Micropropagation of the Ornamental Aquatic Plant, Aponogeton ulvaceus, from Immature Tuber Explants.

The aquatic monocot, Aponogeton ulvaceus Baker, is endemic to Madagascar and is a commercially valuable ornamental aquarium plant. Members of the genus Aponogeton contain a spectrum of phytochemicals associated with a broad range of biological activities. However, much remains to be known about this genus, and the A. ulvaceus population is declining due to anthropogenic activities and climate change. To address these challenges, adopting plant tissue culture technology will be a viable solution for the sustainable production of pest- and pathogen-free plants to meet the demands of the ornamental aquatic plant trade, for conservation and research purposes. A simple micropropagation protocol for A. ulvaceus is described here, starting with seeds to establish sterile stock plants, from which immature tubers were acquired as explants for indirect organogenesis.

In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

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.

Phytochemical Profiling and Assessment of Anticancer Activity of Leptocarpha rivularis Extracts Obtained from In Vitro Cultures.

Plant cell culture is a source of plant material from which bioactive metabolites can be extracted. In this work, the in vitro propagation of Leptocarpha rivularis, an endemic Chilean shrub with anticancer activity, is described. Different media were tested and optimized for the introduction, propagation, and rooting steps of the micropropagation process. At the end of this process, 83% of plants were successfully acclimatized under greenhouse conditions. Callus induction from the internodal stem segment was performed using various combinations of phytohormones. Green-colored, friable, and non-organogenic callus was generated with a callus induction index higher than 90%. The chemical composition of extracts and callus, obtained from clonal plants, was assessed and the results indicate that the phytochemical profiles of extracts from micropropagated plants are like those found for plants collected from natural habitats, leptocarpine (LTC) being the major component. However, no LTC was detected in callus extract. HeLa and CoN cells, treated with LTC or extract of micropropagated plants, exhibit important diminution on cell viability and a drastic decrease in gene expression of IL-6 and mmp2, genes associated with carcinogenic activity. These effects are more important in cancer cells than in normal cells. Thus, micropropagated L. rivularis could be developed as a potential source of efficient antiproliferative agents.

Application of In Vitro Plant Tissue Culture Techniques to Halophyte Species: A Review.

Halophytes are plants able to thrive in environments characterized by severe abiotic conditions, including high salinity and high light intensity, drought/flooding, and temperature fluctuations. Several species have ethnomedicinal uses, and some are currently explored as sources of food and cosmetic ingredients. Halophytes are considered important alternative cash crops to be used in sustainable saline production systems, due to their ability to grow in saline conditions where conventional glycophyte crops cannot, such as salt-affected soils and saline irrigation water. In vitro plant tissue culture (PTC) techniques have greatly contributed to industry and agriculture in the last century by exploiting the economic potential of several commercial crop plants. The application of PTC to selected halophyte species can thus contribute for developing innovative production systems and obtaining halophyte-based bioactive products. This work aimed to put together and review for the first time the most relevant information on the application of PTC to halophytes. Several protocols were established for the micropropagation of different species. Various explant types have been used as starting materials (e.g., basal shoots and nodes, cotyledons, epicotyls, inflorescence, internodal segments, leaves, roots, rhizomes, stems, shoot tips, or zygotic embryos), involving different micropropagation techniques (e.g., node culture, direct or indirect shoot neoformation, caulogenesis, somatic embryogenesis, rooting, acclimatization, germplasm conservation and cryopreservation, and callogenesis and cell suspension cultures). In vitro systems were also used to study physiological, biochemical, and molecular processes in halophytes, such as functional and salt-tolerance studies. Thus, the application of PTC to halophytes may be used to improve their controlled multiplication and the selection of desired traits for the in vitro production of plants enriched in nutritional and functional components, as well as for the study of their resistance to salt stress.

Picloram-induced enhanced callus-mediated regeneration, acclimatization, and genetic clonality assessment of gerbera.

BACKGROUND: Gerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. RESULTS: The fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5-1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. CONCLUSIONS: The results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.

Transcriptomic Changes in Internode Explants of Stinging Nettle during Callogenesis.

Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 µM) and naphthaleneacetic acid (0.5 µM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

Production of Doubled Haploid Plants in Cucumber (Cucumis sativus L.) Through Anther Culture.

As in any other economically important crop, the possibility of producing fully homozygous, doubled haploid lines in cucumber allows for faster and cheaper breeding. At present, the fastest way to doubled haploidy is the production of cucumber haploid plants and duplication of their chromosomes to make them doubled haploid. In this chapter, we describe a complete protocol to successfully produce cucumber doubled haploid plants, including the evaluation of their ploidy level by flow cytometry. Briefly, this protocol involves a first step of anther culture to induce microspores to divide and proliferate forming calli. The calli produced are isolated from anthers and transferred first to a liquid medium and then to a solid medium to induce organogenesis. Organogenic shoots will eventually give rise to entire DH plants.

Anther Culture in Cucurbita Species.

Production of homozygous pure parental lines is the first stage of hybrid vegetable breeding. Unfortunately, producing pure lines takes a long time by classical breeding methods, especially in open-pollinated vegetable species, and this period can be up to 8-10 years. Recently, doubled haploid (DH) technology, as a set of biotechnological methods, has emerged as an alternative to classical breeding methods and allows for the generation of 100% homozygous pure double haploid lines in 1 or 2 years. Although haploid plants were successfully produced via irradiated pollen technique and gynogenesis in some Cucurbita species, haploid plants have not been obtained from some lines due to genotype dependency, and haploidy frequency is still not sufficient for use in a breeding program. Thus, anther culture technique has emerged as an alternative technique in the DH process. The main objective of this chapter is to provide explanatory information on anther culture technique applied in the Cucurbita genus. For this purpose , key points and details of methods and protocols of the anther culture technique are described in summer squash (Cucurbita pepo L.), pumpkin (Cucurbita moschata Duch.), and winter squash (Cucurbita maxima Duch.).

Gynogenesis in Cucurbita Species.

The development of F1 hybrid vegetable varieties emerges as a result of a great effort, long time, investment, knowledge, and advanced technology. The first stage of hybrid vegetable breeding is obtaining pure lines. It is possible to obtain homozygous parent lines used in the production of hybrid varieties with traditional breeding methods. This period takes 8-10 years, especially in some vegetables which are highly open-pollinated, such as Cucurbita spp. Androgenetic- and/or gynogenetic-based dihaploidization methods provide 100% homozygous pure haploid lines in 1-2 years and save time and effort.The DH frequency by irradiated pollen technique and anther culture strongly depends on the genotypic response, whereby their practical use in a breeding program is still limited. As a possible alternative technique, gynogenesis (unfertilized ovule/ovarium cultures) switches on to produce haploid plants in some Cucurbita species. In the Cucurbita genus, gynogenesis has been one of the most studied and popular DH techniques and presented remarkable results in recent years.

Use of polylactic acid microvessel to obtain microplantlets of Eucalyptus microcorys through indirect organogenesis.

Microplants of Eucalyptus microcorys were produced through indirect organogenesis, and the interaction of plant growth regulators (PGRs) (TDZ-thidiazuron and NAA-α-naphthalene acetic acid), juvenile tissues (cotyledon and hypocotyl) and different types of polylactic acid (PLA) microvessels on plant production were evaluated. Cotyledon-derived callus induction increased by 30-60% in all tested combinations of TDZ and NAA concentrations compared the absence of PGRs. Hypocotyl-derived callus induction was improved in most tested combinations of TDZ and NAA concentrations. Moreover, 100% callus induction from both tissues was achieved with TDZ (1, 2 and 3 mg L-1) + NAA (0 mg L-1). Bud induction from cotyledon tissues was improved with TDZ (1 and 3 mg L-1) + NAA (0 mg L-1) and from hypocotyl with TDZ (1 and 2 mg L-1) + NAA (0 mg L-1). Shoot elongation from cotyledon tissues was not improved from any combination of PGRs, whereas TDZ (1 mg L-1) + NAA (0 mg L-1), TDZ (1 mg L-1) + NAA (4 mg L-1), TDZ (2 mg L-1) + NAA (4 mg L-1) and TDZ (3 mg L-1) + NAA (2 mg L-1) improved shoot elongation from hypocotyl tissues. Adventitious rooting and acclimatization of microcuttings ranged from 40 to 70% in three of the tested microvessels. The acclimatized microcuttings had low genetic variability. Successful production of E. microcorys microplants was achieved in this study using hypocotyl tissue and cultivated a culture medium supplemented with TDZ and NAA, using PLA-based microvessels.

WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn.

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous ArabidopsisWUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

Identification of lupeol produced by Vernonanthura patens (Kunth) H. Rob. leaf callus culture.

The lupeol detection in callus of Vernonanthura patens (Kunth) H. Rob. leaves is discussed. Leaf segments previously treated with sodium hypochlorite, ethanol, and distilled water were placed in MS basal medium (Murashige and Skoog) for 7 days. Next, callus induction were done in two complemented MS medium for 6 weeks. Then, callus propagation were performed in MS medium supplemented with 1.0 mg/L of benzylaminopurine (BAP) and 0.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) for 50 days. Fresh callus were extracted every 10 days in an ultrasonic bath using ethyl acetate (1.0 g/10 mL). The identification was carried out by Gas Chromatography-Mass Spectrometry (GC-MS) using selected ion monitoring (SIM) acquisition mode with characteristic ions of lupeol. The results obtained indicate the occurrence of lupeol in callus extract after twenty days of proliferation. These findings could be use in subsequent scale-up studies for biomass production containing this active compound in order to replace conventional methods.

The Effect of Temperature and Growth Regulators on Callus Induction in Watermelon Anthers

HIGHLIGHTS Callogenesis was induced from watermelon anthers The auxin 2,4-D at 2.0 and 5.0 μM concentrations induced callus formation. Anthers' responses to the pre-treatment at 4 °C varied according to the watermelon genotype.

Abstract Callus induction is one of the pathways required for haploid plant regeneration through anther culture. Pollen viability, as well as the effect of growth regulators and cold pretreatment on anthers of two watermelon lines (Smile and Sugar Baby) to induce callus formation were herein evaluated. Pollen viability was estimated through the staining technique using 2% acetic carmine. Male flower buds were collected and disinfested to allow removal anthers. These anthers were placed on Murashige and Skoog medium, which was supplemented with 2,4-dichlorophenoxyacetic (2,4-D) at 0.0, 0.5, 1.0, 2.0 or 5.0 μM or with 6-benzylaminopurine at 0.0, 0.5, 1.0, 1.5, or 2.0 μM, in combination with 2.0 μM of 2,4-dichlorophenoxyacetic. Anthers were pretreated at 4 °C, for two days and then placed in vitro. Both watermelon lines provided high pollen viability rates (from 93 to 98%). The 2.0 and 5.0 μM concentrations of 2,4-D stimulated higher friable callus formation. The optimal concentration of 2,4-D was estimated at 3.78 μM and 4.17 μM, which had callus induction rates of 64% and 52%, respectively. The combination of 2.0 μM of 2,4-D and 6-benzylaminopurine did not lead to increased anther response to callus induction. The pre-treatment applied to flower buds at 4 °C enabled callus induction and the anther response to callus induction was genotype-dependent.

Selection of somaclonal variants of maracuyá (passiflora Edulis var Flavicarpa. Deneger) tolerants to water deficit / Selección de variantes somaclonales de maracuyá (Passiflora edulis var flavicarpa. Deneger) tolerantes a déficit hídrico

