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Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely used as a gene activation system exploiting its ability in recruiting various transcriptional activation domains (TADs) for enhancing expression of the target gene(s). Drought tolerant and resource-efficient crops like millets have the potential for mitigating effects of climate change and for enhancing food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence in the identification of a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring water logging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer water logging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome for the identification and characterization of the ortholog from Setaria italica. Soft berry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a useful example of how to advance in genomics research including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species, through the utilization of the available genome sequence.

Rhizoctonia is a widespread soilborne fungus in different agroecosystems worldwide. It has been reported as a destructive fungal pathogen that caused various types of diseases on a wide variety of crops. The aim of this study was to characterize Rhizoctonia spp. isolated from various crops. We obtained 37 Rhizoctonia isolates from rice, corn, spinach, chrysanthemum, and chili grown in various locations in Peninsular Malaysia. A total of 35 isolates were classified under Rhizoctonia solani (multinucleate) and two isolates belonged to binucleate Rhizoctonia (BNR) according to the number of nuclei and morphological characteristics. Based on rDNA-ITS sequences and PCR profiling using AG group-specific primers, these isolates were identified as R. solani AG-1 IA (75.6%), AG-1 ID (10.8%), AG-2-2 IV (2.7%), AG-4 HG-I (5.4%), BNR AG-Fa (2.7%), and AG-A (2.7%). A specific primer of binucleate Rhizoctonia AG-Fa was designed and its specificity was evaluated by PCR. Further investigation using tef-1α gene confirmed the AG group identities for these isolates. Clustering analysis of molecular markers (OPA13 and OPE-6, 2080 iPBS, and 2249 iPBS and ISSR 3) supported the AG concept rather than geographical origin or plant host. Pathogenicity tests on the leaves of rice and corn showed that R. solani AG-1 IA rice isolates cause necrotic lesions greater than AG-1 IA corn isolates. Rhizoctonia solani AG-1 IA, AG-2-2IV, and AG-4 HG-I isolates cause necrotic lesions greater than BNR isolates on seedling of cabbage, cauliflower, and radish. To the best of our knowledge, this is the first study on morphology and molecular characterization of Rhizoctonia spp. isolated from various crops and regions in Malaysia.

The production of alkaloids in the intact plant is usually low, and some of them are tissue-specific and growth stage–specific. In this study, the total alkaloid content (TAC) in Polyalthia bullata was determined using different extraction solvents, and the extracts were subjected to LC-MS analysis. The manipulation of alkaloid production in P. bullata callus was further carried out by growing the callus on two basal media (Murashige and Skoog (MS) and Woody Plant Medium (WPM)) containing auxins (2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)), precursors (L-phenylalanine, L-tyrosine, and L-tryptophan), and elicitors (methyl jasmonate (MeJA), salicylic acid (SA), and chitosan). The methanolic extracts exhibited the highest alkaloid content in leaf, stem, and root tissues (4.61 ± 0.04 to 7.71 ± 0.04 mg/g DW), and nine alkaloids were detected in P. bullata. The callus grown on MS + 30 μM 2,4-D exhibited the highest alkaloid accumulation (31.07 ± 0.05 μg/mg DW) after 6 weeks of culture compared to other media: MS media containing auxins, MS media containing auxins and precursors, and MS media containing auxins and elicitors. Therefore, we conclude that the MS + 30 μM 2,4-D is potent in enhancing alkaloid production in P. bullata callus.

