summary-mutagenesis response

Mon Jun 10 18:12:53 EST 1996

        In response to several requests, I am posting a summary of the
responses to the questions I asked two weeks ago concerning ems

1.  In response to my query as to the smartest way to titer the
mutagenesis, the most frequent (and probably best) answer was to look for
chlorophyll-deficient sectors in M1 plants (5-10% is considered good) and
reconfirm by looking for chlorophyll-deficient plants in the M2
(suggestions varied from 0.5% to 5%).  Most responses considered seedling
lethality to be too variable to use as a reproducible assay, but others
used it successfully.  Theoretical considerations would dictate 37%
survival as optimal.  In practice, 20%-50% is used.

2.  As to my question concerning pool size, harvesting individual M1
families was considered impractical by all respondents.  A pool size made
to fit the container (a pot; a flat; a petri dish) was recommended.  Small
pools were recommended if it is likely that the desired phenotype will
result in sterility.  In this way, it is not impossible to go back to the
pool and pick up the phenotype in a heterozygote.  Large pools (hundreds to
thousands) were recommended if this is not a concern.  Screening 25 times
the number of M2s as there are M1s in the pool yields a 96.5% chance of
recovering all possible recessive mutations from the pool at least once
(the calculation assumes Redei's number of 2 genetically effective cells in
Arabidopsis).  However, this regiment virtually assures that one will pick
up the same mutations many times.  Screening a few times the number of M2s
as there are M1s in the pool minimizes this concern.  Screening
100,000-125,000 M1s is considered necessary for saturation; however, it was
suggested that mutations can be obtained routinely from screening as little
as 1,000-3,000.

        Other useful suggestions were avoiding the use of bleach (don't
sterilize the seed--it interferes with the mutagenesis); do all mutagenesis
from the same bottle of ems (lot to lot variability was reported); and lack
of M1 fertility indicates overmutagenesis.  Concentrations of ems in use
varied from 20 mM to 100 mM, however, because the level of mutagenesis also
depends on the time exposed to mutagen and the lot to lot variability, this
parameter needs to be determined empirically.

        Several papers were also recommended.  I found the following one
especially useful:

Christianson, M. (1991) Fun with mutants:  applying genetic methods to
problems of weed physiology.  Weed Science 39: 489-496.

        The following papers go into the theory of mutagenesis in Arabidopsis:

Li and Redei (1969) Estimation of mutation rate in autogamous
diploids. Radiation Botany 9:125-131

Korneef, Dellaert and van der Veen (1982) EMS-and radiation-induced
mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh.
Mutation Research, 93:  109-123.

Mednik I.G. (1988) On methods evaluating the frequencies of induced
mutations in Arabidopsis based on embryo-test data. Arabidopsis
Information Service 26:67-72

        The following were also recommended, but I can't comment on their
utility, because Berkeley's library doesn't carry them:

Plant gene isolation: principles and practice / edited by Gary D. Foster
and David Twell, John Wiley & Sons, Chichester, New York, Brisbane,
Toronto, Singapore,1996.

Haughn and Somerville (1987) Selection for herbicide resistance at
the whole-plant level. In: Biotechnology in Agriculture (H.M.
LeBaron, R.O. Mumma, R.C. Hneycutt, J.H. Duesing, eds). American
Chemical Society. pp. 98-108

        Thank you very much to everyone who responded.

Allan Shapiro

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