Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

No abstract

Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli. ARTICLE HISTORY

Background: In humans, various diseases are associated with the accumulation of free radicals. The antioxidants can scavenge free radicals and reduce their impact; thus, the search for effective natural antioxidants of plant origin is indispensable. The present study aims to determine, for the first time, the flavonoid compounds profile and to investigate the free radical scavenging and antioxidant properties of the methanolic extract of Taxus baccata L. from Algeria. Methods:The determination of the flavonoid compound profile of the methanolic extract of Taxus baccata L. was established using high-performance liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS). The total flavonoid content (TFC) was performed according to the aluminum chloride colorimetric method, while the free radical scavenging and antioxidant activities were carried out using three methods, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assay, 2,2'-azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical assay and ferric reducing antioxidant power (FRAP) Assay.Results: A total of 26 compounds including flavon-3-ols, flavanonols, flavones, flavonols and bioflavonoids were characterized and identifiedusing HPLC-DAD-ESI-MS/MS analysis, five were reported for the first time such as taxifolin, apigenin, apigenin 7-O-glucoside, isorhamnetin 3-O-rutinoside and robustaflavone. The plant extract exhibited high total flavonoid content (TFC = 204.26 ± 6.02 mg RE/g dry extract) which corresponded to its strong radical scavenging activities [(DPPH IC 50 = 35.31 ± 0.29 µg/ml and ABTS IC 50 = 8.27 ± 0.52 µg/ml)] as compared to the synthetic antioxidant BHT [(DPPH IC 50 = 78.96 ± 5.70 µg/ml and ABTS IC 50 = 13.56 ± 0.06 µg/ml)]. However, the methanolic extract of T. baccata showed the lowest ferric reducing ability as compared to the positive controls (BHT, BHA, ascorbic acid, trolox and quercetin). Conclusion:Our results imply that the Taxus Baccata L. might be a potential source for the isolation of natural antioxidant compounds.

Description of the subject. The hyoscyamine, a tropane alkaloid, widely used in medicine, can be produced from Datura sp. (Solanaceae). However, its content in the spontaneous roots remains low; therefore, hairy roots (HRs) were envisaged as a potential alternative to improve its biosynthesis. The hairy roots are characterized by a good genetic stability and a rapid growth. Indeed, Datura stramonium HRs have widely been studied in the perspective of improving the yield of hyoscyamine. This study is part of this same perspective. Objectives. This paper aims to study the effects of polyploidization of HRs induced by colchicine in synergy with elicitation (with acetylsalicylic [ASA] or salicylic acids [SA]) on the hyoscyamine content in D. stramonium. Method. Colchicine was applied at different concentrations and periods, on a selected hairy root line (LDS) of D. stramonium obtained by infection with Agrobactrium rhizogenes strain A4. The selection of tetraploid HR lines was performed by the cytogenetic analysis using light microscopy. The effect of polyploidization and elicitation was studied on the biomass (dry weight) and hyoscyamine content of HRs. Results. The untreated HR line (control) shows a diploid level with 2n = 24 chromosomes. However, the HR lines treated with colchicine show, in most cases, an endoreduplication of their genetic material. The survival rate of endoreduplicated lines varies between 30% and 93%, depending on concentration and exposure time to colchicine. Moreover, the tetraploid HR line shows an increase in its biomass and hyoscyamine content in comparison to the diploid HR line (LDS). Further, elicitation of HRs by ASA or AS at the 10-4 M concentration causes a low decrease or increase in dry weight, respectively. However, the same treatments show a significant increase in the yield of hyoscyamine in elicited HR lines. Consequently, our work indicates that the combination of polyploidy and elicitation can lead to significant improvements in hyoscyamine biosynthesis and content due to their synergistic effects. Conclusions. Elicitation of tetraploid hairy root lines improves significantly their content of hyoscyamine.