Epigenetic changes and their relationship to somaclonal variation: a need to monitor the micropropagation of plantation crops.

Chromatin modulation plays important roles in gene expression regulation and genome activities. In plants, epigenetic changes, including variations in histone modification and DNA methylation, are linked to alterations in gene expression. Despite the significance and potential of in vitro cell and tissue culture systems in fundamental research and marketable applications, these systems threaten the genetic and epigenetic networks of intact plant organs and tissues. Cell and tissue culture applications can lead to DNA variations, methylation alterations, transposon activation, and finally, somaclonal variations. In this review, we discuss the status of the current understanding of epigenomic changes that occur under in vitro conditions in plantation crops, including coconut, oil palm, rubber, cotton, coffee and tea. It is hoped that comprehensive knowledge of the molecular basis of these epigenomic variations will help researchers develop strategies to enhance the totipotent and embryogenic capabilities of tissue culture systems for plantation crops.

Improvement of Drought Tolerance in Rice (Oryza sativa L.): Genetics, Genomic Tools, and the WRKY Gene Family.

Drought tolerance is an important quantitative trait with multipart phenotypes that are often further complicated by plant phenology. Different types of environmental stresses, such as high irradiance, high temperatures, nutrient deficiencies, and toxicities, may challenge crops simultaneously; therefore, breeding for drought tolerance is very complicated. Interdisciplinary researchers have been attempting to dissect and comprehend the mechanisms of plant tolerance to drought stress using various methods; however, the limited success of molecular breeding and physiological approaches suggests that we rethink our strategies. Recent genetic techniques and genomics tools coupled with advances in breeding methodologies and precise phenotyping will likely reveal candidate genes and metabolic pathways underlying drought tolerance in crops. The WRKY transcription factors are involved in different biological processes in plant development. This zinc (Zn) finger protein family, particularly members that respond to and mediate stress responses, is exclusively found in plants. A total of 89 WRKY genes in japonica and 97 WRKY genes in O. nivara (OnWRKY) have been identified and mapped onto individual chromosomes. To increase the drought tolerance of rice (Oryza sativa L.), research programs should address the problem using a multidisciplinary strategy, including the interaction of plant phenology and multiple stresses, and the combination of drought tolerance traits with different genetic and genomics approaches, such as microarrays, quantitative trait loci (QTLs), WRKY gene family members with roles in drought tolerance, and transgenic crops. This review discusses the newest advances in plant physiology for the exact phenotyping of plant responses to drought to update methods of analysing drought tolerance in rice. Finally, based on the physiological/morphological and molecular mechanisms found in resistant parent lines, a strategy is suggested to select a particular environment and adapt suitable germplasm to that environment.

Antioxidant Enzyme Activities and Secondary Metabolite Profiling of Oil Palm Seedlings Treated with Combination of NPK Fertilizers Infected with Ganoderma boninense.

Oil palm (Elaeis guineensis Jacq) is one of the major sources of edible oil. Reducing the effect of Ganoderma, main cause of basal stem rot (BSR) on oil palm, is the main propose of this study. Understanding the oil palm defense mechanism against Ganoderma infection through monitoring changes in the secondary metabolite compounds levels before/after infection by Ganoderma under different fertilizing treatment is required. Oil palm requires macro- and microelements for growth and yield. Manipulating the nutrient for oil palm is a method to control the disease. The 3-4-month-old oil palm seedlings were given different macronutrient treatments to evaluate induction of defense related enzymes and production of secondary metabolite compounds in response to G. boninense inoculation. The observed trend of changes in the infected and uninfected seedlings was a slightly higher activity for ß-1,3-glucanases, chitinase, peroxidase, and phenylalanine ammonia-lyase during the process of pathogenesis. It was found that PR proteins gave positive response to the interaction between oil palm seedlings and Ganoderma infection. Although the responses were activated systematically, they were short-lasting as the changes in enzymes activities appeared before the occurrence of visible symptoms. Effect of different nutrients doses was obviously observed among the results of the secondary metabolite compounds. Many identified/unidentified metabolite compounds were presented, of which some were involved in plant cell defense mechanism against pathogens, mostly belonging to alkaloids with bitter-tasting nitrogenous-compounds, and some had the potential to be used as new markers to detect basal stem rot at the initial step of disease.

