However, for edible vaccines,fruit crops are preferred since they can be consumed directly without cooking,else heat during cooking may destroy the protein antigen. Many plant species including maize, carrot, tomato, soybean, lettuce, potato, and alfalfa are used now, since they offer better yields with no toxic compounds, making possible oral immunization using raw plant materials. Papaya and banana are good candidates for rapidly producing cheap vaccines in developing nations since they have high quantity of vitamin “A” and have sterile condition as in banana. Also,the genes are not transferred from one plant to another . Plants can be transformed with antigen containing recombinant vector through direct gene delivery -mediated protoplast transformation or by vector-mediated nuclear or by plastid transformation.Chloroplast transformation is a rapid and low-cost production technique due to high copy number of plastids in a plant cell, has potential to express multiple genes in plastids, ensures site-specific integration of transgene in the plastid genome, eliminates gene silencing effect and ensures natural transgene containment.

However, functional heterologous proteins requiring complex post-translational modifications cannot be produced by chloroplast transformation since glycosylation process does not occur in plastids. Vaccines derived from chloroplast were developed against bacterial diseases such as cholera, lyme disease, anthrax, tetanus, and plague, and against viral diseases such as rotavirus and canine parvovirus. Subcellular targeting signal sequences such as vacuolar or cell wall targeting signals improve antigen accumulation. Agrobacterium-mediated method is widely used for plant transformation to create edible vaccines or plant-based vaccines.Another modification of Agrobacterium-based transformation is agroinfiltration.Using agroinfiltration methods such as syringe and vacuum infiltration, A. tumefaciens suspension is infiltrated into the intracellular spaces of desired parts of the plants for transient expression of desired protein or transgene. The expression level of antigen protein in the plant cells is increased .Stable plant transformation for production of vaccines is a time-consuming process, absorbing much time to generate the plants and has containment issues such as transgene escape etc.

Transient expression of antigens and use of plant cell culture bioreactors as well as greenhouse-grown plants are alternative methods to express vaccine or therapeutic proteins in plants. Proliferation of the transformed cells through a selective condition in the presence of a selective agent, to regenerate plants from successfully transformed cells is important. In vitro conditions including plant growth regulators and culture conditions such as light, temperature etc. that direct the regeneration processes or morphogenetic response of the tissue is to be optimized for the selected plant. Somatic embryogenesis and organogenesis are the two pathways for plant regeneration. In direct somatic embryogenesis, the embryo is formed directly from a cell or group of cells without the production of an intervening callus,while in the indirect somatic embryogenesis, callus is first produced from the explants. In organogenesis, organs are produced from callus or explant. Regeneration steps are avoided in transient expression systems, where whole plants are used and the transgene or DNA is not stably integrated in the genome norinherited, but expressed temporarily in the host. Enzyme-linked immunosorbent assay and western blot assays are used to quantify the foreign protein or antigen in the plant. Immunogenicity of the plant vaccine is assessed in the preclinical level, when test animals are subjected to a defined immunization scheme and antibody levels and proliferation of specific immune cells are often evaluated by ELISA and splenocyte proliferation assays. Immuno-protective potential of the vaccine is evaluated by scoring of deaths in vaccinated and unvaccinated test animal groups or by measuring humoral or cellular immune responses. A small number of vaccines or candidates have been tested for immunogenicity in humans. Clinical trials utilizing transgenic plants for vaccines mostly consist of either the leaves or fruits from the plants .Generation of transformed plant takes much time.

Molecular farming technologies other than transgenics include plant cell suspension cultures, plant virus-based transient expression systems and use of plant viruses as vaccines. Plant cell suspension culture using protoplast or cell culture, or hairy root culture,combines the advantages of higher eukaryotic cells , with the use of simple and inexpensive growth media, to make them suitable for the production of recombinant proteins.Plant cell suspension cultures, rather than whole plants are cultivated under aseptic conditions using classical fermentation technology, are easy to scale-up for manufacturing, and the regulatory requirements are similar to the existing regulations, established for well-characterized production systems based on microbial and mammalian cells.