Survival and growth of pea protoplasts after transformation by electroporation

Abstract

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.

Gene Transfer in Legumes

“…Transient gene expression following protoplast electroporation was reported only seldom in pea (Hashimoto et al 1992;Puonti-Kaerlas et al 1992;Ochatt et al 2005), and the determination of the optimum electrical parameters permitting entry of the foreign information into protoplasts whilst permitting their subsequent viability and proliferation competence was a prerequisite for success in this domain. Conversely, there are quite a few examples of electroporation applied for transient and (less frequently) stable gene transfer in other legume species, as will be discussed crop-wise below.…”

“…Several conclusions were obtained in experiments focused on the utilisation of various genes for resistance to antibiotics as selective genes, particularly kanamycin. Puonti-Kaerlas (1992), De Kathen and Jacobsen (1990) as well as Schroeder et al (1993) found kanamycin less effective for selection than hygromycin and phosphinotricin. On the other hand, Grant et al (1998) deemed kanamycin in higher concentrations and in combination with successive selection steps as effective.…”

“…Plant-derived pharmaceuticals can be used against various human diseases, including cancer (Pujol et al 2007), hepatitis B and C, measles, cholera or AIDS (Floss et al 2008), as well as for veterinary purposes. In peas, there are several reports about pea-derived vaccines against rabbit haemorrhagic disease virus (Mikschofsky et al 2009 Puonti- Kaerlas et al (1992) in pigs (Novoplant 2007; http://www.gmo-safety.eu) and coccidiosis in chickens (Zimmerman et al 2009). For efficient and large-scale production of recombinant proteins in plants, transient expression by agroinfection has a number of advantages over stable transformation, as simple manipulation, rapid analysis and high expression efficiency are possible.…”

“…Puonti-Kaerlas et al (1992) stably transformed protoplasts from two different pea cultivars (Belman and Filby) by direct gene transfer using electroporation; they recovered transgenic calli when hygromycin resistance was used as the selective trait, but no transformants were obtained when kanamycin resistance was used as selective marker. Gus gene was used to assess transformation efficiency using histochemical staining, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.…”

The biotechnology of crop legumes

“…Protoplasts from two pea cultivars were stably transformed by direct DNA transfer using electroporation (Puonti-Kaerlas et al, 1992a). Transgenic calli were obtained after selection with hygromycin, whereas no transformants were obtained when kanamycin was used for selection.…”