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LT19 Arabidopsis
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Arabidopsis genetics and transgenesis
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Arabidopsis thaliana 9 recognised species – temperate plants indigenous to Europe, also found in Asia,
North America
15-20 cm high when mature, seeds 0.5mm long
Readily germinates on nutrient agar medium or in soil
- Easily cultivated in restricted space – germinate thousands in petri dishes, grow
on in illuminated incubators or greenhouses
- Prolific seed production (>5000 seeds per plant)
- Seeds collected, stored in seed banks
Genetics and Genomics
Rapid life cycle ~6 weeks from seed to seed
Self-pollinates as bud open – crossed by applying pollen to stigma
Mutant plants produced y chemical treatment of seeds and screening for aberrant
phenotypes
Regressive mutations identified by allowing mutagenized plants to self-fertilise
2000 mutant phenotypes described
Genome diploid with 5 chromosomes 2n = 10
Used over 50 years for classical genetic analysis – detailed genetic map, molecular
maps based on DNA polymorphisms between ecotypes molecular markers allow
precise mapping
Small genome size – haploid 125 Mbp of DNA, complete genome sequence – 25 000
genes representing 11 000 gene families, very little repetitive DNA
Physical map from BAC and cosmid clones, detailed cDNA database, cDNA clones
available
Transformation of Arabidopsis
Agrobacterium method – regeneration of whole plants from leaf tissue using
phytohormones can lead to somaclonal variations – spontaneous mutations unrelated
to T-DNA insertion
“Floral dip” – plants with developing flowers dipped into Agrobacterium suspension
- Suspension contains surfactant Silwet L77 which promotes uptake of
Agrobacterium into female gametes
- Plants then allowed to self and set seed
- Seeds planted on selective medium to select for transformants – quick, easy,
efficient
Forward Genetics identify mutant phenotype to unknown gene
, Make tagged mutants by insertional mutagenesis
DNA elements used for tagging: T-DNA or activated maize transposon
Creates stable, null mutants (usually recessive)
Tagged genes can be isolated without any genetic knowledge beyond knowning the
mutation and tag co-segregate in cross
Observed mutation not always linked to T-DNA – might be due to abortive T-DNA
integration or an unrelated mutation
1. T-DNA as a tag
- Create T-DNA construct which contains whole plasmid
vector (eg. pBR322)
- pBR322 DNA contains origin of replication and selectable
marker for E.coli
- T-DNA also contains plant selectable marker
(eg. nptII) and the 2 elements are separated by unique restriction
enzyme (RE) site
a) Transform Arabidopsis using Agrobacterium
containing T-DNA construct
b) T-DNA integrates randomly into genome
c) Screen 10 000s of transgenic seedlings for
desired phenotype
d) Confirm that phenotype co-segregates with
Km resistance in crosses
e) Then use plasmid rescue to clone the unknown
gene
2. Using a transposon as a tag
- No natural active transposons in Arabidopsis, but maize
Ds transposon can be introduced by Agrobacterium to
create lines with Ds located a different loci
- Activation of Ds is achieved by crossing to a second
transgenic line carrying the Ac transposase gene
- Transposition tends to result in movement of Ds to closely linked sites in genome
– hence random mutagenesis of a “localised” chromosomal region
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