Parameswari Namasivayam

Acquisition of embryogenic competence during somatic embryogenesis

This review focuses on investigation in acquisition of embryogenic competence during somatic embryogenesis in the last five decades. In tissue culture, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells during the induction phase. These embryogenic cells are important because they differentiate to form somatic embryos at a later time. Various molecular and structural markers for detecting embryogenic cells or enhancing embryogenic competence are summarized and implications of the findings are discussed.

Overexpression of monodehydroascorbate reductase from a mangrove plant (AeMDHAR) confers salt tolerance on rice.

Monodehydroascorbate reductase (MDHAR), an important enzyme of the ascorbate-glutathione cycle, is involved in salt tolerance of plants through scavenging of reactive oxygen species (ROS). In this study, a cDNA encoding MDHAR from the mangrove plant Acanthus ebracteatus was introduced into rice to examine its role in salt tolerance. Three stable transgenic lines (MT22, MT24 and MT25) overexpressing AeMDHAR were selected in vitro using hygromycin and confirmed by PCR, quantitative reverse-transcription (qRT) PCR and enzyme assay. The transgenic line MT24 was predicted to possess a single copy of the transgene while the other two transgenic lines were predicted to have multiple transgene integrations. The AeMDHAR transcripts were detected only in transgenic rice lines but not in untransformed rice. The abundance of AeMDHAR transcripts in transgenic lines MT22 and MT25 was approximately 2.75 times the amount found in MT24. The transgenic rice lines overexpressing AeMDHAR showed a significant increase in MDHAR enzyme activity compared to untransformed plants under both NaCl and control conditions. All transgenic lines showed better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also showed tolerance to salt at germination and seedling stages. The transgenic line MT24, which harbors a single copy of AeMDHAR, displayed a lower rate of sterility, a higher number of tillers and longer panicle compared to untransformed plants when subjected to salt stress.

Identification of a potential structural marker for embryogenic competency in the Brassica napus spp. oleifera embryogenic tissue.

The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.

Development of expressed sequence tag resources for Vanda Mimi Palmer and data mining for EST-SSR.

Vanda Mimi Palmer (VMP) is a highly sought as fragrant-orchid hybrid in Malaysia. It is economically important in cosmetic and beauty industries and also a famous potted ornamental plant. To date, no work on fragrance-related genes of vandaceous orchids has been reported from other research groups although the analysis of floral fragrance or volatiles have been extensively studied. An expressed sequence tag (EST) resource was developed for VMP principally to mine any potential fragrance-related expressed sequence tag-simple sequence repeat (EST-SSR) for future development as markers in the identification of fragrant vandaceous orchids endemic to Malaysia. Clustering, annotation and assembling of the ESTs identified 1,196 unigenes which defined 966 singletons and 230 contigs. The VMP dbEST was functionally classified by gene ontology (GO) into three groups: molecular functions (51.2%), cellular components (16.4%) and biological processes (24.6%) while the remaining 7.8% showed no hits with GO identifier. A total of 112 EST-SSR (9.4%) was mined on which at least five units of di-, tri-, tetra-, penta-, or hexa-nucleotide repeats were predicted. The di-nucleotide motif repeats appeared to be the most frequent repeats among the detected SSRs with the AT/TA types as the most abundant among the dimerics, while AAG/TTC, AGA/TCT-type were the most frequent trimerics. The mined EST-SSR is believed to be useful in the development of EST-SSR markers that is applicable in the screening and characterization of fragrance-related transcripts in closely related species.

Terpenoid, benzenoid and phenylpropanoid compounds in the floral scent of vanda mimi palmer.

Vanda Mimi Palmer is the product of a cross between Vanda Tan Chay Yan and Vanda tessellata. The flower of this hybrid produces a sweet-smelling fragrance during day time at the open-flower stage. This study aimed to investigate the floral scent constituents in Vanda Mimi Palmer. Scent emission analysis of this orchid was carried out at different time points in a 24-h cycle and also at different floral developmental stages. A comparison was also made on the volatiles emitted by Vanda Mimi Palmer and both of its parents. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the scent of Vanda Mimi Palmer was dominated by terpenoid, benzenoid, and phenylpropanoid compounds. The identified terpenoids were ocimene, linalool oxide, linalool, and nerolidol; while the benzenoid and phenylpropanoid compounds were methylbenzoate, benzyl acetate, phenylethanol, and phenylethyl acetate. The emission of terpenoid, benzenoid, and phenylpropanoid compounds was developmentally and temporally regulated. Comparison of the volatiles emitted by both of its parents showed that the scent of Vanda Mimi Palmer is dissimilar to that of its fragrant parent, V. tessellata.

