Modification of gibberellin biosynthesis in apple (Malus) for an improved dwarfing habit

Bulley, Sean M. W. (2002). Modification of gibberellin biosynthesis in apple (Malus) for an improved dwarfing habit. PhD thesis The Open University.



It would be desirable to have dwarfed bittersweet apple cultivars that could be grafted onto semi-vigorous rootstocks, without needing supplementary applications of chemical growth retardants that inhibit gibberellin (GA) biosynthesis. Therefore studies were undertaken in order to manipulate gibberellin biosynthesis in Greensleeves apple and a protocol was developed for genetic transformation of Michelin, a bittersweet variety. Two homologous GA 20-oxidase sequences (94% nucleotide identity) were isolated by RACE from Greensleeves shoot tip mRNA using primers based on a 20-oxidase gene fragment (named 20oxl) previously obtained by degenerate PCR. These sequences named MdGA20oxlA and MdGA20oxlB are highly expressed in developing embryo, at lower levels in shoot tips and young leaves, and at very low levels in ovaries 5 days after pollination. MdGA20oxlA is almost identical to a cDNA previously isolated from Fuji apple (AB07114). Both MdGA20oxlA and MdGA20oxlB were demonstrated to encode enzymes with GA 20-oxidase activity by heterologous expression in Escherichia coli and incubation with [14C]GA12. Using primers based on another fragment isolated by degenerate PCR, a separate GA 20-oxidase cDNA named MdGA20ox2 was obtained by a combination of genome walking and RACE from unpollinated ovary cDNA. It shares 67% and 68% amino acid identity to MdGA20oxlA/B and is expressed in stamens and less so in sepals and unpollinated ovary. Transcripts for a homologous gene were found in unpollinated and pollinated parthenocarpic Wellington Bloomless ovaries 5 days after anthesis. Anti-sense and cosuppression using the original 20oxl fragment driven by the CaMV35S promoter produced dwarf Greensleeves plants. The main effects were reduced intemode length and number. In shoot tissues of one dwarf line the concentration of GA1 was lower, and that of the precursor GA19 higher compared to the control. These results show that it should be feasible to engineer a dwarf habit in bittersweet apple cultivars in the near future.

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