Flower development of Lilium longiflorum - The Lilium information ...

Ascribing genetic function from lily using heterologous system
Table 1. Phenotypical segregation of ap2-like characteristics conferred by the overexpression of LLAG1
in Arabidopsis.
Transformed
background a
T0 T1
n b WT
Mild
ap2 -
Strong
ap2 -
ag - n c
Ler-0 +/? 48 2 2 7 0 11
Col-0 +/+ 16 32 13 4 0 49
Col-0/Ler-0 +/- 39 20 0 2 6 28
a Arabidopsis genetic background used for transformation: + means wild-type AG allele, - means the defective ag-1
allele and ? means undetermined allele (since the plants presented wild-type phenotype, the defective allele was
in the population). Ler-0 is the Landsberg erecta-0 ecotype, Col-0 is the Columbia-0, whereas Col-0/Ler-0 is a
hybrid between the two ecotypes.
b indicates the number of plants used for floral dip transformation.
c indicates the total number of siblings studied in the population.
After the first transformation experiment, we realised that the Ler-0 F1
population was segregating the recessive homozygous ag-1 mutants at much lower
frequencies than expected from truly heterozygous parents, as ordered from the Seed
Bank (Mendelian segregation ratio of 1 out of 4). Among 12 non-transgenic plants
tested, only one presented the ag-1 phenotype and, among the remaining 11 plants,
only one plant showed segregation of the ag-1 allele in the next generation. Hence, the
seeds from this heterozygous plant were used for further experiments.
Molecular characterization of the allele ag-1 was described by Lenhard et al.
(2001) based on enzymatic digestion of a 317-bp PCR amplification product detailed
in the Material and Methods section. In this system, the amplified fragment derived
from the wild-type allele remains uncut, since there is no restriction site for the
endonuclease used, whereas the defective allele is divided into two fragments of 297-
and 20-bp because a point mutation created a HindIII site in the amplified region.
Attempts were done to identify the differences between 317- and 297-bp fragments in
agarose gels but, given the resolution of the technique, no satisfactory results could be
obtained.
Arabidopsis ecotype Col-0 has a higher competence for Agrobacterium
transformation by the floral dip method than the Ler-0 ecotype (Clough and Bent,
1998). In an attempt to increase the transformation frequency, the heterozygous Ler-0
plant identified by progeny analysis was used as a pollen donor to cross with wild-type
Col-0 plants. Thirteen hybrid lines were analysed in the F2 progeny to identify
heterozygous genotypes, in which 8 plants showed segregation of the ag-1 allele.
Segregation of leaf morphology and inflorescence architecture was noticeable as well.
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