Germination Response of Six Sweet Basil (Ocimum basilicum) Cultivars to Temperature Dongfang Zhou, Jacob Barney, Monica A. Ponder and Gregory E. Welbaum* ABSTRACT Sweet basil (Ocimum basilicum) is a warm-season herbaceous plant typically propagated from seed. Establishment of direct-seeded basil is oen difficult because seed germination may be limited, particularly in cold soils. To determine base, optimum, and ceiling germination temperatures and possible genetic variation, seeds of six cultivars of sweet basil, Italian Large Leaf, Italian Large Leaf 63-X, Nufar, Genovese, Genovese Compact Improved, and Aroma 2 were tested on a one dimensional thermogradient table from 0–45 °C. e average optimum germination temperature range was 34.5– 39.0 °C and did not differ among cultivars. Germination rates increased from about 0.05 seeds d−1 near the base temperature to nearly 1.0 seeds d−1 at optimum temperatures for most cultivars. e ceiling temperatures varied little among cultivars, with an average of 43 ± 1.3 °C. e thermal times to germination averaged 29 °h for new crop seeds but were 40 °h for seeds of Italian Large Leaf 63-X stored for 5 y under ambient conditions. Italian Large Leaf 63-X seeds also exhibited lower vigor, germination, and ceiling temperature, compared with the new crop seeds. Base temperatures ranged among cultivars from 9.8–13.2 °C and were statistically significant, suggesting there is genetic variation that can be exploited to improve the low temperature germination performance of basil. INTRODUCTION
Sweet basil (Ocimum basilicum L.; Lamiaceae) is a frost-sensitive, annual, herbaceous plant. Basil is native to southern Asia but grown extensively in regions with temperate to hot climates throughout the world. In the United States, basil is a significant commercial crop in Arizona, California, New Mexico, Florida, North Carolina and Virginia, and is sometimes grown under cooler conditions to meet increasing demand (Simon, 1998). Sweet basil is oen cultivated at temperatures between 7–27 °C under long days in full sun on well-drained soils, and takes 65–80 d or more to mature (Simon, 1998). It can be produced in greenhouses year round. ere are more than 60 cultivars differing in appearance and taste (Putievsky, 1983). Sweet basil has many uses; it is grown as an ornamental bedding plant, as a fresh culinary herb, and for its attractive foliage, flowers, and highly fragrant leaves. e dried/frozen leaves are added to foods as a condiment or spice. A Dongfang Zhou, Research Assistant of Horticulture, Virginia Tech, Blacksburg, VA; Jacob Barney, Assistant Professor of Plant Pathology, Physiology, & Weed Science, Virginia Tech, Blacksburg, VA; Monica A. Ponder, Associate Professor of Food Science and Technology, Virginia Tech, Blacksburg, VA; Gregory E. Welbaum, Professor of Horticulture, Virginia Tech Corporate Research Center, Blacksburg, VA. *Corresponding author (
[email protected]). Received 25 Sept. 2015.
