Agrobacterium -mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB).

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain EHA105 containing the binary vector pVGSUN with the als gene as a selectable marker and an intron-containing the gusA gene as a reporter gene was used for transformations. The herbicide Glean was used as a selection agent. Two hundred putative transformants were recovered, of which a set of 16 was tested by histochemical analysis for GUS expression and by Southern blot analysis with a probe for the gusA gene. The plants were positive for GUS expression and integration of the gusA gene. Two of the transformants were grown to maturity under greenhouse conditions. Bananas were harvested to test GUS expression by histochemical analysis. The fruit from both transgenics tested positive for GUS expression.

Stable transformation of tomato cell cultures after bombardment with plasmid and YAC DNA.

Stable transformants were obtained after microprojectile particle bombardment of tomato cell suspensions (Lycopersicon esculentum cv VFNT Cherry and L. pennellii). The suspensions were bombarded with tungsten particles coated with either plasmid (∼6.3 kb) or yeast artificial chromosome (YAC) (80 kb) DNA containing the ß-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. The YAC DNA contained an insert of approximately 50 kb of DNA from VFNT Cherry. L. pennellii suspensions were more amenable to transformation than VFNT Cherry; more kanamycin-resistant calli were recovered from L. pennelli after bombardment with plasmid DNA, and only L. pennellii cells produced transformants after bombardment with YAC DNA. DNA gel blot analysis confirmed the presence of the nptll and GUS genes. This analysis also confirmed the integration of YAC DNA into the genome of the kanamycin-resistant calli and suggested that the level of intactness of the integrated YAC DNA was fairly high in four of the five transformants examined. Microprojectile bombardment of regenerable cultures with YACs may ultimately aid in map-based cloning of agriculturally-important genes.