Polyalthia bullata is a woody medicinal plant that contains antioxidant compounds. Finding a suitable solvent is important to obtain a high yield of antioxidants in the phenolic, flavonoid, and terpenoid families. In this study, from different solvent extracts, the leaf methanolic extract exhibited the highest total phenolic content (TPC), total flavonoid content (TFC), total terpenoid content (TTC), and total antioxidant activity. For woody parts of stem and roots, methanol was the best solvent for all phytochemicals except for phenolics, which accumulated in the roots and were extracted more efficiently using ethanol. However, the methanolic extracts from both tissues displayed the best antioxidant capacity. Gas chromatography-mass spectrometry (GC-MS) profiling data showed the presence of antioxidant compounds such as thymol, phytol, and neophytadiene in the leaf; trans-farnesol, n-hexadecanoic acid, and 9-Octadecenamide in the stem; and fatty acid (cis-vaccenic) and its methyl ester (11-Octadecanoic acid, methyl ester and [1,1’-bicyclopropyl]-2-octanoic acid, 2’-hexyl-methyl ester) in the roots. These findings reveal important compounds that are present in different plant parts of P. bullata.

An approach to metabolomics profiling of non-infected and Ganoderma boninense (G. boninsense) infected oil palm roots crude extracts that utilize gas chromatography-mass spectrometry (GC–MS) and multivariate statistics of principal component analysis (PCA) have been tested. This combination has provided a rapid approach in investigating the changes in the metabolite variations of non-infected and infected oil palm roots at 14 and 30 days post-infection. The extracts were prepared by using 80% (v/v) of methanol. In identifying the metabolites responsible for each differentiation, PCA model was generated in loading bi-plot. Dimethyl benzene-1,4-dicarboxylate, methyl 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate, ergost-5-en-3-ol, (3β), stigmast-5-en-3-ol, (3β), stigmasterol, methyl hexadecanoate, methyl (9Z,12Z)-octadeca-9,12-dienoate, methyl octadecanoate, 2-(hydroxymethyl)-2-nitropropane-1,3-diol, methyl (Z)-octadec-6-enoate and (E)-icos-5-ene were found more abundant in G. boninense infected roots than in non-infected roots. Steroidal compounds and fatty acid derivatives which has been determined in the non-infected and G. boninense infected roots regulate a variety of responses to the G. boninense. The abundant of these metabolites in G. boninense infected roots are due to the crucial roles in pathogen defence.

Drought-responsive element binding (DREB) is involved in the regulation of stress-responsive gene expressions in plants through abscisic acid (ABA)-independent pathway. In this study, constitutive expression of oil palm (Elaeis guineensis) EgDREB1 driven by double strength cauliflower mosaic virus 35S promoter in tomato (Solanum lycopersicum) reduced seed number, produced parthenocarpic fruits, changed morphology of leaves, and increased root biomass of transgenic plants. Early flowering and fruiting of the transgenic lines were observed in the culture vessels. EgDREB1 was specifically expressed in the fruits and its expression was not detected in vegetative tissues (leaves and roots). Altered expression of several endogenous tomato genes involved in the biosynthesis of phytohormones including jasmonic acid, ethylene, auxin, cytokinin, gibberellin (GA) and ABA were observed compared to wild type plants. The expression of AP2-like-ethylene transcription factor (LeAP2), allene oxide synthase (LeAOS), allene oxide cyclase (LeAOC), aminocyclopropane-1-carboxylic acid synthase (LeACS), 1-aminocyclopropane-1-carboxylate oxidase 1 (LeACO), auxin responsive factor 8 (LeARF8), auxin/indole-3-acetic acid (LeAux/IAA), cytokinin oxidase/dehydrogenase-like (LeSlCKX1), adenylate isopentenyltransferase (LeSlIPT1), gibberellin 2-oxidase 2 (LeGa2ox2), gibberellin 20-oxidase 4 (LeGa20ox4) and ABA-aldehyde oxidase (LeAAO) were different in fruits with reduced seed number compared to parthenocarphic fruits. These results suggest that their expression has significant effects on fruit development in transgenic tomato. EgDREB1 may mediate the expression of some of these genes as dehydration-responsive element binding (DRE) motif were found in their promoter sequences. These data indicate that the EgDREB1 controls fruit development in trsngenic plants by regulating the expression of hormone-associated genes.