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potent antioxidant, basil oil is extracted from plants and used for its anticancer, antiviral, antimicrobial and aromatic properties (Chiang et al., 2005; Bozin et al., 2006; Almeida et al., 2007). Basil is an important traditional Chinese medicine used in treatment of cardiovascular diseases, including hypertension (Umar et al., 2010). In India, basil is used to treat stress, asthma and diabetes (Duke, 2008). To meet the growing demand for ornamental, culinary and medicinal basil, high quality seeds and a greater understanding of the environmental requirements for optimal germination and stand establishment are needed to maximize production and quality. Temperature is an important factor in basil seed germination, but the base, optimum and ceiling temperatures have not been extensively characterized. In one study, sweet basil germinated well at day/night temperature regimes between 13–30 °C, and the optimum temperature was 25 °C without saline stress (Ramin, 2006). Sweet basil germination was rapid and high at temperatures between 21–30 °C, germinating above 80% aer 4 d. At all other temperatures, either percentage or speed of germination was reduced (Putievsky, 1983). ermogradient tables have been widely used to evaluate germination responses to temperature for a number of different plant species (Orozco-Segovia et al., 1996; Schwember and Bradford, 2005; Medany and Hegazy, 2007). To determine the base, ceiling, and optimal temperatures for basil seeds, six cultivars were germinated over a range of temperatures on a thermogradient table. MATERIALS AND METHODS Plant material New crop seed of five diverse sweet basil cultivars, ‘Italian Large Leaf ’ (ILL), ‘Nufar’, ‘Genovese’, ‘Genovese Compact Improved’ (GCI) and ‘Aroma 2’, were purchased in spring 2011 from Johnny’s Selected Seeds Company (Winslow, ME, USA) and germination-tested. Seeds of ‘Italian Large Leaf 63-X’ (ILL63-X), a proprietary selection of ILL, were included in the test for comparison. ILL63-X seeds were produced in 2006 and provided by a California agent who reported low seed vigor. ILL is high yielding with a sweeter, less clove-like scent and taste compared to Genovese. Nufar is a Fusarium-resistant ILL type for field, greenhouse, and hydroponic production. Genovese is a traditional Italian basil with strong flavor. Aroma 2 is a Fusarium-resistant Genovese type and F-1 hybrid for greenhouse and field production. All seeds were sealed in plastic self-sealing bags or glass containers and stored in a refrigerator at 4 °C and 9% water content (DWB) prior to testing. ermogradient table An insulated and enclosed one-dimensional, linear, thermogradient table was used, constructed by welding square tubes to the underside of a 6.4 mm thick × 1 m wide × 1.2 m long aluminum plate. Warm and cool ethylene glycol solutions were circulated by baths (Brookfield AMETEK, Inc., Middleboro, MA, USA) through opposite ends of the table to passively establish a continuous temperature gradient along the length of the table. Across the width of the table, temperatures were stable and varied by less than 1 °C, so replications at a particular temperature could be placed across the table.
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Germination tests Germination experiments were based on AOSA procedures for basil (AOSA, 2015), but under a broader range of temperatures, and were continued until no radicle emergence occurred for three consecutive days (Elias et al., 2012). Four replications of 25 seeds each per cultivar were placed equidistant on top of two thicknesses of germination blue blotter paper (Anchor Paper Co., Saint Paul, MN, USA), inside 110 × 110 × 35 mm sealed clear acrylic boxes (Hoffman Manufacturing, Inc., Jefferson, OR, USA), moistened with 15 mL distilled water, and randomized within each temperature on the gradient table in dark. Two Watch Dog, button-style, temperature loggers (Temp 2K, B-Series; Spectrum Technologies, Inc., Aurora, IL, USA) were placed among seeds on top of the germination blotter paper in at least two boxes in each row. Temperatures in germination experiments were recorded hourly and averaged for each temperature reported. Germination was scored at 24-h intervals, and seeds with protruding radicles of at least 1 mm were recorded as germinated, then removed from the boxes with forceps (Jett and Welbaum, 1996). ILL63-X and Nufar were tested in one experiment in March–June 2011, at 5, 10, 14, 18, 23, 27, 32, 35, 39, 43 and 45 °C. ILL, Genovese, GCI and Aroma 2 were tested in a separate experiment in September–December 2011, at germination temperatures of 7, 11, 13, 17, 19, 22, 26, 29, 32, 35, 38, 42 and 45 °C. Subtle differences in temperature occurred between the two experiments due to variation in room temperature, substrate moisture, bath performance and other environmental factors. Germination data analysis e standard error of the mean was calculated from four replications of 25 seeds each to compare final germination percentages at each temperature. Mean time to germination (MTG) was calculated as: MTG = ∑ (NiTi ) / ∑ (Ni ) where Ni is the number of newly germinated seeds at time Ti recorded as days aer imbibition. Germination rate (GR) was calculated as the inverse of MTG (GR = 1 · t −1) and expressed as seeds d−1 (Nerson and Paris, 1988). Mean minimum or base temperatures (Tb) for seed germination were determined by extrapolating plots of mean GR versus temperature (T) to the intercept on the abscissa (Gummerson, 1986). e slopes of the regression lines are the reciprocal of the thermal times to germination (1/θ T), allowing for a comparison of the speed at which different seed populations progressed toward germination at different temperatures. Since the plot of GR versus T was not linear at the highest germination temperatures, the mean maximum or ceiling temperature (Tm) was estimated as the temperature that reduced the germination percentage to 50% (Jett and Welbaum, 1996). Optimal temperatures (To) for germination were those where the highest germination percentage and GR occurred simultaneously. Germination percentages and rates were plotted using SigmaPlot 12.0 (Systat Soware, Inc., San Jose, CA, USA). Base, optimal and ceiling temperatures and thermal time were compared by analysis of variance with mean separation by LSD (p ≤ 0.05) (SAS Institute, Inc., Raleigh, NC, USA).
