Phytochemical Profiles and Cytotoxic Activity of Bursera fagaroides (Kunth) Engl. Leaves and Its Callus Culture.

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL-1 Naphthaleneacetic Acid (NAA) and 1 mgL-1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of µ = 0.059 days-1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g-1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7',8'-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL-1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL-1) being the most sensitive to the callus culture extract.

Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 µM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

Callus Type, Growth Regulators, and Phytagel on Indirect Somatic Embryogenesis of Coffee (Coffea arabica L. var. Colombia).

Coffee is a crop of global relevance. Indirect somatic embryogenesis has allowed plants of different coffee genotypes to be massively regenerated. The culture medium composition can affect the calli characteristics that are generated and their ability to form somatic embryos. This research aimed to determine the influence of the type of callus, growth regulators, and phytagel concentration on the embryogenic capacity of the Colombia variety. Leaf explants were cultured on Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5-1.0 mg L-1), benzylaminopurine (BAP, 1.0 mg L-1), and phytagel (2.3-5.0 g L-1). The explants generated two types of calli: friable (beige, soft, watery, easy disintegration, polyhedral parenchyma cells) and compact (white, hard, low water content, difficult disintegration, elongated parenchyma cells). About 68% of the total callus generated was compact; this type of callus produced a greater number of embryos (71.3) than the friable one (29.2). The number of differentiated embryos was significantly affected by the concentration of phytagel; higher concentrations (5.0 g L-1) resulted in larger quantities (73.7). The highest number of embryos (127.47) was obtained by combining 1.0 mg L-1 2,4-D, 1.0 mg L-1 BAP, 5.0 g L-1 phytagel, and compact callus.

In vitro micropropagation and tiliroside production in Paratecoma peroba (Record) Kuhlm, an endemic and endangered Brazilian tree.

In vitro tissue culture can be an alternative method for endangered species propagation, biodiversity conservation and secondary metabolite studies. Paratecoma peroba (Record) Kuhlm. (Bignoniaceae) is an endemic and endangered Brazilian species. This work aimed to establish in vitro morphogenesis and callus induction and to perform a phytochemical analysis of P. peroba callus extract. Higher seed germination (43%) was obtained in Wood Plant Medium culture without activated charcoal (AC). Combination of 5 µM benzyladenine + 10 µM gibberellic acid, without AC, resulted in a higher number of shoots (2 shoots/explant). A callus culture was stabilised from zygotic embryos using 2,4-dichlorophenoxyacetic acid. A callus methanolic extract was used for phytochemical analysis. The isolated substance was identified as tiliroside (kaempferol 3-O-ß-D-(6''-O-E-p-coumaroyl)-glucopyranoside) by NMR and quantified in callus and leaf extracts by HPLC. This study adds to the chemical knowledge of this species and it is the first report of a flavonol in Paratecoma.

Phenolic Compounds from Wild Plant and In Vitro Cultures of Ageratina pichichensis and Evaluation of Their Antioxidant Activity.

Ageratina pichichensis, is commonly used in traditional Mexican medicine. In vitro cultures were established from wild plant (WP) seeds, obtaining in vitro plant (IP), callus culture (CC), and cell suspension culture (CSC) with the objective to determine total phenol content (TPC) and flavonoids (TFC), as well as their antioxidant activity by DPPH, ABTS and TBARS assays, added to the compound's identification and quantification by HPLC, from methanol extracts obtained by sonication. CC showed significantly higher TPC and TFC than WP and IP, while CSC produced 2.0-2.7 times more TFC than WP, and IP produced only 14.16% TPC and 38.8% TFC compared with WP. There were identified compounds such as epicatechin (EPI), caffeic acid (CfA), and p-coumaric acid (pCA) in in vitro cultures that were not found in WP. The quantitative analysis shows gallic acid (GA) as the least abundant compound in samples, whereas CSC produced significantly more EPI and CfA than CC. Despite these results, in vitro cultures show lower antioxidant activity than WP, for DPPH and TBARS WP > CSC > CC > IP and ABTS WP > CSC = CC > IP. Overall, A. pichichensis WP and in vitro cultures produce phenolic compounds with antioxidant activity, especially CC and CSC, which are shown to be a biotechnological alternative for obtaining bioactive compounds.

