*5.3.5 Lettuce and Medicago truncatula*

*Genetic Engineering - A Glimpse of Techniques and Applications*

sion therapy [134, 135].

*5.3.4 Amaranth*

this represented an increase of up to 13-fold in the inhibitory activity when compared with the protein hydrolysates of non-transformed fruits [135]. These two results suggest the possible application of tobacco plant cell suspension cultures and transgenic tomato fruits for massive production of this engineered version of amarantin, which could be especially used as an alternative hyperten-

Although amaranth has not been genetically modified to produce ACEI peptides, the feasibility of developing a modified amarantin acidic subunit has been widely assessed [129, 134–139]. Recently, the in vivo effect of an *E. coli*-modified amarantin protein, four units of Val-Tyr dipeptides (VY) in tandem, and one of Ile-Pro-Pro tripeptides (IPP) incorporated in the amarantin acidic subunit (AMC3) was evaluated in SHRs in a one-time oral administration experiment. This study showed that enzymatic hydrolysates of AMC3-containing ACEI peptide (4xVY and IPP) sequences had significant in vivo antihypertensive action [138]. The positive reports of amarantin expression in *E. coli* [136, 138, 139] along with the sustained expression of amarantin-modified proteins in tobacco [134] and tomato [135] prospect the

successful production of ACEI peptide fusion proteins in amaranth.

*Schematic representation of the technologies involved in different plant platforms for the production of therapeutically important proteins and peptides. Plastid transformation by particle bombardment can result in regeneration of transplastomic plants, revealing high-yield heterologous production, with the possibility of protein/peptide oral delivery or purification. Nuclear transformation can be accomplished by particle bombardment or by Agrobacterium-mediated transformation, resulting in the regeneration of stable transgenic plants. Finally, the technology based on transient expression, here with the example of agroinfiltration. We present Medicago truncatula and lettuce as examples: (a) M. truncatula co-culture of leaf explants with Agrobacterium, (b) and (c) plant regeneration via somatic embryogenesis according to Araújo et al. [145], (d) and (e) establishment of a cell suspension culture from callus for protein/peptide production [146, 147], (f) lettuce leaf explant co-culture with Agrobacterium, (g) and (h) plant regeneration via shoot organogenesis at PCB lab, (i) lettuce transgenic plants which can be used for oral delivery, (j) and (k) agroinfiltration of lettuce leaf explants according to Negrouk et al. [74], and (l) example of a control explant (left) and transient* 

*expression of a 35S::GUS(int) cassette in lettuce leaves (right).*

**64**

**Figure 2.**

Lettuce (*Lactuca sativa*) is a commercially important crop belonging to the Asteraceae family. It is a diploid (2n = 18), autogamous species with a genome size of 2.7 Gb [140]. This crop is particularly suitable for oral delivery of therapeutics as its raw leaves are consumed by humans, and the time to obtain an edible product is only weeks, compared to the months needed for crops such as tomato or potato. Therefore, recently lettuce has been investigated as a production host for edible recombinant therapeutics [66, 67, 141]. Furthermore, the fact that stable transformation procedures for both nuclear [142] and plastid genomes [64], and transient expression [74], are widely available, is also an advantage. Lettuce has been used as production host for several recombinant therapeutics, virus-like particles (VLPs) and monoclonal antibodies [143], antigens [142, 144], and human therapeutic proteins [66, 69].

*Medicago truncatula* is a model plant from the legume family. It is a diploid (2n = 16), autogamous species, with a relatively small genome and short life cycle of 3–5 months. These characteristics enable this species to be used in molecular genetic studies and expression of foreign genes [145]. The phylogenetic distance to economically important crops is crucial in the choice of this plant by many researchers and funding agencies, since it allows comparative studies within the legume family. The methodologies for the establishment of long-term cell suspension culture are well recognized [146], and the potential of *M. truncatula* as expression host has also been established for the production of feed additives [20, 87], human hormones [90], and human enzymes [147].

The use of these two species in molecular pharming is at the center of a recent collaboration between the Plant Cell Biotechnology (PCB) Laboratory (ITQB UNL), the Cell Differentiation and Regeneration Laboratory (iBiMED UA), and the Institute of Plant Genetics (IPG PAS). This cooperation foresees the usage of these two species as exceptional hosts for the heterologous production and/or delivery of ACEI peptides, and a resume of this ongoing project is here schematically presented (**Figure 2**). This figure also provides an overview of the technologies involved in different plant platforms discussed in this chapter.
