Tissue-Specific Natural Synthesis of Galanthaminein Zephyranthes Species and Its Accumulation in Different In Vitro-Grown Organs Following Methyl Jasmonate Treatment.

Galanthamine is an immensely valuable alkaloid exhibiting anti-cancer and antiviral activity. The cultivation of plant tissues in in vitro conditions is a good source for the synthesis and enrichment of secondary metabolites of commercial interest. In this study, the Amaryllidaceae alkaloid galanthamine was quantified in three Zephyranthes species, such as Zephyranthes candida, Zephyranthes grandiflora, and Zephyranthes citrina, and the impact of the methyl jasmonate (MJ) signaling molecule on galanthamine accumulation was monitored in in vitro-derived plant tissues. This is the first ever study of the MJ-regulated accumulation of galanthamine in in vitro-grown Zephyranthes tissues. Shoot regeneration was obtained in all three Zephyranthes species on Murashige and Skoog (MS) medium containing 2.0 mgL-1 benzylaminopurine (BAP) + 0.5 mgL-1 naphthalene acetic acid (NAA). The regenerated shoots were rooted on a medium containing 2.0 mgL-1 indole butyric acid (IBA). A GC-MS study of Zephyranthes extracts revealed the presence of 34 phyto-compounds of varied levels with therapeutic activities against diseases. The galanthamine content was quantified in plant parts of the three Zephyranthes species using high-performance thin layer chromatography (HPTLC); the maximum was found in Z. candida bulb (2.41 µg g-1 dry wt.), followed by Z. grandiflora (2.13 µg g-1 dry wt.), and then Z. citrina (2.02 µg g-1 dry wt.). The galanthamine content showed bulb > leaf > root source order. The in vitro-generated plantlets were treated with different MJ concentrations, and the galanthamine yield was measured in bulb, leaf, and root tissues. The highest galanthamine content was recorded in bulbs of Z. candida (3.97 µg g-1 dry wt.) treated with 150 µM MJ, showing an increase of 64.73% compared to the control. This accumulation may be attributed to MJ-induced stress, highlighting the potential commercial synthesis of galanthamine in vitro.

Phytocompounds and Regulation of Flavonoids in In Vitro-Grown Safflower Plant Tissue by Abiotic Elicitor CdCl2.

In this study, a Gas chromatography-mass spectrometry (GC-MS) investigation of embryogenic callus and somatic embryo regenerated shoots of Carthamus tinctorius revealed the presence of a variety of sugars, sugar acids, sugar alcohols, fatty acids, organic acids, and amino acids of broad therapeutic value. The in vitro developed inflorescence contained a wide range of active compounds. In embryogenic calluses, important flavonoids like naringenin, myricetin, kaempferol, epicatechin gallate, rutin, pelargonidin, peonidin, and delphinidin were identified. To augment the synthesis of active compounds, the effect of cadmium chloride (CdCl2) elicitation was tested for various treatments (T1-T4) along with a control (T0). Varying concentrations of CdCl2 [0.05 mM (T1), 0.10 mM (T2), 0.15 mM (T3), and 0.20 mM (T4)] were added to the MS medium, and flavonoid accumulation was quantified through ultra-high-pressure liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS). The flavonoids naringenin, kaempferol, epicatechin gallate, pelargonidin, cyanidin, and delphinidin increased by 6.7-, 1.9-, 3.3-, 2.1-, 1.9-, and 4.4-fold, respectively, at T3, whereas quercetin, myricetin, rutin, and peonidin showed a linear increase with the increase in CdCl2 levels. The impacts of stress markers, i.e., ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), on defense responses in triggering synthesis were also evaluated. The maximum APX and SOD activity was observed at T3, while CAT activity was at its maximum at T2. The impact of elicitor on biochemical attributes like protein, proline, sugar, and malondialdehyde (MDA) content was investigated. The maximum protein, proline, and sugar accumulation was noted at high elicitor dose T4, while the maximum MDA content was noted at T3. These elevated levels of biochemical parameters indicated stress in culture, and the amendment of CdCl2 in media thus could be a realistic approach for enhancing secondary metabolite synthesis in safflower.

The Foliar Anatomy and Micromorphology of Cyphostemma hypoleucum (Vitaceae).

