Determination of soybean cultivar resistance to soybean cyst nematode with quantitative polymerase chain reaction

Heterodera glycines, the soybean cyst nematode, is the major pathogen of Glycine max (soybean). Effective management of this pathogen is contingent on the use of resistant cultivars, thus screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative Polymerase Chain Reaction (qPCR), a prelude to differentiation of resistance levels in soybean cultivars. Two experiments were conducted. In the first one, a consistent inoculation method was developed using to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 and 1000 J2/mL. Twenty-four hours after infestation, the roots were surface sterilized and DNA was extracted with the DNA FastKit (MP Biomedicals, Santa Ana, CA)). For the qPCR assay, primer pair for single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines DNA amplification within soybean roots. In the second experiment, compatible Lee 74, incompatible Peking and cultivars with different levels of resistance to H. glycines were inoculated with 0 and 1,000 J2/seedlings. Twenty-four hours post inoculation they were transplanted into pasteurized soil. Subsequently they were harvested at 1, 7, 10, 14 and 21 days post inoculation for DNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice; the qPCR method can replace the traditional one and improve precision in determining infection levels.

The effects of PVP(FE(III)) catalyst and polymer molecular weight on gene delivery via biodegradable crosslinked polyethylenimine

Human gene therapy has faced many setbacks due to the immunogenicity and oncogenity of viruses. Safe and efficient alternative gene delivery vehicles are needed to implement gene therapy in clinical practice. Polymeric vectors are an attractive option due to their availability, simple chemistry, and low toxicity and immunogenicity. Our group has previously reported biodegradable polyethylenimines (PEI) that show high transfection efficiency and low toxicity by cross-linking 800 Da PEI with diacrylate cross-linkers using Michael addition. However, the synthesis was difficult to control, inconsistent, and resulted in polymers with a narrow range of molecular weights. In the present work, we utilized a heterogenous PVP(Fe(III)) catalyst to provide a more controllable PEI crosslinking reaction and wider range of biodegradable PEIs. The biodegradable PEIs reported here have molecular weights ranging from 1.2 kDa to 48 kDa, are nontoxic in MDA-MB-231 cells, and show low toxicity in HeLa cells. At their respective optimal polymer:DNA ratios, these biodegradable PEIs demonstrated about 2-5-fold higher transfection efficiency and 2-7-fold higher cellular uptake, compared unmodified 25 kDa PEI. The biodegradable PEIs show similar DNA condensation properties as unmodified PEI but more readily unpackage DNA, based on ethidium bromide exclusion and heparan sulfate competitive displacement assays, which could contribute to their improved transfection efficiency. Overall, the synthesis reported here provides a more robust, controlled reaction to produce cross-linked biodegradable PEIs that show enhanced gene delivery, low toxicity, and high cellular uptake and can potentially be used for future in vivo studies.