Phosphate (Pi) fertilizer is applied in huge amount due to the poor absorption of Pi by crops especially in acidic tropical soil. As the major global source of oil and fats, the mechanism of Pi transport in oil palm needs to be studied to maintain good productivity for sustainable palm oil production. Ten Elaeis guineensis Phosphate Transporter1 (EgPHT1) and three Elaeis guineensis Phosphate Starvation Response (EgPHR) genes were identified in this study. All EgPHT1 proteins contain GGDYPLSATIxSE, the signature sequence of PHT1. All EgPHR have MYB-binding domain and coiled-coil domain characteristic of PHR at their C-terminal regions and one unique SOG2 domain for EgPHR1. The expression of four of the EgPHT1 and two of the EgPHR under low Pi (LP) and Pi starvation (-P) was studied by real-time quantitative PCR (qPCR). All genes showed enhanced expression in roots at −P compared to +P but no detectable change in the leaves. The expression profile of EgPHR2 which showed significant upregulation at LP compared to +P and further increase at −P correlated with EgPHT1;4 and EgPHT1;7 that possess P1BS motif in their promoter sequences, the binding site for PHR. PHR2, as a potential early transcriptional regulator for phosphate starvation was proven to be nuclear localized by subcellular localization experiment. Altogether, this study suggests all four analyzed EgPHT1 and two EgPHR play critical role in responding to Pi deprivation in oil palm. EgPHT1;4 and EgPHT1;7 which possess the P1BS motif are potentially upregulated by EgPHR2 as an early response mechanism against phosphate starvation.

Inorganic phosphate (Pi) starvation is an important abiotic constraint that affects plant cellular homeostasis, especially in tropical regions with high acidic soil and less solubilizable Pi. In the current work, oil palm seedlings were hydroponically maintained under optimal Pi-supply and no Pi-supply conditions for 14 days, and metabolites were measured by gas chromatography–mass spectrometry (GC–MS), from leaves and roots, after seven and 14 days of treatment, to investigate biochemical pathways in relation to P-utilizing strategy. After seven days of limited Pi, plant leaves showed increased levels of most soluble sugars, and after 14 days, the sugars’ level decrease, except for erythritol, mannose, fructose, and glucose, which showed the highest levels. Rather in root samples, there were different but overlapping alterations, mainly on sugars, amino acids, and organic acids. The leaf sample was shown to have the highest response of sugars with myo-inositol playing a vital role in the redistribution of sugars, while maltose levels increased, indicating active degradation of starch in the root. High levels of glycerol and stearate in both roots and leaves suggest the metabolism of storage lipids for cellular energy during Pi-deficient conditions.

Local government (LG) is an agency that has been empowered through legal provisions to impose the assessment tax. Thus, it directly causes the affairs of tax administration to be parked under the scope and jurisdiction of the LGs. Assessment tax arrear is a long-standing issue that has been faced by the LGs. From time to time, the crisis of assessment tax arrears is considered as getting worse and it requires various proactive actions to be taken including re-evaluating the current administrative process of assessment tax as it is practicising by the LGs. The main purpose of this study is to identify the factors which related to tax administration that has influenced the existence of assessment tax arrears based on the perspective of taxpayers. This study also aims to assess the strength of the relationship between each of the factors involved. The required data were collected through a survey method and a set of related questionnaire form was used as a research instrument. The respondents of this study are consisting of property owners who are subject to pay the assessment tax. In overall, this study has involved 261 respondents. The data collected were analyzed using descriptive and correlation methods. The findings of the study show that there are 8 administrative related factors that have influenced the existence of assessment tax arrears, namely taxation equality and justice, tax beliefs, tax expertise, staff adequacy, payment facility, transparency of services, tax incentives and tax communication. Through this study as well, it was found all the factors have a significant relationship with most of other factors.

