Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods

24 p.-4 fig.-6 tab.In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz., Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5 % (v/v) NaOCl was found to be most effective, about 97 % of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6- benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number 12-BIO2919-02.Peer reviewe

Nitrogen availability prevents oxidative effects of salinity on wheat growth and photosynthesis by up-regulating the antioxidants and osmolytes metabolism, and secondary metabolite accumulation.

Funder: NorthWest A&F University Yaangling, Shaanxi, China.BACKGROUND: Salinity is one of the damaging abiotic stress factor. Proper management techniques have been proposed to considerably lower the intensity of salinity on crop growth and productivity. Therefore experiments were conducted to assess the role of improved nitrogen (N) supplementation on the growth and salinity stress tolerance in wheat by analyzing the antioxidants, osmolytes and secondary metabolites. RESULTS: Salinity (100 mM NaCl) stress imparted deleterious effects on the chlorophyll and carotenoid synthesis as well as the photosynthetic efficiency. N supplementation resulted in increased photosynthetic rate, stomatal conductance and internal CO2 concentration with effects being much obvious in seedlings treated with higher N dose. Under non-saline conditions at both N levels, protease and lipoxygenase activity reduced significantly reflecting in reduced oxidative damage. Such effects were accompanied by reduced generation of toxic radicals like hydrogen peroxide and superoxide, and lipid peroxidation in N supplemented seedlings. Antioxidant defence system was up-regulated under saline and non-saline growth conditions due to N supplementation leading to protection of major cellular processes like photosynthesis, membrane structure and function, and mineral assimilation. Increased osmolyte and secondary metabolite accumulation, and redox components in N supplemented plants regulated the ROS metabolism and NaCl tolerance by further strengthening the antioxidant mechanisms. CONCLUSIONS: Findings of present study suggest that N availability regulated the salinity tolerance by reducing Na uptake and strengthening the key tolerance mechanisms

GC-MS Analysis: In Vivo

Liver disease is a worldwide problem. It represents one of the main causes of morbidity and mortality in humans. Achillea biebersteinii is used as herbal remedy for various ailments including liver diseases. But the scientific basis for its medicinal use remains unknown. Thus, this research was undertaken to evaluate the efficiency of A. biebersteinii essential oil (ABEO) (0.2 mL/kg) in the amelioration of CCl4-induced hepatotoxicity in rodent model. Moreover, the chemical content of the oil was investigated using GC and GC-MS. The following biochemical parameters were evaluated: serum glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamma-glutamyl-transpeptidase (γ-GGT), alkaline phosphatase (ALP), and total bilirubin. Furthermore, lipid profile, malondialdehyde (MDA), nonprotein sulfhydryl (NP-SH), and total protein (TP) contents in liver tissue were estimated. 44 components (92.0%) of the total oil have been identified by GC-MS analysis where α-terpinene and p-cymene were the most abundant. The high serum enzymatic (GOT, GPT, GGT, and ALP) and bilirubin concentrations as well as the level of MDA, NP-SH, and TP contents in liver tissues were significantly reinstated towards normalization by the ABEO. Histopathological study further confirmed these findings. In addition, ABEO showed mild antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid assays

Establishment of an Efficient In Vitro Propagation Method for a Sustainable Supply of Plectranthus amboinicus (Lour.) and Genetic Homogeneity Using Flow Cytometry and SPAR Markers

Plectranthus amboinicus (Lour.) Spreng is a medicinally important aromatic perennial herb used for the treatment of skin diseases, constipation, asthma, flu, fever, cough, and headache as well as a flavoring ingredient in traditional drinks, food, and meat stuffing. In this study, a high-performance in vitro propagation system of P. amboinicus through direct shoot organogenesis was developed using axillary node explants cultured on MS (Murashige and Skoog) medium augmented with 0.5, 2.5, 5.0, 7.5, and 10.0 µM of 6-benzyladenine (BA) or kinetin (Kin), alone or with 0.1, 0.5, 2.5, and 5.0 µM of indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). To optimize the regeneration potential of node explants, the effects of basal media strength and pH were also investigated. After 8 weeks of culture, explants cultured in full strength MS basal medium (pH 5.7) with 5.0 µM BA and 2.5 µM NAA exhibited the highest percentage (97.1%) of regeneration and the maximum number (19.3) of shoots per explant. Individual elongated shoots were rooted on half strength MS basal medium containing 0.25 µM indole 3-butyric acid (IBA) after 4 weeks of culture, producing 5.3 roots/shootlets with a root induction frequency of 93.7%. First time genetic stability of in vitro raised P. amboinicus plants was determined using SPAR markers, such as DAMD and ISSR, as well as flow cytometric tests, assuring the availability of authenticated raw materials for commercial production of the plant and its bioactive components

Thidiazuron Induced In Vitro Plant Regeneration, Phenolic Contents, Antioxidant Potential, GC-MS Profiles and Nuclear Genome Stability of <i>Plectranthus amboinicus</i> (Lour.) Spreng

