INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 21–0044/2021/26–2–251–256 DOI: 10.17957/IJAB/15.1831 http://www.fspublishers.org Full Length Article Expression Changes of Genes Related to Germination Based on EST Database under Priming Treatment by Gibberellic Acid in Perilla frutescens (Korean Perilla) Eun Soo Seong1†, Byeong Ju Kang2†, Ji Hye Yoo3, Jae Geun Lee4, Na Young Kim5 and Chang Yeon Yu2* 1Department of Medicinal Plant, Suwon Women’s University, Suwon 16632, Republic of Korea, South Korea 2Department of Bio-Resource Sciences, Kangwon National University, Chuncheon 24341, South Korea 3Bioherb Research Institute, Kangwon National University, Chuncheon 24341, South Korea 4Research Institute of Biotechnology, Hwajin Biocosmetics, Hongcheon 25142, South Korea 5Hotel Culinary Arts, Songho University, Hoengseong 25242, South Korea *For correspondence: [email protected] †Contributed equally to this work and are co-first authors Received 12 January 2021; Accepted 30 April 2021; Published 10 July 2021 Abstract It is very important to establish an optimal seed priming process in order to increase the vitality of the seeds and promote the metabolism for the germination of the seeds. The optimum concentrations and species of priming agents to improve seed germination of both medicinal plants were also estimated. To improve the germination rate of Perilla frutescens (Korean perilla) seeds, various seed priming agents were used to analyze seed germination rates in the Saeyeopsil, Okdong and 141 collection Korean perilla cultivars. The agents used for seed priming were CaCl2, Ca(NO3)2, NaCl, K3PO4, polyethylene glycol, and gibberellic acid (GA3). When 0.1 mM GA3 was used for seed priming, germination rates of Okdong, and the 141 collection showed a greater than 70% increase compared to the controls. Nine genes were selected for expression analysis by searching for genes related to seed germination and plant development in the EST (Expressed Sequence Tag) database of the Korean perilla cDNA library. GA3 priming treatment for 1 d induced higher transcriptional levels of genes related to germination and plant development than controls treated with water only. These genes were identified as protochlorophyllide reductase-like, magnesium-chelatase subunit ChlI, heme-binding protein 2-like, glyceraldehyde 3-phosphate dehydrogenase A, Chlorophyll a-b binding protein 6, B2 protein, 2-Cys peroxiredoxin BAS1, and 21 kDa protein. From these results, we suggest that when priming Korean perilla seeds with GA3, a large number of genes involved in plant development at early stages of seed germination play a role in improving the seed germination rate. Also, these induced genes are ideal candidate biomarkers for seed priming of Korean perilla. Specially, protochlorophyllide reductase-like is thought to be a potential gene for future molecular marker. © 2021 Friends Science Publishers Keywords: EST database; GA3; Germination rate; Perilla frutescens; Seed priming Introduction terpenoids and phenolic acids have been extracted from seeds of Korean perilla and studied, with several studies Perilla frutescens is a plant native to regions of Southeast reporting on the importance of flavonoids and phenolic Asia and has various uses such as an ingredient in natural compounds in relation to biological activity (Ozturk et al. products and food, and as a medicinal pigment (Seong et al. 2010; Kim et al. 2019). 2009). This plant has long been utilized as a raw material for Seed priming technology using Ca(NO3)2, KNO3, oil extraction and is commonly known as “Dlggae” in MgSO4, NaNO3, KCl, K3PO4, NH4NO3 and PEG 6000PEG Korea. Recently, consumption of perilla has increased (polyethylene glycol) involves pretreatment of seeds with significantly in Korea; more than 60% of the total different agents with varying concentration, duration, or unsaturated fatty acids (FAs) in perilla seeds comprises α- temperature conditions, with the goal of improving seed linolenic acid (Ichikawa 2006), an essential FA required for production under given environmental conditions (Park et human growth and development, in addition to its known al. 2013). The success of priming is strongly involved in the major role in preventing and treating blood vessel diseases hydration of the metabolism and process by which the (Shahidi and Miraliakbari 2005). Many flavonoids, sterols, seed absorbs a limited amount of water (Rahimi 2013). To cite this paper: Seong ES, BJ Kang, JH Yoo, JG Lee, NY Kim, CY Yu (2021). Expression changes of genes related to germination based on EST database under priming treatment by gibberellic acid in Perilla frutescens (Korean perilla). Intl J Agric Biol 26:251‒256 Seong et al. / Intl J Agric Biol, Vol 26, No 2, 2021 The complex network involved in seed metabolism is priming treatment were CaCl2, Ca(NO3)2, NaCl, K3PO4, dependent on the agent used, duration, and temperature of PEG 6000 and GA3. The concentrations of CaCl2, Ca(NO3)2, the priming treatment, as