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Single Radicle Emergence Count for Predicting Vigour of Marigold (<I Ilbi, Powell and Alan (2020). Seed Science and Technology, 48, 3, 381-389. https://doi.org/10.15258/sst.2020.48.3.06 Single radicle emergence count for predicting vigour of marigold (Tagetes spp.) seed lots Hulya Ilbi1*, Alison A. Powell2 and Ozlem Alan3 1 Ege University, Research and Technology Center for Seed Science, 35100 Bornova, Izmir, Turkey 2 University of Aberdeen, School of Biological Science, Cruickshank Building, 23 St Machar Drive, Aberdeen, AB25 3UU, United Kingdom 3 Ege University, Odemis Vocational School, 35750 Izmir, Turkey * Author for correspondence (E-mail: [email protected]) (Submitted June 2020; Accepted September 2020; Published online October 2020) Abstract This study was carried out to determine whether a single radicle emergence count (RE) during germination can be used for vigour assessment to estimate field emergence of marigold (Tagetes spp.) seed lots. Six marigold seed lots (Tagetes erecta and T. patula) with normal germination above 75% were germinated using the standard ISTA germination test and radicle emergence (production of 2 mm radicle) counted at regular intervals from 25 to 169 hours. The seed lots were also sown in the field with final seedling emergence assessed after 25 days. Seedling emergence was highly correlated with the radicle emergence count after 49 and 66 hours germination (r = 0.90 and r = 0.91, respectively; P < 0.01) but not with germination percentage (r = 0.26). Thus, the radicle emergence counts at 49 and 66 hours accounted for 81 or 83% of the variation in field emergence, respectively. The results indicate that a 49- or 66-hour count of RE during germination could be used as a vigour test to estimate field emergence for marigold seed lots. Keywords: field emergence test, marigold seed, radicle emergence test, Tagetes spp., vigour test Introduction Seed vigour is the sum total of those properties of the seed or seed lot which determines the level of activity and performance of the seed or seed lot during germination and seedling emergence (ISTA, 2020). Seed vigour is a comprehensive characteristic that can reflect the field emergence and storage performance of seed lots. The major cause of differences in vigour is seed ageing, which ultimately leads to loss of the ability to germinate (Powell, 2006). A seed lot with high germination that has undergone little ageing performs well in the field and has good storage potential, i.e. it has high vigour. However, a seed lot © 2020 Ilbi et al. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0 381 HULYA ILBI, ALISON A. POWELL AND OZLEM ALAN having a higher degree of deterioration due to ageing may germinate well under optimum conditions but perform poorly in field conditions and have poor storage potential, i.e. it is low in vigour. Differences in the level of deterioration or ageing of seed lots can be identified by vigour tests (Powell, 2006) which differentiate the potential performance of seed lots more sensitively than the standard germination test. There are five vigour tests validated by ISTA, namely the conductivity test for grain legumes, accelerated ageing and tetrazolium tests for soybean (Glycine max L. Merr.), controlled deterioration test for Brassica spp. and the radicle emergence test (RE) for maize (Zea mays L.), radish (Raphanus sativus L.), wheat (Triticum aestivum L. subsp. aestivum) and oil seed rape (Brassica napus subsp. napus) (ISTA, 2020). The RE test is based on differences in the mean germination time (MGT) of seed lots. The mean germination time (MGT) is calculated by the regular counts of germinated seeds (Ellis and Roberts, 1980) and describes the average time for a seed to germinate, or the delay (lag period) from the start of imbibition to radicle emergence (Mattthews and Khajeh Hosseini, 2007). The more deteriorated a seed lot, the higher the MGT, that is, the greater the lag period from the start of imbibition to radicle emergence due to the need for more time for metabolic repair during the imbibition period (Guy and Black, 1998; Bailly et al., 2002; Matthews and Khajeh Hosseini, 2007). Thus, the germination progress curves and MGT describe the extent of deterioration in a seed lot, and hence seed vigour. Indeed, MGT has been shown to predict differences in vigour in many species including rice (Oryza sativa L.; Luo et al., 2017), maize (Z. mays L.; Matthews et al., 2011), chickpea (Cicer arietinum L.; Akbarpour, et al., 2019), Elymus nutans Griseb and Avena sativa L. (Lv et al., 2016), cress (Lepidium sativum L.; Demir et al., 2019), three cucurbit species (Mavi et al., 2010), several flower species (Demir et al., 