The aim of this work was to develop a broadly applicable in vitro regeneration and
transformation method for pepper (C. annuum L.). Therefore, several parameters
affecting regeneration and transformation were examined. The following investigations
for the development of an efficient regeneration and transformation system
were performed:
(1) Twelve different pepper genotypes were analyzed with regard to their efficiency
for regeneration and transformation in vitro.
(2) The effects of the explant source for de novo-regeneration were investigated.
(3) Optimized culture media for in vitro regeneration and transformation were
developed.
The following results were obtained:
A highly efficient protocol for in vitro regeneration of twelve pepper genotypes was
established. The most successful genotype for regeneration was Conquistador.
Cotyledons were identified as the most suitable tissues for de novo regeneration.
Shoots were induced from tissue explants using MS basal medium supplemented
with auxin and zeatin. Early transfer of initiated shoots to soil was of major
importance for successful rooting and elongation of shoots. The time course of
regeneration from tissue culture to mature plants was about eight months. Selfed
regenerates were more than 95.0% fertile.
Transient gene expression was achieved for twelve genotypes using A. tumefaciens
or particle bombardment. Reporter genes GUS and GFP were used to mark the
progression of transformation. Putative transgenic tissues were analyzed by GUSenzyme
assay and using PCR. Among twelve different pepper genotypes
Conquistador was the most suitable genotype with regard to its amenability for
genetic transformation by A. tumefaciens while R113 was the most suitable genotype
for particle bombardment. Cotyledons were identified as the most promising tissues
for transformation: two days of pre-conditioning before inoculation with Agrobacterium,
followed by co-cultivation on medium supplemented with DTT and Nathiosulfate
for two days. This increased the transformation efficiency slightly. Addition
of acetosyringone during co-cultivation with A. tumefaciens extended reporter gene |
