Functional characterization of flower morphogenesis and senescence related genes using virus induced gene silencing technique in petunia hybrida

In floricultural crops, flower morphology, such as large petals and double flower formation, and flower longevity are important factors that influence their quality. Petunia has been proved to be an excellent model plant for the study of flower development and senescence. However, even in petunia, there are a lot of genes whose function in flower development and senescence have not yet been characterized. Recently, techniques using virus induced gene silencing (VIGS) have been developed as efficient reverse genetics tools to test gene function. In this study, VIGS system that visualizes silencing induced-flower was established in petunia. Using this system, functional characterization of petunia candidate genes involved in flower morphogenesis and senescence was conducted. In parallel, identification and expression analysis of flower development related-genes that had not yet been identified in petunia was performed. Disadvantage of VIGS is that silencing is induced in a chimeric manner and it is sometimes difficult to identify flowers on which silencing is induced. To overcome the disadvantage of VIGS, system that use silencing of the chalcone synthase (CHS) gene, one of genes regulating anthocyanin biosynthesis, as a reporter to visualize silencing induced-flower was established. Tandem pTRV2 constructs containing a Ph-CHS fragment and target gene fragment(s) are prepared and agrobacterium cultures possessing these constructs are infiltrated onto petunia leaves of 2 to 3 weeks old seedlings. Five different petunia cultivars with blue or purple flowers, 'Cutie Blue', 'Fantasy Blue', 'Picobella Blue', 'Mambo Blue' and 'Mambo Purple' were infected with the construct and differences in silencing patterns. was observed among cultivars. In 'Cutie Blue' and 'Fantasy Blue' complete white flowers were observed as a results of silencing of Ph-CHS; however, no white color was noted in flowers or tissues in 'Picobella Blue', 'Mambo Blue' and 'Mambo Purple'. This indicated that effectiveness of this VIGS system differs dependent on flower genetic background. It is also revealed to be dependent on environmental factors such as temperature to get complete silencing phenotypes. Second, using the VIGS system, redundant function of two C-class MADS-box genes, pMADS3 and FBP6 genes on flower morphogenesis was revealed. In flowers induced by either pMADS3-VIGS or FBP6-VIGS, only small changes in commercial appearance were recognized regardless of cultivar, whereas in those induced by pMADS3/FBP6-VIGS, complete conversion of stamens into petaloid tissues and marked enlargement of upper limb- like tissues were observed, resulting in a decorative appearance in all the four cultivars. Moreover, cultivar-dependent conversion of carpels into new flowers was noted in pMADS3/FBP6-VIGS flowers. Of the four cultivars, only 'Mambo Purple' exhibited the development of new flowers instead of carpels and the emergence of ectopic new flowers from the axil of petaloid organs, which created an ornamental appearance with a high commercial value. Further, investigation of large and small petaloid stamens induced by pMADS3/FBP6-VIGS and pMADS3 -VIGS, respectively, revealed only small differences in cell size compared to the large difference in total surface area indicating that the sie of petaloid stamens in C-class genes suppressed flower was determined at early stage of flower development and the suppressed C-class gene function at the late stage of flower development has little influence on the final size of petaloid tissue. Third, function of genes encoding ethylene signal components, Ethylene Insensitive 2 (EIN2) and EIN3 like (EIL), on flower senescence was characterized. A cDNA encoding EIN2 (Ph-EIN2) and five cDNAs encoding EILs (Ph-EILJ to 5) were cloned from petunia petals and VIGS of these genes together with Ph-CHS were conducted. VIGS of all Ph-EILs were attempted at once by using a conserved region of the Ph-EILs. The flower longevity of VIGS-induced flowers was compared to those of non-VIGS flowers under the condition with or without pollination. The results showed prolonged longevity of VIGS-induced flowers indicating the involvement of Ph-EIN2 and Ph-EILs on petal senescence. When flowers were treated with propylene, an ethylene analogue, marked delay in petal senescence was observed in Ph-EIN2-VIGS flowers in comparison to non-VIGS flower. Thus, it has been clarified that Ph-EIN2 is functioning as ethylene signaling factor and involved in the petal senescence. Fourth, petunia orthologues of pollen formation-related genes Tapetum Determinant 1 (Ph-TPDJ) and Excess Male Sporocytes] (Ph-EMS]) were identified and their expression patterns during flower bud development were determined. Quantitative real time analysis of Ph-TPD] in stamen of flower buds at four different developing stages (5 mm to 15 mm in length buds) showed that Ph-TPDJ expression can be detected only in the youngest stage. Expression of Ph-EMS 1 was detected in stamen at all stages with highest expression at the youngest stage. Analysis in various tissues of the buds (sepal, petal, stamen and style) at youngest stage showed that Ph-TPDJ and Ph-EMS] are expressed not only in stamen but also in developing pistils and petals. In sepal, expression of Ph-TPD] was not detected, whereas expression of Ph-EMS] was detected at lower level than in the other tissues. These results suggest that Ph-TPDJ and Ph-EMS] cloned in this study interact with each other to regulate early steps of pollen development. It is also indicated that they are involved in development of style. The findings obtained in this study would be valuable for breeding new cultivars and developing technology to improve quality of petunia and other floricultural crops

