Establishment of embryogenic cell suspension cultures and Agrobacterium -mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Abstract

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation
of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation
of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male
flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained
was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50mg settled cell volume (SCV)
was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried
out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression
of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration
of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation
with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The
present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of
disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist
in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.