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
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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. Field emergence was assessed in the experimental fields of the Faculty of Agriculture, Ege University, Izmir (38°25'N; 27°8'E; 14 m a.s.l.). The soil was sandy loam (62% sand; 20% clay) with 1% organic matter, 3% lime and pH 7.3. In the field emergence test (FE),
4 × 100-seeds of each lot were sown by hand in a randomised block design on 5 May 2017
383 HULYA ILBI, ALISON A. POWELL AND OZLEM ALAN
with 10 mm-spacing. The average minimum and maximum temperatures were 14.9 and
24.4°C and total rainfall was 25.9 mm during the field experiment. Seedling emergence percentage was calculated after 25 days. Pearson correlation analysis with two tailed and linear regression analysis was carried out using IBM SPSS statistics programme version 25.
Results
The germination capacity of the six seed lots ranged between 78 and 90% which is above the commercially required level of 75%. However, the field emergence of the seed lots varied from 34 to 87% (table 1) and there were large differences in the field emergence of lots with similar normal germinations (% normal seedlings). For example, lots T2 and T6 with field emergence of 34 and 87%, respectively. The field emergences of the lots were significantly correlated with their MGT values (r = 0.85, P ≤ 0.05). Seed lots having the lowest field emergence (T1 and T2) had the highest MGT, while the more vigorous lots (T5 and T6), with high field emergence, had the lowest MGT.
Table 1. Normal and abnormal seedlings, mean germination time (MGT) and radicle emergence (RE) assessed in the standard germination test at 20/30°C (16 hours / 8 hours), and field emergence percentages of six marigold (Tagetes spp.) seed lots.
Normal Abnormal RE counts RE counts Field Lot MGT Species seedlings seedlings (%) (%) emergence No. (days) (%) (%) at 49 hours at 66 hours (%)
T1 T. patula 78 12 2.84 44 74 56
T2 T. patula 87 8 2.84 44 75 34
T3 T. erecta 81 16 2.29 71 82 68
T4 T. patula 90 9 2.28 72 91 79
T5 T. patula 83 13 1.75 90 92 83
T6 T. erecta 88 10 2.18 82 91 87
MEAN 85 11 2.36 67 84 68
Comparison of the germination progress curves of the seed lots, from which the MGT was calculated, showed that differences between lots were particularly clear in the first 97 hours (figure 1). The RE values between 49 and 97 hours were significantly correlated with the field emergence of the lots, with the correlations between RE at 49 and 66 hours and field emergence being highly significant (r = 0.90 and 0.91, respectively, P < 0.01; table 2). The R2 values of 0.81 and 0.83 (figure 2) indicated that 81% (49 hours RE)
384 SINGLE COUNTS OF RADICLE EMERGENCE FOR MARIGOLD
100
80
T1
60 T2 T3 T4 40 T5 T6 20 Radicle emergence (%) emergence Radicle
0 0 20 40 60 80 100 120 140 160 180 Time from being set to germinate (hours) Figure 1. Germination (radicle emergence) progress curves of six seed lots of Tagetes spp. Each point is the mean of four replicates of 100 seeds.
Table 2. Correlation coefficient (r) values for the relationship between the field emergence of six seed lots of marigold (Tagetes spp.) and both their radicle emergence assessment after different periods during germination and their normal germination. * P < 0.05, ** P < 0.01. Time (hours) of RE assessment Correlation coefficient (%) 25 0.57 42 0.63 49 0.90** 66 0.91** 73 0.84* 90 0.86* 97 0.81* 114 0.75 121 0.74 138 0.67 145 0.65 162 0.61 169 0.64
Normal germination (%) 0.21
385 HULYA ILBI, ALISON A. POWELL AND OZLEM ALAN and 83% (66 hours RE) of the variation in field emergence was accounted for by the regression on RE. Thus the seed lots with low field emergence (low vigour), such as lots T1 and T2 could be identified by the low RE counts after both 49 and 66 hours germination (table 1) in comparison with the lots with high emergence (high vigour; lots T5 and T6) which had high RE counts. In contrast, only 6% of the variation in the field emergence was accounted for by the regression on standard germination (r = 0.25, 2 R = 0.063; table 2).
100 0 408 4 6422 0 R 0 81 80
0 (%) ce 60
erge n 50
40
30 ield em ield 20
10
0 0 10 20 30 40 50 60 0 80 0 100 Radicle emergence after hours (%)
100
0 2 1802 115 6 80 R 0 83
0 (%) ce 60
erge n 50
40
30 ield em ield 20
10
0 0 10 20 30 40 50 60 0 80 0 100 Radicle emergence after hours (%)
Figure 2. The relationship between the radicle emergence count after (A) 49 and (B) 66 hours during a standard germination test at 20 / 30°C (16 hours / 8 hours) and the field emergence (%) of six lots of marigold seeds. **P < 0.01
386 SINGLE COUNTS OF RADICLE EMERGENCE FOR MARIGOLD
Discussion
A single count of radicle emergence at either 49 or 66 hours during the germination test revealed differences in the vigour of marigold seed lots as expressed in their field emergence. The RE test clearly illustrated differences in quality that were not evident in the standard germination test, as the final normal germination count was not correlated with the emergence of the seed lots. The low RE associated with low field emergence was also associated with a higher MGT which has been shown to be a consequence of seed ageing in maize (Matthews and Khajeh Hosseini, 2007). The higher MGT reveals a longer delay or lag period before RE and has been interpreted as being due to the need for more time for metabolic repair in deteriorated seeds before germination processes can begin (Matthews et al., 2011). Differences in the level of deterioration or ageing of seed lots can be determined by the ISTA validated vigour tests based on the whole process of ageing i.e. the CD test and AA test (Powell and Matthews, 2005). However, these tests usually take a long time and the RE test is a simple and quick way to predict vigour differences in a wide range of species. Furthermore, the test could be incorporated into routine germination testing and hence assess germination and vigour within one test. In the current work, we used two species of Tagetes, T. erecta and T. patula, but as these are often considered merged (USDA, Agricultural Research Service, National Plant Germplasm System, 2020), we believe that our work provides evidence that the same conditions for the RE vigour test can be applied to both species. There is a limited amount of data available on the prediction of vigour in flower species and until now, there has been no data available on the potential for a vigour test to reveal differences in vigour of marigold seed lots. We have shown that vigour differences in marigold seed lots are revealed by MGT (lag period) and that a single count of RE at 49 or 66 hours predicts the vigour of marigold seed lots as reflected in their field emergence.
Acknowledgement
The authors thank Rita Zecchinelli, ISTA Flower Commitee Chair for providing seed material.
References
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387 HULYA ILBI, ALISON A. POWELL AND OZLEM ALAN
Artola, A., Garcia de los Santos, G. and Carrillo Castañeda, G. (2003). A seed vigour test for birdsfoot trefoil (Lotus corniculatus L.). Seed Science and Technology, 31, 753-757.
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Mavi, K., Demir, I. and Matthews, S. (2010). Mean germination time estimates the relative emergence of seed lots of three cucurbit crops under stress conditions. Seed Science and Technology, 38, 14-25.
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