Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses.

Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress conditions like drought, salt, heat and cold stress adversely affect the productivity and quality of mulberry leaves. Quantitative real time PCR (qPCR) is a reliable and widely used method to identify abiotic stress responsive genes and molecular mechanism in different plant species. Selection of suitable reference genes is important requirement for normalizing the expression of genes through qRT-PCR study. In the present study, we have selected eight candidate reference genes in mulberry for analyzing their expression stability in different abiotic stress treatments including drought, salt, heat and cold stresses. The expression stability of these reference genes was determined using geNorm, NormFinder and RefFinder statistical algorithms. The results showed that Ubiquitin and protein phosphatase 2A regulatory subunit A (PP2A) were the most stable genes across all the treatment samples. However, analysis of individual stresses revealed different expression profiles and stability of reference genes. Actin3 and PP2A were most stable in drought and salt conditions respectively. RPL3 most preferred in heat stress and Ubiquitin was most stable in cold stress. We propose the ubiquitin and PP2A are the preferred reference genes for normalization of gene expression data from abiotic stresses. In addition, Actin3 are preferred for drought stress, PP2A for salt stress, RPL3 for heat stress and Ubiquitin for cold stress studies.

Biotechnological Perspectives to Combat the COVID-19 Pandemic: Precise Diagnostics and Inevitable Vaccine Paradigms.

The outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause for the ongoing global public health emergency. It is more commonly known as coronavirus disease 2019 (COVID-19); the pandemic threat continues to spread aroundthe world with the fluctuating emergence of its new variants. The severity of COVID-19 ranges from asymptomatic to serious acute respiratory distress syndrome (ARDS), which has led to a high human mortality rate and disruption of socioeconomic well-being. For the restoration of pre-pandemic normalcy, the international scientific community has been conducting research on a war footing to limit extremely pathogenic COVID-19 through diagnosis, treatment, and immunization. Since the first report of COVID-19 viral infection, an array of laboratory-based and point-of-care (POC) approaches have emerged for diagnosing and understanding its status of outbreak. The RT-PCR-based viral nucleic acid test (NAT) is one of the rapidly developed and most used COVID-19 detection approaches. Notably, the current forbidding status of COVID-19 requires the development of safe, targeted vaccines/vaccine injections (shots) that can reduce its associated morbidity and mortality. Massive and accelerated vaccination campaigns would be the most effective and ultimate hope to end the COVID-19 pandemic. Since the SARS-CoV-2 virus outbreak, emerging biotechnologies and their multidisciplinary approaches have accelerated the understanding of molecular details as well as the development of a wide range of diagnostics and potential vaccine candidates, which are indispensable to combating the highly contagious COVID-19. Several vaccine candidates have completed phase III clinical studies and are reported to be effective in immunizing against COVID-19 after their rollout via emergency use authorization (EUA). However, optimizing the type of vaccine candidates and its route of delivery that works best to control viral spread is crucial to face the threatening variants expected to emerge over time. In conclusion, the insights of this review would facilitate the development of more likely diagnostics and ideal vaccines for the global control of COVID-19.

Novel coronavirus disease (COVID-19): a pandemic (epidemiology, pathogenesis and potential therapeutics).

The coronavirus disease (COVID-19) is highly pathogenic viral infection caused by SARS-CoV-2. Currently, COVID-19 has caused global health concern. It is assumed that COVID-19 has zoonotic origin based on the large number of infected people who were exposed to the wet market in Wuhan City, China. The phylogenetic analysis has revealed that SARS-CoV-2 has significant sequence similarity with severe acute respiratory syndrome-like (SARS-like) bat viruses, thus bats could be primary possible reservoir. The intermediate host and there subsequent transfer is not known yet, although human to human transfer is widely confirmed. The transmission of COVID-19 infection from one person to another resulted in the isolation of patients who were subsequently given a variety of treatments. To monitor the current outbreak, robust steps have been taken around the globe to reduce the transmission of COVID-19 infection particularly banning international and domestic flights, inducting lockdowns in vulnerable areas, social distancing etc. No clinically approved antiviral drug or vaccine against COVID-19 is reported yet. However, in clinical trials, few broad-spectrum antiviral drugs were evaluated against COVID-19 infection which resulted in clinical recovery. In this article emergence and pathogenicity of COVID-19 infection along with potential therapeutic strategies are analyzed to combat the COVID-19 pandemic.

Indirect regeneration and genetic fidelity analysis of acclimated plantlets through SCoT and ISSR markers in Morus alba L. cv. Chinese white.

A reliable protocol was developed for in vitro micro propagation of Morus alba L.cv. Chinese white. Initially, friable callus was induced (242.8 and 128.5 mg) from in vivo leaf and nodal explants on Murashige and Skoog's (MS) medium amended with 4.0 µM/L of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3.0 µM/L of Naphthalene acetic acid (NAA) respectively within 3 weeks. Shoot regeneration (12.2 and 8.6) was obtained from leaf and node derived callus on 6-benzylaminopurine (BAP) + Thidiazuron (TDZ) at 2.5 + 2.0 and 7.5 + 2.0 µM/L concentrations respectively, after 4 weeks of incubation. In vitro shoots were rooted (90 %) on half strength MS medium with 7.5 µM/L indole-3 butyric acid (IBA) and plantlets were hardened in plastic pots contained farmyard manure, sand and garden soil in 1:1:2 ratio. The genetic stability of plantlets were confirmed by start codon targeted (SCoT) and inter simple sequence repeats (ISSR) primers based molecular analysis.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.