Callus Induction, Proliferation, Enhanced Secondary Metabolites Production and Antioxidants Activity of Salvia moorcroftiana L. as Influenced by Combinations of Auxin, Cytokinin and Melatonin

Abstract: Tissue culture technique is one of the best methods to reproduce salvia plant Therefore, the aim of this research was to enhance the in-vitro callus proliferation and production of secondary metabolites of S. moorcroftiana using different combinations of auxin, cytokinin and melatonin. Initially, callus induction was optimized using indole acetic acid (IAA), 2, 4-dichlorophenoxy acetic acid (2,4-D), and naphthalene acetic acid (NAA) applied at different concentrations in combination with 1 mg L-1 of 6-benzylaminopurine (BAP). The results indicates that earliest days to callus induction (14.67 days) was occurred in the media fortified with 2, 4-D+BAP (2.0+1.0 mgL-1). Whereas the highest callus initiation (100%) was induced on MS medium incorporated with 2,4-D+BAP (1+1mgL-1). Furthermore, maximum fresh weight was obtained when 2,4- D + BAP at the rate of (1+ 1mg L-1) was incorporated and dry weight was attained when 2,4- D + BAP at the rate of (2+1 mg L-1) was added to MS media. The maximum fresh and dry weight was obtained when melatonin at rate of 1.5 mg L-1 was supplemented with MS media including 2,4-D + BAP (1+1mg L-1), moreover the maximum DPPH scavenging activity, total phenolic and flavonoid content was noted when supplemented with melatonin at rate of 1.5 mg L-1. In conclusion, among various concentrations of plant growth regulators, 2,4- D + BAP at the rate of (1+ 1mg L-1) along with 1.5 g L-1 melatonin was the best for callus growth and production of secondary metabolites of S. moorcroftiana.

A smart diagnostic tool based on deep kernel learning for on-site determination of phosphate, calcium, and magnesium concentration in a hydroponic system.

Calcium, phosphate, and magnesium are essential nutrients for plant growth. The in situ determination of these nutrients is an important task for monitoring them in a closed hydroponic system where the nutrient elements need to be individually quantified based on ion-selective electrode (ISE) sensing. The accuracy issue of calcium ISEs due to interference, drift, and ionic strength, and the unavailability of phosphate and magnesium ISEs makes the development of these ion detecting tools hard to set up in a hydroponic system. This study modeled and evaluated a smart tool for recognising three ions (calcium, phosphate, and magnesium) based on the automatic multivariate standard addition method (AMSAM) and deep kernel learning (DKL) model. The purpose was to improve the accuracy of calcium ISEs, determining phosphate through cobalt electrochemistry, and soft sensing of magnesium ions. The model provided better performance in on-site detecting and measuring those ions in a lettuce hydroponic system achieving root mean square errors (RMSEs) of 12.5, 12.1, and 7.5 mg L-1 with coefficients of variation (CVs) below 5.0%, 7.0%, and 10% for determining Ca2+, H2PO4 -, and Mg2+ in the range of 150-250, 100-200, and 20-70 mg L-1 respectively. Furthermore, the DKL was implemented for the first time in the third platform (LabVIEW) and deployed to determine three ions in a real on-site hydroponic system. The open architecture of the SDT allowed posting the measured results on a cloud computer. This would help growers monitor their plants' nutrients conveniently. The informative data about the three mentioned ions that have no commercial sensors so far, could be adapted to the other components to develop a fully automated fertigation system for hydroponic production.

Melatonin Mitigates the Infection of Colletotrichum gloeosporioides via Modulation of the Chitinase Gene and Antioxidant Activity in Capsicum annuum L.

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most damaging pepper (Capsicum annum L.) disease. Melatonin induces transcription of defense-related genes that enhance resistance to pathogens and mediate physiological activities in plants. To study whether the melatonin-mediated pathogen resistance is associated with chitinase gene (CaChiIII2), pepper plants and Arabidopsis seeds were treated with melatonin, then CaChiIII2 activation, hydrogen peroxide (H2O2) levels, and antioxidant enzymes activity during plant-pathogen interactions were investigated. Melatonin pretreatment uncoupled the knockdown of CaChiIII2 and transiently activated its expression level in both control and CaChiIII2-silenced pepper plants and enhanced plant resistance. Suppression of CaChiIII2 in pepper plants showed a significant decreased in the induction of defense-related genes and resistance to pathogens compared with control plants. Moreover, melatonin efficiently enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhanced the activities of antioxidant enzymes, which possibly improved disease resistance. The activation of the chitinase gene CaChiIII2 in transgenic Arabidopsis lines was elevated under C. gloeosporioides infection and exhibited resistance through decreasing H2O2 biosynthesis and maintaining H2O2 at a steady-state level. Whereas melatonin primed CaChiIII2-overexpressed (OE) and wild-type (WT) Arabidopsis seedlings displayed a remarkable increase in root-length compared to the unprimed WT plants. Using an array of CaChiIII2 knockdown and OE, we found that melatonin efficiently induced CaChiIII2 and other pathogenesis-related genes expressions, responsible for the innate immunity response of pepper against anthracnose disease.

