Preparation of bio-based modified starch film and analysis of preservation mechanism for sweet cherry.

This study aimed to synthesize packaging films using bioactive ingredients. The composite film was prepared by blending octenyl succinate cassava starch ester (OSCS) with chitosan (CS) nano-ZnO and then adding ε-polylysine (ε-PL). The study also explored the effect of different concentrations of ε-PL on OSCS/CS/ZnO films. Fourier infrared spectroscopyand fluorescence microscopy revealed that the composite film was formed by both hydrogen bonding and a Schiff base reaction. The diffraction peaks of the original materials in X-ray diffraction disappeared after film formation, indicating good miscibility between the materials. Scanning electron microscope showed that the density of its structure increased with increasing the ε-PL content. The thermogravimetric analysis showed that the addition of ε-PL improved the thermal stability of the composite film to some extent. When used in cherry preservation, the bio-based modified starch film effectively reduced cherry decay, stem dryness, and weight loss, maintained surface color, and increased the soluble solid content.

Identification and characterization of drought-responsive CC-type glutaredoxins from cassava cultivars reveals their involvement in ABA signalling.

BACKGROUND: CC-type glutaredoxins (GRXs) are plant-specific glutaredoxin, play regulatory roles in response of biotic and abiotic stress. However, it is not clear whether the CC-type GRXs are involve in drought response in cassava (Manihot esculenta), an important tropical tuber root crop. RESULTS: Herein, genome-wide analysis identified 18 CC-type GRXs in the cassava genome, of which six (namely MeGRXC3, C4, C7, C14, C15, and C18) were induced by drought stress in leaves of two cassava cultivars Argentina 7 (Arg7) and South China 124 (SC124). Exogenous abscisic acid (ABA) application induced the expression of all the six CC-type GRXs in leaves of both Arg7 and SC124 plants. Overexpression of MeGRXC15 in Arabidopsis (Col-0) increases tolerance of ABA on the sealed agar plates, but results in drought hypersensitivity in soil-grown plants. The results of microarray assays show that MeGRXC15 overexpression affected the expression of a set of transcription factors which involve in stress response, ABA, and JA/ET signalling pathway. The results of protein interaction analysis show that MeGRXC15 can interact with TGA5 from Arabidopsis and MeTGA074 from cassava. CONCLUSIONS: CC-type glutaredoxins play regulatory roles in cassava response to drought possibly through ABA signalling pathway.

pXST, a novel vector for TA cloning and blunt-end cloning.

BACKGROUND: With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired. RESULTS: A bifunctional vector pXST has been constructed. The pXST vector harbors a XcmI-ccdB-XcmI cassette and restriction site SmaI. Digestion the vector with XcmI generates a single thymidine (T) overhang at 3' end which facilitates TA cloning, and SmaI gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 × 106 transformants/µg) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs). CONCLUSIONS: We describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses XcmI-ccdB-XcmI cassette and restriction site SmaI, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

BACKGROUND: Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. METHODS: Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. RESULTS: In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. CONCLUSION: We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Two new terpenoids from the stems of Manihot esculenta.

A new eremophilane sesquiterpene, sporogen AO-2 (1), and a new beyerane diterpene, thecacorin C (2), together with two known compounds, longifoamide-B (3) and methylcholestane-3ß,5α,6ß-triol (4), were isolated from the stems of Manihot esculenta. The structures of the two new compounds were determined by spectroscopic techniques (UV, IR, MS, 1D, and 2D NMR). Antimicrobial assay showed that compound 3 possessed modest inhibitory effects on Saphylococcus aureus and methicillin-resistant S.aureus, diameters of inhibition zones of which were 7.5 and 8.0 mm, respectively. Compound 4 possessed modest inhibitory effect on S. aureus, the diameter of inhibition zone of which was 6.8 mm.

Isopentenyl transferase gene (ipt) downstream transcriptionally fused with gene expression improves the growth of transgenic plants.

This research reports a promising approach to increase a plant's physiological cytokinin content. This approach also enables the increase to play a role in plant growth and development by introducing the ipt gene to downstream transcriptionally fuse with other genes under the control of a CaMV35S promoter, in which the ipt gene is far from the 35S promoter. According to Kozak's ribosome screening model, expression of the ipt gene is reduced by the terminal codon of the first gene and the internal untranslated nucleotides between the fused genes. In the transgenic plants pVKH35S-GUS-ipt, pVKH35S-AOC-ipt, and pVKH35S-AtGolS2-ipt, cytokinins were increased only two to threefold, and the plants grew more vigorously than the pVKH35S-AOC or pVKH35S-AtGolS2 transgenic plants lacking the ipt gene. The vigorous growth was reflected in rapid plant growth, a longer flowering period, a greater number of flowers, more seed product, and increased chlorophyll synthesis. The AOC and AtGolS2 genes play a role in a plant's tolerance of salt or cold, respectively. When the ipt gene transcriptionally fuses with AOC or AtGolS2 in the frame of AOC-ipt and AtGolS2-ipt, slight cytokinin increases were obtained in their transgenic plants; furthermore, those increases played a positive role in improvements of plant growth. Notably, an increased cytokinin volume at the physiological level, in concert with AtGolS2 expression, enhances a plant's tolerance to cold.

CaCl2 enhanced somatic embryogenesis in Manihot esculenta Crantz.

Primary cassava somatic embryos were induced on a medium without CaCl(2), however, no or only a few secondary somatic embryos were formed from them. With 15 mM CaCl(2) in the medium for induction of cassava primary embryos, more secondary somatic embryos were produced from them, and they were much effective in maintaining their embryogenic capacity than the controls of embryos which were induced without CaCl(2).

[Application of ICP-MS to detection of mineral elements and heavy metals in Cassava's byproducts].

Cassava is a main cultivated tropical crop in China, its rich starch roots are often used to produce fuel ethanol in recent years, so it's a kind of hot biomass energy crops. But cassava's byproducts such as leaves, stems and peels are regarded as waste, and are not fully utilized. Cassava's byproducts contain many nutrients, and can be used to process high value food products. The contents of mineral elements and heavy metals in cassava's byproducts were studied by ICP-MS. The results showed that cassava's byproducts contained many elements necessary to human health, the sequence of macroelements was K>Ca>P> Mg>S>Mn>Zn>Na>Fe>B>Cu, particularly, the contents of Fe, Mn, Zn and B ranged from 10 to 800 microg x g(-1) (DW), while the contents of microelements including Mo, Co, Se and Ge ranged from 0.01 to 0.2 microg x g(-1) (DW), which are important to human health. Besides macroelements and microelements, the contents of heavy metals (As, Cr, Pb and Hg) were also important to identify the quality of farm products, and the results showed that cassava's byproducts contained little heavy metals except Pb (2.19 microg x g(-1) (DW) in stalk peels). All the data showed that cassava's byproducts accorded with the national hygiene standards.