Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 205

An attempt to use gene technology as an alternative method to protect
plants from microbial diseases was made by Sawahel and Hagran (2006) in
sunflower. The human lysozyme gene, which had been proven to be a simple
and effective approach for genetic engineering disease resistance against
phytopathogenic bacteria and fungi, only requires the introduction of a
single transgene (Nakajima et al. 1997). Sunflower plants were transformed
via cocultivation of previously bombarded hypocotyls explants with
Agrobacterium tumefaciens harboring the plasmid pNGL that contained the
human lysozyme gene (Sawahel and Hagran 2006). Two regenerants from
one primary transformant that expressed the human lysozyme gene were
confirmed to be resistant against Sclerotinia sclerotiorum .
Other researchers used H. annuus as well as Nicotiana tabacum to test the
function of their genes (Almoguera et al. 2002; Hewezi et al. 2006). Using
degenerate primers derived from NBS domains an NBS-LRR homologous
sequence (PLFOR48) was isolated from sunflower, which cosegregated with
a downy mildew disease resistance locus (Bouzidi et al. 2002). The antisense
sequence of PLFOR48 under the control of the constitutive CaMV 35S
promoter was introduced via transformation into sunflower and tobacco to
assess loss of function. However, the construct caused major developmental
abnormalities in sunflower as well as in tobacco so that it is assumed that
PFLOR48 may be also involved in regulating developmental pathways apart
from a role in disease resistance.
Rojas et al. (1999) performed successfully transient expression analyses
in sunflower embryos by bombardment of microprojectiles coated with a
DNA plasmid mixture, including reference, effector and reporter plasmids.
Each plasmid combination was bombarded at least 25 times (five replications
in each of five independent experiments). These transient expressions
showed that ABI3, a seed-specific transcription factor from Arabidopsis , also
activated the chimeric gene with the Ha hsp17.7 G4 promoter. Trans -activation
with LpHSFA1, a heat shock factor from tomato, reproduced the activation
patterns of wild type and mutant promoters observed with ABI3 (Rojas et al.
1999). Using LpHsfA1 and LpHsfA2, another well characterized heat stress
transcription factors (Hsfs) from tomato, transcription activation of the Ha
hsp17.6 G1 promoter was analysed by transient expression analyses (Rojas
et al. 2002). By the same co-bombardment of sunflower embryos, Díaz-Martín
et al. (2005) analyzed the interaction of the two transcription factors
HaHSFA9 and HaADREB2 that could be shown to synergistically trans -
activate the Ha hsp17.6 G1 promoter.
6.4.2 Use of Heterologous Systems to Analyze Genes Isolated
from Sunflower
As sunflower proved to be so difficult in transformation experiments
heterologous systems, e.g., Arabidopsis thaliana or Nicotiana tabacum , have