Lesão na região metacarpiana do membro torácico esquerdo de bezerra Gir decorrente de contenção para pesagem / Injury in the metacarpinal region of the left thoracic limbs resulting from retention for weighing in a Gir calf

Fraturas ósseas e luxações são mais comuns em animais jovens e, na maioria das vezes, essas fraturas ocorrem devido ao manejo incorreto desses animais, podendo levar a uma queda de produção a curto ou longo prazo, gerando perdas econômicas e produtivas de animais de alto padrão genético. Nesse contexto, o objetivo deste trabalho foi relatar a ocorrência de uma fratura na região metacarpiana do membro torácico esquerdo de uma bezerra Gir de 12 dias de idade, causada por uma contenção ineficaz durante a pesagem. O animal proveniente de uma fazenda em Umirim/CE foi encaminhado ao Hospital Veterinário de Grandes Animais do Centro Universitário INTA em Sobral/CE. Ao passar pelo exame radiográfico, foi constatada a fratura na região metacarpiana do membro torácico esquerdo. O animal foi imobilizado de forma manual, seguindo-se métodos semiológicos para minimizar o estresse. Para a imobilização do membro, foi utilizada a muleta de Thomas modificada e bandagens, associadas ao controle da dor com as drogas Flunixin meglumine (1,1mg/kg) e Fenilbutazona (4,4mg/kg). Depois da imobilização, o animal seguiu internado no HOVET-GA e, após 18 dias, foi realizada uma nova radiografia para ver o progresso a partir do tratamento adotado. Constatou-se a formação de um calo ósseo, com a ossificação da fratura, não sendo necessário o encaminhamento cirúrgico do animal. Assim, o tratamento com muleta de Thomas modificada foi efetivo para a recuperação do membro fraturado, além de ser um tratamento de baixo custo e fácil aplicação, tendo o animal apresentado uma boa resposta ao tratamento terapêutico para controle da dor.

Bone fractures and dislocations are more common in young animals and mostly occur due to incorrect handling of these animals, which can lead to a short or long-term drop in production, generating economic and productive losses of animals of high genetic standard. This study aimed to report the occurrence of a fracture in the metacarpal region of the left thoracic limb of a 12-day-old Gir heifer, caused by an ineffective restraint during weighing. The animal from a farm in Umirim/CE was sent to the Veterinary Hospital of Large Animals of the University Center INTA in Sobral/CE. The radiographic examination found a fracture in the metacarpal region of the left thoracic limb. The animal was manually immobilized following semiological methods to minimize stress. For the limb immobilization, a modified Thomas crutch and bandages associated with pain control with the drugs Flunixin meglumine (1.1mg/kg) and Phenylbutazone (4.4mg/kg) were used. After immobilization, the animal remained hospitalized at HOVET-GA and after 18 days a new radiograph was performed to evaluate the progress achieved with the treatment adopted. It was found the formation of a bone callus with the ossification of the fracture, not requiring the surgical referral of the animal. Thus, the treatment with the modified Thomas crutch was effective for the recovery of the fractured limb, in addition to being a low-cost and easy-to-apply treatment. The animal showed a good response to the therapeutic treatment for pain control.

Regulatory roles of noncoding RNAs in callus induction and plant cell dedifferentiation.

KEY MESSAGE: Plant regulatory noncoding RNAs (ncRNAs) have emerged as key modulators of gene expression during callus induction. Their further study may promote the design of innovative plant tissue culture protocols. The use of plants by humans has recently taken on a new and expanding insight due to the advent of genetic engineering technologies. In this context, callus cultures have shown remarkable potential for synthesizing valuable biomolecules, crop improvement, plant micropropagation, and biodiversity preservation. A crucial stage in callus production is the conversion of somatic cells into totipotent cells; compelling evidence indicates that stress factors, transcriptional regulators, and plant hormones can trigger this biological event. Besides, posttranscriptional regulators of gene expression might be essential participants in callus induction. However, research related to the analysis of noncoding RNAs (ncRNAs) that modulate callogenesis and plant cell dedifferentiation in vitro is still at an early stage. During the last decade, some relevant studies have enlightened the fact that different classes of ncRNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs) are implicated in plant cell dedifferentiation through regulating the expression levels of diverse gene targets. Hence, understanding the molecular relevance of these ncRNAs in the aforesaid biological processes might represent a promising source of new biotechnological approaches for callus culture and plant improvement. In this current work, we review the experimental evidence regarding the prospective roles of ncRNAs in callus induction and plant cell dedifferentiation to promote this field of study.

