Journal of Applied Biosciences (J. Appl. Biosci.) [ISSN 1997 - 5902]
Volume 30: 1845 - 1860. Published June 9, 2010.
Transformation of cowpea (Vigna unguiculata L. Walp.) by Agrobacterium infiltration
Adesoye, A.I.1,2j ,Togun, A.O.1, Machuka, J.2,
1. Department of Crop Protection and Environmental Biology, University of Ibadan, Nigeria.
2. Biotechnology Research Unit, International Institute of Tropical Agriculture (IITA), Ibadan, NigeriaCorresponding Author Email:aadesoye@yahoo.com
ABSTRACT
Objectives: Until recently, stable genetic transformation of cowpea through tissue culture technique could not be established. The aim of this work was to optimize inoculation and cocultivation medium factors for cowpea transformation and avoid in vitro regeneration procedures in obtaining transgenic cowpea through vacuum infiltration of embryos.
Methodology and results: Using plasmid pCAMBIA 1301, influence of inoculation and cocultivation media compositions on transient gene expression were determined. Embryos were inoculated on Murashige and Skoog (MS) or yeast extract broth (YEB) solutions supplemented with either acetosyringone, 0.05% silwet L-77, both or none and then cocultivated on MS solid medium for 24 hours. Gus assays showed that inoculating explants in MS containing acetosyringone and 0.05% silwet L-77 gave the highest transformation frequency (55.3%). When tested, the presence of acetosyringone in the cocultivation medium increased transformation frequency by 15.35%. When untransformed control seeds were screened on hygromycin and phosphinotricin 100% shoot and root growth inhibition were obtained at 50mg/l and 5mg/l, respectively. Secondly, cowpea embryos were transformed with Agrobacterium cells carrying two plasmids, ptjk 142 and pCAMBIA 1301, by inoculating them in media optimized as above and subjecting to two rounds of vacuum infiltration. Thereafter they were cocultivated for nine days on MS selection-free medium where they germinated and produced mature plantlets. T1 seeds were selected on antibiotic concentrations determined as above. Plants surviving both phosphinotricin and hygromycin showed gus positive reactions when subjected to gus histochemical assay and amplified the gus primer during molecular analysis. Phosphinotricin resistant plants also amplified bar primers. Percentage transformation based on total number of T1 seeds and number of plants with positive PCR reaction using both primers was 2.5 %forpCAMBIA 1301 and 3.9 %forptjk 142 plants.
Conclusion and application of findings: The integration of transgenes into cowpea by Agrobacterium infiltration of embryos germinating on selection free medium has been demonstrated. This technique has the potential of rapidly producing stably transformed cowpea and avoiding all the limitations imposed by de novo regeneration in tissue culture.
Key words: cowpea, genetic engineering, Agrobacterium infiltration,
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