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.

The genus Narcissus has several endemic, rare and/or threatened species in the Iberian Peninsula and North Africa. In vitro propagation is a useful tool for threatened plants conservation used in ex situ strategies. Thus, the aim of this work was to study the propagation in vitro of bulb scale explants of five endemic, rare and/or endangered Narcissus species from the Iberian Peninsula, treated with different PGR combinations. Initiation was achieved in half-strength Murashige and Skoog (MS) basal salts and vitamins, 10 g/L sucrose, 500 mg/L casein hydrolysate, 2 mg/L adenine, 10 mg/L glutathione and 5.5 g/L plant agar. In the multiplication phase, the highest bulblet proliferation was obtained in MS medium supplemented with 30 g/L sucrose and the combination of 10 μM 6-Benzylaminopurine (BAP) + 5 μM α-Naphthaleneacetic acid (NAA) in N. alcaracensis, N. eugeniae and N. hedraeanthus; 20 μM BAP + 5 μM NAA in N. jonquilla and N. yepesii. The highest rooting was obtained with 5 μM NAA + 1 μM Indole-3-butyric acid (IBA) for all species (>75%) and more than 80% of the produced bulblets were successfully acclimatized.