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

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

Chapter 4 A C 1 2 3 4 5

Chapter 4 A C 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 B 1 2 3 4 5 6 Figure 3. Southern and northern analyses of plants transformed with LLAG1 (T1 generation). Southern blot with genomic DNA derived from Col-0 Arabidopsis digested with HindIII showing multiple insertions of NPTII (A) and LLAG1 (B). Lane 1 was loaded with DNA derived from a non-transgenic plant, whereas lanes 2 to 6 were derived from plants selected for kanamycin resistance. Plant 2 showed wild-type characteristics and plants 3 to 6 showed ap2-like phenotype. (C) Northern blot of flower RNA isolated from transformed Ler-0 Arabidopsis plants and probed with 3' portion of LLAG1, indicating its active transcription in transgenic plants. Lane 1 was loaded with RNA derived from a non-transgenic plant, whereas plants 2 showed a wild-type phenotype; plant number 3 showed a mild ap2-like phenotype, and plants 4 to 10 showed a strong phenotype. Lower panel shows RNA loading quantities manifested by ethidium bromide gel staining. 56

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. 57

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