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

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

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