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.

Abstract Mechanical damage to scales of Hippeastrum × hybr. bulbs leads to the formation of phytoalexin-like compounds which redden the wounded tissue. The reaction is accompanied by an increase in methyl jasmonate (JA-Me). Applying 2-(4-isobutylphenyl) propionic acid, a jasmonate biosynthesis inhibitor, decreases the level of endogenous jasmonates and decreases the plant's ability to produce the red pigment. Experimental results indicate that jasmonates are involved in the defense response to wounding in Hippeastrum, which is manifested in the formation of red pigment, a compound of chalcones and flavans with phytoalexin-like properties.