Arabidopsis : a laboratory manual by Detlef Weigel; Jane Glazebrook

By Detlef Weigel; Jane Glazebrook

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Screening the T l generation has the advantage that any phenotype observed is likely to be dominant, since T l plants are normally hemizygous for a T-DNA insertion (Ye et al. 1999) (see Vacuum Infiltration of Arabidopsis in Chapter 5). The dominant behavior of a mutation should be confirmed in subsequent generations. As with any other insertional mutant, it must be confirmed that the dominant mutation is linked genetically to a T-DNA insertion. Adjacent sequences can then be recovered either by TAIL-PCR (thermal asymmetric interlaced-polymerase chain reaction) (see Chapter 6) or by plasmid rescue.

Trends Plant Sei. 5: 2229. , and Dean C. 1992. Development of an efficient two-element transposon tagging system in Arabidopsis thaliana. Mol. Gen. Genet. 233: 449-461. , and Haseloff J. 1998. Stomata patterning on 38 \: ARABIDOPSIS: A LABORATORY MANUAL the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Dev. Biol. 194: 226-234. R. 1993. Control of flower development in Arabidopsis thaliana by A PETA I Al and interacting genes. Development 119: 721-743.

1 % agarose, and use a Pasteur pipette to sow the seeds on soil (see Chapter 1). If collecting M7 seeds from individual Mj plants, grow the plants at a density of 50-100 per 2-ft2 flat. If collecting M, seeds in pools, grow the plants at a density of 500-1000 per 2-ft2 flat. Collect all of the M, seeds when the plants have reached maturity. 2% EMS exposure for 15 hours generally gives a reasonable level of mutagenesis. However, the efficiency of mutagenesis does vary, so it may be desirable to vary the severity of the treatment by altering the EMS concentration; EMS is unstable over the course of 15 hours, so it is difficult to control the effects of varying times of exposure.

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