2011; Guloksuz and Demir, 2012) and pepper (Capsicum sativum L.; Demir et al., 2008). In spite of this, application of MGT as a vigour test, or indeed any assessment of rate of germination that involves many counts of germination over time, is not practical as a routine test in a seed testing laboratory. However, MGT can be predicted by a single radicle emergence count during the early stages of germination of many species (Khajeh Hosseini et al., 2009; Mavi et al., 2010; Guloksuz and Demir, 2012; Lv et al., 2016; Akbarpour et al., 2019). Furthermore, a single radicle emergence count has predicted seed vigour in a wide range of species (Artola et al., 2003; McLaren et al., 2010; Matthews and Powell, 2011; Matthews et al., 2011; Ermis et al., 2015; Luo et al., 2015, 2017; Ozden et al., 2017; Akbarpour et al., 2019; Demir et al., 2019). These observations led to the development of the radicle emergence vigour test, its validation by ISTA and the inclusion of the test in the ISTA Rules (ISTA, 2020), currently for the four species noted above. Marigold (Tagetes spp.) is native to Mexico and Guatamala although some species have become naturalised around the world. Most commonly cultivated are the so-called African marigolds (Tagetes erecta L.) and French marigolds (T. patula L.) although the two species are often merged (USDA, Agricultural Research Service, National Plant Germplasm System, 2020). Marigolds are an important annual flower crop grown for both ornamental and commercial purposes. The oil is used in perfumery (Kaul et al., 2000), the ground florets in the poultry industry to enhance the colour of chicken skins and egg yolks, as a colourant in human foods and as a dye, and there is a wide range of uses in 382 SINGLE COUNTS OF RADICLE EMERGENCE FOR MARIGOLD herbal medicine (Bown, 1995; Gopi et al., 2012). The flowers have cultural importance in India, Thailand and Mexico, plant extracts have been shown to have insecticidal properties (Santos et al., 2016) and the whole plant is often used in companion planting (Afzal et al., 2012; Kumar et al., 2014). Due to the commercial use of marigold in some industries in recent years, there has been increasing interest in cultivation of marigold. The crop is produced through transplants or is direct seeded and uneven germination, slow emergence and poor stand establihsment often occur (Afzal et al., 2012). Rapid and uniform seedling emergence is important for growers to ensure production of uniform transplants and to avoid gaps in the field. Hence, seed quality is important to achieve successful emergence. Germination of Tagetes spp. is tested at 20°C or alternating 20/30°C over a period of 14 days (ISTA, 2020), with a commercially required minimum germination of 75%. However, even when germination is high, plant establishment of marigold seeds is often slow and poor, particularly under hot or cool field conditions (Bosma et al., 2003; Afzal et al., 2009, 2012). This indicates that there is a vigour problem in seed lots of Tagetes spp. with high germination. The use of a vigour test could be beneficial for estimating plant establishment, but there is no vigour test method developed for application to marigold seed lots. The aim of this study was to determine whether MGT and subsequently a single radicle emergence count (RE) can be used as a vigour assessment method to predict differences in field emergence, and hence to develop a protocol for an RE test for marigold (Tagetes spp.) seed lots. Materials and methods Six marigold (T. erecta and T. patula) seed lots were obtained from Vilmorin Seed Company (four lots) and from Rita Zecchinelli, ISTA Flower Commitee Chair (two lots). Each lot came from a different variety, all of which were open pollinated. The seed lots were kept at 5 ± 1°C for two weeks (four lots) or three months (two lots) before they were used. For the standard germination (SG) test, 4 × 100 seeds of each lot were sown in Petri dishes between moistened papers, and placed into an incubator at the start of the dark phase of a 20°C dark / 30°C light (16 hours / 8 hours) regime. They were kept in these conditions for 14 days after which, the percentage of normal seedlings was assessed (ISTA, 2020). During the SG test, radicle emergence (RE, assessed as production of a 2 mm radicle) was recorded at 25, 42, 49, 66, 73, 90, 97, 114, 121, 138, 145, 162 and 169 hours after sowing. The RE counts were used to produce a germination progress curve and to calculate the mean germination time (MGT) (Ellis and Roberts, 1980). MGT is calculated as: ∑ (nt) / ∑ n, where n is the number of seeds newly germinated (2 mm-long radicle) at time, t days from when seeds were set to germinate.
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