Additional file 3:. Electrophoretic mobility shift assay (EMSA) of EgERF113 with DRE/CRT probe. Lane 1 to 3 consist of EBNA control system. Lane 4 to 8 consist of EgERF113 test system. Lane 1 and 8 are the blank for EBNA and EgERF113 systems, respectively. Lane 2 is the positive control for EMSA. EMSA shows direct binding of EgERF113 to DRE/CRT probe in lane 6. EgERF113 is unable to bind to untransformed yeast and biotinylated mutant DRE/CRT (mDRE/CRT) probe in lane 4 and 5, respectively. Successful binding of 200-fold molar excess of unlabelled DRE/CRT (competitor) probe is shown in lane 7.

Additional file 1:. Electrophoretic mobility shift assay (EMSA) of EgJUB1 with SNBE1 probe. Lane 1 to 3 consist of EBNA control system. Lane 4 to 8 consist of EgJUB1 test system. Lane 1 and 8 are the blank for EBNA and EgJUB1 systems, respectively. Lane 2 is the positive control for EMSA. EMSA shows direct binding of EgJUB1 to SNBE1 probe in lane 6. EgJUB1 is unable to bind to untransformed yeast and biotinylated mutant SNBE1 (mSNBE1) probe in lane 4 and 5, respectively. Successful binding of 200-fold molar excess of unlabelled SNBE1 (competitor) probe is shown in lane 7.

BackgroundHemibiotrophic pathogen such as the fungal pathogenGanoderma boninensethat is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection.ResultsThe present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings againstG. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation ofJUNGBRUNNEN 1(EgJUB1) during the fungal biotrophic phase whileEthylene Responsive Factor 113(EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells.ConclusionOur findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

Additional file 2:. Electrophoretic mobility shift assay (EMSA) of EgERF113 with GCC-box probe. Lane 1 to 3 consist of EBNA control system. Lanes 4 to 8 consist of EgERF113 test system. Lane 1 and 8 are the blank for EBNA and EgERF113 systems, respectively. Lane 2 is the positive control for EMSA. EMSA shows direct binding of EgERF113 to GCC-box probe in lane 6. EgERF113 is unable to bind to untransformed yeast and biotinylated mutant GCC-box (mGCC-box) probe in lane 4 and 5, respectively. Successful binding of 200-fold molar excess of unlabelled GCC-box (competitor) probe is shown in lane 7.

Background Phosphorus (P), in its orthophosphate form (Pi) is an essential macronutrient for oil palm early growth development in which Pi deficiency could later on be reflected in lower biomass production. Application of phosphate rock, a non-renewable resource has been the common practice to increase Pi accessibility and maintain crop productivity in Malaysia. However, high fixation rate of Pi in the native acidic tropical soils has led to excessive utilization of P fertilizers. This has caused serious environmental pollutions and cost increment. Even so, the Pi deficiency response mechanism in oil palm as one of the basic prerequisites for crop improvement remains largely unknown. Results Using total RNA extracted from young roots as template, we performed a comparative transcriptome analysis on oil palm responding to 14d and 28d of Pi deprivation treatment and under adequate Pi supply. By using Illumina HiSeq4000 platform, RNA-Seq analysis was successfully conducted on 12 paired-end RNA-Seq libraries and generated more than 1.2 billion of clean reads in total. Transcript abundance estimated by fragments per kilobase per million fragments (FPKM) and differential expression analysis revealed 36 and 252 genes that are differentially regulated in Pi-starved roots at 14d and 28d, respectively. Genes possibly involved in regulating Pi homeostasis, nutrient uptake and transport, hormonal signaling and gene transcription were found among the differentially expressed genes. Conclusions Our results showed that the molecular response mechanism underlying Pi starvation in oil palm is complexed and involved multilevel regulation of various sensing and signaling components. This contribution would generate valuable genomic resources in the effort to develop oil palm planting materials that possess Pi-use efficient trait through molecular manipulation and breeding programs.

