Diego Silva Batista

In vitro photoautotrophic potential and ex vitro photosynthetic competence of Pfaffia glomerata (Spreng.) Pedersen accessions

This study aimed to investigate whether the in vitro photoautotrophic potential and 20 hydroxyecdysone (20E) production of six Pfaffia glomerata accessions could be correlated to photosynthetic performance and biomass accumulation under ex vitro conditions in a greenhouse environment. Our results showed that P. glomerata accessions displayed varying photosynthetic rates that may have resulted in differential biomass accumulation both in vitro and ex vitro. Accessions A4, A13 and A43 showed the highest dry weight under in vitro photoautotrophic conditions, coinciding with the higher photosynthetic rates of the same accessions in greenhouse. They also showed the highest 20E mass per plant ex vitro. Production of 20E in vitro also varied among accessions, with the highest total mass achieved by accessions A4 and A43. In addition to having the potential for optimizing propagation by reducing the duration of the in vitro culture stage and lessening death rate during acclimatization, the use of these genotypes with higher in vitro photoautotrophic potential can guarantee highly productive plants ex vitro because the biomass accumulation pattern observed in vitro matched that under greenhouse conditions.

Suppression of ethylene levels promotes morphogenesis in pepper ( Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.

Induced polyploidization increases 20-hydroxyecdysone content, in vitro photoautotrophic growth, and ex vitro biomass accumulation in Pfaffia glomerata (Spreng.) Pedersen

The present study aimed to verify the effects of induced polyploidization on Pfaffia glomerata regarding its 20-hydroxyecdysone (20E) production both in vitro and under greenhouse conditions, its in vitro photoautotrophic potential, and its ex vitro biomass accumulation and photosynthetic performance. Synthetic polyploidization efficiently produced individuals with increased in vitro photoautotrophic potential and ex vitro biomass accumulation, although photosynthetic rates per leaf area did not vary between diploids and tetraploids. Among the five tetraploids tested (P28, P60, P68, P74, and P75), P28 showed significantly increased biomass both in vitro and ex vitro when compared with diploid plants, whereas the other tetraploids did not differ significantly from the diploids in terms of biomass accumulation. Although photosynthetic rates per unit leaf area remained constant among all the plants tested, P28 showed a significantly greater total leaf area, which may have resulted in an increase in net photosynthesis on a whole-plant basis. Under greenhouse conditions, the 20E content in the tetraploid P28 was 31% higher than that in diploid plants, and the final 20E mass per plant produced by P28 ex vitro was approximately twice that produced by diploid plants. Accumulation of 20E in vitro did not follow the same pattern observed among the plants ex vitro; instead, greater accumulation was observed in diploid plants. The induction of polyploidy in P. glomerata appears to be a promising strategy for producing plants with higher biomass accumulation and 20E production ex vitro, in addition to its higher in vitro photoautotrophic potential.

Light quality in plant tissue culture: does it matter?

The primary issues regarding the lack of protocol reproducibility among laboratories are environmental factors. Light (quantity and particularly quality), is one of those main factors, and studies seldom present the spectral quality of the light sources used. With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and morphogenetic responses, as directed by the light used in the culture shelves. A wide array of LED lights with different spectra are currently available and light is important in large-scale propagation, especially liquid bioreactor systems. LED technology continues to evolve rapidly and has created additional possibilities. This laboratory has dedicated extensive efforts to implement photoautotrophic propagation, and light is a key component of the system. This review presents relevant topics on the influence of light in various plant tissue culture-based techniques.

