Development of transgenic banana expressing the Salmonella typhi gene encoding flagellar protein antigens

Plant tissue and cell culture experiments on banana cv. Lakatan and Latundan, tomato (cv. Apollo White, Marikit, Caraibo and Improved Pope) as a model system have concentrated on the optimization of plant growth regulator combinations on MS [Murashige and Skoogs] medium. The protocols that were used were those available from the literature for micropropagation, multiple shoot formation, scalp and callus induction and initiation of embryogenic cell suspension cultures. Multiple shoot formation was efficient on MS + 5 ppm BAP [benzyl-amino purine]. Latundan generated more scalps (50% more) per explants than Lakatan at MS + 50 ppm BAP, undisturbed for at least two months. However, callus induction was observed only from the meristem and inflorescence of Lakatan on MS + 3 ppm 2, 4-D. Scalps transferred to 1/2 strength MS media supplemented with various combinations of growth hormones did not develop into embryogenic cell clusters largely due to excessive browning of the liquid medium. Polyphenols generated from explants inhibit cell proliferation. Transfer to semi-solid medium was also evaluated. Tomato seeds were generated on 1/2 strength MS supplemented with 10 ppm BAP. Callus induction from meristem was obtained on MS plus combinations of 10 ppm BAP. Callus induction from meristem was obtained on MS plus combinations of 1 ppm NAA [naphthalene acetic acid], IAA [indole acetic acid] and 2, 4-D [dichlorophenoxy acetic acid] with in 4-6 weeks. The response was cultivar dependent. Scalps and callus induced to form ECS [embryogenic cell suspension] were hampered by browning of the liquid medium-due to polyphenotic compounds released by the explant. Transfer to semi-solid medium and addition of anti-oxidants were required. Initiation of ECS in tomato using friable callus was more successful. One week after culture induction, tiny cream cells were observed in suspension, steadily increasing in cell number in two weeks. The heavier embryongenic cells could be easily separated from the lighter non-embryogenic cells by sedimentation. Homogeneous suspension culture can persist up to two months from date of culture induction. Shoot formation from tomato leaf discs was found to be optimum in MSB (MS + 5 ppm BAP, 1 ppm IAA) and MSD (MS + 2.5 ppm BAP, 1 ppm IAA) for Apollo white, Improved Pope, Marikit, Caraibo, P8, TM9 and 56 04. For the construction of the expression vector, the hypervariable regions 4-6 of the H1-d flagelin gene was amplified by PCR. The approx 0.565 kb amplicon was inserted into pGEM-T-Easy for DNA sequencing. The construct was designated as pGTE-PCR 11p23. This construct was them ligated into the yeast expression vector pPICZ alpha A as an EcoRI /Xba 1 fragment. The resulting construct was designated as pEO. A stable transformant (A sub 20) carrying this construct was selected for expression in Pichia pastories X-33. The aim of the study was to over express the prokaryotic hypervariable regions 4-6 in a eukaryote.