Transgenic Sunflower - Genetics, Genomics and Breeding of Crop Plants - page 290

(Hewezi et al. 2002), and cotransferring of the ipt gene from Agrobacterium
vitis (Molinier et al. 2002). Two detailed sunflower transformation protocols,
one for highly responding genotypes and a second one for recalcitrant
genotypes, were published by Hewezi et al. (2004). Gene transfer, earlier
recognized as an instrument to overcome sexual incompatibility in
interspecific hybridization (Skoric 1992), was used for transformation of
sexual (Pugliesi et al. 1993) and somatic interspecific hybrids (Fambrini et
al. 1996; Henn et al. 1998; Taski-Ajdukovic 2006); but plant regeneration
was genotype-dependent and of low efficiency (Kallerhoff and Alibert 1996;
Faure et al. 2002b). The partial genome transfer from perennial Helianthus
species performed by Binsfeld et al. (2000) emerged as a potential tool for
transferring polygenic traits or alien genes. Advances in sunflower genomics
made since 1995 have greatly enhanced the development and application
of new tools for crop improvement (Paniego et al. 2007).

9.3.2 General Procedures to Transform Sunflower

Detailed techniques for sunflower transformation have been patented ( www.
patentstorm.us ; www.freepatentsonline.com ). A typical expression vector contains
prokaryotic DNA elements and an antibiotic resistance gene, a cloning site
for insertion of the exogenous DNA sequence, eukaryotic DNA elements
such as a promoter, an optional enhancer, a transcription termination/
polyadenylation sequence, and sequences that are necessary to allow for
the eventual integration of the vector into a chromosome. The vector also
contains a gene encoding a selection marker, which is functionally linked to
promoters that control transcription initiation. The promoter sequences can
be constitutive or inducible, environmentally- or developmentally-regulated,
or cell- or tissue-specific. A preferred selection marker gene for sunflower
transformation is the neomycin phosphotransferase II ( npt II) gene, which
confers resistance to kanamycin when placed under the control of plant
regulatory signals. Another useful marker, which allows quantification or
visualization of the spatial pattern of expression of the DNA sequence in
specific tissues, is the reporter gene -glucuronidase ( GUS ). Methods for

introducing DNA into sunflower cells comprise bacterial infection, artificial
chromosome vectors, and direct delivery of DNA. Co-cultivation with
Agrobacterium tumefaciens or combined with biolistic methods is used for
sunflower transformation. Transformed tissues include hypocotyls, apical
meristems, and protoplasts ( Table 9-1 ). Cotyledons can often give rise to
shoots without an intervening callus stage, producing whole plants more
rapidly and efficiently. Regeneration from shoot meristems usually leads to
the formation of chimaeras; this can be avoided through transformation
protocols based on non-meristematic regeneration (Rao and Rohini 1999;
Mohamed et al. 2003). Some examples are hypocotyl regenerants, which