well as vigor, dehydration, and NaCl and K3PO4 used were 100, 300 and 500 mM, storage conditions of primed seeds (Dezfuli et al. 2008). respectively, and 0.6 and -0.9 MPa for PEG 6000. GA3 was Seeds priming to enhance seed quality show increase pattern used at concentrations of 50, 100, 300 and 500 μM. Among of germination rate which result in high levels of abiotic priming techniques, osmotic priming and biopriming are the stress resistance. All these characteristics directly correlate most widely used. Chemicals related to osmotic priming to seed vigour, plant genotype and physiology controlled by include CaCl2, Ca(NO3)2, NaCl, K3PO4, and PEG 6000, and multiple genetic and environmental factors (Jisha et al. GA3, a metabolite related to biopriming, was also selected 2013). Priming method is generally used to treat vegetables and applied to the experiment. Treatment for concentrations seeds such as carrot, celery, lettuce, pepper and tomato of priming agents and seeds were preceded at 20°C for 3 (Paparella et al. 2015). However, the establishment of seed days in a dark condition (Park et al. 2013). priming techniques for medicinal crops is extremely limited. Therefore, it is necessary to improve the germination rate, Germination of primed Korean perilla seeds shorten the number of days it takes to germinate, and establish optimal priming conditions for uniform seedling Korean perilla seeds were sterilized with 70% ethanol for 5 production in medicinal crops. min and 1% hydrogen peroxide for 5 min, and then dried The effect of priming has been proven to improve seed naturally for 1 hour to achieve moisture balance of seeds. germination and seedling growth using numerous chemical Next, 100 mL of priming solution and 5 g of sterilized factors in various crops such as wheat, beans, sunflower, perilla were placed in an Erlenmeyer flask. Priming corn, and brassica (Cho et al. 2011a). For instance, the treatment with CaCl2, Ca(NO3)2, NaCl, K3PO4 and PEG germination characteristics of corn seeds were improved 6000 was carried out for 3 days, at 20°C in the dark on a after gibberellic acid (GA3) or hydropriming treatment shaking incubator. Priming treatment with GA3 was (Subedi and Ma 2005). performed under dark condition at 20°C for 1 day. Gibberellic acid (GA3) is essential for seed germination and flower development; for example, Gene selection and primer design from the EST Arabidopsis exhibiting a deficiency in GA3 content showed databases of Korean perilla defects in seed germination and organ formation (Kim et al. 2014). In addition, loss-of-function studies have identified In our previous study, we analyzed and reported the many genes involved in GA3-induced seed germination metabolic classification for genes from the EST database (Cao et al. 2006). Genetic markers responding to GA3 may contained in the Korean perilla cDNA library (Seong et al. be used to assess the specificity, which AtGA3ox1(GA4) 2015). As a result of Seong et al. (2015), nine genes related was downregulated by GA3 activity (Silverstone et al. to seed germination were selected for analysis and are 2001). Gene expression regulated by GA3 during the shown in Table 1, with numbers and the annotation of the germination process has also been studied, helping to EST database (Seong et al. 2015). To analyze the explain the GA3 response mechanism (Cao et al. 2006). expression patterns of the 9 selected genes, RT-PCR were The purpose of this study was to establish an optimal performed with 20-mer primers designed using the PICK germination system for Korean perilla through priming primer program on the Bioneer homepage treatment, using various agents such as CaCl2, Ca(NO3)2, (https://www.bioneer.co.kr/index.php/). NaCl, K3PO4, polyethylene glycol (PEG), and GA3. In this report, we investigated the germination ratios resulting from RNA extraction from Korean perilla treated with GA3 the application of all the agents used in the seed priming treatments. Furthermore, this study aimed to reveal the The prepared samples were placed in a pre-frozen pestle genetic relationship between seed germination and GA3 bowl with liquid nitrogen and ground to a fine powder using response, using gene expression data from the Perilla a stick. The ground sample was placed in a tube with frutescens EST database generated in our previous study TRIzol® Reagent (Thermo Fisher Scientific, USA), allowed (Seong et al. 2015). to stand at room temperature for 5 min, with shaking, for thorough mixing. The samples were separated using a Materials and Methods centrifuge at 13000 rpm and the supernatant transferred to a new tube, chloroform was added and left for 10 min, with Priming treatments for Korean perilla seeds using shaking. The sample was again centrifuged at 13000 rpm various agents and the supernatant transferred to a new tube. The supernatant was slowly mixed with 2‒3 times volume of All seeds used in this study were stored at 4°C and the isopropanol and stored overnight at -20°C.