Background Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at later stage of infection. Results The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data resulting in 106 upregulated and 108 downregulated TFs being identified. DEGs involved in four established defense-related pathways are presented which are responsible in cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear localized based on subcellular localization experiment using onion epidermal cells. Conclusion Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose EgJUB1 and EgERF113 as key TFs in orchestrating the defense mechanisms during biotrophic and during the subsequent transition to necrotrophic phase, respectively. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

Basal stem rot (BSR) caused by Ganoderma boninense is a major threat to sustainable oil palm production especially in Southeast Asia and has brought economic losses to the oil palm industry around the world. With no definitive cure at present, this study introduces a new fertilizer technology called GanoCare®, as an effort to suppress BSR incidence in oil palm. Experiments were carried out to evaluate the effect of GanoCare® on growth, physiology, and BSR disease suppression using sitting technique in the oil palm nursery stage. A follow-up using similar treatments was carried out in the field to test on severity of Ganoderma using baiting technique under natural condition. Treatments tested were 10 g/month and 30 g/three months given as pretreatment only or continuous treatment. Results showed that GanoCare® increased the height, bulb diameter, leaf area, chlorophyll content, photosynthesis rate, and fresh and dry weight of the leaf, bole, and root of oil palm seedlings in the nursery trial. Seedlings treated with GanoCare® exhibited reduced percentage of disease severity, incidence, and dead seedlings, compared to the control. In nursery and field, lowest percentage of dead seedlings due to Ganoderma was found in seedlings given combination of pretreatment and continuous treatment of 30 g/three months (T4) with 5.56 and 6.67%, while control seedlings significantly marked the maximum percentage of 94.45 and 93.33%. The most successful treatment in both nursery and field was T4 with disease reductions of 77.78 and 82.36%, respectively, proving that nutrients contained in GanoCare® are essential in allowing better development of a strong defense system in the seedlings.

Tocotrienols are forms of vitamin E that are present in several important food crops. Compared to tocopherols, less research has been conducted on these compounds because of their low bioavailability and distribution in plant tissues. Both tocotrienols and tocopherols are known for their antioxidant and anticancer activities, which are beneficial for both humans and animals. Moreover, tocotrienols possess certain properties which are not found in tocopherols, such as neuroprotective and cholesterol-lowering activities. The contents of tocotrienols in plants vary. Tocotrienols constitute more than 70% and tocopherols less than 30% of the total vitamin E content in palm oil, which is the best source of vitamin E. Accumulation of tocotrienols also occurs in non-photosynthetic tissues, such as the seeds, fruits and latex of some monocotyledonous and dicotyledonous plant species. The use of biotechnological techniques to increase the tocotrienol content in plants, their biological functions, and benefits to human health are discussed in this review.

Extraction of RNA of high quality and integrity is essential for gene expression studies and all downstream RNA-based techniques. The leaves of 16 merit Malaysian rice varieties were used to isolate total RNA using five different methods. The quantity, quality and integrity of extracted RNA were confirmed using three different means. The ratios of A260/280 ranged from 2.12 to 2.20. Electrophoresis (1.5% agarose gel) was performed, illustrating intact and sharp bands representing the 28S, 18S, 5.8S and 5S ribosomal subunits of RNA, presenting intact RNA. RNA quality was verified using semi-quantitative polymerase chain reaction (sqPCR). The objective of this study was to identify different genes involved in the resistance of rice plants using high-quality RNA extracted 31 h after inoculation of Magnaporthe oryzae pathotype P7.2. The expression levels of eight blast resistance genes, Pikh, Pib, Pita, Pi21, Pi9, Os11gRGA8, OsWRKY22 and OsWRKY45, were evaluated by real-time PCR (RT-PCR). Real-time PCR was performed to identify candidate genes using RNA extracted by the TRIzol method, which showed the highest score compared with other methods in terms of RNA quantity, purity and integrity. In addition, the results of real-time PCR confirmed that the up-regulation of seven blast resistance genes may confer stronger resistance for the MR 276 variety against M. oryzae pathotype P7.2.