Combination of Multivariate Standard Addition Technique and Deep Kernel Learning Model for Determining Multi-Ion in Hydroponic Nutrient Solution.

Ion-selective electrodes (ISEs) have recently become the most attractive tools for the development of efficient hydroponic systems. Nevertheless, some inherent shortcomings such as signal drifts, secondary ion interferences, and effected high ionic strength make them difficult to apply in a hydroponic system. To minimize these deficiencies, we combined the multivariate standard addition (MSAM) sampling technique with the deep kernel learning (DKL) model for a six ISEs array to increase the prediction accuracy and precision of eight ions, including NO3-, NH4+, K+, Ca2+, Na+, Cl-, H2PO4-, and Mg2+. The enhanced data feature based on feature enrichment (FE) of the MSAM technique provided more useful information to DKL for improving the prediction reliability of the available ISE ions and enhanced the detection of unavailable ISE ions (phosphate and magnesium). The results showed that the combined MSAM-feature enrichment (FE)-DKL sensing structure for validating ten real hydroponic samples achieved low root mean square errors (RMSE) of 63.8, 8.3, 29.2, 18.5, 11.8, and 8.8 mg·L-1 with below 8% coefficients of variation (CVs) for predicting nitrate, ammonium, potassium, calcium, sodium, and chloride, respectively. Moreover, the prediction of phosphate and magnesium in the ranges of 5-275 mg·L-1 and 10-80 mg·L-1 had RMSEs of 29.6 and 8.7 mg·L-1 respectively. The results prove that the proposed approach can be applied successfully to improve the accuracy and feasibility of ISEs in a closed hydroponic system.

Identification of Pepper CaSBP08 Gene in Defense Response Against Phytophthora capsici Infection.

Little information is available on the role of Squamosa promoter binding protein (SBP)-box genes in pepper plants. This family of genes is known to have transcription characteristics specific to plants and to regulate plant growth, development, stress responses, and signal transduction. To investigate their specific effects in pepper (Capsicum annuum), we screened pepper SBP-box family genes (CaSBP genes) for Phytophthora capsici (P. capsici) resistance genes using virus-induced gene silencing. CaSBP08, CaSBP11, CaSBP12, and CaSBP13, which are associated with plant defense responses against P. capsici, were obtained from among fifteen identified CaSBP genes. The function of CaSBP08 was identified in pepper defense response against P. capsici infection in particular. CaSBP08 protein was localized to the nucleus. Silencing of CaSBP08 enhanced resistance to P. capsici infection. Following P. capsici inoculation, the malondialdehyde content, peroxidase activity, and disease index percentage of the CaSBP08-silenced plants decreased compared to the control. Additionally, the expression levels of other defense-related genes, especially those of CaBPR1 and CaSAR8.2, were more strongly induced in CaSBP08-silenced plants than in the control. However, CaSBP08 overexpression in Nicotiana benthamiana enhanced susceptibility to P. capsici infection. This work provides a foundation for the further research on the role of CaSBP genes in plant defense responses against P. capsici infection.

The CaChiVI2 Gene of Capsicum annuum L. Confers Resistance Against Heat Stress and Infection of Phytophthora capsici.

