Micropropagation and In Vitro Inflorescence of Pentas lanceolata

In this study, different concentrations of 6-benzyladenine (BA) on in vitro shoot and inflorescence inductions of P. lanceolata were investigated. The in vivo and in vitro floral characteristics of this plant were also compared. Nodal explants of P. lanceolata were cultured vertically with the cut ends inserted into semi-solid Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1, 2, 4, and 8 mg L–1 BA. The results showed that the explants formed the highest numbers of shoots even when cultured in MS basal medium without any addition of BA, while the shoots formed in the explants cultured in MS medium supplemented with 1 mg L–1 BA were the longest. No inflorescence was found in the shoots cultured in MS medium supplemented with 8 mg L–1 BA, while the highest percentage of inflorescence induction was found in the shoots cultured in the medium supplemented with 0.5 mg L–1 BA. The apperances of in vivo and in vitro flowers of P. lanceolata were the same in many aspects except that the number of flower/inflorescence formed was different. In addition, water accumulation was observed only inside the in vitro flowers. Water deposit in the long tubular structure of P. lanceolata flower could cause anther injury, suggesting that flowers developed in vitro may not always produce pollen.

In Vitro Propagation, Genetic Assessment, and Medium-Term Conservation of the Coastal Endangered Species Tetraclinis Articulata (Vahl) Masters (Cupressaceae) from Adult Trees

Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (<17%). Addition of 0.9 g L−1 AC improved rooting (40%) but rooting performance was optimal (66.7%) after a pulse treatment consisting of 4 h immersion in liquid SH medium without growth regulators, followed by 8 weeks of cultivation. Rooted microplants were successfully acclimatized (93.33%) in a peat moss and vermiculite mixture (1:1 v/v ratio). The genetic stability of the in vitro regenerated plantlets was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Explant survival and growth remained higher than 90% after 28 weeks of cold storage at both 4 °C and 10 °C. The protocol presented here allows for largescale T. articulata production and could be applied for both ex situ conservation strategies and industrial purposes.

Assessment of Domestic Wastewaters as Potential Growth Media for Chlorella vulgaris and Haematococcus pluvialis

Domestic wastewater contains chemical compounds that can be used as nutrients for microalgae. Removing these chemical compounds from wastewater by microalgae might help in reducing the operation cost of wastewater management while minimizing the cultivation cost for large-scale microalgae metabolite production. In this study, domestic wastewater collected from Indah Water Konsortium (IWK), Kuala Lumpur, Malaysia, was assessed as growth media for two types of microalgae, namely Chlorella vulgaris and Haematococcus pluvialis. The biomass growth and nutrient removal efficiency of total nitrogen (TN), total phosphorus (TP), and total ammonia (TAN) in different concentrations of diluted wastewater were measured. The results showed that biomass concentration (0.227 g/L), biomass productivity (0.029 g/L/day), and specific growth rate (0,284 d-1) yielded by C. vulgaris in 14 days of 80% wastewater were comparable to those microalgae grew in standard Bold’s Basal medium (BBM). Besides, C. vulgaris grew in 50% wastewater to remove TN, TP, and TAN with the highest removal efficiency (>88%). For H. pluvialis, the biomass concentration in all wastewater concentrations was lower than BBM. The removal efficiencies of TN and TP were lower than 55%, but more than 80% for removal efficiency of TAN in 50% and 80% wastewater. Hence, C. vulgaris has better growth performance and nutrient removal efficiency than H. pluvialis. These findings indicated that IWK domestic wastewater could be used as growth media for microalgae, especially C. vulgaris.

Development of An Efficient Protocol for Rapid Propagation of Curculigo orchioides Using Leaf Explant Culture