De novo assembly and transcriptome of Pfaffia glomerata uncovers the role of photoautotrophy and the P450 family genes in 20-hydroxyecdysone production

Pfaffia glomerata is a medically important species because it produces the phytoecdysteroid 20-hydroxyecdysone (20-E). However, there has been no ready-to-use transcriptome data available in the literature for this plant. Here, we present de novo transcriptome sequencing of RNA from P. glomerata in order to investigate the 20-E production as well as to understand the biochemical pathway of secondary metabolites in this non-model species. We then analyze the effect of photoautotrophy on the production of 20-E genes phylogenetically identified followed by expression analysis. For this, total messenger RNA (mRNA) from leaves, stems, roots, and flowers was used to construct indexed mRNA libraries. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 164,439 transcripts were annotated. In addition, the effect of photoautotrophy in two genes putatively involved in the 20-E synthesis pathway was analyzed. The Phantom gene (CYP76C), a precursor of the route, showed increased expression in P. glomerata plants cultured under photoautotrophic conditions. This was accompanied by increased production of this metabolite indicating a putative involvement in 20-E synthesis. This work reveals that several genes in the P. glomerata transcriptome are related to secondary metabolism and stresses, that genes of the P450 family participate in the 20-E biosynthesis route, and that plants cultured under photoautotrophic conditions promote an upregulated Phantom gene and enhance the productivity of 20-E. The data will be used for future investigations of the 20-E synthesis pathway in P. glomerata while offering a better understanding of the metabolism of the species.

Regulation of cell wall development in Brachypodium distachyon in vitro as affected by cytokinin and gas exchange

Brachypodium distachyon is successfully used as a model grass species. The present study used molecular and chemical approaches to investigate the effects of the addition of the cytokinin 6-benzyladenine (BA) and two levels of gas exchange on the transcriptional regulation of lignin biosynthesis in B. distachyon. It was observed that the control of lignin biosynthesis is affected by both BA and the in vitro gas exchange rate, by sealing the vial with absence or presence of membranes. These observations are supported by anatomical and antioxidant enzyme activity analyses. However, the hyperhydricity caused by BA did not affect cell wall properties in B. distachyon. Interestingly, the CCR1 gene presented 70-fold increase in expression under the effect of BA and without membranes in addition to a significant increase in the peroxidases activity and total lignin content. Treatments with gas exchange showed higher qualitative accumulation of lignin S/G. Lignin pathway genes were transcriptionally coordinated according to the imposed treatment with apparent co-expression. Low expression of these genes was observed in the sealing treatment without membranes, while the opposite was verified in the condition of sealing with two membranes. CESA4 and CESA7 genes responded positively to gas exchange, without altering the cell wall cellulose/lignin ratio in B. distachyon. These results contribute to a better understanding of how cell wall-related gene expression is regulated by exogenous factors, as well as the many possibilities of cell wall manipulation due to its plasticity in adaptive responses.

Ethylene Responses and ACC oxidase Gene Expression in Lippia alba (Verbenaceae) Chemotypes with Varying Ploidy Levels

Lippia alba (Mill.) N. E. Br ex Britton & P. Wilson is a species of great economic importance due to the medicinal activity conferred by its essential oils. Ethylene (ET) is a gaseous hormone that affects several processes in plant growth and development. This hormone is synthesized through a reaction in which 1-aminocyclopropane-1-carboxylic acid (ACC) is converted to ET by the action of the enzyme ACC oxidase (ACO). The ET activity in three L. alba chemotypes (BGEN-01, triploid; BGEN-02, diploid; and BGEN-42, hexaploid) was evaluated by culturing plants in vitro in the presence of the ET precursor (ACC), scavenger mercury perchloride (MP), the inhibitors aminoethoxyvinylglycine (AVG) and silver thiosulfate (STS), and a CO2-enriched atmosphere. Leaf abscission rate, ET accumulation, and ACO gene expression (LaACO) were investigated. Leaf abscission was higher in BGEN-42, regardless of the precursor/inhibitor. ACC had the highest ET accumulation in BGEN-02 and BGEN-01, whereas BGEN-42 presented lower ET accumulation than the control. MP and AVG were effective in preventing ET production when compared to the control. LaACO was upregulated in BGEN-01 and BGEN-42 treated with ACC and in BGEN-01 treated with STS. In the CO2 enrichment experiments, LaACO expression was higher in BGEN-42 in the treatments without forced ventilation. Thus, the expression of this gene in L. alba can be altered by elevating CO2 levels and also by the addition of ACC. This is the first report of ET interactions in L. alba cultured in vitro.