ABSTRACT Climate change will have an impact on the Colombian agricultural sector, by 2050 increases in temperature and distribution of erratic rainfall are expected. Passion fruit cultivation does not tolerate water deficit, it reduces flower induction, generates fruit drop and defoliation. To tackle this problem, somaclonal variants (VS) of passion fruit were selected in-vitro, seeking tolerance to water deficit. Four phases were developed: I) callogenesis, II) direct and indirect organogenesis, II) Induction and evaluation of the water deficit with Polyethylene glycol 6000 (PEG 6000) and IV) in vitro selection of VS by morphometric measurements, chlorophyll and total sugars contents. Differences in callogenesis were found with different concentrations of 2,4-D, the concentration of 2 mg L-1 presented better results producing calluses in less time and in greater quantity (8 days, 90% of the leaf area). In indirect and direct organogenesis the medium MS + ANA + BAP (0.3: 0.6), showed significant statistical differences with respect to other means, for the variables root length (15.14 cm), stem (16.72 cm) and leaves ( 14.51 cm) and root thickness (0.76 cm) stem (1.25) and leaf width (6.75). The influence of PEG 6000 showed significant differences, the treatment with 30 g L-1 showed the smallest leaf width, the greatest width was found in 25 g L1. Statistical differences were found in chlorophyll levels and total sugar contents, the highest contents were recorded in the VS 25VS1, showing the possibility of obtaining seedlings tolerant to the water deficit of passion fruit by inducing somaclonal variation.

RESUMEN El cambio climático tendrá impactos en el sector agropecuario colombiano, para el 2050 se prevén aumentos de temperatura y distribución de lluvias erráticas. El cultivo de maracuyá no tolera el déficit hídrico, este disminuye la inducción floral, genera caída de frutos y defoliación. Para abordar esta problemática se seleccionaron in-vitro variantes somaclonales (VS) de maracuyá, buscando tolerancia al déficit hídrico. Se desarrollaron cuatro fases: I) callogénesis, II) organogénesis directa e indirecta, III) Inducción y evaluación del déficit hídrico con Polientilenglicol 6000 (PEG 6000) y IV) selección in vitro de VS por mediciones morfométricas, contenidos de clorofila y azúcares totales. Se hallaron diferencias en callogénesis con diferentes concentraciones de 2,4-D, la concentración de 2 mg-L-1 presentó mejores resultados produciendo callos en menor tiempo y en mayor cantidad (8 días, 90% del área foliar); en organogénesis indirecta y directa el medio MS + ANA + BAP (0.3:0.6), mostró diferencias estadísticas significativas respecto a otros medios, para las variables longitud de raíz (15.14 cm), tallo (16.72 cm) y hojas (14.51 cm) y grosor de raíz (0.76 cm) tallo (1.25) y ancho de hojas (6.75). La influencia de PEG 6000 mostró diferencias significativas, el tratamiento con 30 g-L-1 mostró menor ancho de hojas, el mayor ancho se encontró en 25 g-L-1. Se hallaron diferencias estadísticas en niveles de clorofila y contenidos de azúcares totales, los mayores contenidos se registraron en el VS 25VS1, mostrando la posibilidad de obtener plántulas tolerantes al déficit hídrico de maracuyá mediante la inducción de variación somaclonal.

A robust workflow for indirect somatic embryogenesis and cormlet production in saffron (Crocus sativus L.) and its wild allies; C. caspius and C. speciosus.

Saffron (Crocus sativus L.) and its wild relatives, Crocus caspius and Crocus speciosus are of considerable significance in the pharmaceutical, nutraceutical, and ornamental bulbs industry. Towards the ultimate goal of the conservation of wild Crocus species and establishment of an efficient workflow for in vitro production of Crocuses, efficient protocols were developed for disinfection and in vitro production of cormlets in C. sativus and its wild allies C. caspius and C. speciosus. Moreover, the differential expression of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene was evaluated as a potential molecular marker during embryogenesis between embryogenic and non-embryogenic calli. A highly efficient disinfection recipe and a low-cost TDZ-free protocol have been successfully developed for in vitro cormlet production in three Crocus species. MS medium containing 10.18 µM 2, 4-D + 4.44 µM BAP was most efficiently induced callus and somatic embryo formation. The highest conversion frequency and maximum cormlet weight were achieved in MS containing 5.37 µM NAA + 8.88 µM BAP. The SERK expression was significantly much higher in embryogenic calli than non-embryogenic in all Crocus species. The current low-cost and easy-to-use recipe suggests a promising in vitro propagation workflow for mass production of uniform pathogen-free cormlets of Crocus species, as well as a platform to better conservation of wild Crocus species and effective gene and genome editing using CRISPR-Cas9 in future studies.

Enhanced production of podophyllotoxin, kaempferol, and quercetin from callus culture of Dysosma pleiantha (Hance) Woodson: An endangered medicinal plant.

