Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method

Centella asiatica is a locally important medicinal plant. It is non-toxic, high in medicinal values, and can serve as a good candidate for genetic manipulation. However, to date no transformation protocol has been developed to fully utilize the potential of this plant. Therefore, this research is to establish an efficient particle bombardment transformation protocol for C. asiatica embryogenic calli. In addition, an attempt to express the haemagglutinin-neuraminidase (HN) protein from Newcastle disease virus (NDV) strain AF2240 in C. asiatica embryogenic calli were carried out using the developed transformation system. The HN protein can serve as a potential vaccine candidate for Newcastle disease (ND) in poultry. The induced embryogenic calli revealed the presence of extracellular matrix layer (ECM) during the microscopy studies. Particle bombardment transformation protocol was developed using the green fluorescent protein (GFP) as reporter. A total of eight parameters mainly different target distance, helium pressure, gold particles size, chamber vacuum pressure, number of bombardment, precipitation agents, post-bombardment incubation time, and plasmid DNA concentration were identified and successfully optimized. Based on the established protocol, transformations of C. asiatica embryogenic calli were performed using the constructed recombinant pMDC32’HN and HBT95:sGFP(S65T)-NOS’HN plasmids. Genomic PCR analysis revealed the presence of HN transgene in the transformed lines. Unfortunately no protein bands were detected during SDS-PAGE and western blotting, indicating low or no HN protein expression. Transformation using recombinant HBT95:sGFP(S65T)-NOS’HN plasmid resulted in very low GFP expression as compared to the positive control. Nonetheless, the mRNA transcripts were detected in the RT-PCR analysis. Positive signal from the dot blot assay further confirmed the presence of the HN protein expression in the transformed lines

Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection.

Scanning DNA sequences for polymorphisms and mutations often involve the mismatch specific cleavage by endonucleases at the mismatch sites and subsequent analysis of the digested product for mutation discovery. One of the limitations of using enzymatic mutation detection methods are the cost and availability of a mismatch specific endonuclease. We report the establishment of Nicotiana tabacum L. Cv. Bright Yellow 2 cells stably expressing the truncated ENDO1 (tENDO1) mismatch specific endonuclease. The 5′-Untranslated region of N. tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of cauliflower mosaic virus (CaMV 35S) promoter was employed to improve the tENDO1 protein yield. To ease the purification process, tENDO1 was secreted into the culture medium and isolated using nickel affinity chromatography. The tENDO1 was estimated to be stably produced in an average of 0.7–0.9 % total soluble protein. Functional test on tENDO1 for mismatch detection demonstrated that tENDO1 retained mismatch specific endonuclease activity resembles its native protein. Further biochemical analysis showed that tENDO1 exhibited mismatch detection specificity and efficiency comparable to other commonly used endonucleases

Analysis of EXO70C2 expression revealed its specific association with late stages of pollen development

Exocyst is an octameric protein complex that mediates the tethering of secretory vesicles at the plasma membrane for exocytosis. In this study, a 1136 bp promoter fragment of exocyst subunit exo70 family protein C2 (EXO70C2) was fused with the β-glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana via Agrobacterium tumefaciens. Detail histochemical analysis of EXO70C2-GUS expression in T3 transgenic A. thaliana lines revealed a strong GUS activity at late stages of pollen development. Strong GUS signals were visible from post meiotic pollen, mature pollen and pollen release stages. There was no detectable expression of EXO70C2-GUS during early pollen development and meiosis stages. Consistent with the GUS assay, EXO70C2 transcript profiling using real-time PCR analysis also showed high expression of EXO70C2 at these late stages of pollen development. Further RNA in situ hybridisation revealed the presence of EXO70C2 signals in pollen and as well as anther tapetum. Taken together, these results indicate that, EXO70C2 is specifically expressed in pollen and anther tapetum during the late stages of pollen development, suggesting its function in regulating essential secretory vesicles to support pollen maturation

Antimicrobial Usage and Resistance in Dairy Cattle Production

Antimicrobial resistance (AMR) has been a public health threat globally, with millions of lives lost due to AMR infections each year. The cases of AMR continue to escalate and cause devastating effect to both humans and animals. AMR contributes to high morbidity and mortality of the livestock, which results in staggering economic losses to the livestock producers. The main factor for AMR to arise in this industry is mainly due to the eagerness of livestock producers to meet high demand by using antimicrobials to promote animal growth and disease prevention. From a public health perspective, AMR in dairy cattle can also jeopardize human population due to the potential dissemination of AMR pathogens to humans via consumption of infected dairy products or direct contact with infected dairy cattle. At the current rate of unrestricted antimicrobial usage, AMR will be expedited and soon we will run out of effective treatment for even the simplest infection. World Health Organization (WHO) has issued a set of guidelines for the use of medically important antimicrobials on animals to mitigate the adverse consequences of AMR on human. Thus, this chapter will explain antimicrobial usage in dairy cattle production and the recent approaches and challenges on AMR

Effects of food wastes on yellow mealworm Tenebriomolitor larval nutritional profiles and growth performances