Antifungal activity of biosynthesized silver nanoparticles mediated by neem leaf extract against aspergillus sp.

Silver nanoparticles (AgNPs) have attracted considerable attraction as excellent antifungal agents against various pathogens. In the present study, AgNPs were biosynthesized using neem leaf aqueous extract, and their antifungal properties were evaluated against Aspergillus sp. The formation of newly synthesized AgNPs was confirmed through visual observation by a change in the color of the solution, followed by an analysis of their surface plasmon resonance via UV-vis spectrophotometer. Further characterization of its crystalline nature and morphology structure was assessed by X-Ray Diffraction (XRD) and Field-emission Scanning Electron Microscope (FESEM), respectively. The result revealed that the synthesized AgNPs showed UV-vis spectra peak around 421 nm, are crystalline in nature, and have a spherical morphology with an average size of 20.13 ± 3.3 nm in diameter. Furthermore, these AgNPs exhibit excellent antifungal activity against the waterborne pathogen Aspergillus sp. on agar well diffusion assay with a maximum 26.54 ± 1.23 mm zone of inhibition. FESEM image revealed hyphal damage and deformation of fungus when treated with AgNPs, causing retardation of fungus growth for further reproduction. The results suggested that this biosynthesis AgNPs from neem leaf extract has great potential as an alternative antifungal agent for use in water treatment

De novo transcriptome dataset of Stevia rebaudiana accession MS007

Stevia rebaudiana (S. rebaudiana) is a herbaceous and perennial plant belonging to Asteraceae family. The genus stevia is well known as a natural producer of sweetener comprising non-caloric and non-carcinogenic steviol glycosides. In recent years, the capability in producing natural sweetner has increased the demand for S. rebaudiana as substitute of processed sugars. Flowering phase of S. rebaudiana has shown to affect the content of steviol glycosides in the leaves. Steviol glycosides level is the highest at the time of flower bud formation and lowest at time preceding and following flower bud formation. Therefore, sequencing and analysing the genes that are involved in flowering phase will provide platform for gene manipulation in increasing steviol glycosides content. The Stevia transcriptome data that include two stages of growth (before flowering and after flowering), were obtained using Illumina RNA-seq technology and can be accessed at NCBI Sequence Read Archive under Accession No. SRX6362785 and SRX6362784

Characterisation of Aquilaria hirta based on morphology evaluation and volatile chemical compound

Aquilaria hirta is one of the agarwood tree species in Malaysia that produces a unique resinous wood and essential oil used in perfumery, medicinal, cosmetic and religious ceremonies. Currently, it has been listed as vulnerable (VU) and threatenad species in the IUCN Red List. This study aims to characterise the morphology of A. hirta via scanning electron microscopic analysis, and to identify the volatile chemical compounds of the wood and its essential oil using gas chromatography-flame ionisation detector and gas chromatography-mass spectrometry analyses. Aquilaria hirta was identified by the presence of hirsute on the abaxial side of leaves and midrib. The field emission scanning electron microscope analysis showed plant vessel pits can be seen in the healthy wood images compared with resinous wood images due to presence of resin and microorganisms. A total of 19 compounds were identified in resinous A. hirta wood consisting of 5.97% sesquiterpenes and 20.32% oxygenated sesquiterpenes. The major compounds are kessane, γ-cadinene, α-caryophyllene, β-caryophylene and caryophyllene oxide. There were 35 compounds found in A. hirta oil comprising 0.45% monoterpenes, 23.51% sesquiterpenes and 19.53% oxygenated sesquiterpenes. Major compounds detected were γ-cadinene, nor-ketoagarofuran, allo-aromadendrene, γ-gurjunene and β-gurjunene. This study provides a reference for the identification of A. hirta species based on morphology evaluation and volatile chemical compounds profile of the essential oil and wood

Transcriptome profiling of stevia rebaudiana ms007 revealed genes involved in flowedevelopment