Ganoderma boninense (G. boninense) has been identified as a major problem in oil palm industry which caused basal stem rot disease. Identification of metabolite variation of healthy and G. boninense-infected oil palm leaves at 14 days postinfection using NMR metabolomics approach followed by characterization of an electrochemical sensor based on a functionalized multiwalled carbon nanotube (MWCNT) layer-by-layer framework on modified screen-printed carbon electrode has been successfully determined. Significant differences from the 1H NMR data were observed between healthy and G. boninense-infected oil palm leaves, according to principal component analysis. Gold nanoparticle-functionalized MWCNT and chitosan-functionalized MWCNT were deposited on a screen-printed carbon electrode and were applied for the electrochemical detection of healthy and G. boninense-infected oil palm leaves. The electrocatalytic activities of a modified electrode towards oxidation of healthy and G. boninense-infected oil palm leaves at a concentration of 100 mg/L were evaluated using cyclic voltammetry and linear sweep voltammetry. The limits of detection of healthy and G. boninense-infected oil palm leaves were calculated to 0.0765 mg/L and 0.0414 mg/L, respectively. The modified electrode shows a good sensitivity and reproducibility due to the unique characteristics of gold nanoparticles, chitosan, MWCNTs, and synergistic interaction between them.

Salinization process occurs when soil is contaminated with salt, which consequently influences plant growth and development leading to reduction in yield of many food crops. Responding to a higher salt concentration than the normal range can result in plant developing complex physiological traits and activation of stress-related genes and metabolic pathways. Many studies have been carried out by different research groups to understand adaptive mechanism in many plant species towards salinity stress. However, different methods of sodium chloride (NaCl) applications definitely give different responses and adaptive mechanisms towards the increase in salinity. Gradual increase in NaCl application causes the plant to have salt stress or osmotic stress, while single step and high concentration of NaCl may result in salt shock or osmotic shock. Osmotic shock can cause cell plasmolysis and leakage of osmolytes in plant. Also, the gene expression pattern is influenced by the type of methods used in increasing the salinity. Therefore, this chapter discusses the adaptive mechanism in plant responding to both types of salinity increment, which include the morphological changes of plant roots and aerial parts, involvement of signalling molecules in stress perception and regulatory networks and production of osmolyte and osmoprotective proteins.

Soil salinity is one of the main factors restricting crop production throughout the world. Various salt tolerance traits and the genes controlling these traits are responsible for coping with salinity stress in plants. These coping mechanisms include osmotic tolerance, ion exclusion, and tissue tolerance. Plants exposed to salinity stress sense the stress conditions, convey specific stimuli signals, and initiate responses against stress through the activation of tolerance mechanisms that include multiple genes and pathways. Advances in our understanding of the genetic responses of plants to salinity and their connections with yield improvement are essential for attaining sustainable agriculture. Although a wide range of studies have been conducted that demonstrate genetic variations in response to salinity stress, numerous questions need to be answered to fully understand plant tolerance to salt stress. This chapter provides an overview of previous studies on the genetic control of salinity stress in plants, including signaling, tolerance mechanisms, and the genes, pathways, and epigenetic regulators necessary for plant salinity tolerance.

CBF/DREB1 is a group of transcription factors that are mainly involved in abiotic stress tolerance in plants. They belong to the AP2/ERF superfamily of plant-specific transcription factors. A gene encoding a new member of this group was isolated from ripening oil palm fruit and designated as EgCBF3. The oil palm fruit demonstrates the characteristics of a climacteric fruit like tomato, in which ethylene has a major impact on the ripening process. A transgenic approach was used for functional characterization of the EgCBF3, using tomato as the model plant. The effects of ectopic expression of EgCBF3 were analyzed based on expression profiling of the ethylene biosynthesis-related genes, anti-freeze proteins (AFPs), abiotic stress tolerance and plant growth and development. The EgCBF3 tomatoes demonstrated altered phenotypes compared to the wild type tomatoes. Delayed leaf senescence and flowering, increased chlorophyll content and abnormal flowering were the consequences of overexpression of EgCBF3 in the transgenic tomatoes. The EgCBF3 tomatoes demonstrated enhanced abiotic stress tolerance under in vitro conditions. Further, transcript levels of ethylene biosynthesis-related genes, including three SlACSs and two SlACOs, were altered in the transgenic plants' leaves and roots compared to that in the wild type tomato plant. Among the eight AFPs studied in the wounded leaves of the EgCBF3 tomato plants, transcript levels of SlOSM-L, SlNP24, SlPR5L and SlTSRF1 decreased, while expression of the other four, SlCHI3, SlPR1, SlPR-P2 and SlLAP2, were up-regulated. These findings indicate the possible functions of EgCBF3 in plant growth and development as a regulator of ethylene biosynthesis-related and AFP genes, and as a stimulator of abiotic stress tolerance.

