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.

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.

Phenotypic trait variation in the North American Tragopogon allopolyploid complex.

PREMISE: Recently formed allopolyploids Tragopogon mirus and T. miscellus and their diploid parental species, T. dubius, T. porrifolius, and T. pratensis, offer a rare opportunity to study the earliest stages of allopolyploidy. The allopolyploid species have also been resynthesized, allowing comparisons between the youngest possible allopolyploid lineages and their natural, established counterparts. For the first time, we compared phenotypic traits on a large scale in Tragopogon diploids, natural allopolyploids, and three generations of synthetic allopolyploids. METHODS: Our large common-garden experiment measured traits in growth, development, physiology, and reproductive fitness. We analyzed trait differences between allopolyploids and their parental species, and between synthetic and natural allopolyploids. RESULTS: As in many polyploids, the allopolyploid species had some larger physical traits and a higher capacity for photosynthesis than diploid species. Reproductive fitness traits were variable and inconsistent. Allopolyploids had intermediate phenotypes compared to their diploid parents in several traits, but patterns of variation often varied between allopolyploid complexes. Resynthesized and natural allopolyploid lines generally showed minor to nonexistent trait differences. CONCLUSIONS: In Tragopogon, allopolyploidy results in some typical phenotypic changes, including gigas effects and increased photosynthetic capacity. Being polyploid did not produce a significant reproductive advantage. Comparisons between natural and synthetic T. mirus and T. miscellus are consistent with very limited, idiosyncratic phenotypic evolution following allopolyploidization.

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.

Chromosomal view of Lippia alba, a tropical polyploid complex under genome stabilization process.

Lippia alba is a phenotypically variable tropical shrub thought to comprise a young autopolyploid complex. Chromosome numbers in L. alba include 2n = 30, 38, 45, 60, and 90. High levels of chemical and phenotypic variation associated with economic and medicinal importance were reported. However, the genetic background including chromosome composition remains under-explored. Furthermore, the occurrence of at least four ploidal levels in L. alba and the lack of data for polyploid plants in tropical areas also merit further study of L. alba. Here we assessed the chromosome composition using two new satellite repeats (CL98 and CL66) applied as FISH probes to mitotic chromosomes, and we proposed to calculate the degree of homozygosis for CL66 satDNA (named as index h) and to associate it to meiotic instability. The CL98 mapping showed few variations in both number of signals and position. However, the levels of structural homozygosity for a satellite repeat CL66 were very variable. The numbers of CL66-bearing-chromosomes were under-represented in tetraploids relative to diploids implying that CL66 arrays have been lost in tetraploid lineages as a result of increased meiotic instability. High percentage of irregularities was observed in meiotic cells, especially in polyploids. L. alba complex comprised a mixture of homomorphic and heteromorphic chromosomes. Overall, the polyploid complex presents features typical of both young and older stable polyploids. It seems that L. alba genome is still in the process of stabilization.

Deciphering ploidal levels of Lippia alba by using proteomics.

Lippia alba (Mill.) N.E. Brown (Verbenaceae), popularly known as "lemon balm" or "bushy matgrass", is widely used in folk medicine due to its anti-inflammatory, antispasmodic, analgesic, and digestive properties. It was described as an autopolyploid complex with five cytotypes (2n = 30, 38, 45, 60 and 90). To enhance our understanding of the biological variation of the species, we investigated, comparatively, the proteomic profile of all ploidal levels (diploid, aneuploid, triploid, tetraploid, and hexaploid). Leaf proteins were extracted with subsequent separation by two-dimensional electrophoresis, spot analysis, and protein identification by mass spectrometry. By comparing the proteomic profile of diploid accession to the profile of the other ploidal levels we identified differential expression between the analysed spots. We identified 34 proteins with differential expression between the ploidal levels in comparison with the diploid. The identified proteins seem to play relevant roles in the primary metabolism of L. alba suggesting that a specific set of proteins was selected during the polyploidization process, being the triploid the most different one. Given that protein composition can substantially affect the desired therapeutic effect, we posit that further combination of proteomic and metabolomic studies may help to unravel genetic variations and phenotypic profiles in L. alba.

