RNA extraction involves several main stages, regardless of the method of extraction: homogenization, effective denaturation of proteins from RNA, inactivation of ribonuclease and removal of any DNA, protein, and some residual contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of polysaccharides and phenols. Several efforts have been made towards the comparison and optimization of extraction and purification methods for RNA from plant tissues. This is dictated by the necessity of obtaining RNA of a good quality and in a sufficient quantity for further molecular analyzes. Plant storage organs (such as bulbs or seeds) rich in polysaccharide and polyphenolic compounds present distinct challenges for total RNA isolation. Such components, considered in this case as contamination, may bind and co-precipitate with nucleic acids and negatively affect later assays. Since standard routine protocols yield unacceptable results in bulbs, we have designed a new method for RNA extraction. We used two modified procedures (based on CTAB and sarkosyl reagents) of RNA extraction from so called “difficult plant material” and compared them to a popular RNA isolation base on the column isolation kit and TriPure reagent. Our modified protocols dealt with problems of both RNA degradation and low yield caused by co-purification with polysaccharides present in plant bulbs. In this study we have shown that improvement of the CTAB and sarkosyl method with a lyophilization step of plant tissues leads to isolation of high quality RNA from difficult material like storage organs of bulbous plants. The main changes in the procedure compared to the previously described methods concerned the different order of lithium chloride and sodium acetate addition, lithium chloride concentration increase and modification of centrifugation conditions. Gel electrophoresis and spectrophotometer analysis confirmed the high quality and integrity of the obtained RNA. The modified procedures allowed for obtaining a satisfying amount of RNA concentration in the range from 280 to 950 ng/μl depending on the plant species. Thus, the demonstrated RNA isolation methods are efficient and can be used for plant material rich in polysaccharides, such as bulbs.

Chrysanthemum stunt viroid (CSVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, a colorimetric loop-mediated isothermal amplification (LAMP) technique was developed. Six LAMP primers were newly designed and tested to determine the optimal conditions using a recombinant plasmid of CSVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 45 min. Under these conditions, a ladder-like pattern of LAMP products was detected along with a change of color from pink to yellow in the positive reactions. Limits of the detection (LOD) of colorimetric LAMP were up to 1 fg ∙ μl–1 of plasmid DNA concentration which was 104 times greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruses and viroids. From detection of actual samples and chrysanthemum plantlets which were obtained from meristem tip culture, the colorimetric LAMP showed effective potential in detecting CSVd. Therefore, the colorimetric LAMP can be used as a main technique to detect CSVd and ensure CSVd-free chrysanthemum plantlet production due to its accuracy, rapidness and sensitivity.