Dysosma pleiantha (Hance) Woodson is one of the endangered traditional Chinese medicinal herbs, highly valued for its medicinal properties by Taiwan's mountain tribes. The present study aims to develop an efficient protocol for callus biomass by optimizing suitable culture medium, carbon source culture condition, and enhanced production of pharmaceutically important podophyllotoxin, kaempferol, and quercetin from callus culture of D. pleiantha under the influence of different additives. Best callus induction was achieved in Gamborg's medium (B5) with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) along with 0.2 mg/L kinetin under dark condition. Tender leaves of D. pleiantha showed the maximum of 86% callus induction among the different explants tested. Highest leaf callus proliferation was noted in B5 medium with 1 mg/L 2,4-D incubated under complete darkness. In addition, it was found that B5 medium with 1 mg/L 2,4-D along with 2 g/L peptone produced more leaf callus biomass and enhanced production of podophyllotoxin (16.3-fold), kaempferol (12.39-fold), and quercetin (5.03-fold) compared to control. Therefore, D. pleiantha callogenesis can provide an alternative source for enhanced production of secondary compounds regardless of the exploitation of its natural plant population.

Induction, biochemical trait and phytochemical screening of calluses of Amburana cearensis (Allemão) A. C. Smith

Amburana cearensisis an arboreal legume of the Fabaceaefamily,with high phytotherapic and medicinal potential due the presence of secondary metabolites. The objective of this study was to evaluate the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-2,5,6-trichloro-2-pyridinecarboxylic acid (picloram) on the in vitroinduction of callogenesis of A. cearensisand analyze the biochemical and phytochemical potential of these calluses. For callus induction, leaf and cotyledon segments were used as explants, which were inoculated in woody plant medium (WPM) supplemented with different concentrations of 2,4-D (0, 5, 10, 20, 40 μM) or picloram (0, 5, 10, 20, 40, 80 μM). The callus growth curve was estimated based on fresh weight, measured at 7-day intervals until 28 days after inoculation. The calluses were analyzed by biochemical tests to quantify the reducing sugars and total proteins. Phytochemical screening and high-performance liquid chromatography were performed to establish the phytochemical profile of extracts from calluses. The concentrations of 21.94 μMand 26.46 μMof 2,4-Dinduced the greatest formation of compact and friable calluses from the leaf and cotyledon segments, respectively. The growth curve had two distinct phases(lag and exponential) for both types of calluses evaluated. The maximum levels of reducing sugars and total proteins in the calluses from leaf and cotyledon segments were obtained on the day of inoculation and after 28 days of cultivation, respectively. The results of the phytochemical analysis identified the presence of coumarin in all the extracts evaluated, this secondary metabolite has high pharmacological potential.

Uprising the antioxidant power of Argania spinosa L. callus through abiotic elicitation.

This study was carried out in order to investigate the ability of tissues of Argania spinosa (L.) to undergo unlimited cell divisions by triggering their proliferative potential via callogenesis. Axenic cultures were efficiently established using axillary buds cultured on half-strength Murashige and Skoog (MS) medium after 20min of surface sterilization with sodium hypochlorite 6% (v/v). The highest callus rate was achieved with 1.0mgL-1 of naphthaleneacetic acid (NAA) and 1.0mgL-1 of 2,4-dichlorophenoxyacetic acid (2,4D) or similarly with 0.01mgL-1 of 6-benzylaminopurine (BAP) and 1.0mgL-1 of 2,4D at pH of 5.8, under dark conditions. The results of this study show also a significant increase in the callus's antioxidant power under abiotic pressure induced by NaCl. Catalase (CAT), peroxidase (PO), and superoxide dismutase (SOD) activities were significantly triggered, which protected the cells from the stimulated oxidative stress, under hydrogen peroxide (H2O2) significant release. This reaction favors subsequently the tissue recover process linked to the low abundance of polyphenol oxidase (PPO) activity and malondialdehyde (MDA) content. This work proves the efficiency of salt stress in boosting the argan cell's antioxidant status, which could be commercially applied in the field of cells regenerative therapy.