Cyphostemma hypoleucum (Harv.) Desc. ex Wild & R.B. Drumm is a perennial climber, indigenous to Southern Africa, and belongs to the Vitaceae. Although there have been many studies of Vitaceae micromorphology, only a few taxa have been described in detail. This study aimed to characterize the micro-morphology of the leaf indumentum and determining its possible functions. Stereo microscope, scanning electron microscope (SEM), and transmission electron microscope (TEM) were used to produce images. Micrographs of stereomicroscopy and SEM showed the presence of non-glandular trichomes. In addition, pearl glands were observed on the abaxial surface using a stereo microscope and SEM. These were characterized by a short stalk and a spherical- shaped head. The density of trichomes decreased on both surfaces of leaves as the leaf expanded. Idioblasts that contained raphide crystals were also detected in tissues. The results obtained from various microscopy techniques confirmed that non-glandular trichomes serve as the main external appendages of the leaves. Additionally, their functions may include serving as a mechanical barrier against environmental factors such as low humidity, intense light, elevated temperatures, as well as herbivory and insect oviposition. Our results may also be added to the existing body of knowledge with regard to microscopic research and taxonomic applications.

Development and Biomechanics of Grewia lasiocarpa E. Mey. Ex Harv. Trichomes Exudate.

Grewia lasiocarpa E. Mey. Ex Harv., Malvaceae (forest raisin) is a tropical small tree or shrub valued for its ecological importance as well as its nutritional, antioxidant, antibacterial, and anti-cancer properties as well as its ecological and ornamental importance. Glandular and non-glandular trichomes are present on the fruits, stem bark and leaves of G. lasiocarpa and these trichomes are the first line of defense. They are important structures that plants use to combat biotic and abiotic stress. The development of G. lasiocarpa trichomes and the biomechanics of the exudates present in the glandular (capitate) trichome were investigated for the first time using advanced microscopy techniques [Scanning electron microscope (SEM) and Transmission electron microscope (TEM)]. The pressurized cuticular striations may play a role in the exudates' biomechanics, i.e., releasing secondary metabolites present in the capitate trichome, which was observed to be multidirectional. The presence of many glandular trichomes on a plant implies an increase in the amount of phytometabolites. A common precursor for the development of trichomes (non-glandular and glandular) was observed to be DNA synthesis associated with a periclinal cell division, thus the final fate of the cell is determined by cell cycle regulation, polarity, and expansion. The glandular trichomes of G. lasiocarpa are multicellular and polyglandular, while the non-glandular (glandless) trichomes are either single-celled or multicellular. Since, trichomes 'house' phytocompounds of medicinal, nutritional, and agronomical benefits; the molecular and genetic study of the glandular trichomes of Grewia lasiocarpa will be beneficial to humanity.

Response of Rowan Berry (Sorbus redliana) Shoot Culture to Slow Growth Storage Conditions.

Slow growth storage can preserve the genetic resources of endangered species such as those of genus Sorbus. Our aim was to study the storability of rowan berry in vitro cultures, their morpho-physiological changes, and regeneration ability after different storage conditions (4 ± 0.5 °C, dark; and 22 ± 2 °C, 16/8 h light/dark). The cold storage lasted for 52 weeks, and observations were made every four weeks. Cultures showed 100% survival under cold storage, and those taken from the storage showed 100% regeneration capacity after the passages. A dormancy period lasting about 20 weeks was observed, followed by intensive shoot growth until the 48th week, which led to the exhaustion of the cultures. The changes could be traced to the reduction of the chlorophyll content and the Fv/Fm value, as well as in the discoloration of the lower leaves and the appearance of necrotic tissues. Long, etiolated shoots (89.3 mm) were obtained at the end of cold storage. Shoot cultures stored in a growth chamber as control (22 ± 2 °C, 16/8 h light/dark) senesced and died after 16 weeks. Explants from stored shoots were subcultured for four weeks. The number and length of newly developed shoots were significantly higher on explants from cold storage compared to those from control cultures if the storage was longer than one week.

Phytotoxicity and Other Adverse Effects on the In Vitro Shoot Cultures Caused by Virus Elimination Treatments: Reasons and Solutions.