Extreme environmental conditions seriously affect crop growth and development, resulting in substantial reduction in yield and quality. However, chitin-binding proteins (CBP) family member CaChiVI2 plays a crucial role in eliminating the impact of adverse environmental conditions, such as cold and salt stress. Here, for the first time it was discovered that CaChiVI2 (Capana08g001237) gene of pepper (Capsicum annuum L.) had a role in resistance to heat stress and physiological processes. The full-length open-reading frame (ORF) of CaChiVI2 (606-bp, encoding 201-amino acids), was cloned into TRV2:CaChiVI2 vector for silencing. The CaChiVI2 gene carries heat shock elements (HSE, AAAAAATTTC) in the upstream region, and thereby shows sensitivity to heat stress at the transcriptional level. The silencing effect of CaChiVI2 in pepper resulted in increased susceptibility to heat and Phytophthora capsici infection. This was evident from the severe symptoms on leaves, the increase in superoxide (O2 -) and hydrogen peroxide (H2O2) accumulation, higher malondialdehyde (MDA), relative electrolyte leakage (REL) and lower proline contents compared with control plants. Furthermore, the transcript level of other resistance responsive genes was also altered. In addition, the CaChiIV2-overexpression in Arabidopsis thaliana showed mild heat and drought stress symptoms and increased transcript level of a defense-related gene (AtHSA32), indicating its role in the co-regulation network of the plant. The CaChiVI2-overexpressed plants also showed a decrease in MDA contents and an increase in antioxidant enzyme activity and proline accumulation. In conclusion, the results suggest that CaChiVI2 gene plays a decisive role in heat and drought stress tolerance, as well as, provides resistance against P. capsici by reducing the accumulation of reactive oxygen species (ROS) and modulating the expression of defense-related genes. The outcomes obtained here suggest that further studies should be conducted on plants adaptation mechanisms in variable environments.

Developing the first halophytic turfgrasses for the urban landscape from native Arabian desert grass.

Climate change is occurring and is influencing biological systems through augmented temperatures, more inconstant precipitation, and rising CO2 in the atmosphere. For sustainable landscaping, it was essential to assess the diversity of native/wild grasses and their suitability for turf and to combat the salinity problem in the region. For this purpose, a native halophytic grass, Aeluropus lagopoides, was investigated by conducting mowing tests on its ecotypes during the year 2014-2016 under desert climatic conditions. The research was carried out in two phases, i.e. Phase-I was for collection and establishment of ecotypes from various parts of UAE, while in Phase-II, mowing tests were conducted. During mowing tests, 50 ecotypes of A. lagopoides were given various mowing treatments (i.e. they were cut back at 1-, 2-, 3-, 4- and 5-cm heights) in field conditions. Significant differences were found among various ecotypes for different agronomic parameters such as ground cover, canopy stiffness, leaf number, clippings fresh and dry weights and internode length. Overall, the grass exhibited better performance at mowing heights of 3 and 4 cm, which are the standard mowing heights for turfgrasses. Ecotypes FA5, RA3, RUDA2, RUDA7 and RUADA1 of A. lagopoides showed the best performance against mowing shock and became the candidates for the turfgrass varieties from the native Arabian flora.

Heat Shock Proteins: Dynamic Biomolecules to Counter Plant Biotic and Abiotic Stresses.

Due to the present scenario of climate change, plants have to evolve strategies to survive and perform under a plethora of biotic and abiotic stresses, which restrict plant productivity. Maintenance of plant protein functional conformation and preventing non-native proteins from aggregation, which leads to metabolic disruption, are of prime importance. Plant heat shock proteins (HSPs), as chaperones, play a pivotal role in conferring biotic and abiotic stress tolerance. Moreover, HSP also enhances membrane stability and detoxifies the reactive oxygen species (ROS) by positively regulating the antioxidant enzymes system. Additionally, it uses ROS as a signal to molecules to induce HSP production. HSP also enhances plant immunity by the accumulation and stability of pathogenesis-related (PR) proteins under various biotic stresses. Thus, to unravel the entire plant defense system, the role of HSPs are discussed with a special focus on plant response to biotic and abiotic stresses, which will be helpful in the development of stress tolerance in plant crops.

Knockdown of the chitin-binding protein family gene CaChiIV1 increased sensitivity to Phytophthora capsici and drought stress in pepper plants.

Phytophthora capsici has been the most destructive pathogen of pepper plants (Capsicum annuum L.), possessing the ability to quickly overcome the host defense system. In this context, the chitin-binding protein (CBP) family member CaChiIV1 regulates the response to P. capsici and abiotic stresses. The relevance of functional characterization and regulation of CaChiIV1 has not been explored in horticultural crops, especially pepper plants. The target gene (CaChiIV1) was isolated from pepper plants and cloned; the encoded protein carries a chitin-binding domain (CBD) that is rich in cysteine residues and has a hinge region with an abundance of proline and glycine residues. Additionally, the conserved regions in the promoter have a remarkable motif, "TTGACC". The expression of CaChiIV1 was markedly regulated by methyl-jasmonate (MeJA), hydrogen peroxide (H2O2), melatonin, mannitol and P. capsici (PC and HX-9) infection. Knockdown of CaChiIV1 in pepper plants increased sensitivity to P. capsici (PC strain). Higher malondialdehyde (MDA) content and relative electrolyte leakage (REL) but lower antioxidant enzyme activities, chlorophyll content, root activity, and proline content were observed in CaChiIV1-silenced plants than in control plants. In conclusion, CaChiIV1-silenced pepper plants displayed increased susceptibility to P. capsici infection due to changes in expression of defense-related genes, thus showing its coregulation affect in particular conditions. Furthermore, antioxidant enzymes and proline content were largely diminished in CaChiIV1-silenced plants. Therefore, this evidence suggests that the CaChiIV1 gene plays a prominent role in the defense mechanism of pepper plants against P. capsici infection. In the future, the potential role of the CaChiIV1 gene in defense regulatory pathways and its coregulation with other pathogen-related genes should be identified.

