Two transformation approaches are commonly used to produce recombinant pharmaceuticals in plants (i) Transient expression and ii) Stable transformation of crop species. There are three major transient expression systems to deliver a gene to plant cells includes delivery of projectiles coated with ‘naked DNA’ by particle bombardment, infiltration of intact tissue with recombinant Agrobacterium (agro infiltration), or infection with modified viral vectors. Stable expressions of transgenes include insertion of genes in the nuclear genome of transgenic plants by two general methodsAgrobacterium-mediated transformation and particle bombardment.
The transient production platform is perhaps the fastest and the most convenient production platform for plant molecular farming (Rybicki, 2010). The systems, which are mainly used for quick validation of expression constructs, are routinely used for the production of considerable amounts of proteins within a few weeks (Vezina et al., 2009). The following methods are commonly used for transient expression in plants.
The agro infiltration method, which was developed by Kapila etal., (1997), involves infiltration of a suspension of recombinant Agrobacterium tumefaciens into tobacco leaf tissue, which facilitates thetransfer of T-DNA to a very high percentage of the cells, where it expresses the transgene at very high levels without stable transformation, as in the case of transgenic crops. This method has now been developed into a very rapid, high-yielding transient expression strategy for producing clinical grade bio-pharmaceuticals (Vézina et al., 2009; Pogue et al., 2010; Regnard et al., 2010).
The viral infection method is dependent on the ability of plant viruses such as tobacco mosaic virus (TMV) and potato virus X (PVX) to be used as vectors to deliver foreign genes into plants, without integration (Porta and Lomonossoff, 2002). Both expression platforms infect tobacco plants and then transiently express a target protein in the plant tissue. Using this expression method Varsani et al., (2006) were able to successfully obtain protein yield as high as 17% of the total protein. The main drawback of this system was a tendency to lose the foreign insert during spread of the virus throughout the plant and the potential environmental issues associated with the presence of infectious recombinant viral particles.However, in the transient expression system the recombinant protein has to be processed immediately to prevent tissue degradation and protein instability.
Stable nuclear transformation involves the incorporation of a foreign gene of interest into the nuclear genome of the plant, thereby altering its genetic makeup, and leading to the expression of the transgene. The stable nuclear transformation has produced most of the recombinant proteins till date. The method has been used to accumulate protein in the dry seeds of cereals, which allows long term storage of the seed at room temperature without degradation of the protein (Horn etal., 2004). These include the following methods
Particle bombardment is one of the most widely used plant transformation method and has been applied to a broad range of species, especially monocots. The process involves the introduction of genetic material into intact cells and tissues through the use of high velocity micro projectiles. The high velocity microprojectiles are used to carry DNA being ‘shot’ into cells which represents a type of biological ballistics, hence the term ‘biolistics’ (Sanford, 1990). Efficient transformation of sugarcane through particle bombardment has been achieved by using embryogenic callus cultures (Bower and Birch, 1992). Based on acceleration of microscopic tungsten or gold particles coated with DNA can be propelled into practically all kind of tissues like callus (Ritala et al., 1994) suspension culture (Gordon-Kamm et al., 1990) inflorescences (Barcelo et al., 1994) shoot apices (Zhong et al., 1996) microspores (Jahne et al., 1994) leaves (Tomes et al., 1990) roots (Seki etal., 1991) or pollen grains (Stoeger et al., 1995) and stable transgeniccereal plants were reported for wheat (Vasil et al., 1992), oat (Somers etal., 1992), barley (Wan and Lemaux, 1994) as well as for rye (Castillo et al., 1994). Although particle bombardment can be used to testrecombinant protein stability before stable transformation, it is unsuitable for the expression of larger amounts of foreign proteins because of low efficiency, reproducibility and regeneration potential was also limited for long term callus cultures (Christou, 1996).
Agrobacterium tumefaciens is a gram-negative soil bacteriumused to transfer foreign genes to plants. The merits include integration of the small copy number of T-DNA into plant chromosomes, and stable expression of transferred genes.Agrobacterium-mediated gene transfer offers potential advantages, including preferential integration of the transgene into transcriptionally active regions of the chromosome (Koncz et al., 1989) with exclusion of vector DNA (Hiei et al., 1997), unlinkedintegration of co-transformed T-DNAs (McKnight et al., 1987), transfer of large DNA fragments (Liu et al., 1999). This method is normally used for the transformation of dicot species (De Block et al., 1985) and has been used in tobacco, alfalfa, pea, tomato and potato (Ma et al., 2003). Monocots are more difficult to transform using Agrobacterium mediatedtransformation. However, monocots have also been transformed by Agrobacterium, and the technology has been optimized for selectedmodel varieties. Agrobacterium has been used to transform the plants of Gladiolus genus (Graves and Goldman, 1987), maize (Ishida et al., 1996), barley (Wu et al., 1998), rice (Dong et al., 1996; Rashid et al., 1996), Asparagus (Hernalsteens et al., 1984), banana (Sreeramanan etal., 2009; May et al., 1995). Agrobacterium-mediated DNA transfer tosugarcane meristems has been attempted and optimized to give better efficiency (Arencibia et al., 1995, EnroAquez-Obregoan et al. 1997). Consequently, herbicide-resistant sugarcane plants have been produced through Agrobacterium transformation (Enriquez Obregon et al., 1998).
The interaction of two compatible plasmids, one containing the vir-region, the other carrying the T-DNA on a wide host-range replicon forms a binary vector system (Hoekema et al., 1983). A binary vector system is generally used for Agrobacterium mediated transformation. Agrobacterium contain the T-DNA, which is stably integrated into the plant genome. Apart from the T-DNA another region of the Ti-plasmid-called the vir-region, is essential for tumour induction. Transfer of the plasmid into an A. tumefaciens strain harbouring the plasmid with the vir-region allows introduction of the manipulated T-DNA into plant cells. Production of recombinant proteins in transgenic plants was initially based on integration of a target gene into the nuclear genome, expression of virurence (vir) genes from the Ti plasmid of Agrobacterium tumefaciens are enhanced by several experimentalfactors, including phenolic compounds (Spencer and Towers, 1988), acetosyringone concentration from 100 to 200 μM, sugars (Shimodaetal., 1990) and pH of co-cultivation media, ranging from pH 5.4 to 5.6(Mondal et al., 2001). The T-DNAs insertion is random into the genome (Ambros, 1986; Wallroth, 1986) and remains stable in the original insertion site through multiple generations (Krysan et al., 1999). Biolistic transformation invariably leads to the genomic insertion of multiple copies of the transgene cassette, which in turn can lead to gene silencing. Hence,Agrobacterium-mediated transformation may be another alternative to reduce somaclonal variation and for overcoming gene silencing.