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.

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.

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.

Stachytarpheta gesnerioides Cham: chemical composition of hexane fraction and essential oil, antioxidant and antimicrobial activities / Stachytarpheta gesnerioides Cham: composición química de la fracción hexánica y aceite esencial, actividades antioxidante y antimicrobiana

In the present study we investigated the chemical composition of hexane fraction and essential oil of Stachytarpheta gesnerioides (Verbenaceae) by GC-MS, total phenol and flavonoid contents. The antioxidant capacity and antimicrobial activity were investigated in five extracts of leaves of S. gesnerioides. Aqueous and 100 percent ethanol extracts were prepared by dynamic maceration. Hexane, ethyl acetate and methanol extracts were prepared by Soxhlet extraction. The essential oil (EO) and hexane fraction (HF) are mainly composed by guaiol. Moreover, the HF is also rich in the monoterpene alpha-pinene. The total phenol content ranged from 0.85 to 22.74 mg gallic acid equivalent /100mg dry extract at Folin–Ciocalteu’s reagent method. The total flavonoid concentration ranged from 0.68 to 13.65 mg rutin equivalent /100mg dry extract, detected using 8 percent aluminium chloride. The ethyl acetate extract (IC50=9.41 ug/ml) showed the highest antioxidant activity. The extracts were found to be effective to inhibit the microorganisms tested.

Se han investigado la composición química de la fracción hexánica (FH) y aceite esencial (AE) de Stachytarpheta gesnerioides (Verbenaceae) por GC-MS, el contenido de fenoles totales y flavonoides. La capacidad antioxidante y actividad antimicrobiana fueron investigadas en cinco extractos de hojas de S gesnerioides. Extractos acuosos y etanolico fueron preparados por la maceración dinámica y extracción continua en Soxhlet con hexano, acetato de etilo y metanol. Las fracciones AE y FH están compuestas principalmente por guaiol. La fracción FH es también rica en alfa-pineno. El contenido de fenoles totales varió desde 0,85 hasta 22,74 mg de ácido gálico/100 mg de extracto seco (Folin-Ciocalteu). La concentración total de flavonoides varió desde 0,68 hasta 13,65 mg en equivalentes de rutina/100 mg de extracto seco, que se detectó mediante reacción con cloruro de aluminio al 8 por ciento. El extracto de acetato de etilo (CI50=9,41 ug/ml) enseño la más grande actividad antioxidante. Los extractos se encontraron eficaces para inhibir los microorganismos ensayados.

Next-generation sequencing and genome evolution in allopolyploids.

PREMISE OF THE STUDY: Hybridization and polyploidization (allopolyploidy) are ubiquitous in the evolution of plants, but tracing the origins and subsequent evolution of the constituent genomes of allopolyploids has been challenging. Genome doubling greatly complicates genetic analyses, and this has long hindered investigation in that most allopolyploid species are "nonmodel" organisms. However, recent advances in sequencing and genomics technologies now provide unprecedented opportunities to analyze numerous genetic markers in multiple individuals in any organism. METHODS: Here we review the application of next-generation sequencing technologies to the study of three aspects of allopolyploid genome evolution: duplicated gene loss and expression in two recently formed Tragopogon allopolyploids, intergenomic interactions and chromosomal evolution in Tragopogon miscellus, and repetitive DNA evolution in Nicotiana allopolyploids. KEY RESULTS: For the first time, we can explore on a genomic scale the evolutionary processes that are ongoing in natural allopolyploids and not be restricted to well-studied crops and genetic models. CONCLUSIONS: These approaches can be easily and inexpensively applied to many other plant species-making any evolutionarily provocative system a new "model" system.