In general, in vitro virus elimination is based on the culture of isolated meristem, and in addition thermotherapy, chemotherapy, electrotherapy, and cryotherapy can also be applied. During these processes, plantlets suffer several stresses, which can result in low rate of survival, inhibited growth, incomplete development, or abnormal morphology. Even though the in vitro cultures survive the treatment, further development can be inhibited; thus, regeneration capacity of treated in vitro shoots or explants play also an important role in successful virus elimination. Sensitivity of genotypes to treatments is very different, and the rate of destruction largely depends on the physiological condition of plants as well. Exposure time of treatments affects the rate of damage in almost every therapy. Other factors such as temperature, illumination (thermotherapy), type and concentration of applied chemicals (chemo- and cryotherapy), and electric current intensity (electrotherapy) also may have a great impact on the rate of damage. However, there are several ways to decrease the harmful effect of treatments. This review summarizes the harmful effects of virus elimination treatments applied on tissue cultures reported in the literature. The aim of this review is to expound the solutions that can be used to mitigate phytotoxic and other adverse effects in practice.

Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions.

MAIN CONCLUSION: Shoot tip necrosis is a physiological condition that negatively impacts the growth and development of in vitro plant shoot cultures across a wide range of species. Shoot tip necrosis is a physiological condition and disorder that can arise in plantlets or shoots in vitro that results in death of the shoot tip. This condition, which can spread basipetally and affect the emergence of axillary shoots from buds lower down the stem, is due to the cessation of apical dominance. STN can occur at both shoot multiplication and rooting stages. One of the most common factors that cause STN is nutrient deficiency or imbalance. Moreover, the presence or absence of plant growth regulators (auxins or cytokinins) at specific developmental stages may impact STN. The cytokinin to auxin ratio within an in vitro plant can be modified by varying the concentration of cytokinins used in the culture medium. The supply of nutrients to in vitro shoots or plantlets might also affect their hormonal balance, thus modifying the occurrence of STN. High relative humidity within culture vessels and hyperhydricity are associated with STN. An adequate supply of calcium as the divalent cation (Ca2+) can hinder STN by inhibiting the accumulation of phenolic compounds and thus programmed cell death. Moreover, the level of Ca2+ affects auxin transport and ethylene production, and higher ethylene production, which can occur as a result of high relative humidity in or poor ventilation of the in vitro culture vessel, induces STN. High relative humidity can decrease the mobility of Ca2+ within a plant, resulting in Ca2+ deficiency and STN. STN of in vitro shoots or plantlets can be halted or reversed by altering the basal medium, mainly the concentration of Ca2+, adjusting the levels of auxins or cytokinins, or modifying culture conditions. This review examines the literature related to STN, seeks to discover the associated factors and relations between them, proposes practical solutions, and attempts to better understand the mechanism(s) underlying this condition in vitro.

In vitro tissue culture of apple and other Malus species: recent advances and applications.

MAIN CONCLUSION: Studies on the tissue culture of apple have allowed for molecular, biotechnological and applied breeding research to advance. In the past 8 years, over 100 papers advancing basic biology, genetic transformation and cryobiology have emerged. Apple (Malus × domestica Borkh.; Rosaceae) is an important fruit crop grown mainly in temperate regions of the world. In vitro tissue culture is a biotechnological technique that has been used to genetically improve cultivars (scions) and rootstocks. This updated review presents a synthesis of findings related to the tissue culture of apple and other Malus spp. between 2010 and 2018. Increasingly complex molecular studies that are examining the apple genome, for example, in a bid to identify the cause of epigenetic mutations and the role of transposable elements in this process would benefit from genetically stable source material, which can be produced in vitro. Several notable or curious in vitro culture methods have been reported to improve shoot regeneration and induce the production of tetraploids in apple cultivars and rootstocks. Existing studies have revealed the molecular mechanism underlying the inhibition of adventitious roots by cytokinin. The use of the plant growth correction factor allows hypothetical shoot production from leaf-derived thin cell layers relative to conventional leaf explants to be determined. This updated review will allow novices and established researchers to advance apple and Malus biotechnology and breeding programs.

Combined inhibition of key potassium currents has different effects on cardiac repolarization reserve and arrhythmia susceptibility in dogs and rabbits.

