Isolation of Protoplasts from Tissues of Mexican Prickly Poppy (Argemone mexicana L.): An Alkaloid-Producing Medicinal Plant.

Protoplasts are plant cells from which the pectocellulosic cell wall has been removed, thus keeping the plasma membrane intact. For plant secondary metabolites research, this system is a powerful tool to study the metabolites' dynamics inside the cells, such as the subcellular localization of proteins, characterization of gene function, transcription factors involved in metabolite pathways, protein transport machinery, and to perform single-cell omics studies. Due to its lack of a cell wall, better images of the interior of the cell can be obtained compared to the whole tissue. This allows the identification of specific cell types involved in the accumulation of specialized metabolites, such as alkaloids, given their autofluorescence properties. Here is a simplified protocol to obtain protoplasts from leaves and in vitro cell cultures from Argemone mexicana, which produces the pharmacologically important alkaloids berberine and sanguinarine.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

Verbascoside production in long-term Buddleja cordata Kunth cell suspension cultures.

Previously, our group reported the establishment of a white callus cell line of Buddleja cordata Kunth that is a high producer of the secondary metabolite, verbascoside (VB, also named acteoside), under suspension culture conditions. Here, we present experimental evidence of the sustained ability of that cellular line to grow and produce high amounts of VB for 5 years of continuous culture. Cellular line profiles were determined at the early (at the beginning) and late stages (at the end of 5 years of continuous subculturing) by analyzing relevant parameters of culture growth, i.e., specific growth rate [µ], doubling time [dt], and growth index [GI], as well as VB production. Late-stage cultures exhibited a 61% faster growth rate than early-stage subcultures, and 25 and 3% lower doubling time and growth index. The extents of growth phases were found to be different. Similar amounts of biomass were found (9.5 g and 9.4 g L-1). Verbascoside production increased parallel to cell growth; maximal yield level occurred in the mid-exponential phase and lasted until the end of the stationary phase (i.e., from the 15th to the 25th day and from the 9th to the 21st day for the early and late stages, correspondingly). The content of VB was higher in the late-stage culture (1.43 ± 0945 g L-1) than in the early-stage culture (1.21 ± 0.0286 g L-1). Productivity values point out the potential use of B. cordata cell line in the biotechnological production of VB and for research focused on the biochemistry of secondary metabolism.

Antimicrobial activity of Anonna mucosa (Jacq.) grown in vivo and obtained by in vitroculture.

Brazilian flora includes numerous species of medicinal importance that can be used to develop new drugs. Plant tissue culture offers strategies for conservation and use of these species allowing continuous production of plants and bioactive substances. Annona mucosa has produced substances such as acetogenins and alkaloids that exhibit antimicrobial activities. The widespread use of antibiotics has led to an increase in multidrug-resistant bacteria, which represents a serious risk of infection. In view of this problem, the aim of this work was to evaluate the antibacterial potential of extracts of A. mucosa obtained by in vitro techniques and also cultured under in vivo conditions. Segments from seedlings were inoculated onto different culture media containing the auxin picloram and the cytokinin kinetin at different concentrations. The calluses obtained were used to produce cell suspension cultures. The materials were subjected to methanol extraction and subsequent fractionation in hexane and dichloromethane. The antimicrobial activity against 20 strains of clinical relevance was evaluated by the macrodilution method at minimum inhibitory and minimum bactericidal concentrations. The extracts showed selective antimicrobial activity, inhibiting the growth of Streptococcus pyogenes and Bacillus thuringiensis at different concentrations. The plant tissue culture methods produced plant materials with antibacterial properties, as well as in vivo grown plants. The antibacterial activity of material obtained through biotechnological procedures of A. mucosa is reported here for the first time.

Antimicrobial activity of Anonna mucosa (Jacq.) grown in vivo and obtained by in vitroculture

Brazilian flora includes numerous species of medicinal importance that can be used to develop new drugs. Plant tissue culture offers strategies for conservation and use of these species allowing continuous production of plants and bioactive substances. Annona mucosa has produced substances such as acetogenins and alkaloids that exhibit antimicrobial activities. The widespread use of antibiotics has led to an increase in multidrug-resistant bacteria, which represents a serious risk of infection. In view of this problem, the aim of this work was to evaluate the antibacterial potential of extracts of A. mucosa obtained by in vitro techniques and also cultured under in vivo conditions. Segments from seedlings were inoculated onto different culture media containing the auxin picloram and the cytokinin kinetin at different concentrations. The calluses obtained were used to produce cell suspension cultures. The materials were subjected to methanol extraction and subsequent fractionation in hexane and dichloromethane. The antimicrobial activity against 20 strains of clinical relevance was evaluated by the macrodilution method at minimum inhibitory and minimum bactericidal concentrations. The extracts showed selective antimicrobial activity, inhibiting the growth of Streptococcus pyogenes and Bacillus thuringiensis at different concentrations. The plant tissue culture methods produced plant materials with antibacterial properties, as well as in vivo grown plants. The antibacterial activity of material obtained through biotechnological procedures of A. mucosa is reported here for the first time.