Evolución de la osteosíntesis de los huesos largos: fijación por enclavado intramedular con clavos rígidos

Se presenta la evolución histórica y científica de la osteosíntesis de huesos largos realizada con clavos rígidos. Mediante una revisión bibliográfica retrospectiva en revistas de Traumatología nacionales y extranjeras, así como libros relevantes de esta especialidad, se pudieron establecer tres momentos claves en la osteosíntesis intramedular con clavos rígidos y caracterizar las diferentes generaciones que aportaron a este procedimiento en la historia de la Ortopedia y la Traumatología. Destacan las innovaciones tecnológicas incorporadas a la práctica quirúrgica y el desarrollo de biomateriales para mejorar la reparación de lesiones e incorporar al paciente a su vida cotidiana. El trabajo demuestra cómo ha avanzado la técnica quirúrgica de fijación intramedular y la consolidación ósea; y gracias a ello los problemas de las fracturas han quedado prácticamente solucionados.

The historical and scientific evolution of osteosynthesis of long bones performed with rigid nails is presented. Through a retrospective bibliographic review in national and foreign Traumatology journals, as well as relevant books of this specialty, it was possible to establish three key moments in intramedullary osteosynthesis with rigid nails and characterize the different generations that contributed to this procedure in the history of Orthopedics and Traumatology. The technological innovations incorporated into surgical practice and the development of biomaterials to improve the repair of injuries and incorporate the patient into their daily lives stand out. The work demonstrates how the surgical technique of intramedullary fixation and bone consolidation has advanced; and how thanks to this the problems of fractures have been practically solved.

Vibration therapy as an effective approach to improve bone healing in diabetic rats.

Objective: To investigate the effects of vibration therapy on fracture healing in diabetic and non-diabetic rats. Methods: 148 rats underwent fracture surgery and were assigned to four groups: (1) SHAM: weight-matched non-diabetic rats, (2) SHAM+VT: non-diabetic rats treated with vibration therapy (VT), (3) DM: diabetic rats, and (4) DM+VT: diabetic rats treated with VT. Thirty days after diabetes induction with streptozotocin, animals underwent bone fracture, followed by surgical stabilization. Three days after bone fracture, rats began VT. Bone healing was assessed on days 14 and 28 post-fracture by serum bone marker analysis, and femurs collected for dual-energy X-ray absorptiometry, micro-computed tomography, histology, and gene expression. Results: Our results are based on 88 animals. Diabetes led to a dramatic impairment of bone healing as demonstrated by a 17% reduction in bone mineral density and decreases in formation-related microstructural parameters compared to non-diabetic control rats (81% reduction in bone callus volume, 69% reduction in woven bone fraction, 39% reduction in trabecular thickness, and 45% in trabecular number). These changes were accompanied by a significant decrease in the expression of osteoblast-related genes (Runx2, Col1a1, Osx), as well as a 92% reduction in serum insulin-like growth factor I (IGF-1) levels. On the other hand, resorption-related parameters were increased in diabetic rats, including a 20% increase in the callus porosity, a 33% increase in trabecular separation, and a 318% increase in serum C terminal telopeptide of type 1 collagen levels. VT augmented osteogenic and chondrogenic cell proliferation at the fracture callus in diabetic rats; increased circulating IGF-1 by 668%, callus volume by 52%, callus bone mineral content by 90%, and callus area by 72%; and was associated with a 19% reduction in circulating receptor activator of nuclear factor kappa beta ligand (RANK-L). Conclusions: Diabetes had detrimental effects on bone healing. Vibration therapy was effective at counteracting the significant disruption in bone repair induced by diabetes, but did not improve fracture healing in non-diabetic control rats. The mechanical stimulus not only improved bone callus quality and quantity, but also partially restored the serum levels of IGF-1 and RANK-L, inducing bone formation and mineralization, thus creating conditions for adequate fracture repair in diabetic rats.

Improvement of cell suspension cultures of transformed and untransformed Carica papaya cell lines, towards the development of an antiparasitic product against the gastrointestinal nematode Haemonchus contortus.