Contribution of transposable elements in the plant's genome.

Plants maintain extensive growth flexibility under different environmental conditions, allowing them to continuously and rapidly adapt to alterations in their environment. A large portion of many plant genomes consists of transposable elements (TEs) that create new genetic variations within plant species. Different types of mutations may be created by TEs in plants. Many TEs can avoid the host's defense mechanisms and survive alterations in transposition activity, internal sequence and target site. Thus, plant genomes are expected to utilize a variety of mechanisms to tolerate TEs that are near or within genes. TEs affect the expression of not only nearby genes but also unlinked inserted genes. TEs can create new promoters, leading to novel expression patterns or alternative coding regions to generate alternate transcripts in plant species. TEs can also provide novel cis-acting regulatory elements that act as enhancers or inserts within original enhancers that are required for transcription. Thus, the regulation of plant gene expression is strongly managed by the insertion of TEs into nearby genes. TEs can also lead to chromatin modifications and thereby affect gene expression in plants. TEs are able to generate new genes and modify existing gene structures by duplicating, mobilizing and recombining gene fragments. They can also facilitate cellular functions by sharing their transposase-coding regions. Hence, TE insertions can not only act as simple mutagens but can also alter the elementary functions of the plant genome. Here, we review recent discoveries concerning the contribution of TEs to gene expression in plant genomes and discuss the different mechanisms by which TEs can affect plant gene expression and reduce host defense mechanisms.

Screening and Expression of a Silicon Transporter Gene (Lsi1) in Wild-Type Indica Rice Cultivars.

Silicon (Si) is one of the most prevalent elements in the soil. It is beneficial for plant growth and development, and it contributes to plant defense against different stresses. The Lsi1 gene encodes a Si transporter that was identified in a mutant Japonica rice variety. This gene was not identified in fourteen Malaysian rice varieties during screening. Then, a mutant version of Lsi1 was substituted for the native version in the three most common Malaysian rice varieties, MR219, MR220, and MR276, to evaluate the function of the transgene. Real-time PCR was used to explore the differential expression of Lsi1 in the three transgenic rice varieties. Silicon concentrations in the roots and leaves of transgenic plants were significantly higher than in wild-type plants. Transgenic varieties showed significant increases in the activities of the enzymes SOD, POD, APX, and CAT; photosynthesis; and chlorophyll content; however, the highest chlorophyll A and B levels were observed in transgenic MR276. Transgenic varieties have shown a stronger root and leaf structure, as well as hairier roots, compared to the wild-type plants. This suggests that Lsi1 plays a key role in rice, increasing the absorption and accumulation of Si, then alters antioxidant activities, and improves morphological properties.

Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice.

Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48, and 72 h after inoculation in transgenic plants, while it was increased in the inoculated control plants. This study successfully clarified that over-expression of the Pikh gene in transgenic plants can improve their blast resistance against the M. oryzae pathotype P7.2.

Towards understanding pre-mRNA splicing mechanisms and the role of SR proteins.

Alternative pre-mRNA splicing provides a source of vast protein diversity by removing non-coding sequences (introns) and accurately linking different exonic regions in the correct reading frame. The regulation of alternative splicing is essential for various cellular functions in both pathological and physiological conditions. In eukaryotic cells, this process is commonly used to increase proteomic diversity and to control gene expression either co- or post-transcriptionally. Alternative splicing occurs within a megadalton-sized, multi-component machine consisting of RNA and proteins; during the splicing process, this complex undergoes dynamic changes via RNA-RNA, protein-protein and RNA-protein interactions. Co-transcriptional splicing functionally integrates the transcriptional machinery, thereby enabling the two processes to influence one another, whereas post-transcriptional splicing facilitates the coupling of RNA splicing with post-splicing events. This review addresses the structural aspects of spliceosomes and the mechanistic implications of their stepwise assembly on the regulation of pre-mRNA splicing. Moreover, the role of phosphorylation-based, signal-induced changes in the regulation of the splicing process is demonstrated.

Suppression Subtractive Hybridization Versus Next-Generation Sequencing in Plant Genetic Engineering: Challenges and Perspectives.

Suppression subtractive hybridization (SSH) is an effective method to identify different genes with different expression levels involved in a variety of biological processes. This method has often been used to study molecular mechanisms of plants in complex relationships with different pathogens and a variety of biotic stresses. Compared to other techniques used in gene expression profiling, SSH needs relatively smaller amounts of the initial materials, with lower costs, and fewer false positives present within the results. Extraction of total RNA from plant species rich in phenolic compounds, carbohydrates, and polysaccharides that easily bind to nucleic acids through cellular mechanisms is difficult and needs to be considered. Remarkable advancement has been achieved in the next-generation sequencing (NGS) field. As a result of progress within fields related to molecular chemistry and biology as well as specialized engineering, parallelization in the sequencing reaction has exceptionally enhanced the overall read number of generated sequences per run. Currently available sequencing platforms support an earlier unparalleled view directly into complex mixes associated with RNA in addition to DNA samples. NGS technology has demonstrated the ability to sequence DNA with remarkable swiftness, therefore allowing previously unthinkable scientific accomplishments along with novel biological purposes. However, the massive amounts of data generated by NGS impose a substantial challenge with regard to data safe-keeping and analysis. This review examines some simple but vital points involved in preparing the initial material for SSH and introduces this method as well as its associated applications to detect different novel genes from different plant species. This review evaluates general concepts, basic applications, plus the probable results of NGS technology in genomics, with unique mention of feasible potential tools as well as bioinformatics.

Genetic Variation, Heritability, and Diversity Analysis of Upland Rice (Oryza sativa L.) Genotypes Based on Quantitative Traits.

Upland rice is important for sustainable crop production to meet future food demands. The expansion in area of irrigated rice faces limitations due to water scarcity resulting from climate change. Therefore, this research aimed to identify potential genotypes and suitable traits of upland rice germplasm for breeding programmes. Forty-three genotypes were evaluated in a randomised complete block design with three replications. All genotypes exhibited a wide and significant variation for 22 traits. The highest phenotypic and genotypic coefficient of variation was recorded for the number of filled grains/panicle and yields/plant (g). The highest heritability was found for photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2, and number of filled grains/panicle and yields/plant (g). Cluster analysis based on 22 traits grouped the 43 rice genotypes into five clusters. Cluster II was the largest and consisted of 20 genotypes mostly originating from the Philippines. The first four principle components of 22 traits accounted for about 72% of the total variation and indicated a wide variation among the genotypes. The selected best trait of the number of filled grains/panicle and yields/plant (g), which showed high heritability and high genetic advance, could be used as a selection criterion for hybridisation programmes in the future.

Highly efficient protocol for callogenesis, somagenesis and regeneration of Indica rice plants.

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.

Differential Gene Expression Reflects Morphological Characteristics and Physiological Processes in Rice Immunity against Blast Pathogen Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae is a serious pathogen that jeopardises the world's most important food-security crop. Ten common Malaysian rice varieties were examined for their morphological, physiological and genomic responses to this rice blast pathogen. qPCR quantification was used to assess the growth of the pathogen population in resistant and susceptible rice varieties. The chlorophyll content and photosynthesis were also measured to further understand the disruptive effects that M. oryzae has on infected plants of these varieties. Real-time PCR was used to explore the differential expression of eight blast resistance genes among the ten local varieties. Blast disease has destructive effects on the growth of rice, and the findings of our study provide evidence that the Pikh, Pi9, Pi21, and Osw45 genes are involved in defence responses in the leaves of Malaysian rice at 31 h after inoculation with M. oryzae pathotype P7.2. Both the chlorophyll content and photosynthesis were reduced, but the levels of Pikh gene expression remained constant in susceptible varieties, with a developed pathogen population and mild or severe symptoms. The Pi9, Pi21, and Osw45 genes, however, were simultaneously upregulated in infected rice plants. Therefore, the presence of the Pikh, Pi9, Pi21, and Osw45 genes in the germplasm is useful for improving the resistance of rice varieties.

