Sufficient yields of high-quality RNA are needed for next-generation sequencing and high-throughput real-time polymerase chain reaction analyses. In the case of strawberry (Fragaria ×ananassa) fruits, successful RNA isolation requires removal of abundant inhibitory substances (polysaccharides and polyphenols) that greatly reduce quality and yield. In this study, we applied various combinations of RNA isolation protocols directed at reproductive organs. The best manual isolation method involved nonionic polymer and modified acid guanidinium thiocyanate-phenol-chloroform treatments followed by phenol/chloroform/isoamyl alcohol extraction. Compared with other methods, this approach gave significantly higher yields [84.0 µg/g fresh weight (FW)] of RNA of greater purity (A260/A280 = 1.99; A260/230 = 1.51). Better-quality RNA (A260/230 = 2.11) was obtained using an automated method, but the yield was lower (18.1 µg/g FW) than that obtained manually. This automated method consisted of pretreatment with nonionic polymer followed by a silica-based system extraction. Although RNA of sufficient quality [RNA Integrity Number (RIN) ≥ 6.5 and 28S/18S ≥ 1.0] for RNA sequencing was obtained from receptacles using both automated and manual methods, the manual method yielded high-quality RNA from achenes and anthers. The automatic method features 6-fold faster high-throughput capacity, whereas the manual method has wider applicability to different tissues.
Misaki Ishibashi, Takeshi Nabe, Yoko Nitta and Yuichi Uno
Ryohei Koyama, Hiromichi Itoh, Syuji Kimura, Ai Morioka and Yuichi Uno
Plants can synthesize some antioxidants, including L-ascorbic acid (AsA) and polyphenol, in response to environmental stresses. Antioxidants detoxify reactive oxygen species in plants and also aid in human health. In this study, we demonstrate that a novel hydroponic treatment can increase leafy vegetable nutritional quality without retarding growth. Leaf lettuce (Lactuca sativa) was grown hydroponically and subjected to rhizosphere drought stress by lowering the water level in the solution tub before harvesting. Appropriate drought stress using this method could increase AsA, polyphenol, and sugar content by 24%, 50%, and 17%, respectively, and decrease nitrate nitrogen content by 18% without reducing yield. Similar effects of drought stress on AsA content were observed in four other plant species. This hydroponic method has a universal potential to increase leafy vegetable quality without reducing yield in controlled environments such as plant factories.
Yusuke Ii, Yuichi Uno, Michio Kanechi and Noboru Inagaki
To determine the sex of asparagus (Asparagus officinalis) at the seedling stage, an easy, economical, and reliable method was developed. We used a modified single-step DNA extraction protocol, which resulted in a crude extract containing sufficient genomic DNA for use as a template. The male-specific marker (Asp1-T7sp) is a dominant marker and may lead to false negatives caused by an incomplete reaction; therefore, a multiplex polymerase chain reaction (PCR) was developed using a ribosomal RNA gene marker. The resulting banding pattern distinguished males from females without false negatives. To determine the best tissue for extraction of template DNA, phylloclades (a specialized stem that resembles and functions like a leaf) or root tips of individual asparagus plants were collected and weighed. A 4.0-mg phylloclade sample or a 0.8-mg root sample provided sufficient DNA for PCR analysis of asparagus. Root excision at day 19 did not affect subsequent growth of asparagus seedlings after 28 days. The method can determine the sex of asparagus at day 19 after seeding. A combination of single-step DNA extraction from root tips and multiplex PCR made for a simple and reliable screening method.