Parasitic diseases have a major impact on human and animal health worldwide. Despite the availability of effective anti-parasitic drugs, their excessive and uncontrolled use has promoted the emergence of drug resistance, severely affecting ecosystems and human health. Thus, developing environmentally friendly antiparasitic treatments is urgently needed. Carica papaya has shown promising effects against infectious diseases. C. papaya embryogenic calluses were genetically modified by our research team to insert immunogenic peptides with the goal of developing an oral anti-cysticercosis vaccine. Among these callus cell lines, one labeled as CF-23, which expresses the KETc7 immunogenic peptide, induced the highest protection levels against experimental cysticercosis. In the process of designing a natural antiparasitic product based on C. papaya that simultaneously induced immunity against cysticercosis, both transformed (SF-23) and untransformed (SF-WT) suspension cultures were produced and optimized. Our results showed a better duplication time (td) for SF-23 (6.9 days) than SF-WT (13.02 days); thus, the SF-23 line was selected for scale-up in a 2-L airlift bioreactor, reaching a td of 4.4 days. This is the first time that a transgenic line of C. papaya has been grown in an airlift bioreactor, highlighting its potential for scale-up cultivation in this type of reactor. Considering the previously reported nematocidal activity of C. papaya tissues, their activity against the nematode Haemonchus contortus of aqueous extracts of SF-WT and SF-23 was explored in this study, with promising results. The information herein reported will allow us to continue the cultivation of the transgenic cell suspension line of C. papaya under reproducible conditions, to develop a new anti-parasitic product.

Somatic Embryogenesis in Banana (Musa spp.).

Bananas (Musa ssp.) are among the world's most important crops. In terms of gross value of production, they are the fourth most important global food crop and have an important socioeconomic and ecological role. Somatic embryogenesis (SE) is a developmental process, in which somatic cells differentiate into embryos which eventually develop and regenerate into plants. SE is exploited to generate a large quantity of very high economic value, genetically identical and disease-free plantlets. In bananas, the use of shoot apexes of axillary buds to induce SE resulted an alternative for plant regeneration through embryogenic cell suspension (ECS). The protocol has been scaled up to commercial laboratories for tissue culture (biofactories) for production of planting materials. The genetic stability of regenerated plants and high yields obtained under field conditions demonstrate the feasibility of scaling up this biotechnological protocol and adapting it to commercial production of planting materials to mitigate a critical bottleneck in the value chain of this important crop.

Citrus Protoplast Isolation and Plant Regeneration Through Somatic Embryogenesis.

Protoplasts are an attractive explant source for biotechnological tools widely used on citrus genetic improvement, such as somatic hybridization and direct genetic transformation. These delicate and responsive materials are subjected to cell proliferation induction and differentiation of somatic embryos which further regenerate into entire plants. The isolation of viable protoplasts followed by regeneration of plants through somatic embryogenesis is an important methodology for breeding applications. The methods presented here can also be used as a reference for protoplast work in other species, followed by protocol optimization for different species/genotypes.

Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia.

Somatic embryogenesis has been obtained in many citrus cultivars; however, the efficiency of the system is genotype dependent and culture synchronization is important to reach more efficient systems. In this chapter we present a detailed protocol of somatic embryogenesis induction from nucellar tissue and the use of an alternative method of callus sieving for culture synchronization and embryo production. This is a simple method which can also be evaluated for other species aiming at better culture efficiency and somatic embryo production.

Cellular responses of oil palm genotypes during somatic embryogenesis involve participation of procambial cells, DNA demethylation, and auxin accumulation.

KEY MESSAGE: Cell markers of somatic embryogenesis initiation from leaf tissues in oil palm involve the participation of procambial cells, DNA demethylation, and auxin accumulation. Low callogenesis and genotype-dependent response have been mentioned in the development of somatic embryogenesis protocols of Elaeis oleifera × E. guineensis elite hybrids, which requires more detailed investigations of the process. Thus, the initial cellular responses of immature leaves of adult genotypes of this hybrid were investigated for the first time, emphasizing histological, epigenetic, and endogenous auxin changes. Leaf segments from two genotypes, one responsive to somatic embryogenesis (B351733) and another non-responsive (B352933), were inoculated in Murashige and Skoog medium with 450 µM of 4-amino-3, 5, 6-trichloropicolinic acid. For anatomical analysis, samples of both genotypes were collected at 0, 20, 90, and 105 days of cultivation. Samples of both genotypes were also taken at different cultivation periods to analyze DNA methylation status (% 5-mC-5 methylcytosine) via ELISA test. Immunolocalization assays were performed with anti-indole-3-acetic acid and anti-5-methyl-deoxycytosine antibodies from samples of hybrid B351733. We distinguished two groups of cells reactive to the induction of embryogenic callogenesis, parenchymatous sheath cells, and procambial cells; however, only the latter are directly involved with the formation of calluses. The data obtained indicate that the formation of calluses in hybrid B351733 is related to DNA hypomethylation, while the non-responsiveness of leaf explants in hybrid B352932 is related to DNA hypermethylation. The in situ immunolocalization enabled the identification of initial markers of the callogenic process, such as IAA accumulation and hypomethylation. Identifying these events brings the possibility of establishing strategies for efficient manipulation of somatic embryogenesis protocols in palm trees.