Curculigo orchioides is one of the most common medicinal plants used by diverse cultures and tribal groups. The roots of the plant are used medicinally in Asian countries. Curculigo orchioides have the ability to regenerate through seeds and tubers, but the regeneration rate is low. Plant tissue culture method was believed to have potential for rapid multiplication of this medicinal plant. An efficient protocol for rapid propagation of Curculigo orchioides, of the family Amaryllidaceae, was developed using leaf explants culture. The leaf explants (1 cm x 1 cm squares) cultured on Murashige and Skoog (MS) basal medium were supplemented with various concentrations and combinations of auxins and cytokinins with temperature 25 ± 2°C, relative humidity 85-90% and photoperiod of 12 hours light (2000-3000 lux). Callus induction was obtained within 4 weeks, 2,4-D at 3 mg/l formed profuse callus and the degree was found to be the highest (+++) among all the treatments. The best response to shoot induction, with maximum shoot number 5.33 (mean number of shoots per explant) was obtained using 1.0 mg/l 6-benzyl aminopurine (BAP) in combination with 1.0 mg/l Kinetin. In vitro shoots were induced for rooting on 0.5 mg/l of NAA supplemented medium. In order for seedlings propagated in vitro to adapt to natural conditions, plants were growned on a substrate coir: husk ash: sand (with the ratio of 0.5: 0.5: 1) in a greenhouse (humidity: 70%, temperature: 28-300C) gave 88.33% survival rate after 8 weeks of culture. With the results received, this is an effective approach to propagating Curculigo orchioides.

Development of Growth Media from Agricultural By-Products for Cultivation of PUFA-Producing Sicyoidochytrium minutum

The demand for novel sources of marine oils, which contain polyunsaturated fatty acids (PUFAs), has increased due to the realization of the importance of PUFAs, e.g., docosahexaenoic acid (DHA), in the human diet. However, the natural supply is limited. By-product peptones (BYPP) intended as a growth medium for the PUFA-producing strain Sicyoidochytrium minutum of family Thraustochytriaceae were produced after several experiments on the pancreatic digestion of bovine lungs and spleens. S. minutum was able to grow in a medium containing BYPP made from the pancreatic digestion of lung and spleen with glycerol, resulting in 1.14 ± 0.03 g cell dry weight (CDW)/L and 1.44 ± 0.24 g CDW/L, respectively, after 5 days of incubation at 25 °C, compared to 1.92 ± 0.25 g CDW/L in Basal Medium (BM) containing tryptone, peptone, and glycerol. The lipid content, obtained after growth in lung BYPP media with glycerol as a carbon source, was significantly higher (28.17% ± 1.33 of dry weight) than in the control basal medium (BM) (21.72% ± 2.45); however, DHA as a percentage of total fatty acids was lower in BYPP than in the control BM (25.24% ± 1.56 and 33.02% ± 2.37, respectively). It is concluded that low-value by-products from abattoirs can be used as ingredients for the cultivation of oligogenic Thraustochytriaceae.

In Vitro Propagation of Aconitum chasmanthum Stapf Ex Holmes: An Endemic and Critically Endangered Plant Species of the Western Himalaya

Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum.

IN VITRO PROPAGATION OF PLUM ROOTSTOCK DOCERA 6

This paper describes research on the application of tissue culture techniques to the micropropagation of interspecific rootstock ʹDocera 6ʹ. The experimental work was carried out in the period 2017-2018, in the in vitro propagation laboratory of the Fruit Growing Institute Plovdiv. Аxillary buds were employed as initial explantеs in two different seasons (March-May; September-October). The action of the mineral medium was studied in the multiplication stage. The best result was obtained on LS medium included BAP 0.5 mg/l and IAA 0.05 mg/l. Тhe obtained average multiplication rate is 3.08. The concentration of auxin applied to the basal medium influence the quality of the root system Treatment with high concentrations of IBA added to the rooting medium gives the best results (V5). Тhe influence of the season on growth and development of micropropagated ʹDocera 6ʹ rootstock during ex vitro acclimatization is also part of our research. The spring acclimatization gives better results than the autumn.

353 SARS-CoV-2 pulmonary vasculitis and Takotsubo myocarditis syndrome. Hypoxia, endothelial dysfunction, and inflammation are the trigger?