Importance of silicon and mechanisms of biosilica formation in plants.

Silicon (Si) is one of the most prevalent macroelements, performing an essential function in healing plants in response to environmental stresses. The purpose of using Si is to induce resistance to distinct stresses, diseases, and pathogens. Additionally, Si can improve the condition of soils, which contain toxic levels of heavy metals along with other chemical elements. Silicon minimizes toxicity of Fe, Al, and Mn, increases the availability of P, and enhances drought along with salt tolerance in plants through the formation of silicified tissues in plants. However, the concentration of Si depends on the plants genotype and organisms. Hence, the physiological mechanisms and metabolic activities of plants may be affected by Si application. Peptides as well as amino acids can effectively create polysilicic species through interactions with different species of silicate inside solution. The carboxylic acid and the alcohol groups of serine and asparagine tend not to engage in any significant role in polysilicates formation, but the hydroxyl group side chain can be involved in the formation of hydrogen bond with Si(OH)4. The mechanisms and trend of Si absorption are different between plant species. Furthermore, the transportation of Si requires an energy mechanism; thus, low temperatures and metabolic repressors inhibit Si transportation.

Serine-rich protein is a novel positive regulator for silicon accumulation in mangrove.

Silicon (Si) plays an important role in reducing plant susceptibility against a variety of different biotic and abiotic stresses; and also has an important regulatory role in soil to avoid heavy metal toxicity and providing suitable growing conditions for plants. A full-length cDNAs of 696bp of serine-rich protein was cloned from mangrove plant (Rhizophora apiculata) by amplification of cDNA ends from an expressed sequence tag homologous to groundnut (Arachis hypogaea), submitted to NCBI (KF211374). This serine-rich protein gene encodes a deduced protein of 223 amino acids. The transcript titre of the serine-rich protein was found to be strongly enriched in roots compared with the leaves of two month old mangrove plants and expression level of this serine-rich protein was found to be strongly induced when the mangrove seedlings were exposed to SiO2. Expression of the serine-rich protein transgenic was detected in transgenic Arabidopsis thaliana, where the amount of serine increased from 1.02 to 37.8mg/g. The same trend was also seen in Si content in the roots (14.3%) and leaves (7.4%) of the transgenic A. thaliana compared to the wild-type plants under Si treatment. The biological results demonstrated that the accumulation of the serine amino acid in the vegetative tissues of the transgenic plants enhanced their ability to absorb and accumulate more Si in the roots and leaves and suggests that the serine-rich protein gene has potential for use in genetic engineering of different stress tolerance characteristics.

Isolation and expression analysis of novel silicon absorption gene from roots of mangrove (Rhizophora apiculata) via suppression subtractive hybridization.

Silicon (Si) is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots' cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes in Rhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2 for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene of serine-rich protein.

Genetic variation, heritability, divergence and biomass accumulation of rice genotypes resistant to bacterial blight revealed by quantitative traits and ISSR markers.