Performance of Aeluropus lagopoides (mangrove grass) ecotypes, a potential turfgrass, under high saline conditions.

Climate change has become a real threat, and its impacts are being felt throughout the world. Temperature is considered one of the significant elements by the recent consequences of climate change and global warming, specially the salinity which is increased at higher temperature. Turfgrasses are adversely affected due to an increasing trend in salinity. The main aim of this investigation was to find out salt-tolerant ecotypes from native species of UAE to mitigate the salinity problem. Performance of a native grass, Aeluropus lagopoides, was investigated under high saline conditions during the year 2014 under the UAE climatic conditions. The experiment was planned under randomised complete block design (RCBD) with two factors and four replications. During the experiment, 50 ecotypes of Aeluropus lagopoides, alongside Paspalum vaginatum (as control), were tested at different salt levels, i.e. 0, 15, 30, 45, 60 and 75 dSm-1. Significant differences were found among various ecotypes as well as salinity levels for different agronomic traits including green cover, canopy stiffness, leaf colour and salinity of leaf rinseates. Most of the ecotypes tolerated salinity up to 30 dSm-1, maintaining the quality, but beyond this level the quality declined. However, some of the ecotypes survived under high salinity, even beyond sea level (75 dSm-1). All the ecotypes, except RUA2, RUA3 and RUA1, showed better performance than P. vaginatum, the prevailing commercial turfgrass in the UAE. Based on their performance, the ecotypes RUDA7, FA5, RA3, RUDA2 and RA2 could be used for turf purposes under saline conditions.

Concentrations, dietary exposure, and human health risk assessment of heavy metals in market vegetables of Peshawar, Pakistan.

The present study was carried out to assess heavy metal concentrations in ready-to-eat vegetables (RTEs) collected from open markets. Samples of RTEs including lettuce, coriander, and carrot were collected from five different local markets of Peshawar, Pakistan including Industrial estate, Board, Agriculture University, Firdos, and Hashtnaghri on four different dates, i.e., 21st March, 04th April, 19th April, and 05th May, 2016. The samples were analyzed through atomic absorption spectrophotometer to see the amount of heavy metals present in them. The elements studied were cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), and lead (Pb). Maximum Cd, Cr, Cu, Ni, and Pb concentrations in samples collected from different markets were 0.68, 5.28, 12.31, 7.61, and 25.04 mg kg-1, respectively, whereas maximum Cd, Cr, Cu, Ni, and Pb concentrations in RTEs collected at different dates were 0.59, 4.08, 14.85, 7.23, and 21.97 mg kg-1, respectively. The mean Cd, Cr, and Pb concentrations exceeded the permissible limits, while Cu and Ni were found within the limits set by FAO/WHO in all studied RTEs. The daily dietary intake and hazard quotient (HQ) showed great variations. The HQ was found > 1 for Pb in all studied vegetables, while it was < 1 for Cd, Cr, Cu, and Ni with few exceptions. It can be concluded from the study that heavy metal concentrations were above the permissible toxicity levels and their continuous consumption may cause several health issues.

Light quality and temperature effects on antirrhinum growth and development.

An experiment was carried out to examine the effects of light quality on the growth and development of antirrhinum under three different temperatures 19 degrees C, 24 degrees C and 27 degrees C in glasshouses. Five different colour filters (i.e. 'Red absorbing', 'Blue absorbing', 'Blue and Red absorbing' and two 'partially Blue absorbing' materials) were tested, with one clear polythene as a control. Plant height, internode length and leaf area were significantly affected by the spectral filters as well as the temperature. Analysis of color filter's effect on presumed photoreceptors to exist indicated that antirrhinum plant height was regulated by the action of a blue acting photoreceptor (BAP) and not the phytochrome. There was no evidence for an effect of phytochrome or BAP on time to flowering, however, increasing temperature levels effectively decreased the time to flowering. To predict the effects of different spectral qualities and temperature, simple models were created from data on plant height, internode length and time to flowering. These models were then applied to simulate the potential benefits of spectral filters and temperature in manipulation of growth control and flowering in antirrhinum.