Aims Nowadays the spread of respiratory infection caused by SARS-CoV-2 results in a global pandemic. The World Health Organization (WHO) declared about 450 000 deaths in more than 200 countries, until June 2020. SARS-CoV-2 pneumonia develops a stress condition through systemic hypoxygenation with activation of adrenergic pathways. Takotsubo syndrome is characterized by a temporary wall motion abnormality of the left ventricle (LV) and has common features with acute coronary syndrome (ACS), representing a form of myocardial infarction without coronary arteries thrombosis. Among possible causes there are several stress conditions, including physical, psychological, and illnesses, for example interstitial pneumonia. Methods and results A 54-years-old man, previous smoker, was admitted to emergency room (ER) complaining of dyspnoea and fever, initially treated with ceftriaxone. Physical examination was characterized by tachypnoea (respiratory rate 30 acts/min), tachycardia and hypotension (arterial pressure 90/60 mmHg), and hypoxaemia at transcutaneous saturation (Sat O2 85%). High-resolution computed tomography (HRCT) showed bilateral interstitial pneumonia with ground glass opacities (visual involvement of 80% of pulmonary parenchymal) (Figure 1). Nasopharyngeal swab was positive for SARS-CoV-2 and ECG revealed Atrio-Ventricular Nodal Reentrant Tachycardia (AVRNT) with heart rate of 140 b.p.m., partially responsive to Valsalva manoeuver (Figure 2). Echocardiogram showed severe ventricular dysfunction [ejection fraction (EF): 30–35%] with hypokinesia of apical region associated with hyperkinesia of medio-basal segments, mild mitral regurgitation, and slight pericardial effusion (Figure 2). Laboratory tests were TnI hs 10 ng/l first detection – 26 ng/l second detection (normal range: 2.3–17.8 ng/l), PCR &gt;250 mg/l (normal range 0.5–5 mg/l), d-dimer 1893 ng/ml (normal range: &lt;500 ng/ml), PCT 3.10 ng/ml (normal range: 0–0.5 ng/ml). The worsening of clinical condition needed an orotracheal intubation and a transfer to Intensive Care Unit (ICU). The patient was treated with many antiviral drugs (darunavir, ritonavir), tocilizumab, steroid therapy, colchicine, and plasmapheresis. We observed a progressive clinical and echocardiography improvement, evidenced by partial recovery of EF (45%). CT scan revealed a normal coronary tree. The patient underwent cardiac magnetic resonance (CMR) that showed typical Takotsubo cardiomyopathy characterized by thinning and hypo-akinesias of apical wall of left ventricle (‘apical ballooning’) and normokinesis of basal/medium segments; no late gadolinium enhancements (LGEs); no oedema in T2 weighted images (Figure 3). Follow-up echocardiogram confirmed recovery of EF (50%) associated with mild hypokinesia of apical segments. Acute myocardial injury, as evidenced by elevated levels of cardiac biomarkers or electrocardiogram abnormalities, was observed in 7–20% of patients with COVID-19 in early studies in China (4). In a multicentre cohort study of 191 patients with COVID-19, 33 patients (17%) had acute cardiac injury, of whom 32 died. Whether typical clinical features of myocarditis were present in patients, who had elevated levels of cardiac troponins during the course of COVID-19 is unclear because most of the early studies did not include echocardiography or MRI data. By contrast, several case reports have described typical signs of myocarditis in patients with COVID-19. A woman aged 53 years with myocardial injury, as evidenced by elevated levels of cardiac biomarkers and diffuse ST segment elevation on the electrocardiogram, had diffuse biventricular hypokinesis on cardiac MRI, especially in the apical segments, in addition to severe LV dysfunction (LVEF = 35%). MRI data also revealed marked biventricular interstitial oedema, diffuse LGE and circumferential pericardial effusion, features that are consistent with acute myocarditis. Furthermore, in a man aged 37 years with chest pain and ST segment elevation, echocardiography revealed an enlarged heart. The Lake-Louise Criteria gave good diagnostic accuracy in patients with suspected myocarditis, evaluating the principle tissue targets in myocarditis, including: myocardial oedema, using T2-based imaging; hyperaemia and capillary leak, using early gadolinium enhancement (EGE) imaging; and myocyte necrosis and fibrosis, using LGE imaging [14]. The presence of myocardial injury was associated with a significantly worse prognosis. In the initial report of 41 patients with COVID-19 in Wuhan, five patients had myocardial injury with elevated levels of high-sensitivity cardiac troponin I (&gt;28 pg/ml), and four of these five patients were admitted to an ICU. Histological evidence of myocardial injury or myocarditis in COVID-19 is also limited. An autopsy of a patient with COVID-19 and ARDS who died of a sudden cardiac arrest showed no evidence of myocardial structural involvement, suggesting that COVID-19 did not directly impair the heart. By contrast, another case report described a patient with low-grade myocardial inflammation and myocardial localization of coronavirus particles (outside of cardiomyocytes), as measured by endomyocardial biopsy, suggesting that SARS-CoV-2 might infect the myocardium directly. In this case report, CMR showed typical Takotsubo cardiomyopathy (with hypokinesis of apical wall of left ventricle and normokinesis of basal/medium segments), but it showed no oedema in T2-weighted images, no hyperaemia nor capillary leakage (no myocardial EGE), no signs of necrosis or fibrosis (no LGE), no pericardial effusion. This case-report CMR images demonstrated the absence of typical myocardial injury caused by myocarditis, evidenced by the absence of the main tissue markers. Conclusions Therefore, the ventricular dysfunction, presented with Takotsubo syndrome typical pattern, could hypothetically be secondary to systemic hypoxygenation and stress condition caused by the systemic inflammation and endothelial dysfunction developed by SARS-CoV-2 interstitial pneumonia. About the current diagnostic possibilities, CMR is a valuable option for the assessment of inflammatory heart diseases.

