terclim by ICS banner
IVES 9 IVES Conference Series 9 Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Optimized protocol for high-quality RNA extraction from grape tissues using sorbitol pre-wash

Abstract

Obtaining high-quality RNA from grape tissues, including berry pulp, berry skins, stems, rachis, or roots, is challenging due to their composition, which includes polysaccharides, phenolic compounds, sugars, and organic acids that can negatively affect RNA extraction. For instance, polyphenols and other secondary metabolites can bind to RNA, making it difficult to extract a pure sample. Additionally, RNA can co-precipitate with polysaccharides, leading to lower extraction yield. Also, sugars and organic acids can interfere with the pH and ionic properties of the extraction buffer. To address these challenges, we optimized a protocol for RNA isolation from grape tissues. Although commercial kits can provide a rapid extraction, they were inefficient for these plant materials. Similarly, protocols that work well for other vegetal tissues were also inefficient and time-consuming on grape tissues. To overcome these limitations, we added a sorbitol pre-wash step to both a three-day long protocol based on LiCl precipitation and a commercial kit. Our results showed that the addition of a sorbitol pre-wash improved multiple parameters: the A260/280 absorbance ratio, integrity and quality (IQ), and RNA integrity number (RIN). Sorbitol played a crucial role in ensuring high-quality RNA extraction from grape tissues. It inhibits RNase, thereby preserving RNA integrity and stability. It also helps in disrupting cellular membranes, facilitating the release of RNA, and maintains the osmotic pressure through hypertonicity, which is beneficial to RNA extraction. By using sorbitol, commercial kits can be used to extract RNA from challenging grape tissues, leading to an efficient and time-saving procedure.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Annalisa Prencipe1, Antonella Salerno1,2, Marco Vendemia2, Carlo Bergamini2, Margherita D’Amico2, Lucia Rosaria Forleo2, Teodora Basile2, Maria Francesca Cardone2, Antonio Domenico Marsico2, Riccardo Velasco2, Mario Ventura1, Flavia Angela Maria Maggiolini2*

1 Department of Biosciences, Biotechnology and Environment, University of Bari “Aldo Moro”, 70125 Bari, Italy
2 Council for Agricultural Research and Economics – Research Center Viticulture and Enology (CREA-VE), Via Casamassima 148-70010 Turi (Ba), Italy

Contact the author*

Keywords

Vitis vinifera, RNA, sorbitol, extraction protocol

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

Using climate services to project grapevine varietal adequation under climate change – application to cv. Tempranillo in the Douro wine region

Vine growth circumstances are becoming warmer and drier because of climate change. Higher temperatures advance ripening to a point in the season less conducive to the production of fine wine, while drought reduces yields (Van Leeuwen et al., 2019). Several wine-producing regions around the world have already recognized threats to their viticultural viability (Santos et al., 2020). An economical and cost-effective strategy for adaptation is the employment of late-ripening, drought-resistant plant material (varieties, clones, and rootstocks).

ePROSECCO: Historical, cultural, applied philosophy analysis and process, product and certification innovation, for the “sustainable original progress and promotion 4.1c” of a historic and famous territory and wine

According to the algorithm “A step back towards the future 4.1C”, (Cargnello,1986a, 1987d, 1988a.b, 1991, 1993, 1994b, 1995, 1999a.e, 2000b, 2007c, 2008a, 2009d, 2013; and according to the principles of “Charter of Sustainable Viticulture BIO‐MetaEthics 4.1CC” of GiESCO (Carbonneau and Cargnello, 2003 2015, 2017), the historical, applied philosophy and productive analysis connected to the innovations and to the “Certification of the Universal Holistic MetaEthical Sustainability 4.1C” “indexed new global production model 4.1C” has always been fundamental, especially for the “Prosecco Territory” and for the “Prosecco Wine” to design and implement their synergistic future “Sustainable and Certificable 4.1CC” according to the principles of the “Charter of Sustainable Viticulture BIO‐MetaEthics 4.1CC” by the GiESCO (Carbonneau and Cargnello, lc, Cargnello et Carbonneau, 2007, 2018), and of the Conegliano Campus 5.1C. (Cargnello, lc). Nowadays, people think that Prosecco is a wine from the Veneto Region (from Conegliano and Valdobbiadene in particular), while it comes from Friuli‐Venezia Giulia Region (in North Eastern Italy, such as Veneto) more precisely from “Prosecco” in the Municipality of Trieste (TS‐Italy), as documented in 1382 and in 1548, when Pier Andrea Mattioli, described “that ancient wine, which is born in Prosecco”, as a wine with the following characteristics “thin, clear, shiny, golden, odorous and pleasant to taste». In 1888 at the “Wine Fair” of Trieste there were the “Sparkling wine Prosecco” by Giovanni Balanc, by Giuseppe Klampferer and that one by Marino Luxa. In the 19th century, many expressed their appreciation for the “Prosecco” of Trieste. In order to implement intra and extra territorial and cross‐border relations, as well as, the “Certification of: Products, Companies, Territory, Bio‐MétaÉthique District 4.1C” of Prosecco, a series of activities and researches were conducted in 8 companies: 5 in the “Territory of Prosecco” (TS) in which the principles of “Charter of Sustainable Viticulture BIO‐MetaEthics 4.1CC” of GiESCO (Carbonneau and Cargnello, lc) have been successfully applied. In particolar: 1‐ new and original “Sustainable 4.1C global production model” developed also to prevent the problems caused by wild boar, roe deer, and birds while safeguarding their “psychophysical wellness”, as well as the “psychophysical wellness 4.1C” of the macro and micro flora and fauna, of the biodiversity, of the landscape, etc. (Cargnello, lc), 1.2‐ chemical weed control and “Non MetaEthics 4.1C” processing with the total grass growing of the ground without or with mowing, better if it is manual to protect grass, air and soil, 2‐ recovery of “Historic”: land, vineyards, vines, biodiversity, landscapes, productions, products, … , 3‐ production of the famous “Prosekar, also rosé, of Prosecco” and “Prosecco di Prosecco”, according to “A step back towards the future 4.1C” 4‐ to offer a deserved psychophysical well‐being to the “Prosecco Territory” and entrepreneurs. 

LIFE Climawin: impacts, risks and opportunities in the transition to sustainable viticulture

The LIFE Climawin project drives the sustainable transformation of the wine sector in response to climate change through the implementation of an innovative management model applied at the demonstrative winery, Bosque de Matasnos.

Screening sensory-directed methodology for the selection of non-saccharomyces wine yeasts based on perceived aroma quality

The present work contributes by developing a rapid sensory-directed methodology for the screening and selection of high quality wines with different sensory profiles Therefore, Verdejo and Tempranillo musts were fermented with 50 different yeasts each under controlled laboratory conditions. Resulting samples were firstly categorized according to five levels of quality by a panel of wine professionals (Sáenz-Navajas, Ballester et al. 2013). Higher quality samples were described by flash profiling by a semi-trained panel
(Valentin, Chollet et al. 2012) and most distinctive samples were screened by gas chromatography-olfactometry (GC-O) (López, Aznar et al. 2002).

Growers’ attitudes towards organic certification: the case of Central Otago, New Zealand

New Zealand viticulture has long been characterised by sustainable grape growing practices as promoted by Sustainable Winegrowing New Zealand (SWNZ) as well as by Organic Viticulture.