segregating Ds insertion lines
C. Robb Flynn
Robb.Flynn at asu.edu
Mon Feb 5 18:36:28 EST 2001
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I have some rudimentary questions on Arabidopsis Ds insertion mutagenesis
that I can't seem to find the answers to. The answers are probably
there, I just don't understand them. I have read both of Dr. Sundarasen's
papers, a number of different websites, and the <i>Arabidopsis protocols</i>
boo, yet I am still unclear on a few things. I would very much appreciate
anyone's time and answers to the following:
<p>1. In regards to the 931 characterized IMA lines described by
Sundarasen that are currently being distributed by NASC, are the seeds
being distributed from the F2 or F3 generation?
<p>2. Can you briefly describe the percent germination numbers expected
for a homozygote, or a heterozygote Ds insertion, at each the F2, F3, and
F4 stages if the seeds are grown on Kan/NAM? Is it possible to tell
the difference between the two based on germ. rates alone?
<p>Basically, I can't yet figure out if I have segregated the seeds enough
to verify that I have a homozygous knock-out phenotype. My germination
numbers are described below (if anyone has the time) but answers to the
above questions should help me figure out where I stand. I realize that
a Southern blot would give me an answer to my question, and I have PCR
data verifying at least one copy of the insertion in the right gene, but
I was hoping germination rates alone could tell me homo vs. hetero.
<p>Thanks,
<p>Robb Flynn
<p>robb.flynn at asu.edu
<br>
<p>Upon receiving a line (~50 seeds) from NASC, I started about 25 seeds
(some on Kan/NAM plates, some on dirt - I did not keep track of numbers
), however only 18 germinated and gave seedlings. All plants were
eventually transferred to dirt and selfed. Seed was collected from
each plant and kept separate (I know which was on dirt and which was on
media). About 200 seeds (1 batch) from each of these plants were
germinated on Kan/NAM plates. 9 of the 10 batches of seeds from plants
initially germinated on dirt were selected against and died (yellow, small,
aborted). The other batch had a ~66% germination rate. 5 of
the 7 batches of seed collected from plants initially germinated on Kan/NAM
showed a germination rate of ~66%, the other two batches had germination
rates of ~75%. Based on this information can you tell me if any of
my seed batches is homozygous for the transosable element/knockout?</html>
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