**2. The strategies of plant transformation**

Plant molecular farming depending on the production of transgenic plants has been operated by two general methods as the following:

#### **2.1. Stable or permanent expression systems**

a. Stable nuclear transformation: Stable nuclear transformation refers to the integration of genes or nominated foreign genes into the nuclear genome of plants, which results in the change of genetic structures and consequent expression of transgenes after integration with the host genomes. The largest amount of recombinant proteins has been produced by one of the most common methods of stable nuclear transformation. A method exploited for aggre‐ gating proteins in dried beans of maize culminates in a long-term storage in the beans at the room temperature without decomposition of proteins [9]. In addition, it has a considerable potential for producing crops like cereals that actually grow everywhere. However, a long production cycle for some crops and their potential collisions with natural species or food products have restricted the wide acceptance of this method [10].

purposes. A vast majority of developing countries cannot afford the high costs of medical treatments resulted from the existing methods. Hence, we need to produce not only the new drugs but also the cheaper versions of the present samples in the market. Molecular farming can offer efficient solutions for the current growing need for the biomedicines [1]. Plants provide an inexpensive and simple system for the production of valuable recombinant proteins on large scale, and compared to the other production systems, they have numerous advantages in terms of economy, safety, and applicability. Though using transgenic plants has entailed some sorts of limitations and concerns, the optimization has been operated for solving the existing problems. Normally, the production of pharmaceutical proteins has been largely concentrated by the technology of molecular farming in plants, also plants can be used for the production of food supplements, biopolymers, industrial enzymes, and proteins in the investigations (avidin, β-glucuronidase, etc.). Prior production systems, including bacteria, microbial eukaryotes (yeasts, double-stranded fungi), animal cells, and transgenic animals, as a result of their limitations, were replaced by transgenic plants. The primary recombinant pharmaceutical proteins, extracted from the plants (hormones of human growth), and the first recombinant antibodies were generated from transgenic plants, respectively, in 1986 and 1989 [2, 3]. In 1997, the first recombinant protein, avidin (egg protein) was produced in a transgenic maize for industrial uses [4]. These applications proved that plants can be converted into bioreactors to produce a wide range of recombinant proteins. Many years had already passed when it was proved that plants were even able to produce several complexes of functional mammal proteins with the pharmacological functions, such as human serum proteins, growth regulators, antibodies, vaccines, hormones, cytokines, and enzymes [5]. An increasing request for the biomedicines was aligned with the high costs and inefficiency of existing production

systems [6] including yeasts, bacteria, animal cells [7], and transgenic animals [8].

**2. The strategies of plant transformation**

by two general methods as the following:

26 Plants for the Future

**2.1. Stable or permanent expression systems**

The aim of this study is to review the technologies of molecular farming, limitations and advantages of plant systems, challenges, bio-security, public acceptance of molecular farming.

Plant molecular farming depending on the production of transgenic plants has been operated

a. Stable nuclear transformation: Stable nuclear transformation refers to the integration of genes or nominated foreign genes into the nuclear genome of plants, which results in the change of genetic structures and consequent expression of transgenes after integration with the host genomes. The largest amount of recombinant proteins has been produced by one of the most common methods of stable nuclear transformation. A method exploited for aggre‐ gating proteins in dried beans of maize culminates in a long-term storage in the beans at the room temperature without decomposition of proteins [9]. In addition, it has a considerable b. Stable plastid transformation: Plastid transformation offers a remarkable solution in comparison to that of nuclear transformation since it has numerous advantages including preventing transgene escape through amphimixis (because plastids are inherited through the maternal tissue in the majority of species.) and absence of chloroplasts in pollen and conse‐ quent improbability of their transfer, which reduces environmental concerns [11, 12]. The transformed transgenic plants with homoplasmic chloroplasts (all chloroplasts carry trans‐ genes) were selected after several generations of plant regeneration from bombarded leaf explants. Selection was conducted on a medium containing spectinomycin or combined with streptomycin. The researchers [13, 14] have already extracted a human pharmaceutical protein, more than 3% to 6%, from the total soluble proteins in the chloroplasts of tobacco. Recently, Oey et al. [75] reported a very high level (70% of an entire soluble protein) for a protein antibiotic with the chloroplast system, which, till today, has been the highest concentration of recombinant proteins. Despite this, the great potential of plastid transformation has some functional limitations. Although this technology has been developed in other species such as tomatoes, lettuce, soy, and eggs [15, 16], in the current situation, chloroplast transformation only in tobacco is practically possible, but unfortunately this plant is inedible and full of poisonous alkaloids; in addition, long lasting storage in refrigerators will bring about changes in protein stability [9].

c. Plant cell suspension culture: This method involves the removal of cell walls and gene transfer to the obtained protoplasts and suspension culture. The purification system and its downstream processing are cheaper and easier [17]. In addition, the use of suspension culture can decrease heterogeneity in proteins and sugar (N-glycans) regarding the uniformity of the type and size of cells [5]. Furthermore, as a fast system there is no need for the production of transgenic plants; however, the cell lines can be produced after a few months [18, 19]. Some samples of plant suspension cultures can be used for producing biomedicines, including vaccines of Newcastle disease virus of chicks approved by the Center for Veterinary Biology and recombinant human glucocerebrosidase for treating diabetes (www.protalix.com) [19]. Though this method is cheaper, safer, and easier in comparison to the other methods of genetic manipulation, cell suspension has not yet been suggested as an optimal production choice of production in plant systems. This is due to a belief that the ultimate products and their usability are constrained by reducing the level of recombinant proteins during the stationary phase because of the enhanced proteolytic activity [20].

#### **2.2. Temporary or transient expression systems**

A transient production may be the fastest system for plant molecular farming [21]. Nowadays, these are the systems routinely applied for verifying expression constructs during a few weeks for a significant amount of proteins [22]. The given systems include the following methods:

