T-DNA Insertion Mutagenesis in Arabidopsis: Prospects and Perspectives

Abstract

Approximately 13 000 independent transformants of Arabidopsis have been generated utilising the Agrobacterium-mediated seed infection/transformation procedure. The average number of functional independent T-DNA insertion events per transformed line is 1.5, thus constituting a population with 19 500 independent inserts. These transformed lines have been screened under two environmental regimes for visible alterations in phenotype. In one screen the lines were grown under greenhouse conditions and examined at weekly intervals. In the second, seeds from each line were plated under axenic conditions in vertically oriented square petri plates and examined 4 and 9 days after germination. The mutants observed were placed into 7 distinct phenotypic classes: seedling-lethal, size variant, reduced-fertility, pigment defect, embryo-defective, dramatic morphological, and physiological. The mutational spectrum of the first 8000 transformed lines has been reported earlier. The mutational spectrum for a further 5000 transformed lines is reported here and, with several notable exceptions, is similar to that observed previously.

Identification of molecular markers of embryogenesis in Arabidopsis thaliana by promoter trapping

“…These frequencies are relatively low compared with those found by us for Z~gus activation in post-embryonic organs such as leaves (28%), roots (30%) or floral organs (22%), which may be a reflection either of the relative number of genes expressed in the respective organs, or of the possible low levels of expression of Agus fusions in early embryos, or of a bias into the types of genes into which T-DNA inserts, as discussed previously . Insertional mutagenic studies by Forsthoefel et al (1992) on large populations of transgenic Arabidopsis found a frequency of embryonic lethals of 1-5.8%, although particular transformants may contain multiple T-DNA inserts, and only a relatively small proportion of mutants may be due to T-DNA insertion events (Errampalli et al, 1991). Further information on the nature of the proteins encoded by the tagged genes is required before we can consider their functions.…”

Auxin Transport Promotes Arabidopsis Lateral Root Initiation

“…All seed were surface sterilized (Forsthoefel et al, 1992) and plated onto Murashige and Skoog (1962) agar (4.3 g/L Murashige and Skoog salts [Sigma, Poole, UK], 1% sucrose, and 1% bacto-agar, pH 6.0, with 1 M KOH), with the exception of transgenic CycB1:1:GUS seed, which were germinated on Hoagland medium with 0.1% sucrose (Beemster and Baskin, 1998). The plates were placed at 4ЊC for 48 hr and then in constant white light at 22ЊC.…”

Anther development: basic principles and practical applications.

“…Although most male sterility genes and their wild-type alleles have not been cloned and studied (Aarts et al, 1993), phenotypic and histological analyses suggest that some male sterility mutations affect the differentiation and/or function of many anther cell types, including those in the stomium, tapetum, endothecium, and the archesporial and sporogenous layers of the anther primordium (Kaul, 1988;Chaudhury, 1993, this issue). Arabidopsis malesterile mutants are easy to identify because they flower for a longer time period, grow taller, and remain in a green or nonsenesced state longer than their male-fertile, wild-type counterparts (Feldmann, 1991;Chaudhury et al, 1992;Forsthoefel et al, 1992;Aarts et al, 1993;Preuss et al, 1993). Because large numbers of mutagenized plants can be screened readily (Feldmann, 1991;Chaudhury et al, 1992;Forsthoefel et al, 1992), genetic studies in Arabidopsis have the potential to dissect gene pathways that control both the histodifferentiation program (phase 1) and the dehiscence and cell degeneration program (phase 2) of anther development (Figure 1).…”

“…Arabidopsis malesterile mutants are easy to identify because they flower for a longer time period, grow taller, and remain in a green or nonsenesced state longer than their male-fertile, wild-type counterparts (Feldmann, 1991;Chaudhury et al, 1992;Forsthoefel et al, 1992;Aarts et al, 1993;Preuss et al, 1993). Because large numbers of mutagenized plants can be screened readily (Feldmann, 1991;Chaudhury et al, 1992;Forsthoefel et al, 1992), genetic studies in Arabidopsis have the potential to dissect gene pathways that control both the histodifferentiation program (phase 1) and the dehiscence and cell degeneration program (phase 2) of anther development (Figure 1).…”

“…Recently, we screened a large number of Arabidopsis lines that were treated with either T-DNA (Feldmann, 1991;Forsthoefel et al, 1992) or ethyl methanesulfonate (EMS) (Koornneef et al, 1982) for male-sterile mutants. We were particularly interested in anther mutants that failed to dehisce and those that had defects in the anther differentiation pathway.…”