Plant secondary biosynthetic pathways are exceedingly inducible by elicitors and facilitate enhanced metabolite production using plant cell tissue and organ cultures. Elicitors can regulate large number of control points and trigger the expression of key genes with increased cellular activities at biochemical and molecular level involving signal compounds. A large number of chemical elicitors viz: jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), acetyl salicylic acid (ASA), ethylene (ET) and ethrel (Ethe), heavy metals (HM), many types of chemical compounds (natural or synthetic) and their combinations are used for elicitation studies. Cell suspensions and hairy roots are commonly used culture systems followed by adventitious roots and multiple shoots for elicitation experiments. Amongst the elicitors and concentrations used 100 µM MeJA was found optimum for secondary metabolite enhancement in majority of experiments compared to SA and JA. Elicitor treatments promoted yield enhancements starting from 1.0 to maximum of 2230-fold across plant species studied. Elicitors singly with media additives, combination of elicitors and elicitors other than signal compounds along with hormones were found beneficial for enhanced secondary metabolite production. Further, a combination of target gene over expression and elicitor treatment also supported higher secondary product yield. The present communication presents information exclusively about the use of chemical elicitors for secondary metabolites production in vitro covering approximately more than a decade of research at one place in one review. Further, this extensive appraisal will be useful for the understanding and manipulation of secondary metabolites for enhanced production in vitro.

Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal

Lycoris aurea (L' Her.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

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Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

Tomato, eggplant, and pepper are three solanaceous crops of outstanding importance worldwide. For hybrid seed production in these species, a fast and cheap method to obtain pure (homozygous) lines is a priority. Traditionally, pure lines are produced by classical inbreeding and selection techniques, which are time consuming (several years) and costly. Alternatively, it has become possible to accelerate the production of homozygous lines through a biotechnological approach: the induction of androgenesis to generate doubled haploid (homozygous) plants. This biotechnological in vitro tool reduces the process to only one generation, which implies important time and costs savings. These facts make androgenic doubled haploids the choice in a number of important crops where the methodology is well set up. Unfortunately, recalcitrant solanaceous crops such as tomato, eggplant, and pepper are still far from an efficient and reliable technology to be applied on a routine basis to different genotypes in breeding programs. In eggplant and pepper, only anther cultures are known to work relatively well. Unfortunately, a more efficient and promising technique, the culture of isolated microspores, is not sufficiently developed yet. In tomato, none of these methods is available nowadays. However, recent advances in the knowledge of embryo development are filling the gaps and opening new ways to achieve the final goal of an efficient protocol in these three recalcitrant species. In this review, we outline the state of the art on androgenic induction in tomato, eggplant, and pepper, and postulate new experimental ways in order to overcome current limitations.

/pdf/androgenesis-in-recalcitrant-solanaceous-crops-5et8n33oan.pdf

Androgenesis in recalcitrant solanaceous crops.

Several plant isoquinoline alkaloids (PIAs) possess powerful pharmaceutical and biotechnological properties. Thus, PIA metabolism and its fascinating molecules, including morphine, colchicine and galanthamine, have attracted the attention of both the industry and researchers involved in plant science, biochemistry, chemical bioengineering and medicine. Currently, access and availability of high-value PIAs [commercialized (e.g. galanthamine) or not (e.g. narciclasine)] is limited by low concentration in nature, lack of cultivation or geographic access, seasonal production and risk of overharvesting wild plant species. Nevertheless, most commercial PIAs are still extracted from plant sources. Efforts to improve the production of PIA have largely been impaired by the lack of knowledge on PIA metabolism. With the development and integration of next-generation sequencing technologies, high-throughput proteomics and metabolomics analyses and bioinformatics, systems biology was used to unravel metabolic pathways allowing the use of metabolic engineering and synthetic biology approaches to increase production of valuable PIAs. Metabolic engineering provides opportunity to overcome issues related to restricted availability, diversification and productivity of plant alkaloids. Engineered plant, plant cells and microbial cell cultures can act as biofactories by offering their metabolic machinery for the purpose of optimizing the conditions and increasing the productivity of a specific alkaloid. In this article, is presented an update on the production of PIA in engineered plant, plant cell cultures and heterologous micro-organisms.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12494

Metabolic engineering for the production of plant isoquinoline alkaloids

Proteomics in commercial crops: An overview.

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 μM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 μM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 μM α-naphthalene acetic acid (NAA) and 5 μM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO3) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO4). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO4 during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).

G. Wyżgolik

Papers

Effects of ethylene on somatic embryogenesis and galanthamine content in Leucojum aestivum L. cultures