Ana Claudia Ferreira da Cruz

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.

Synergism and negative interference during co-infection of tomato and Nicotiana benthamiana with two bipartite begomoviruses

In Brazil, at least eight begomoviruses including Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) infect tomatoes. ToYSV symptoms in tomato and Nicotiana benthamiana appear earlier and are more severe compared to those of ToRMV. We investigated the role of several factors in this differential adaptation. To analyze infection kinetics, a single leaf was inoculated and subsequently detached after different periods of time. Viral DNA accumulation was quantified in plants, viral replication was analyzed in protoplasts, and tissue tropism was determined by in situ hybridization. Results indicate that ToYSV establishes a systemic infection and reaches a higher concentration earlier than ToRMV in both hosts. ToRMV negatively interferes with ToYSV during the initial stages of infection, but once systemic infection is established this interference ceases. In N. benthamiana, ToYSV invades the mesophyll, while ToRMV is phloem-restricted. During dual infection in this host, ToYSV releases ToRMV from the phloem.

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species.

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.

Somatic Embryogenesis in Annatto (Bixa orellana L.)

Our research group has pioneered the work on somatic embryogenesis of Bixa orellana (annatto), and since then we have directed efforts in understanding several aspects of this morphogenic pathway in annatto. Here, we present a synthetic description of such works, emphasizing anatomical analyzes and the characterization of the cellular alterations that occur in the process, and the association of the SERK gene expression and somatic embryogenesis. These results are unprecedented and contribute to a better understanding of the processes involving somatic embryogenesis in the species. Advances in this area will facilitate the improvement of the mass propagation, genetic manipulation of the carotenoid biosynthetic pathway, and the overall breeding perspectives of the genus.

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.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

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.