Identification of differentially expressed sequences in pre-embryogenic tissue of oilseed rape by suppression subtractive hybridisation (SSH)

A subtracted cDNA library comprising of 576 clones were constructed to isolate differentially expressed sequences in the pre-embryogenic tissue (PEC) of winter oilseed repe. After differential screening, only 16 clones were identified as potential positives. Eventually, only three clones: BNPE DG3, BNPE AE4 and BNPE EG1 were confirmed by Northern analysis as upregulated in␣PEC of the oilseed rape embryogenic culture. This is the first study to report Ae4, Dg3 and Eg1 sequences as preferentially expressed in the oilseed␣rape PEC and associated with the induction of somatic embryogenesis in B.napus secondary embryogenesis. Ae4 encodes a putative proline-rich protein, Dg3 encodes a lipid transfer-like protein and Eg1 encodes a napin, member of the BNMNAP subfamily.

EgRBP42 encoding an hnRNP-like RNA-binding protein from Elaeis guineensis Jacq. is responsive to abiotic stresses

AbstractContents RNA-binding proteins (RBPs) have been implicated as regulatory proteins involved in the post-transcriptional processes of gene expression in plants under various stress conditions. In this study, we report the cloning and characterization of a gene, designated as EgRBP42, encoding a member of the plant heterogeneous nuclear ribonucleoprotein (hnRNP)-like RBP family from oil palm (Elaeis guineensis Jacq.). EgRBP42 consists of two N-terminal RNA recognition motifs and a glycine-rich domain at the C-terminus. The upstream region of EgRBP42 has multiple light-responsive, stress-responsive regulatory elements and regulatory elements associated with flower development. Real-time RT-PCR analysis of EgRBP42 showed that EgRBP42 was expressed in oil palm tissues tested, including leaf, shoot apical meristem, root, female inflorescence, male inflorescence and mesocarp with the lowest transcript level in the roots. EgRBP42 protein interacted with transcripts associated with transcription, translation and stress responses using pull-down assay and electrophoretic mobility shift assay. The accumulation of EgRBP42 and its interacting transcripts were induced by abiotic stresses, including salinity, drought, submergence, cold and heat stresses in leaf discs. Collectively, the data suggested that EgRBP42 is a RBP, which responds to various abiotic stresses and could be advantageous for oil palm under stress conditions. Key message EgRBP42 may be involved in the post-transcriptional regulation of stress-related genes important for plant stress response and adaptation.

Biosynthesis of agar in red seaweeds: A review

Agar is a jelly-like biopolymer synthesized by many red seaweeds as their major cell wall component. Due to its excellent rheological properties, it has been exploited commercially for applications in food, cosmetic, pharmaceutical, biomedical and biotechnology industries. Despite its multiple uses, the biosynthesis of this phycocolloid is not fully understood. The current knowledge on agar biosynthesis is inferred from plant biochemistry and putative pathways for ulvan and alginate biosynthesis in green and brown seaweeds, respectively. In this review, the gaps in our current knowledge on agar biosynthetic pathway are discussed, focusing on the biosynthesis of agar precursors, elongation of agar polysaccharide chain and side chain modification. The development of molecular markers for the screening of desired seaweeds for industrial exploitation is also discussed.

A Novel Transcript of Oil Palm (Elaeis guineensis Jacq.), Eg707, is Specifically Upregulated in Tissues Related to Totipotency

In this study, we report the molecular characterization of clone Eg707 isolated from cell suspension culture of the oil palm. The deduced polypeptide of clone Eg707 is highly similar to an unknown protein from Arabidopsis thaliana. The presence of an Ald-Xan-dh-C2 superfamily domain in the deduced protein sequence suggested that Eg707 protein might be involved in abscisic acid biosynthesis. Eg707 might be present as a single copy gene in the oil palm genome. This gene is highly expressed in tissue cultured materials compared to vegetative and reproductive tissues, suggesting a role of this gene during oil palm somatic embryogenesis or at the early stages of embryo development. Expression analysis of Eg707 by RNA in situ hybridization showed that Eg707 transcripts were present throughout somatic embryo development starting from proembryo formation at the embryogenic callus stages till the maturing embryo stages. Since proembryo formation within the embryogenic callus is one of the first key factors in oil palm somatic embryo development, it is suggested that Eg707 could be used as a reliable molecular marker for detecting early stage of oil palm somatic embryogenesis.

Factors affecting yield and gelling properties of agar

Agar, a gelatinous polysaccharide in the cell wall of many red algal species, is widely used as a gelling, thickening and stabilizing agent. The commercial value of seaweed is judged by their agar content and gel quality. Seaweed materials with higher agar yield and better gelling properties are desired due to the growing demand for agar in the global market. Agar biosynthesis in seaweeds is affected by genetic variations, developmental stages and environmental conditions, while different agar extraction techniques can also affect the yield and quality of agar. In this paper, the effects of different physiological states of seaweed, abiotic and biotic factors, seaweed storage and agar extraction techniques on the agar yield and gelling characteristics, are reviewed. This information is important as a guide for marine aquaculture of potential agarophytes and the possible effects of climate change on the stock of this natural resource.