Agrobacterium-mediated transformation of yellow lupin to generate callus tissue producing HBV surface antigen in a long-term culture

Abstract

The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 microg per g of fresh tissue.

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in Zoysia japonica

“…Firstly, it facilitates the transgenic program by providing a sustainable material for gene transfer which is continuously available with only minor input and consistent between experiments. The importance of this point is underlined by the level of research into such tissue culture systems, in order that the production of multiple transgenic lines does not require the costly and time-consuming process of initiating fresh callus for each round of transformation (Zhang et al 2000;Sharma et al 2005;Pniewski et al 2006). Furthermore, using the somaclonal culture as the target material should mean that the resulting transgenic lines have a more uniform genetic background.…”

Is An Oral Plant-based Vaccine against Hepatitis B Virus Possible?

“…Plant-expressed S-HBsAg had buoyant density comparable to the antigen from human plasma or recombined in yeast [35,45,49,52,54,56,58]. Western blot analyses showed that S-HBsAg in plant cell was of proper size of 24 kDa and non-glycosylated, similarly to the yeast-derived antigen [41,46,50,51,59,71]. Yet, glycosylation of S-HBsAg as well as M and L antigens in plants was also reported [54,56,67,70], although putatively the antigens were modified according to the plant glycosylation pattern.…”

“…Antigen content was comparable in plants transformed using both optimised and simple vectors, i.e. containing the nonmodified HBsAg coding sequence under control of the regular 35S promoter [37,39,40,46,52,63,[67][68][69][70] and ranged usually from 0.01 to several micrograms per g FW (which approximately corresponds to tens ng/mg TSP), rarely 10 g/g FW and exceptionally several tens of g/g FW ( Table 1). It may be supposed that intensive transcription caused by highly active regulatory elements of HBsAg cassettes could be rather conducive to gene silencing, instead of efficient translation.…”

Gene Transfer in Legumes

“…Kanamycin-resistant plants obtained expressed b-glucuronidase activity, and integration of the nptII and b-glucuronidase genes into the genome was confirmed by non-radioactive DNA-DNA hybridisation. Pniewski et al (2006) developed an improved transformation protocol with seedlings and hypocotyls of yellow lupin, reaching 44 % efficiency rate, which they used to produce calli and tumours that produced a small surface antigen of Hepatitis B Virus, the final goal being the production of an oral vaccine that could be administered as a portion of plant tissue. Finally, Zinn et al (2009) produced transgenic white lupin and Arabidopsis and studied deletion and mutation constructs linked to the b-glucuronidase (gus) reporter gene, in order to understand the structure of the LaSAP1 promoter and explore the role of the P1BS motif.…”