Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.

Synthetic Tetraploid of Oncidium crispum Lodd. (Orchidaceae).

In ornamental plants, artificial polyploidization has enabled the creation of new cultivars. Due to their high commercial value in the international flower market and their ornamental characteristics, such as the shape, size, color, and durability of their flower, orchids have received great attention in studies of artificial polyploidization. Here we described the protocol used for polyploid induction in Oncidium crispum, an epiphyte species native of southeastern Brazil, of great ornamental interest and widely sold in flower shops. The species stands out for having inflorescence with large flowers, brown in color with yellow spots. In addition, O. crispum has great potential for use in genetic improvement programs since the species is widely used in interspecific crosses. Closed capsules containing mature O. crispum seeds were subjected to running sterilized water for 10 min and then to a 1.5% sodium hypochlorite solution for 10 min. Small portions of seeds were introduced into 50 mL of water-soluble fertilizer with macro- and micronutrients (B>M) plus 0.7% agar. Explants originating from seeds previously in vitro germinated were submitted to 0.05% and 0.1% of colchicine for 4 days and 8 days. Flow cytometry and chromosome counts confirmed that the protocol successfully produced synthetic polyploid plants.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Salicylic Acid and Water Stress: Effects on Morphophysiology and Essential Oil Profile of Eryngium foetidum.

The exogenous application of bioregulators, such as salicylic acid (SA), has exhibited promising outcomes in alleviating drought stress. Nevertheless, its impact on culantro (Eryngium foetidum L.) remains unexplored. Thus, the aim of this study was to assess how SA impacts the growth, morphophysiology, and essential oil composition of culantro when subjected to drought. To achieve this, culantro plants were grown under three different watering regimes: well-watered, drought-stressed, and re-watered. Additionally, they were either treated with SA (100 µM) or left untreated, with water serving as the control. SA application did not mitigate the effects of drought in biomass production but increased biomass, leaf number, leaf area, and photosynthetic pigments under well-irrigated and re-watered conditions. After a drought period followed by re-watering, plants recovered membrane integrity independently of SA application. Water stress and the exogenous application of SA also modulated the profile of essential oils. This is the first report about SA and drought affecting growth and essential oil composition in culantro.

Phenotypic variation seems not to be associated with the genetic profile in Zygopetalum (Orchidaceae): a case study of a high-elevation rocky complex.

BACKGROUND: Hybridization associated with polyploidy studies is rare in the tropics. The genus Zygopetalum (Orchidaceae) was investigated here as a case study of Neotropical plants. In the rocky highlands of the Ibitipoca State Park (ISP), southeast Brazil, individuals with intermediate colors and forms between the species Z. maculatum and Z. triste were commonly identified. METHODS AND RESULTS: Chromosomal analysis and DNA quantity showed a uniform population. Regardless of the aspects related to the color and shape of floral structures, all individuals showed 2n = 96 chromosomes and an average of 14.05 pg of DNA. Irregularities in meiosis associated with chromosome number and C value suggest the occurrence of polyploidy. The genetic distance estimated using ISSR molecular markers revealed the existence of genetic variability not related to morphological clusters. Morphometric measurements of the flower pieces revealed that Z. maculatum shows higher variation than Z. triste although lacking a defined circumscription. CONCLUSION: The observed variation can be explained by the polyploid and phenotypic plasticity resulting from the interaction of the genotypes with the heterogeneous environments observed in this habitat.

Water stress modulates terpene biosynthesis and morphophysiology at different ploidal levels in Lippia alba (Mill.) N. E. Brown (Verbenaceae).

