Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen

Abstract

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65-70 μg LTB g(-1) tissue whilst hairy roots of tomato contained ~10 μg LTB g(-1). Antigen at ~600 ng ml(-1) was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.

Tomato tolerance to abiotic stress: a review of most often engineered target sequences

“…Hairy root culture appeared as an alternative system for producing biopharmaceutical compounds in tomato plants. De Guzman et al (2011) achieved production of the Escherichia coli B-subunit heat labile toxin antigen in tomato hairy root cultures (approximately 10 µg/g blotted weight, BW). Unfortunately, numerous attempts to obtain regenerated plants from hairy root cultures were unsuccessful.…”

Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine

“…This can be further exacerbated by the use of strong constitutive promoters such as CaMV35S, to drive expression of the transgene, as was recently demonstrated in hairy root cultures of Solanaceous sp. expressing the model recombinant antigen LTB-the B-subunit of the heat labile toxin from Escherichia coli (De Guzman et al 2011). Inducible and/ or tissue specific promoters may provide a means of addressing this limitation by allowing for controlled temporal and spatial expression (Gatz and Lenk 1998;Tang et al 2004;Garoosi et al 2005).…”

“…2.3 kb of the NtQPT2 promoter sequence from N. tabacum cv Xanthi (Accession AJ748263) was used to replace the CaMV35S promoter of the LTB gene construct in pTH210 Mason et al 1998 son et al 1987) or empty pBinPlus vector were used as positive and negative controls respectively. A. rhizogenes strains were used to generate clonal hairy roots of tobacco (N. tabacum), petunia (P. parodii) and tomato (S. lycopersicum) and were selected for binary vector T-DNA by growth in kanamycin-containing media as described previously (De Guzman et al 2011). For initial characterisation, at least 10 independent clonal hairy root cultures of each species were established containing T-DNA from each binary vector.…”

“…For initial characterisation, at least 10 independent clonal hairy root cultures of each species were established containing T-DNA from each binary vector. The presence of the LTB gene was confirmed by PCR analysis of genomic DNA as described previously (De Guzman et al 2011). Clonal hairy roots containing a CaMV35S-uidA (GUS) construct were also confirmed to contain GUS by histochemical staining with X-Gluc (Jefferson et al 1987).)…”

“…Clonal hairy roots containing a CaMV35S-uidA (GUS) construct were also confirmed to contain GUS by histochemical staining with X-Gluc (Jefferson et al 1987).) All root samples were analysed using blotted dry weights (designated BW in figures) of fresh tissues in order to ensure effective extraction and quantification of LTB protein in lg/g (blotted weight) as described previously (De Guzman et al 2011).…”