Cytogenetic characterization of Aloysia virgata Ruiz and Pavan (Verbenaceae)

Since previous cytogenetic reports of Aloysia have only described the meiotic behavior and chromosomal number of some species, the aim of this work was to provide detailed cytogenetic description of Aloysia virgata that would contribute to the understanding of the taxonomical organization of the Verbenaceae. Aloysia virgata had a karyotype with 2n = 36 metacentric chromosomes, all with similar size. The large amount of heterochromatin seen after Giemsa staining was confirmed by C-banding. Four nucleolar organizer regions (NORs) were detected with an rDNA 45S probe in two homologous pairs and two sites of 5S rDNA located on one chromosomal pair were detected by fluorescence in situ hybridization. The interphase nucleus was classified as semi-reticulate. Meiotic analysis showed a normal chromosomal behavior, with 18 bivalents in some parts of prophase I and in metaphase I. The number of chromosomes, NORs and 5S rDNA segments did not exclude a possible polyploid origin.

O gênero Aloysia reúne aproximadamente 30 espécies, porém sua circunscrição tem sido motivo de controvérsia. São poucos os estudos citogenéticos para o gênero, relatando apenas o número e o comportamento cromossômico. O presente trabalho teve como objetivo, identificar os caracteres citogenéticos de Aloysia virgata da flora brasileira que possam ser utilizados para um melhor entendimento e caracterização dos aspectos citogenéticos da família Verbenaceae, que possam contribuir para a organização taxonômica do grupo. Aloysia virgata apresentou cariótipo com 2n = 36 pequenos cromossomos, todos com o mesmo tamanho. Grande quantidade de heterocromatina foi observada com Giemsa e confirmada pela técnica de banda C. Foram detectadas quatro regiões organizadoras de nucléolo (NORs) e dois sítios de rDNA 5S pela técnica de hibridação in situ fluorescente (FISH). O núcleo interfásico foi classificado como semireticulado. Foi realizada a caracterização meiótica, na qual os cromossomos apresentaram comportamento normal, com a presença de 18 bivalentes na prófase I e na metáfase I. O número de cromossomos, de NORs e de segmentos de DNAr 5S não excluem uma possível origem poliplóide.

Mitodepressive and clastogenic effects of aqueous extracts of the lichens Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae) on meristematic cells in plant bioassays

The cytotoxicity effect of aqueous extracts of the lichens species Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae) were evaluated using meristematic cells of lettuce (Lactuca sativa) and maize (Zea mays). The seasonal effect was also evaluated. Extracts of M. lindmanii and C. texana inhibited root growth and/or percentage germination, possibly due to alterations in the cell cycle. The M. lindmanii extract showed anti-mitotic effects and blocked the cell cycle in metaphase so that c-mitosis and cells with chromosome duplication were produced. The C. texana extract appeared to hinder cell division, increasing the number of interphase cells. In addition, both extracts caused an increase in percentage of cell death. Clastogenic effects were also observed, such as sticky chromosomes, bridges, fragments and later segregation. Both lichen species are thus potential sources of biologically active substances with possible applications in biology, medicine and agronomy.

Cytogenetic characterization of Lippia alba and Lantana camara (Verbenaceae) from Brazil.

The aim of this work was to determine the cytogenetic characteristics of Brazilian Lippia alba (Mill) N. E. Brown and Lantana camara Plum. that could be useful for future characterization of these genera. Our analyses revealed that Li. alba has 2n=30 chromosomes consisting of ten metacentric and five submetacentric pairs, while La. camara has 44 metacentric chromosomes. The large blocks of heterochromatin seen in both species suggest an apomorphic condition. Six 45S rDNA sites were detected in both species by fluorescence in situ hybridization (FISH). Two and four 5S rDNA sites were observed in Li. alba and La. camara, respectively. Meiotic analysis revealed a normal chromosomal behaviour. The number of chromosomes and the presence of 45S rDNA and 5S rDNA sites do not exclude a possible polyploid origin. The cytogenetic differences between La. camara and Li. alba may be useful markers for differentiating these species.