Monoterpenes are the main component in essential oils of Lippia alba. In this species, the chemical composition of essential oils varies with genome size: citral (geraniol and neral) is dominant in diploids and tetraploids, and linalool in triploids. Because environmental stress impacts various metabolic pathways, we hypothesized that stress responses in L. alba could alter the relationship between genome size and essential oil composition. Water stress affects the flowering, production, and reproduction of plants. Here, we evaluated the effect of water stress on morphophysiology, essential oil production, and the expression of genes related to monoterpene synthesis in diploid, triploid, and tetraploid accessions of L. alba cultivated in vitro for 40 days. First, using transcriptome data, we performed de novo gene assembly and identified orthologous genes using phylogenetic and clustering-based approaches. The expression of candidate genes related to terpene biosynthesis was estimated by real-time quantitative PCR. Next, we assessed the expression of these genes under water stress conditions, whereby 1% PEG-4000 was added to MS medium. Water stress modulated L. alba morphophysiology at all ploidal levels. Gene expression and essential oil production were affected in triploid accessions. Polyploid accessions showed greater growth and metabolic tolerance under stress compared to diploids. These results confirm the complex regulation of metabolic pathways such as the production of essential oils in polyploid genomes. In addition, they highlight aspects of genotype and environment interactions, which may be important for the conservation of tropical biodiversity.

Water stress and exogenous carnitine on growth and essential oil profile of Eryngium foetidum L.

Water stress influences plant growth and metabolism. Carnitine, an amino acid involved in lipid metabolism, has been related to responses of plants to abiotic stresses, also modulating their metabolites. Culantro (Eryngium foetidum L.) is a perennial herb, rich in essential oils, native to Latin America, commonly used due to its culinary and medicinal properties. Here, we investigated the effect of exogenous carnitine on morphophysiology and the essential oil profile of culantro plants under water stress. For this, plants were grown under three water conditions: well-watered, drought stress, and re-watered; and sprayed with exogenous carnitine (100 µM) or water (control). Culantro growth was impaired by drought and enhanced by re-watering. Carnitine, in turn, did not reverse drought effects on growth, and impaired the growth of re-watered plants, also improving photosynthetic pigment content. Water conditions and carnitine application changed the essential oil profile of the plants. Drought and re-watering improved the production of eryngial, which was even increased with exogenous carnitine in re-watered plants. In addition, hydroquinone was only produced with the combination of re-watering and carnitine application. The application of exogenous carnitine can be a strategy to induce the production of essential oil compounds with cosmetic and pharmaceutical importance in culantro. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03757-y.

Aneuploids and its increment on diversity of Lippia alba polyploid complex: genetic aspects and origin.

BACKGROUND: Aneuploidy is associated with add or lack of individual chromosomes. The knowledge regarding aneuploidy is still rare in wild and tropical populations. Lippia alba is a tropical polyploid complex naturally formed, with 2x, 3x, 4x, 6x, and aneuploid individuals. The species presents pharmacological and medicinal importance, due to its essential oil compounds, which are related to the ploidal level. Considering the singularity of aneuploids emergence and stability, we proposed to investigate putative cytotypes involved in the aneuploids formation. METHODS AND RESULTS: Molecular, cytogenetic, reproductive, and chemical approaches were adopted. The results showed that the aneuploids possibly have independent origin considering the genetic, chemical and karyotypical profiles. The chemical composition of aneuploids is related to genetic similarity. The aneuploid origin may involve 2x and 3x cytotypes being possible to rise four scenarios of crosses to explain that. CONCLUSIONS: The results, in general, contribute to the comprehension of the origin of aneuploids and highlight the genetic profile of these accessions as a key element on the understanding of the chemical profile of L. alba accessions.

Validation of reference genes for quantitative gene expression in the Lippia alba polyploid complex (Verbenaceae).

Lippia alba (Verbenaceae) is one of the most studied species of the genus Lippia, mainly due to its medicinal properties. The species was described as a polyploid complex with five cytotypes. The comparison of gene expression in species with several ploidal levels needs to be conducted carefully due to possible changes in gene regulation. Quantitative reverse transcription PCR (qRT-PCR) is a widely used method for transcript abundance analyses in plants. Besides being an extremely powerful technique, relative quantification by Real-Time quantitative PCR (RT-qPCR) needs the normalization with a stable reference gene. We evaluated the stability of nine candidate reference genes in Lippia alba with different ploidal levels using NormFinder, geNorm, and RefFinder software. The product of each primer showed a single peak in the melting curve. The R2 value ranged from 0.998 to 1000 and primers efficiency ranged from 98.95% to 129%. The CIT gene came up as a stable housekeeping gene, being appropriate for studies in polyploid accessions of Lippia alba. Considering that polyploidy is widely documented in Angiosperms, the results can be used not only for further gene expression studies in L. alba but also as a possible reference gene for other polyploid complexes. Differential stability among different genes highlights the importance of the validation of reference genes used for RT-qPCR approach in polyploid studies.