A reliable assessment of the pro-arrhythmic potential for drugs in the development phase remains elusive. Rabbits and dogs are commonly used to create models of pro-arrhythmia, but the differences between them with respect to repolarizing potassium currents are poorly understood. We investigated the incidence of drug-induced torsades de pointes (TdP) and measured conventional ECG parameters and the short-term variability of the QT interval (STVQT) following combined pharmacological inhibition of IK1+IKs and IK1+IKr in conscious dogs and anesthetized rabbits. A high incidence of TdP was observed following the combined inhibition of IK1+IKs in dogs (67% vs. 14% in rabbits). Rabbits exhibited higher TdP incidence after inhibition of IK1+IKr (72% vs. 14% in dogs). Increased TdP incidence was associated with significantly larger STVQT in both models. The relatively different roles of IK1 and IKs in dog and rabbit repolarization reserve should be taken into account when extrapolating the results from animal models of pro-arrhythmia to humans. A stronger repolarization reserve in dogs (likely due to stronger IK1 and IKs), and the more human-like susceptibility to arrhythmia of rabbits argues for the preferred use of rabbits in the evaluation of adverse pro-arrhythmic effects.

Models and tools for studying drought stress responses in peas.

The pea (Pisum sativum L.) is an important pulse crop but the growing area is limited because of its relatively low yield stability. In many parts of the world the most important abiotic factor limiting the survival and yield of plants is the restricted water supply, and the crop productivity can only be increased by improving drought tolerance. Development of pea cultivars well adapted to dry conditions has been one of the major tasks in breeding programs. Conventional breeding of new cultivars for dry conditions required extensive selection and testing for yield performance over diverse environments using various biometrical approaches. Several morphological and biochemical traits have been proven to be related to drought resistance, and methods based on physiological attributes can also be used in development of better varieties. Osmoregulation plays a role in the maintenance of turgor pressure under water stress conditions, and information on the behaviour of genotypes under osmotic stress can help selection for drought resistance. Biotechnological approaches including in vitro test, genetic transformation, and the use of molecular markers and mutants could be useful tools in breeding of pea. In this minireview we summarized the present status of different approaches related to drought stress improvement in the pea.

Degradation of ochratoxin A and other mycotoxins by Rhizopus isolates.

Several filamentous fungi representing the genera Rhizopus and Mucor were examined for their ability to degrade ochratoxin A (OTA), aflatoxin B1, zearalenone and patulin in a liquid medium. While none of the isolates exhibited aflatoxin degrading activity, ochratoxin A, zearalenone and patulin were decomposed by several isolates. Ochratoxin A was successfully degraded by Rhizopus stolonifer, R. microsporus, R. homothallicus and two R. oryzae isolates, and by four unidentified Rhizopus isolates. Kinetics of ochratoxin A detoxification of selected Rhizopus isolates was also examined. Rhizopus isolates were able to degrade more than 95% of ochratoxin A within 16 days. A R. stolonifer isolate could also effectively decompose ochratoxin A on moistened wheat. Further studies are in progress to identify the enzymes and genes responsible for ochratoxin detoxification and to transfer these genes to other Rhizopus isolates or microbes which could be used safely for decontamination of cereal products.

Growth and developmental responses of potato to osmotic stress under in vitro conditions.

The effect of mannitol on different genotypes of potato was studied in callus and plantlet culture. In vitro responses of five potato genotypes with well-known field behaviour to water deficit were analysed. After a 4-week-long cultivation on media containing mannitol up to 0.8 M, different morpho-physiological parameters were determined and statistically analysed. The useful concentration of mannitol for in vitro screening the osmotic tolerance of different genotypes depended on the type of culture; it was 0.4 M in plantlet-test and 0.8 M in callus-test. In callus-test the relative increase of callus mass was a useful parameter for determination of osmotic tolerance of genotypes at cellular level. In plantlet culture, stress index calculated from the rate of surviving in vitro shoots, number and length of roots per surviving explant and the rate of rooted explants were applicable to determine three groups according to the tolerant, medium tolerant and sensitive categories in agreement with the field behaviour of these genotypes. Under in vitro stress conditions we were able to distinguish the examined genotypes with different drought tolerance.