Antimicrobial activity of Anonna mucosa (Jacq) grown in vivo and obtained by in vitroculture

Brazilian flora includes numerous species of medicinal importance that can be used to develop new drugs. Plant tissue culture offers strategies for conservation and use of these species allowing continuous production of plants and bioactive substances. Annona mucosa has produced substances such as acetogenins and alkaloids that exhibit antimicrobial activities. The widespread use of antibiotics has led to an increase in multidrug-resistant bacteria, which represents a serious risk of infection. In view of this problem, the aim of this work was to evaluate the antibacterial potential of extracts of A. mucosa obtained by in vitro techniques and also cultured under in vivo conditions. Segments from seedlings were inoculated onto different culture media containing the auxin picloram and the cytokinin kinetin at different concentrations. The calluses obtained were used to produce cell suspension cultures. The materials were subjected to methanol extraction and subsequent fractionation in hexane and dichloromethane. The antimicrobial activity against 20 strains of clinical relevance was evaluated by the macrodilution method at minimum inhibitory and minimum bactericidal concentrations. The extracts showed selective antimicrobial activity, inhibiting the growth of Streptococcus pyogenes and Bacillus thuringiensis at different concentrations. The plant tissue culture methods produced plant materials with antibacterial properties, as well as in vivo grown plants. The antibacterial activity of material obtained through biotechnological procedures of A. mucosa is reported here for the first time..(AU)

The expression of the 14D9 catalytic antibody in suspended cells of Nicotiana tabacum cultures increased by the addition of protein stabilizers and by transference from Erlenmeyer flasks to a 2-L bioreactor.

The effect of two protein stabilizers (polyvinylpyrrolidone [PVP] and gelatine) on growth and 14D9 yield of Nicotiana tabacum cell suspension cultures (Ab-KDEL and sec-Ab) was analyzed. The addition of PVP at a concentration of 1.0 g L(-1) produced the highest total 14D9 yield (biomass + culture medium) in the Ab-KDEL line (4.82% total soluble protein [TSP]). With the addition of gelatine, the highest total 14D9 yield (2.48% TSP) was attained in the Ab-KDEL line at 5.0 g L(-1) gelatine. When the Ab-KDEL suspended cells were cultured in a 2-L bioreactor, the highest 14D9 yield was 8.1% TSP at a 5% w/v inoculum size, which was the best 14D9 yield so far obtained in the platforms tested (E. coli, N. tabacum leaves and seeds, N. tabacum hairy roots, and cell suspension cultures).

Stimulation of trans-resveratrol biosynthesis in Vitis vinifera cv. Kyoho cell suspension cultures by 2, 3-dihydroxypropyl jasmonate elicitation

Background: Plant cell suspension culture of Vitis vinifera is a promising technology for investigating different factors that are able to induce and/or modify stilbenes biosynthesis. Jasmonates have been reported to play an important role in a signal transduction pathway that regulates defence responses as well as the production of secondary metabolites. In this study, 2, 3-dihydroxypropyl jasmonate (DHPJA) was used to investigate its effect on stimulating trans-resveratrol (t-R) accumulation and the plant defence responses in Vitis vinifera cv. Kyoho cell suspension cultures for the first time. Results: It demonstrated that DHPJA had superior effects on stilbenoids accumulation over methyl jasmonate (MeJA). The optimal condition was 150 uM DHPJA added on day 15 of cultivation period, with the highest level of t-R accumulation which was increased 1.8-fold and 1.3-fold compared with the control and 150 uM MeJA respectively. DHPJA induced stronger plant defence responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL) than MeJA. H2O2 generation induced by DHPJA played a significant role in enhancing t-R accumulation. Adding a specific inhibitor of H2O2 signalling pathway inhibited DHPJA-induced t-R accumulation, but had no effects on DHPJA-induced other metabolites accumulation, which resulted in regulations of product diversity. Conclusions: This study demonstrated that DHPJA was an efficient elicitor to enhance t-R accumulation by activating stronger oxidative burst, and H2O2 signalling pathway could regulate product diversity in DHPJA-induced V. vinifera cv. Kyoho cell suspension cultures.