Stevia rebaudiana is a medicinal plant recommended to diabetic or obese patients as an alternative sweetener owing to its low-calorie property. Previous studies have found that the stevioside level is highest at the time of flower bud formation and lowest at the time of preceding and following flower bud formation. Hence, this study aims to identify the genes involved in the flowering of local S. rebaudiana accession MS007 by investigating the transcriptomic data of two stages of growth, before flowering (BF) and after flowering (AF) that were deposited under accession number SRX6362785 and SRX6362784 at the NCBI SRA database. The transcriptomic study managed to annotate 108299 unigenes of S. rebaudiana with 8871 and 9832 genes that were differentially expressed in BF and AF samples, respectively. These genes involved in various metabolic pathways related to flower development, response to stimulus as well as photosynthesis. Pheophorbide A oxygenase (PAO), eukaryotic translation initiation factor 3 subunit E (TIF3E1), and jasmonate ZIM domain-containing protein 1 (JAZ1) were found to be involved in the flower development. The outcome of this study will help further research in the manipulation of the flowering process, especially in the breeding programme to develop photo-insensitive Stevia plant

Effect Of Temperature And Formic Acid Concentration On Delignification Of Leucaena Leucocephala

Delignification is essential for wide range of technologies especially in paper industry and ethanol as transportation fuel.Researches have been made by thermal, mechanical, chemical and biology treatment. Recently, L. Leucocephala has been in centre of attraction for paper making industry while formic acid, a typical organosolv system has been given attention by the researchers due to effectiveness on the ability to break down and modify the lignin structure. Hence, delignification of L. Leucocephala was studied using chemical treatment with formic acid. The effect of reaction conditions including reaction temperature and formic acid concentration were investigated at atmospheric pressure for 6 hours of reaction time. Delignification percentage approximately 70–75%(v/w) with a pulp yield of 40–62%(w/w) was obtained and the optimal reaction temperature is 90oC with 90% of formic acid concentration, respectively. Therefore, temperature and formic acid concentration affected significantly on the delignification process of L. Leucocephal

Characterization of aquilaria malaccensis callus cells using SEM and somatic embryogenesis associated genes identification

Aquilaria malaccensis or gaharu is one of Malaysia's top tropical resources that is being protected and conserved. Somatic embryogenesis is a plant tissue culture method used most extensively in commercial micropropagation systems and conservation purposes. This study aims to optimize callus induction techniques for somatic embryogenesis study using Scanning Electron Microscope (SEM) and Polymerase Chain Reaction (PCR) analysis. In this study, callus was induced on Murashige and Skoog's (MS) medium supplemented with a combination of plant growth regulators and optimized parameters (explant used, sterilization techniques, media content, pH media, plant growth regulators, incubation condition, and incubation temperature). The cells were subcultured for long-term callus maintenance and subjected to SEM analysis for somatic embryogenesis confirmation. Four set of genes associated to somatic embryogenesis (SERK, BBM, LEC1, and WOX) were studied based on National Centre for Biotechnology Information database and literatures. The data obtained were used for primer design and gene amplification using 3 different tissues (leaf, stem, callus).The highest frequencies of callus induction were observed on Murashikage and Skoog medium supplemented with 6-Benzylaminopurine and 1-Naphthaleneacetic acid together with optimized growth parameters. SEM analysis showed embryogenic characteristics in cells of the yellow compact calli evidenced by the presence of small and isodiametric cells. Only SERK gene was successfully amplified and enable to proceed with in silico analysis. This study provides fundamental results for genetic conservation effort of A. malaccensis related to somatic embryogenesis study

Study on Soil Properties Towards Formation of High-Quality Agarwood Resin in Aquilaria Crassna

Agarwood has been used for its fragrance and medicinal properties in Asian culture for centuries. In recent years, agarwood gains its popularity in the west due to its usage in perfume formulation. Unfortunately, the supply does not meet the augmentation market demand. This is mostly because of depleting nature agarwood reservoir and lack of induction technique that can produce agarwood consistently in the plantation. In this study, we look into a case where artificial inducing technique successfully produced high quality agarwood. To assure its quality, agarwood chip was analysed by Gas Chromatography for its chemical profiles. Discovered compounds were identified as sesquiterpene group which also had been characterized as major agarwood compound listed on previous studies. Few compounds that are detected such as δ-cadinene (0.20%), jinkoh-eremol (22.09%), epi-α-cadinol (4.74%), agarospirol (3.75%) and others. Soil condition that contributes into this agarwood formation; soil analysis on physical properties, chemical properties and nutrients content of the soil have been analyzed. Based on the findings, soil condition is an important factor to successfully induced high quality agarwood