Homogentisate geranylgeranyl transferase (HGGT) and homogentisate phytyltransferase (HPT) are the two key enzymes involved in condensation of homogentisic acid (HGA) with a prenyldiphosphate to produce tocotrienols and tocopherols in plants, respectively. The partial cDNAs encoding HGGT and HPT enzymes were successfully isolated from the two oil palm species, Elaeis guineensis and Elaeis oleifera by PCR amplification using degenerate primers. Subsequently, full length cDNA sequences were completed by rapid amplification of cDNA ends (RACE) and further annotated using various bioinformatics tools. The analysis revealed the presence of an UbiA prenyltransferase conserved domain in all four deduced amino acid sequences and suggested that oil palm HGGT and HPT are more evolutionarily related to their counterparts from other monocot plant species. Quantitative gene expression analysis was carried out to elucidate the transcript profiles of the oil palm HGGT and HPT in different oil palm tissues and at different developmental stages of the mesocarp. The HPT was constitutively expressed in all analyzed tissues except in 15w.a.a kernel whereas oil palm HGGT showed preferential expression in mesocarp and kernel tissues. However, HPT was highly expressed at the fruit ripening stage of 17w.a.a mesocarp when active oil deposition occurs. Genome-walking PCR successfully amplified the promoter regions of HGGT and HPT from E. guineensis. Computational analysis using PlantCare and PLACE databases revealed several cis-regulatory elements including phytohormone-responsive, light-responsive and abiotic factor-responsive elements which may be involved in coordinating expression of both genes. Taken together, this study provides useful information about important features of the cDNA and promoter sequences as well as an insight into the transcriptional regulation of these key vitamin E genes for future genetic improvement efforts.

Imidazolinone-herbicide-resistant Clearfield® (CL) rice permits the selective chemical control of weedy rice (Oryza sativa), a major weed problem in South-East Asian rice growing countries. However, there is major concern involving resistant individuals resulting from gene flow as the cultivated and weedy rice live side by side in the fields. An experiment was conducted in the rice fields of Kuala Rompin, Pahang, Malaysia to determine which Clearfield® rice cultivars and weedy rice cultivars are more prone to hybridization, and the effect on distance between the pollen donor and receptor plants. The experiment was piloted in a split plot design with four replications. Encircled population technique was used to determine the distance between the Clearfield® rice and detection of hybrids (F1). Resistance of progeny was determined after spraying with OnDuty™ and the confirmation of hybrids was done using the SSR primer RM251. Higher survival rate was recorded with cv. CL2 which was significantly different from cv. CL1. Weedy rice cultivar V1 and V2 in CL1 plots differed significantly from the same cultivar from CL2 plots. However, no significant difference was observed between weedy rice cultivars of V3 and V4, either in CL1 or CL2 plots. No survivors were found after second spraying. Suspected hybrids were found up to 5 m however the rate was much lower compared to only 1 m from the CL plots.