In this study, nutritional profiles and growth performances of yellow mealworm, Tenebriomolitor larvae (TML) were assessed cultivated using common food wastes i.e. watermelon rinds, broilers’ eggshells and banana peels. Nutritional profiles and growth performance of TML were evaluated after 28-day feeding trial. Post-feeding proximate analysis showed significant increment of nutritional contents compared to the control groups; whereby TML demonstrated highest level of crude protein (43.38%±2.71), moisture (9.74%±0.23) and ash (4.40%±0.22) in the group treated with watermelon wastes. On the other hand, TML showed highest level of crude fibre (8.73%±0.05) when treated with broilers’ eggshells; and higher level of crude fat (40.13%±4.66) with banana wastes. Nitrogen-free extract (NFE) contents were also noticed higher in the group treated with banana wastes (4.46%±5.30). In terms of growth performance, TML administrated with watermelon wastes demonstrated superior in specific growth rate (2.50%±0.43) and feed conversion efficiency (0.10%±0.01). Interestingly, TML grown with banana wastes showed highest survival rate (97.5%) among all. In short, TML cultivation using watermelon and banana wastes showed a promising result on nutritional fortification and growth enhancement

A review on induced mutagenesis of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni in the Asteraceae family is commercially valuable and cultivated throughout the world due to the great demand for its steviol glycosides (SGs) contents particularly rebaudioside A. Previous studies confirmed that maximal content of SGs in stevia was achieved at or just before flowering, and delayed flowering with long days provide longer duration for steviol glycosides accumulation. However, there is no suitable stevia variety to be cultivated in Malaysia due to her short day length. Mutation induction, including gamma irradiation, had been shown to be useful for generating genetic variations as well as developing new plant varieties from which desired mutants were successfully selected. The use of mutagens, both physical and chemical, has helped in creating mutants that expressed the selected desirable traits. This paper presents some selected essential data available in extant scientific studies on stevia with the focus on application of gamma irradiation on stevia. Both established achievements and recent publications of gamma radiation on stevia were reviewed. Emphasis is on the exceptional potential of stevia through induced mutation approach especially by using gamma rays

Generation of transgenic rice expressing cyclotide precursor Oldenlandia affinis kalata B1 protein

Golden apple snail (Pomacea canaliculata) is a devastating pest on rice that causes heavy economic losses in South East Asia. In this study, we transformed mature seed-derived rice callus with plasmid containing Oldenlandia affinis kalata B1 (Oak1) gene encoding precursor's protein for potent molluscicidal agent of cyclotidekalata B1 targeting the golden apple snails. A total of 11 independent T0 transformants were recovered and 7 were positive Oak1transformants according to genomic PCR analysis. The Oak1 mRNA transcript was successfully detected on all the tested T0 transformants using real-time PCR. Further immunoprecipitation experiment using specific Oak1 antibodies confirmed the presence of Oak1 protein expression in the transformants. We report, for the first time, the generation of transgenic rice plants expressing Oak1 as a potential crop protection strategy against the golden apple snail pest

Heat and hydrolytic enzymes treatment improved the Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.)

Reported Agrobacterium transformation efficiency of indica rice shoot apices varied from 0.4 to 13.8 %. In order to improve the transformation efficiency, modification of transformation protocols through heat and hydrolytic enzyme treatments on rice shoot apices were carried out. Transient expression study using reporter genes revealed that shoot apices heat treated for 3 min at 42 °C during bacterial immersion showed improved GFP (63.0 %) and GUS (42.5 %) expressions per plant as compared to standard protocol (34.0 % GFP and 36.25 % GUS). Shoot apices pre-treated with hydrolytic enzymes containing macerase, pectinase and cellulase at concentration ratio of 1:1:1 (w/v) also demonstrated high percentage of transient GFP (40.0 %) and GUS (35.0 %) expressions per plant. PCR analyses further confirmed the presence of GFP and GUS genes in the transformants. Stable expressions of GFP and GUS were also obtained in multiple shoots of regenerated shoot apices after 4 weeks of culturing in shoot proliferation media without hygromycin. In conclusion, the transformation efficiencies were improved significantly when heat (15.83 %) and hydrolytic enzymes (16.67 %) were applied as individual treatments as compared to the standard transformation method which only accounted for 5.83 %

MicroRNAs in Bone Diseases: Progress and Prospects

With 19–25 nucleotides long, microRNAs (miRNAs) are small noncoding RNA molecules which play crucial roles in major cellular functions such as cell cycle control, apoptosis, metabolism, cell proliferation, and cell differentiation. Changes in the expression of miRNAs can cause significant effects to normal and aberrant cells. The dysregulation of miRNAs has been implicated in various human diseases such as brain tumor, osteoarthritis, schizophrenia, and breast cancer. Generally, miRNAs negatively regulate gene expression by binding to their specific mRNAs, thereby blocking their translation of the mRNAs. However, a few studies have reported that miRNAs could also upregulate the translation of certain proteins. This shows the important roles of miRNAs in various cell functions. This chapter will focus on the role of miRNAs in normal osteoblast and osteosarcoma cells. In addition, the great potential of miRNA as a new therapeutic approach to treat human bone diseases will also be discussed

A Review on Induced Mutagenesis of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni in the Asteraceae family is commercially valuable and cultivated throughout the world due to the great demand for its steviol glycosides (SGs) contents particularly rebaudioside A. Previous studies confirmed that maximal content of SGs in stevia was achieved at or just before flowering, and delayed flowering with long days provide longer duration for steviol glycosides accumulation. However, there is no suitable stevia variety to be cultivated in Malaysia due to her short day length. Mutation induction, including gamma irradiation, had been shown to be useful for generating genetic variations as well as developing new plant varieties from which desired mutants were successfully selected. The use of mutagens, both physical and chemical, has helped in creating mutants that expressed the selected desirable traits. This paper presents some selected essential data available in extant scientific studies on stevia with the focus on application of gamma irradiation on stevia. Both established achievements and recent publications of gamma radiation on stevia were reviewed. Emphasis is on the exceptional potential of stevia through induced mutation approach especially by using gamma rays