Molecular cloning and functional studies of silicon-responsive serine-rich protein transcripts from mangrove plant, rhizophora apiculata (Blume)

Silicon (Si) is one of the most plentiful elements found in the soil. Silicon plays an important role in decreasing susceptibility of plants against variety of different biotic and abiotic stresses. Mangrove plant (Rhizophora apiculata) is able to accumulate, and process Si to generate biosilica. Therefore, it would be a beneficial source for genetic manipulation of susceptible plants in the stress conditions. The objectives of the study were (i) to identify and characterize of a Si responsive gene in mangrove,(ii) to analyze the expression levels of a gene encoding serine-rich protein, and (iii) Functional studies of serine-rich protein in Arabidopsis thaliana. Three different methods and RNeasy plant mini kit were used to extract nucleic acids. The Suppression Subtractive Hybridization (SSH) technique was used to remove transcripts from proteins which were not involved in Si accumulation. Specific primer was designed to get full-length CDS of serine-rich protein. Semi-quantitative RT-PCR and real-time PCR were performed to examine its expression level under the control and treatment conditions. The Gateway Technology was used to construct entry and the expression vectors. Transformation of Arabidopsis thaliana with serine-rich protein gene was performed using Agrobacterium-mediated transformation by the floral-dip method. Energy-dispersive X-ray spectroscopy and high performance liquid chromatography were used to measure the quantity of Si and serine amino acid, respectively. Modified CTAB and SDS were quick and reliable methods for isolation of total RNA from the roots and leaves of mangrove, respectively. Of the sequences obtained from cDNA library, four were 97% similar to serine-rich protein gene of groundnut(Arachis hypogaea). Full-length of the serine-rich protein cDNA obtained through amplification of the cDNA template using specific primers. The expression levels of serine-rich protein transcript were generally higher in the Si treated mangrove plants than untreated plants. The amount of serine amino acid of transgenic Arabidopsis has increased significantly from 1.02 mg g-1 in wild-type plants to 37.76 mg g-1. In addition, concentration of Si in the leaves and roots of transgenic plant was significantly higher than that in the wild type (P<0.01). This study successfully determined the Si responsive transcript related to serine-rich protein in mangrove plant (R. apiculata)

Genetic analysis of rust resistance genes in global wheat cultivars: an overview

Rust is the most devastating fungal disease in wheat. Three rust diseases, namely, leaf or brown rust caused by Puccinia triticina Eriks, stem or black rust caused by Puccinia graminis f. sp. tritici West, and stripe or yellow rust caused by Puccinia striiformis f. Tritici Eriks, are the most economically significant and common diseases among global wheat cultivars. Growing cultivars resistant to rust is the most sustainable, cost-effective and environmentally friendly approach for controlling rust diseases. To date, more than 187 rust resistance genes (80 leaf rust, 58 stem rust and 49 stripe rust) have been derived from diverse wheat or durum wheat cultivars and the related wild species using different molecular methods. This review provides a detailed discussion of the different aspects of rust resistance genes, their primitive sources, their distribution in global wheat cultivars and the importance of durable resistant varieties for controlling rust diseases. This information will serve as a foundation for plant breeders and geneticists to develop durable rust-resistant wheat varieties through marker-assisted breeding or gene pyramiding

Suppression subtractive hybridization technique in wheat for the identification of disease resistance differentially expressed genes

A resistant variety with high yielding potential is key for increasing crop production to fulfill the food requirement of the ever increasing world populations. Consequently, the aim of plant breeders is to develop high yielding varieties or cultivars that are resistant or tolerant to specific diseases or insects. For developing a resistant variety, it is enormously indispensable to incorporate or introgress the specific resistant genes of that particular disease into the recipient. Suppression subtractive hybridization (SSH) is a powerful technique for the identification of disease specific differentially expressed genes that are expressed in a resistant or susceptible variety. This paper presents a brief review on the SSH technique with examples focusing on the identification of the wheat disease specific differentially expressed genes and their defense mechanisms against fungal pathogens in global wheat cultivars. This review is helpful for wheat researchers for the updated information on the SSH technique for the identification of differentially expressed genes in the global wheat cultivars and varieties. Eventually, the identified genes could be used to develop the disease resistance variety through marker-assisted backcrossing programme or conventional breeding

Phenotypic and molecular effects of chronic gamma irradiation on Curcuma alismatifolia

Mutation breeding is one of the methods for generating genetic variation and obtaining new cultivars of ornamental plants during the past decades. In present study, the effects of four doses (0, 14.6, 33, and 87.4 Gy) of chronic gamma irradiation on three cultivars of Curcuma alismatifolia and one Curcuma hybrid were investigated. Morphological aberrations from non-treated plants were observed by exposing growing plants. Higher doses induced phenotypical variations and significantly affected the plant growth parameters and flowering capacity. In terms of genetic variation, among the irradiated cultivars, the number of presumed alleles revealed by SSR analysis ranged from two to five with a mean value of 3.1 to 3.7 alleles per locus for radiation doses. The average value of the effective number of alleles, Nei’s gene diversity, and Shannon’s information index were 2.42–2.66, 0.50–0.56, and 0.90–1.03, respectively. Heat map hierarchical clustering divided 52 studied individuals into four major clusters. Results of this study showed that chronic gamma irradiation efficiently can enhance the phenotypical and genetic variations in C. alismatifolia cultivars at doses of 33 Gy and 84.6 Gy. In addition, SSR markers will likely accelerate the progress of selection of desired mutants during mutation breeding programs