Ilex paraguariensis: the effect of genotypes and growth phase on biomass, secondary metabolism and antioxidant activity of in vitro cultivated calli / Ilex paraguariensis: el efecto de los genotipos y la fase de crecimiento sobre la biomasa, metabolismo secundario y la actividad antioxidante de los callos cultivados in vitro

Yerba mate ( Ilex paraguariensis ) produces several secondary metabolites of interest to the phar maceutical industry, such as chlorogenic acids and methylxanthines. These compounds have been produced in vitro by callus culture from different species. However, for I. paraguariensis , no studies upon the production of these compounds in vitro have been p erformed to date. In this work, we show that the concentration of secondary metabolites from I. paraguariensis callus is possible and highly dependent on the callus growth phase. We observed that the best phase for the production of secondary compounds in calli of yerba mate is the stationary growth phase on both genotypes tested. In this phase, higher levels of phenolic compounds, chlorogenic acid and 3,5 - dicaffeoylquinic acid and greater antioxidant activity were observed. Chlorogenic acid and 3,5 - dicaffe oylquinic acid presented positive correlation with antioxidant activity. For the first time, secondary compounds were reported in yerba mate calli cultivated in vitro .

La yerba mate ( Ilex paraguariensis ) produce varios metabolitos secundarios de interés para la industria farmacéutica, como los ácidos clorogénicos y las metilxantinas. Estos compuestos se han producido in vitro mediante cultivo de ca llos de diferentes especies. Sin embargo, para I. paraguariensis , hasta la fecha no se han realizado estudios sobre la producción de estos compuestos in vitro . En este trabajo, mostramos que la concentración de metabolitos secundarios desde callos de I. pa raguariensis es posible y altamente dependiente de la fase de crecimiento del callo. Observamos que la mejor fase para la producción de compuestos secundarios en callos de yerba mate es la fase de crecimiento estacionario en ambos genotipos probados. En es ta fase se observaron niveles más altos de compuestos fenólicos, ácido clorogénico y ácido 3,5 - dicafeoilquínico y una mayor actividad antioxidante. El ácido clorogénico y el ácido 3,5 - dicafeoilquínico presentaron correlación positiva con la actividad antio xidante. Por primera vez, se reportaron compuestos secundarios en callos de yerba mate cultivados in vitro .

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7 days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Effect of UV-B Radiation on Flavonoids and Phenols Accumulation in Tempisque (Sideroxylon capiri Pittier) Callus.

Tempisque (Sideroxylon capiri Pittier) is classified as a threatened species and has been reported with a high content of phenols and flavonoids in the leaves. The use of abiotic elicitors such as radiation has been reported due to the changes it produces in the metabolism of plants by activating their defense mechanisms and increasing the biosynthesis of bioactive compounds with antioxidant capacity such as phenols and flavonoids. Therefore, the aim of this work was to evaluate the effect of UV-B radiation on growth parameters and the synthesis of bioactive compounds in in vitro culture of tempisque callus. For the callus induction, we used thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.5 and 1 mg/L. Calluses were exposed to UV-B radiation (0, 1, 2, 3 and 4 h/day) for two and four weeks. The highest callus formation index was obtained with TDZ and 2,4-D at 1 mg/mL. The greatest increase in the concentration of phenols and flavonoids was detected in the fourth week with 4 h of exposure per day. The highest concentrations of quercetin (230 µg/g dry weight), kaempferol (235 µg/g dry weight) and gallic acid (240 µg/g dry weight) were found in callus obtained from leaves explants.