A field experiment was carried out in order to evaluate genetic diversity of 41 rice genotypes using physiological traits and molecular markers. All the genotypes unveiled variations for crop growth rate (CGR), relative growth rate (RGR), net assimilation rate (NAR), yield per hill (Yhill(-1)), total dry matter (TDM), harvest index (HI), photosynthetic rate (PR), leaf area index (LAI), chlorophyll-a and chlorophyll-b at maximum tillering stage. The CGR values varied from 0.23 to 0.76 gm cm(-2) day(-1). The Yhill(-1) ranged from 15.91 to 92.26 g, while TDM value was in the range of 7.49 to 20.45 g hill(-1). PR was found to vary from 9.40 to 22.34 µmol m(-2) s(-1). PR expressed positive relation with Yhill(-1). Significant positive relation was found between CGR and TDM (r = 0.61**), NAR and CGR (r = 0.62**) and between TDM and NAR (r = 0.31**). High heritability was found in RGR and Yhill(-1). Cluster analysis based on the traits grouped 41 rice genotypes into seven clusters. A total of 310 polymorphic loci were detected across the 20 inter-simple sequence repeats (ISSR) markers. The UPGMA dendrogram grouped 41 rice genotypes into 11 clusters including several sub-clusters. The Mantel test revealed positive correlation between quantitative traits and molecular markers (r = 0.41). On the basis of quantitative traits and molecular marker analyses parental genotypes, IRBB54 with MR84, IRBB60 with MR84, Purbachi with MR263, IRBB65 with BR29, IRBB65 with Pulut Siding and MRQ74 with Purbachi could be hybridized for future breeding program.

Comparison of soil phosphorus tests for assessing plant availability of phosphorus in an ultisol amended with water-soluble and phosphate rock sources.

The effectiveness of different soil tests in assessing soil phosphorus (P) in soils amended with phosphate rocks (PRs) is uncertain. We evaluated the effects of triple superphosphate (TSP) and PRs on extractable P by conventional soil tests (Mehlich 3 [Meh3] and Bray-1 [B1]) and a nonconventional test (iron oxide-impregnated paper, strip). Extracted amounts of P were in the order: Meh3 >B1 > strip. All the tests were significantly correlated (p = 0.001). Acidic reagents extracted more P from TSP than PRs, while the strip removed equal amounts from the two sources. The P removed by the three tests was related significantly to dry matter yield (DMY), but only in the first harvest, except for B1. Established critical P levels (CPLs) differed for TSP and PRs. In PR-fertilized soils, CPLs were 27, 17, and 12 mg P kg(-1) soil for Meh3, B1, and strip, respectively, and 42, 31, and 12 mg P kg(-1) soil, respectively, in TSP-fertilized soils. Thus, the strip resulted in a common CPL for TSP and PRs (12 mg P kg(-1) soil). This method can be used effectively in soils where integrated nutrient sources have been used, but there is need to establish CPLs for different crops. For cost-effective fertilizer P recommendations based on conventional soil tests, there is a need to conduct separate calibrations for TSP- and PR-fertilized soils.

Phosphorus leaching in an acid tropical soil "recapitalized" with phosphate rock and triple superphosphate.

With high rates of phosphorus applied to increase "capital P" as a stock for plant uptake over several years, the question of P leaching is inevitable. We conducted an intact soil column experiment in the field to evaluate P leached from soils treated with triple superphosphate (TSP) and Gafsa phosphate rock (GPR) at 300, 600, and 900 kg P ha-1 with and without integration of cattle manure. The lysimeters, made from PVC tubes of 30-cm length, were inserted into the soil up to the 25-cm depth. The tubes were fitted with a resin bag containing a mixture of cation and anion exchange resin (50:50) at the lower end of the tube inserted into the soil. The tubes, arranged in a completely randomized design, were sampled randomly at 10-week intervals for 12 months. Phosphorus extractable from the top- and subsoil at the end of experiment and leached P were determined. More P was leached out from TSP (threefold) compared to GPR, and the amount of P leached increased with increasing rates of P fertilizer applied. Application of manure intensified the amounts of P leached from TSP, particularly at the 6-month sampling time. There was hardly any substantial P leached from the soil treated with GPR. Thus, for effective and efficient long-term P fertilizer management strategies, choosing the right P fertilizer source and monitoring P losses through leaching has to be done for enhanced fertilizer use efficiency and thus reducing P pollution of ground waters.