Conviron Growth
chambers.
Microscopy and Measurements
Developing seeds were cleared with Hoyer's medium and observed with DIC optics
with a Leica microscope (DM600). Images were recorded with a Snapshot camera
and processed with Metamorph for morphometric measurements. For confocal
microscopy, material was prepared and observed as described previously [1].
Experimental Strategy
In order to evaluate the relationship between seed size and parental inheritance
of met1 we performed a series of four experiments. We produced crosses between
wild type and met1-3/+ plants grown in the same conditions and obtained two
populations of 900 seeds with inheritance of met1 from the mother or from the
father. We visually separated seeds according to size categories in each population
and tested BASTA resistance in a subset representing the largest or smallest seeds.
In a second series of crosses we produced crosses between wild type emasculated
plants and wild-type or met1-3/+ plants or met1-3/met1-3 plants grown together.
The seeds obtained were imaged and seed size was measured as detailed below and
the data are reported in Figure 1 and Table 1. We obtained a third series of crosses
from single plants in order to have an ideal wild type control to compare seed size
with and to establish correlation with BASTA resistance. The dataset is reported
in Figure 2.