TAAAAT and a novel motif, GCTTCA found in the oil palm stearoyl-ACP desaturase (SAD1) promoter are involved in regulating mesocarp-specific expression. Two key fatty acid biosynthetic genes, stearoyl-ACP desaturase (SAD1), and acyl-carrier protein (ACP3) in Elaeis guineensis (oil palm) showed high level of expression during the period of oil synthesis in the mesocarp [12–19 weeks after anthesis (w.a.a.)] and kernel (12–15 w.a.a.). Both genes are expressed in spear leaves at much lower levels and the expression increased by 1.5-fold to 2.5-fold following treatments with ethylene and abscisic acid (ABA). Both SAD1 and ACP3 promoters contain phytohormone-responsive, light-responsive, abiotic factors/wounding-responsive, endosperm specificity and fruit maturation/ripening regulatory motifs. The activities of the full length and six 5′ deletion fragments of the SAD1 promoter were analyzed in transiently transformed oil palm tissues by quantitative β-glucuronidase (GUS) fluorometric assay. The highest SAD1 promoter activity was observed in the mesocarp followed by kernel and the least in the leaves. GUS activity in the D3 deletion construct (− 486 to + 108) was the highest, while the D2 (− 535 to + 108) gave the lowest suggesting the presence of negative cis-acting regulatory element(s) in the deleted − 535 to − 486 (49 bp). It was found that the 49-bp region binds to the nuclear protein extract from mesocarp but not from leaves in electrophoretic mobility shift assay (EMSA). Further fine-tuned analysis of this 49-bp region using truncated DNA led to the identification of GCTTCA as a novel motif in the SAD1 promoter. Interestingly, another known fruit ripening-related motif, LECPLEACS2 (TAAAAT) was found to be required for effective binding of the novel motif to the mesocarp nuclear protein extract.

Balanced nutrient elements in fertilizer play a critical role in oil palm seedling successful growth and development, and at the same time reduces of fertilizer losses in the environment. This study examines the effect of different levels of N, P2O5 and K2O for oil palm seedlings in solution culture on growth traits, nutrient uptake in plant tissues and biomass accumulation under nursery conditions. Five concentration levels of N (50, 100, 300, 600 and 900 mg L–1), P2O5 and K2O (15, 30, 60, 90 and 120 mg L–1) were used in a completely randomized design (CRD) with five replications for each. Parameters measured during the growing period include - plant height, leaf number/plant, stem diameter, SPAD chlorophyll value, and at harvest – total leaf area, root dry weight, shoot dry weight and total dry weight. Different levels of N, P2O5 and K2O showed significant effects on all the parameters studied. The highest values for diameter, plant height, leaf number/plant, total leaf area, root dry weight, shoot dry weight and total dry biomass were obtained using 100, 90 and 300 mg/L levels of N, P2O5 and K2O, respectively. Most of the growth parameters, declined with lower levels of N, P2O5 and K2O. The results of this study provide a new knowledge to produce oil palm plant with better nutrient management at the nursery under solution culture.

In the present study, we have reported a simple, fast and efficient regeneration protocol using mature embryos as explants, and discovered its effective applicability to a range of Indica rice genotypes. We have considered the response of six varieties in the steps of the regeneration procedure. The results showed that calli were variably developed from the scutellar region of seeds and visible within 6-20 days. The highest and lowest calli induction frequency (70% and 51.66%) and number of induced calli from seeds (14 and 10.33) were observed in MR269 and MRQ74, respectively. The maximum and minimum number (7.66 and 4) and frequency of embryogenic calli (38.33% and 20%) were recorded in MR219 and MRQ74, respectively. However, the highest browning rate was observed in MR84 (87%) and the lowest rate in MRQ50 (46%). The majority of plants regenerated from embryogenic calli were obtained from MRQ50 (54%) and the minimum number of plants from MR84. In this study, the maximum numbers of plantlets were regenerated from the varieties with highest rate of embryogenic calli. Also, various varieties, including MRQ50, MR269, MR276 and MR219, were satisfactorily responding, while MRQ74 and MR84 weakly responded to the procedure. Such a simple, successful and generalized method possesses the potential to become an important tool for crop improvement and functional studies of genes in rice as a model monocot plant.

An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.