Anatomy, Flow Cytometry, and X-Ray Tomography Reveal Tissue Organization and Ploidy Distribution in Long-Term In Vitro Cultures of Melocactus Species.

Cacti have a highly specialized stem that enables survival during extended dry periods. Despite the ornamental value of cacti and the fact that stems represent the main source of explants in tissue culture, there are no studies on their morpho-anatomical and cytological characteristics in Melocactus. The present study seeks to address the occurrence of cells with mixed ploidy level in cacti tissues. Specifically, we aim to understand how Melocactus stem tissue is organized, how mixoploidy is distributed when present, and whether detected patterns of ploidy change after long periods of in vitro culture. To analyze tissue organization, Melocactus glaucescens and Melocactus paucispinus plants that had been germinated and cultivated in vitro were analyzed for stem structure using toluidine blue, Xylidine Ponceau, Periodic Acid Schiff, ruthenium red, and acid floroglucin. To investigate patterns of ploidy, apical, medial, and basal zones of the stem, as well as, periphery, cortex, and stele (vascular tissue and pith) regions of the stem and root apexes from four- and ten-year old cultured in vitro were analyzed by flow cytometry. X-ray micro-computed tomography (XRµCT) was performed with fragments of stems from both species. The scarcity of support elements (i.e., sclereids and fibers) indicates that epidermis, hypodermis, and wide-band tracheids present in cortical vascular bundles and stele, as well as water stored in aquifer parenchyma cells along the cortex, provide mechanical support to the stem. Parenchyma cells increase in volume with a four-fold increase in ploidy. M. glaucescens and M. paucispinus exhibit the same pattern of cell ploidy irrespective of topophysical region or age, but there is a marked difference in ploidy between the stem periphery (epidermis and hypodermis), cortex, stele, and roots. Mixoploidy in Melocactus is not related to the age of the culture, but is a developmental trait, whereby endocycles promote cell differentiation to accumulate valuable water.

Development of microsatellite markers for Lippia alba and related Lippia species.

Microsatellite primers were developed in Lippia alba complex to better understanding the origins and evolution of the species. We sought to increase the numbers of available simple sequence repeat (SSR) markers. We performed low-coverage (~ twofold) genomic DNA sequencing of a diploid accession and generated a de novo assembly comprising 175,572 contigs. Sixteen SSR loci were selected and of these 13 SSR loci were successfully amplified in 20 L. alba tetraploid accessions and in 12 other Lippia species. Only one SSR locus was monomorphic, whereas 12 loci were polymorphic, yielding one to nine alleles. The heterozygosity was similar among markers, with values of 0.274-0.485; the polymorphism information content values varied from 0.237 to 0.367. These markers were successfully amplified in related species with 85% of transferability on average. Thus, we demonstrate the utility of including a de novo assembly step to obtain SSR markers from low-coverage genomic datasets.

Induction of Synthetic Polyploids and Assessment of Genomic Stability in Lippia alba.

Polyploidy is widely recognized as a major evolutionary force in plants and has been reported in the genus Lippia (Verbenaceae). Lippia alba, the most studied species, has been documented as a polyploid complex involving at least four ploidal levels. L. alba presents remarkable chemical and genetic variation and represents a model for understanding genome organization. Although the economic and medicinal importance of the species has been widely described, no established polyploid induction protocol has been reported so far. Here, we describe the production of synthetic polyploid plants of L. alba using colchicine. The ploidal levels were estimated by flow cytometry and chromosome counting. In addition, FISH and molecular markers approaches were used to confirm the stability of the synthetic polyploids. The major component of the essential oils was estimated by GCMS to compare with the natural individuals. Tetraploids and triploids were produced providing new opportunities for investigating medicinal, pharmacological, and economic applications as well as addressing intrinsic questions involved in the polyploidization process in tropical plants.

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.