Air layering in Caryocar brasiliense - effect of stem diameter / Alporquia em pequizeiro - efeito do diâmetro do caule

Seed propagation of pequizeiro (Caryocar brasiliense) has many disadvantages, including low germination rates and high genetic variability of the resulting plants. Therefore, vegetative propagation techniques, such as the air layering process, are potentially important for this species. This study evaluated the effects of stem diameter and parental plant on the air layering efficacy of pequizeiro. The air layering process was carried out in mid-December 2019, in the following stem diameters: less than 15 mm; 15-19 mm; 20-24 mm; 25-29 mm and above 29 mm. The treatments comprised four replicates of 10 air layers. After 3 months, the percentages of survival, callus formation and rooting were evaluated, in addition to the rooting vigor. The percentages of survival and callus formation observed in the air layering, regardless the parental plant, were higher than 85%, showing the high potential of this technique in the propagation of pequizeiro. The callus differentiation rate in roots varied between the parental plants. Considering the evaluated parameters, stem diameters ranging from 20 to 24 mm are the most suitable ones for the air layering process in pequizeiro. These results indicated the high potential of the air layering technique for the commercial propagation of pequizeiro.

A propagação seminal de pequizeiro acarreta grande variabilidade na taxa de germinação e no genótipo das mudas produzidas, mostrando a necessidade do desenvolvimento de técnicas para a propagação vegetativa dessa espécie, como a alporquia. Os objetivos deste estudo foram avaliar a eficácia da alporquia, bem como o efeito do diâmetro do caule e da planta-mãe na sobrevivência, no calejamento e no enraizamento de alporques de pequizeiro. O processo de alporquia foi realizado em meados de dezembro de 2019, nos seguintes diâmetros de caule: inferior a 15 mm; 15-19 mm; 20-24 mm; 25-29 mm e superior a 29 mm. Os tratamentos foram testados em quatro repetições de 10 alporques. Após três meses, avaliaram-se as porcentagens de sobrevivência, de calejamento e de enraizamento, além do vigor de enraizamento. As porcentagens de sobrevivência e de calejamento nos alporques, independentemente da planta matriz, foram superiores a 85%, mostrando o grande potencial dessa técnica na reprodução de pequizeiro. A taxa de diferenciação de calos em raízes variou entre as matrizes. Considerando os parâmetros avaliados, diâmetros de caule de 20 a 24 mm são os mais adequados para a alporquia em pequizeiro. Esses resultados indicam o potencial da técnica de alporquia para a produção comercial de mudas de pequizeiro.

Optimized somatic embryogenesis and plant regeneration in elite Argentinian sugarcane (Saccharum spp.) cultivars.

BACKGROUND: Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. RESULTS: Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. CONCLUSIONS: This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.

Arnica montana Cell Culture Establishment, and Assessment of Its Cytotoxic, Antibacterial, α-Amylase Inhibitor, and Antioxidant In Vitro Bioactivities.

Arnica montana cell suspension culture could be a sustainable source of a vegetal material producer of secondary metabolites (SMs) possessing biological effects. Different plant growth regulator concentrations (0-5 mg/L) were tested in foliar explants to induce a callus that was used to establish a cell suspension culture. Growth kinetics was carried out for 30 days. A methanolic extract obtained from biomass harvested at 30 days of growth kinetics was fractionated, and three fractions were tested for bioactivities. We induced a callus with 1 mg/L of picloram and 0.5 mg/L of kinetin in foliar explants, which allowed for the establishment of a cell suspension culture, and the latter had the highest total SMs contents at day 30. Three fractions showed differences in total SMs contents, with the highest values per gram as follows: 270 mg gallic acid equivalent for total phenolic content, 200 mg quercetin equivalent for total flavonoid content, 83 mg verbascoside equivalent for total phenolic acid content, and 396 mg parthenolide equivalent for total sesquiterpene lactone content. The best bioactivities were 2-6 µg/mL for the 50% inhibition of 2,2-diphenyl-1-picrylhydrazyl radical, 30% cellular viability of lymphoma cells at 40 µg/mL, 17% inhibition against Escherichia coli and Staphylococcus aureus at 8 µg/disk, and α-amylase inhibition at 12% with 10 µg/mL. The total SMs contents were correlated with bioactivities.