Somatic embryogenesis from young inflorescences of the giant bamboo Dendrocalamus asper (Schult f.) Backer ex Heyne

Bamboos are an important worldwide non-timber forest product with current rising interest due to their environmentally friendly applications. Besides the consolidated uses of the sweet shoots and culms for structural uses, Dendrocalamus asper is an imposing ornamental bamboo for horticulture. The present work aimed to establish in vitro calli culture and plant regeneration through somatic embryogenesis starting from young inflorescences of the giant bamboo, D. asper. Pre-anthesis inflorescences were collected, disinfested, and subjected to callus induction on MS basal medium supplemented by 0 µM, 9 µM, 18 µM, 27 µM, and 36 µM of 2,4-D in combination with 9 µM of 2-iP or 9 µM Kin. The different obtained calli types were characterized and subcultured in 0 µM, 4.5 µM, 9 µM, and 18 µM of 2,4-D in combination with 9 µM of both cytokinins for multiplication and differentiation. Additionally, the explant incision and its inoculation orientation onto culture media were tested for callus induction improvement. The 2,4-D was essential for callus induction, and its combination with both cytokinins resulted in embryogenic callus induction and further somatic embryos regeneration. The subsequent reduction of this auxin to 4.5 µM resulted in somatic embryo maturation. Somatic embryos transferred to a plant growth regulator-free medium resulted in plantlet conversion. The present work showed the feasibility of using inflorescences as explants and the efficiency of using the 2-iP in combination with 2,4-D to callus induction and in vitro bamboo plant regeneration through somatic embryogenesis.

Lippia graveolens (Lamiales: Verbenaceae) and Oryganum vulgare (Lamiales: Lamiaceae) obtained by means of the in vitro propagation technique

Objective: The objective of this study was to establish the method of propagation of Oryganum vulgare and Lippia graveolens employing a plant tissue culture technique that decreased the phenolization percentages and increased the multiplication coefficients. Design/ methodology/ approach: The in vitro germination percentage was evaluated in both MS and MS medium + activated carbon. Microcuttings (small shoots) of both species were established in base medium added with different antioxidant agents to decrease the phenolization of explants; the treatments were arranged in a completely randomized block design. For the propagation phase, a completely randomized factorial design was used, where the auxin/cytokinin phytoregulators, type of explants (axillary buds and leaves), and the species (Lippia graveolens and Oryganum vulgare) were considered as factors. Results: maximum germination (63.3% ±12.5) was obtained on day 15 ​​in both culture media for L. graveolens and O. vulgare. The use of antioxidant agents mainly activated carbon, increased the in vitro establishment and activation of vegetative buds in both species by up to 90%. There were significant differences in the variables evaluated regarding the treatments, the explant, and the species in the multiplication phase. The combination 1.0/ 0.5 mg L-1 BA/AIB induces callus formation for both species. When used as leaf explants, callus formation was potentiated. Study Limitations / Implications: The results presented are advances from a long-term experiment. Findings/conclusions: The germination of L. graveolens seeds can be achieved in MS medium after 15 days. Microcuttings of both L. graveolens and O. vulgare were successfully established in MS basal medium enriched with 1 g L-1 charcoal that showed low oxidation percentages and induced up to 90% the production of shoots in the explants. The mixture of 1.0/0.5 mg L-1 BA/AIB induces callus formation for both species; when this medium is in contact with leaves as an explant, its formation is potentiated, achieving diameters up to 15 mm. In order to achieve the induction of shoots and roots, buds should be established in MS medium enriched with 0.5 mg L-1 IBA for both species; this mixture encreased the multiplication coefficients