Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

BackgroundCapsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established.ResultsIn this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 ?m of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant.ConclusionsWe have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants

Identification of differentially expressed genes during somatic embryogenesis of Axonopus compressus by restriction fragment differential display-coupled FSD

We present an approach to profile the expression of gene family members during somatic embryogenesis of Axonopus compressus through the modification of a recently developed RNA fingerprinting method. The protocol combines restriction fragment differential display technology with primers directed to a gene family specific domain signature on mRNA and hence is termed ‘RFDD-coupled FSD’. By using this method, two differentially expressed mRNAs, AC1 and AC2, containing the chromo-like domain and the serine/threonine protein kinase signatures, respectively, were isolated. Sequence analysis revealed that AC1encoded a MIP (plasma membrane protein) meanwhile AC2 shared low homology to other serine/threonine protein kinases. The abundance of these two transcripts appeared to be down-regulated in the embryogenic and non-viable embryogenic tissues when examined on Northern and semi-quantitative RT-PCR analyses, suggesting possible developmental arrest. This preliminary differential expression examination has somewhat provided a first insight into the molecular events underlying A. compressus somatic embryo development. Thus, RFDD-coupled FSD has been shown to be an efficient procedure for identifying differential regulated transcripts that correspond to their defined gene family domain signature sequences

Transcriptome-wide identification and characterization of the Rab GTPase family in mango

© 2020, Springer Nature B.V. The Rab GTPase family plays a vital role in several plant physiological processes including fruit ripening. Fruit softening during ripening involves trafficking of cell wall polymers and enzymes between cellular compartments. Mango, an economically important fruit crop, is known for its delicious taste, exotic flavour and nutritional value. So far, there is a paucity of information on the mango Rab GTPase family. In this study, 23 genes encoding Rab proteins were identified in mango by a comprehensive in silico approach. Sequence alignment and similarity tree analysis with the model plant Arabidopsis as a reference enabled the bona fide assignment of the deduced mango proteins to classify into eight subfamilies. Expression analysis by RNA-Sequencing (RNA-Seq) showed that the Rab genes were differentially expressed in ripe and unripe mangoes suggesting the involvement of vesicle trafficking during ripening. Interaction analysis showed that the proteins involved in vesicle trafficking and cell wall softening were interconnected providing further evidence of the involvement of the Rab GTPases in fruit softening. Correlation analyses showed a significant relationship between the expression level of the RabA3 and RabA4 genes and fruit firmness at the unripe stage of the mango varieties suggesting that the differences in gene expression level might be associated with the contrasting firmness of these varieties. This study will not only provide new insights into the complexity of the ripening-regulated molecular mechanism but also facilitate the identification of potential Rab GTPases to address excessive fruit softening

Variation in turfgrasses demonstrated by amplified fragment length polymorphism (AFLP)

The classification of turfgrass cultivars is difficult as it has mostly relied on morphological characteristics that are multigenic, quantitative, and susceptible to modification by environmental factors. In this study, we employed an Amplified Fragment Length Polymorphic (AFLP) technique to distinguish between several turfgrass samples (including two ‘offtypes’) that were collected from Malaysian golf courses in comparison to four Australian Cynodon samples. The results clearly demonstrated the applicability of AFLP as a method to identify and fingerprint turfgrass samples and their ‘offtypes’. The agreement of the three dendrograms generated by AFLP, Arbitrary Primed Polymorphic DNA (AP-PCR) and Internal Transcribed Spacer (ITS) gives good confidence in the use of the AFLP data for identification and analysis of relationships between the Australian Cynodon samples. The occurrence of ‘offtypes’ at one of the Malaysian golf courses is possibly a result from somatic mutation (TD6) or sod contamination at planting (TW4)

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

Bioengineering of the plant culture of Capsicum frutescens with vanillin synthase gene for the production of vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand towards vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L.

Background Capsicum is a genus of important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava via a biolistic particle delivery system. In addition, in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results In this biolistic system study, explant target distance, bombardment helium (He) pressure and the size of microcarrier were the key parameters to be investigated. The optimized parameters based on screening of GFP expression were determined to be 6 cm target distance, 1350 psi of helium pressure and 1.6 μm of gold particle (microcarrier) size. The greatest number of shoots were obtained from hypocotyl as explant using Murashige and Skoog medium supplemented with 5.0 mg/L BAP and 0.1 mg/L NAA. An average of 5 shoots per explant were formed. Out of which, one shoot managed to form root and developed into whole plant. Conclusions We have obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of the chili plants

Characterization of Southeast Asia mangoes (Mangifera indica L) according to their physicochemical attributes

Mango (Mangifera indica L.) is an economically important fruit crop grown in the tropics. One of the important traits of mango for successful commercial production is the storage quality of the fruit. This study was conducted to evaluate the postharvest qualities of three mango (Mangifera indica) varieties namely ‘Chokanan’, ‘Golden phoenix’ and ‘Water lily’ grown in Southeast Asia regions. The study found that variety and ripening stage had an impact on the postharvest qualities. In general, an increase in weight loss, L* value and soluble solids concentration (SSC) along with a reduction in titratable acidity (TA), firmness and hue value as ripening progressed were observed irrespective of the variety. Analysis of variance and multivariate analysis were used to characterize the ripening process. This study provides useful information for devising strategies in postharvest handling and implementation of breeding programs for mango crop improvement

Comparative proteomic analysis on fruit ripening processes in two varieties of tropical mango (Mangifera indica)

Mango (Mangifera indica L.) is an economically important fruit. However, the marketability of mango is affected by the perishable nature and short shelf-life of the fruit. Therefore, a better understanding of the mango ripening process is of great importance towards extending its postharvest shelf life. Proteomics is a powerful tool that can be used to elucidate the complex ripening process at the cellular and molecular levels. This study utilized 2-dimensional gel electrophoresis (2D-GE) coupled with MALDI-TOF/TOF to identify differentially abundant proteins during the ripening process of the two varieties of tropical mango, Mangifera indica cv. ‘Chokanan’ and Mangifera indica cv ‘Golden Phoenix’. The comparative analysis between the ripe and unripe stages of mango fruit mesocarp revealed that the differentially abundant proteins identified could be grouped into the three categories namely, ethylene synthesis and aromatic volatiles, cell wall degradation and stress-response proteins. There was an additional category for differential proteins identified from the ‘Chokanan’ variety namely, energy and carbohydrate metabolism. However, of all the differential proteins identified, only methionine gamma-lyase was found in both ‘Chokanan’ and ‘Golden Phoenix’ varieties. Six differential proteins were selected from each variety for validation by analysing their respective transcript expression using reverse transcription-quantitative PCR (RT-qPCR). The results revealed that two genes namely, glutathione S-transferase (GST) and alpha-1,4 glucan phosphorylase (AGP) were found to express in concordant with protein abundant. The findings will provide an insight into the fruit ripening process of different varieties of mango fruits, which is important for postharvest management