Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 204

6.4 Verification of Candidate Genes and Functional Analyses
of Genes

6.4.1 Attempts of Functional Analyses by Transgenic
Approaches in Sunflower
Any approach to use sunflower for transformation experiments to confirm
functions of the candidate genes isolated has been severely hampered by
the fact that sunflower has proven to be recalcitrant in tissue culture due to
its low regeneration potential (Hahne 2001). Efforts have been made to select
genotypes from interspecific hybrids with superior regeneration potential
as compared to the public lines (Weber et al. 2000). Most of the successful
transformation approaches applied to produce transgenic offspring used
the shoot apical meristems (Bidney et al. 1992; Knittel et al. 1994; Malone-
Schoneberg et al. 1994; Grayburn and Vick 1995; Lucas et al. 2000). However,
these approaches were inefficient and unreliable as most of the regenerants
proved to be chimeric as transformation events giving rise to regenerated
shoots predominantly occurred in preexisting meristems (Burrus et al. 1996).
Improved transformation protocols using macerating enzymes (0.1%
cellulase and 0.05% pectinase) prior to Agrobacterium incubation of apical
shoot meristems (Weber et al. 2003) or successive excisions of the apical and
axillary shoots and cultivating the split embryonic axes on medium
containing 0.1 mg l -1 6-benzylaminopurine (Hewezi et al. 2003) have
increased the rates of stable transformation events. Mohamed et al. (2006)
also used juvenile split apical meristems from two important high oleic
sunflower genotypes (Capella and SWSR2) to obtain transformants in
sunflower using gus and gfp as reporter genes. Transformation was
performed via Agrobacterium and particle bombardment.
However, laboratories still stick with their own protocols and a few
reports have been made on transforming sunflowers for characterization or
functional analyses of genes isolated from sunflower. Rousselin et al. (2002)
successfully transformed sunflower using an interspecific hybrid line STR1/
95 derived from a cross between Helianthus annuus and H. strumosus and a
chimaeric construct consisting of the coding sequence of 9-stearoyl-(acyl

carrier protein) desaturase from Ricinus communis under the control of the
sunflower promoter Ha ds10 . Two independent transformants could be
obtained containing three and six copies of the T-DNA. Transcript analysis
of the

9-stearoyl-(acyl carrier protein) desaturase under the control of the
seed-specific promoter Ha ds10 confirmed the tissue-specific expression in
developing embryos and not in leaves. Some of the transgenic lines produced
oil with a significantly reduced stearic acid content (Rousselin et al. 2002).