Effect of four different salts on seed germination and morphological characteristics of Oryza sativa L. cv. MR219

The response of Oryza sativa L. cv. MR219 to NaCl, KCl, MgCl2 and MgSO4 at different salinity levels (0, 50, 100, 150, 200 and 250 mM) was studied with emphasis on seed germination and early seedling stage. High salinity delayed mean germination time of seeds and increased biomass, relative injury rate and seedling height reduction. Seeds are more tolerant to NaCl among four salts even at the highest salinity. Results showed that 50mM KCl enhanced the root growth with more roots developed at this salinity. Abnormal seed germination was found in MgCl2 and MgSO4 due to inhibition of root growth. This study proposes that degree of tolerance of MR219 to salts from morphological results is NaCl>KCl>MgCl2>MgSO4. This study might be useful for further research of salinity effect on growth and physiological processes at advanced stage of MR 219 growth

TILLING, high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.Peer reviewe

Comparative study of lignin in roots of different oil palm progenies in relation to Ganoderma basal stem rot disease

Basal stem rot (BSR) disease caused by Ganoderma boninense Pat., a serious constraint to oil palm (Elaeis guineensis Jacq.) in South-east Asia, namely Malaysia and Indonesia, is also becoming a threat in Africa and Latin America. Currently, no complete management is available although Ganoderma tolerant oil palm is likely to have a crucial role in the management of the disease in the future. In this study, oil palm progenies exhibiting different reactions to Ganoderma BSR were investigated. Lignin in their roots was detected by phloroglucinol-HCl. The intensity of burgundy red colour developed after staining varied among progenies, indicating differences in lignin content but this was a subjective qualitative approach. A quantitative approach following the modified Klason method was then used. Uninoculated progenies TK 714 and TK 716 were significantly different in lignin content at six to seven months but 10 months later, lignin contents of all uninoculated progenies were similar, while that of inoculated plants exhibited some small but significant differences. Nevertheless, the accumulation of lignin did not correlate well with susceptibility or tolerance to BSR and consequently, lignin content may not be a reliable trait to characterise oil palm progenies for Ganoderma tolerance or susceptibility in screening tests

A critical review of the concept of transgenic plants: insights into pharmaceutical biotechnology and molecular farming

Using transgenic plants for the production of high-value recombinant proteins for industrial and clinical applications has become a promising alternative to using conventional bioproduction systems, such as bacteria, yeast, and cultured insect and animal cells. This novel system offers several advantages over conventional systems in terms of safety, scale, cost-effectiveness, and the ease of distribution and storage. Currently, plant systems are being utilised as recombinant bio-factories for the expression of various proteins, including potential vaccines and pharmaceuticals, through employing several adaptations of recombinant processes and utilizing the most suitable tools and strategies. The level of protein expression is a critical factor in plant molecular farming, and this level fluctuates according to the plant species and the organs involved. The production of recombinant native and engineered proteins is a complicated procedure that requires an inter- and multi-disciplinary effort involving a wide variety of scientific and technological disciplines, ranging from basic biotechnology, biochemistry, and cell biology to advanced production systems. This review considers important plant resources, affecting factors, and the recombinant-protein expression techniques relevant to the plant molecular farming process

Analysis of SSR markers linked with brown planthopper resistance genes (Bph) using high-resolution melting (HRM) in rice

Developing rice cultivars with host-plant resistance is widely considered the best strategy for the long-term control of the brown planthopper (BPH). The use of molecular markers in many aspects of rice ('Oryza sativa' L.) studies, such as the genetic analysis of insect and disease resistance, is increasing. In the present study, 110 simple sequence repeat (SSR) markers that are associated with Bph resistance genes were selected from the Gramene database and used to develop SSR marker-based strategies for the reliable selection of BPH-resistant genotypes. Fifty-seven of the best polymorphic markers were used to identify the segregation ratio in 176 individual F2 rice progeny from a MR276 (susceptible) x Rathu Heenati (resistant) interspecific cross. Thirty-five SSR markers, including RM544, RM547, and RM8213, showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, p <= 0.05) in chi-square (x2) analyses. The remaining markers did not fit the expected Mendelian segregation ratios. The genetic information generated in this research will be useful in rice breeding programmes to provide varieties with durable resistance to BPH. Additionally, this research showed that high-resolution melting analysis (HRM) is powerful and applicable for accurately and quickly genotyping many samples

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