Plectranthus amboinicus (Lour.) Spreng is a perennial plant from the mint family with aromatic, succulent leaves and several health benefits. Multiple shoot regeneration was accomplished in vitro using nodal segments (NS) explants of P. amboinicus pretreated with 0, 0.5, 5, 25, 50, and 100 μM thidiazuron (TDZ) for 4 h, then transferred to a growth regulator-free media. After 8 weeks of growth, NS explants pre-treated with 25 μM TDZ for 4 h and then transferred to TDZ-free Murashige and Skoog (MS) media produced the greatest number of shoots (27.3 per NS) with the longest average shoot length (4.9 mm) in 97.2% of cultures. On the same medium, regeneration of roots in most of the P. amboinicus shoots occurred spontaneously. The in vitro-regenerated P. amboinicus plantlets were adequately hardened off and adapted to the ex-vitro environment with a 90% survival rate. Total phenolic, tannin, and flavonoid contents, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging/antioxidant activity, were significantly higher in in vitro-regenerated plants than in ex vitro-plants. Flow cytometry (FCM) analysis validated the nuclear genome stability of the in vitro generated plants, which assessed their nuclear DNA content and found it to be comparable in genome size to that of the field-grown plants. The study found a quick and efficient method for in vitro multiplication of P. amboinicus which can aid to increased availability and accessibility of this plant species for various purposes. The genetic and phytochemical analysis of the in vitro propagated plants can also provide valuable insight into the plant’s properties and potential applications, which can further assist in its preservation and sustainable usage

Artificial microRNA-Based RNA Interference and Specific Gene Silencing for Developing Insect Resistance in Solanum lycopersicum

RNA Interference (RNAi), which works against invading nucleic acids or modulates the expression of endogenous genes, is a natural eukaryotic regulating system, and it works by noncoding smaller RNA molecules. Plant-mediated gene silencing through RNAi can be used to develop plants with insect tolerance at transcriptional or post-transcriptional levels. In this study, we selected Myzus persicae&rsquo;s acetylcholinesterase 1 gene (Ace 1) as a silencing target to develop transgenic Solanum lycopersicum L. plants&rsquo; resistance to aphids. An RNAi plasmid vector containing an artificial microRNA (amiRNA) sequence was engineered and successfully transformed into Jamila and Tomaland, two elite tomato cultivars. A northern blot analysis and PCR were carried out to check the efficacy of Agrobacterium-mediated transformation in T0 transgenic plants. The quantitative PCR data showed a substantial downregulation of the Ace 1 gene in aphids fed in clip cages on T1 transgenic plants. Furthermore, there was a substantial drop in aphid colonies that were fed on T1 transgenic plants of both the cultivars. These findings strongly suggest that transgenic plants that express amiRNA could be an important tool for engineering plants resistant to aphids and possibly for the prevention of viral disease in other plant-infested pests

Inoculation with arbuscular mycorrhizal fungi alleviates harmful effects of drought stress on damask rose

This study was conducted to examine the role of arbuscular mycorrhiza fungi (AMF) in alleviating the adverse effects of drought stress on damask rose (Rosa damascena Mill.) plants. Four levels of drought stress (100, 75, 50, and 25% FC) were examined on mycorrhizal and non-mycorrhizal plants in pots filled with sterilized soil. Our results showed that increasing drought stress level decreased all growth parameters, nutrient contents, gas exchange parameters, and water relations indicators. Under different levels of drought stress, mycorrhizal colonization significantly increased all studied parameters. Pn, gs, and E of the mycorrhizal plants was higher than those of non-mycorrhizal plants under different levels of drought stress. The increase in those rates was proportional the level of the mycorrhizal colonization in the roots of these plants. Majority of growth, nutrition, water status and photosynthetic parameters had a great dependency on the mycorrhizal colonization under all levels of drought stress. The results obtained in this study provide a clear evidence that AMF colonization can enhance growth, flower quality and adaptation of rose plants under different drought stress levels, particularly at high level of drought stress via improving their water relations and photosynthetic status. It could be concluded that colonization with AMF could help plants to tolerate the harmful effects caused by drought stress in arid and semi-arid regions. Keywords: AMF, Gas exchange, Glomus, Pigments content, Rosa damascena Mill., Drought stres

Oxidative Modification of LDL by Various Physicochemical Techniques: Its Probable Role in Diabetes Coupled with CVDs

Background. Pro- and antiatherogenic properties of oxidised low density lipoprotein (Ox-LDL) are responsible for different chronic diseases including diabetes and cardiovascular diseases (CVD). The constant attack on the body from oxidative stress makes the quantification of various oxidation products necessary. In this study, the oxidative stress causing the structural and chemical changes occurring in the LDL molecule is comprehensively done. Moreover, the prevalence of the autoantibodies against the oxidised LDL is also determined. Methods. Our study made an attempt to see the effect of Ox-LDL as an enhancer of type 2 diabetes mellitus (T2DM) coupled with CVD. Primarily, we detected the oxidation of LDL with different concentration of Fenton reaction. The biochemical parameters were assessed for the changes occurring in the LDL molecule. In a clinical set up, 20 sera samples were taken from patients who are healthy, 30 from those with diabetes, 20 from those with CVD, and 30 from diabetes with CVD patients. Results. In biochemical assays there were markedly increased TBARS, carbonyl, and HMF content in Ox-LDL as compared to native LDL. The prevalence of autoantibodies against the T2DM was recorded to be 36%, while for CVD it was recorded to be 29%. However, it was found that 50% of the sera samples showed autoantibodies against oxidized LDL in the sera of T2DM with CVD complications as compared to the native analogue. Conclusion. There is significant change in the LDL molecule as revealed by various physicochemical analysis. The change in the LDL macromolecule as a result of oxidation triggered the development of the autoantibodies against it

Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5–7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite™ with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation