Cleber Witt Saldanha

Photoautotrophic propagation of Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen]

Pfaffia glomerata (Spreng.) Pedersen is a medicinal species of great interest because it produces the phytoecdysteroid 20-hydroxyecdysone (20E). Generally, because of atypical growing conditions, in vitro propagated plants function less efficiently as autotrophs and have poorly developed morphological structures. This study analyzed the autotrophic potential of P. glomerata propagated in vitro and evaluated the influence that this has on 20E biosynthesis. Physiological and structural parameters of plants subjected to heterotrophic, photomixotrophic and photoautotrophic growth conditions were evaluated. Levels of 20E were measured by HPLC. Plants were acclimatized in a mixture of soil, sand and substrate, in a greenhouse. Conditions that provided higher carbon input led to an increase in plant growth, and the presence of sucrose was critical, in closure systems without a gas permeable membrane, for normal anatomical development of the micropropagated plants. The absence of sucrose increased photosynthesis and conditions that enhanced photoautotrophy induced greater levels of 20E. The increase of 20E levels by the photoautotrophic system offers new prospects for increasing the commercial production of this species, and for studies that could elucidate the biosynthetic pathway of phytoecdysteroids in plants.

A low-cost alternative membrane system that promotes growth in nodal cultures of Brazilian ginseng [ Pfaffia glomerata (Spreng.) Pedersen]

In vitro propagated plants under conditions of low gas exchange generally show morphological and physiological anomalies that lead to high mortality rates during ex vitro acclimatization. The use of gas-permeable membranes increases natural ventilation in culture vessels, photosynthesis and growth rates. However, commercial membranes are expensive, which limits their application. In this study, low-cost, simple to manufacture, alternative membranes were developed to promote gas exchange in jars used for in vitro plant tissue culture. The membranes were developed using polytetrafluoroethylene film and two or three layers of microporous tape (Missner & Missner®), and were designed to increase the growth of nodal cultures of Pfaffia glomerata (Brazilian ginseng). Conditions that provided higher gas exchange led to an increase in plant growth and content of photosynthetic pigments compared to a closed system without a gas-permeable membrane. The alternative membranes showed similar results for water vapor loss rate and photosynthetic pigments when compared to a commercial membrane. The alternative membranes were also an efficient barrier against contamination and remained intact after being autoclaved multiple times. Among the membranes tested, the traits of the P. glomerata in vitro-derived plants were similar when propagated using the alternative membrane with three layers of microporous tape or the commercial membrane. However, the alternative membrane has a unit cost that is ten times lower than the commercial membrane.

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.

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.

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.

Massa é fator determinante na qualidade fisiológica de sementes de Parapiptadenia rigida

This study aimed to assess the physiological quality of Parapiptadenia rigida (Benth.) Brenan seed weight classes subjected to accelerated aging under laboratory and greenhouse conditions. A lot of P. rigida seeds was split in three seed weight classes (thousand seed weight): TSW1 (original lot; control, 19.23 g), TSW2 (25.45 g) and TSW3 (13.58 g), which were subjected to three accelerated aging times (0,24 and 48 h). First count, weak and strong normal seedlings, percentage of accumulated germination, percentage of death seed, electrical conductivity, fresh and dry weight and shoot length were assessed in laboratory. Seedling emergence, emergence velocity and emergence velocity index were assessed in greenhouse. P. rigida seeds showing higher weight displayed higher percentage of germination and average vigor characteristics. Seeds with higher weight indicated high tolerance to accelerated aging conditions and produced more vigorous plants. Therefore, we suggest that seed lots of P. rigida should be composed considering TSW classes. Massa é fator determinante na qualidade fisiológica de sementes de Parapiptadenia rigida Resumo – O presente estudo teve por objetivo avaliar em laboratório e em casa de vegetação a qualidade fisiológica de classes de massa de sementes de Parapiptadenia rigida (Benth.) Brenan, submetidas a condições de envelhecimento acelerado. Um lote de sementes de P. rigida foi separado em três grupos de massa de sementes (peso de mil sementes): PMS1 (lote original; controle, 19,23 g), PMS2 (25,45 g) e PMS3 (13,58 g); e submetido a três tempos de envelhecimento acelerado (0, 24 e 48 h). Em laboratório, foram avaliadas: primeira contagem, plântulas normais fracas e fortes, porcentagem de germinação acumulada, porcentagem de sementes mortas, condutividade elétrica, massas fresca e seca e comprimento da parte aérea das plântulas. Em casa de vegetação, foram avaliadas a emergência de plântulas, a velocidade de emergência e o índice de velocidade de emergência. Sementes de P. rigida com maior massa apresentaram maiores valores de porcentagem de germinação e características de vigor médias. Sementes com maior massa apresentaram indicativos de alta tolerância a condições de envelhecimento acelerado e possibilitaram a obtenção de plântulas mais vigorosas. Portanto, sugere-se que lotes de semente de P. rigida sejam constituídos por sementes classificadas pelo PMS. e-ISSN: 1983-2605

Parapiptadenia rigida MYCORRHIZATION WITH SPORES OF Scleroderma citrinum

A inoculacao de fungos ectomicorrizicos em mudas florestais beneficia o estabelecimento e crescimento das plantas no campo. Os objetivos do trabalho foram: determinar a capacidade de micorrizacao do fungo ectomicorrizico Scleroderma citrinum em mudas de Parapiptadenia rigida (angico-vermelho) e avaliar a viabilidade da tecnica de inoculacao micorrizica de mudas florestais atraves do uso de esporos. Esporos maduros foram inoculados ao substrato (75% solo e 25% casca de arroz carbonizada), totalizando 1,5 gramas de esporos de fungo por litro de substrato. Sementes de P. rigida foram semeadas em substratos inoculados ou nao com esporos de S. citrinum, na presenca ou ausencia dos oleos essencias de Pinus echinata e Eucalyptus citriodora: nao inoculado (T1), inoculado (T2), inoculado mais adicao de oleo essencial de pinus (T3), inoculado mais adicao de oleo essencial de eucalipto (T4). Mudas de Pinus elliottii foram utilizadas para controle positivo da inoculacao micorrizica, inoculadas (T5) e nao inoculadas com esporos ectomicorrizicos (T6). Aos 90 dias apos a semeadura, foram determinados o diâmetro do colo, a altura, as massas fresca e seca da parte aerea e das raizes, o percentual de colonizacao radicular e o Indice de Dickson. Observou-se presenca de estruturas fungicas no interior das raizes de P. rigida e P. elliotti inoculadas com esporos de S. citrinum, evidenciando a ocorrencia de associacao ectomicorrizica. A aplicacao dos oleos essenciais de pinus e de eucalipto no substrato elevou o percentual de colonizacao ectomicorrizica nas mudas de angico-vermelho. A adicao de esporos maduros de S. citrinum ao substrato de producao das mudas representa uma pratica viavel de inoculacao ectomicorrizica, com possibilidade de utilizacao em viveiros florestais em programas de micorrizacao controlada.