Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes.
We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws.
Ivo
Nayche L
NL
Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, Asa Norte, Brasília, DF, 70770-200, Brazil.
Nascimento
Cristina P
CP
Vieira
Lívia S
LS
Campos
Francisco A P
FA
Aragão
Francisco J L
FJ
D000095:Acetolactate Synthase / Q000235:genetics; D019470:Biolistics; D018744:DNA, Plant / Q000235:genetics; D007887:Fabaceae / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter; D007093:Imidazoles / Q000494:pharmacology; D040582:Inheritance Patterns; D008854:Microscopy, Electron; D009525:Niacin / Q000031:analogs & derivatives / Q000494:pharmacology; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics*; D046509:Tissue Culture Techniques; D014170:Transformation, Genetic; D019076:Transgenes
D018744:DNA, Plant; D007093:Imidazoles; D009525:Niacin; C076656:imazapyr; D000095:Acetolactate Synthase