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Figure 1. Germination of six sweet basil cultivars, Italian Large Leaf (ILL), Italian Large Leaf 63-X (ILL 63X), Nufar, Genovese, Genovese Compact Improved (GCI) and Aroma 2, at different temperatures. Mean ceiling temperatures (Tm ± SE) were estimated graphically as those that reduced germination to 50%, and are summarized in Table 1.
RESULTS AND DISCUSSION Germination of all cultivars increased sharply when temperature increased from 7–14 °C (Fig. 1). Over a range of 18–35 °C, germination was greater than 90% for ILL, Nufar, GCI and Aroma 2, and 85% for ILL63-X and Genovese (Fig. 1). e five cultivars grown from new seed germinated above 60% at 42 °C, but values declined sharply and no germination was observed at temperatures above 45 °C (Fig. 1). For stored seeds of ILL63-X, germination percentages were lower across the same range of temperatures and ceased above 43 °C (Fig. 1). GR increased linearly over a range from approximately 10 °C to a maximum at 35 °C. Values were similar for all cultivars and then declined sharply at higher temperatures (Fig. 2). e decline in GR at high temperatures was not linear. At most temperatures, the GR of stored ILL63-X seeds was lower compared to the five cultivars purchased in 2011, including ILL, whose GRs were similar (Fig. 2). Single Tb values for each of the six sweet basil cultivars were calculated by linear regression over a temperature range of 10–35 °C, and mean Tb values (Table 1) were the average derived from regression lines of the four replications. Mean Tb varied significantly from 9.8–13.2 °C, with Nufar having the
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Figure 2. Mean germination rate (GR ± SE) of six sweet basil cultivars, Italian Large Leaf (ILL), Italian Large Leaf 63-X (ILL 63X), Nufar, Genovese, Genovese Compact Improved (GCI) and Aroma 2, across temperatures. Optimal temperature(s) (To) for germination are summarized in Table 1, and determined as the temperatures where the highest germination percentage and GR occurred simultaneously.
Table 1. Base (Tb), ceiling (Tm) and optimal (To) temperatures, and thermal time (θ T), for germination of six sweet basil cultivars, Italian Large Leaf (ILL), Italian Large Leaf 63-X (ILL 63X), Nufar, Genovese, Genovese Compact Improved (GCI) and Aroma 2. All means were calculated graphically from four replications of 25 seeds per replication, and treatment effects tested by ANOVA. Cultivar
ILL ILL63-X Nufar Genovese GCI Aroma 2 Mean F test
Tb
To
Tm
θT
(°C)
(°C)
(°C)
(°h)
11.9b 12.3b 9.8d 10.9c 10.8c 13.2a — **
35–38 35–39 35–39 35–42 35–38 32–38 34.5–39.0 NS
43.6 40.4 44.1 43.4 43.3 43.3 43.0 NS
29 40 27 25 26 29 29.3 NS
NS, **Not significant, and significant (p ≤ 0.01), respectively. Mean separation following significant F tests was by LSD (p ≤ 0.05).
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Figure 3. Representative linear regression equations from one replication, calculated over the range of temperatures, for all six sweet basil cultivars, Italian Large Leaf (ILL), Italian Large Leaf 63-X (ILL 63X), Nufar, Genovese, Genovese Compact Improved (GCI) and Aroma 2, and used in the calculation of mean base temperatures (Tb) and thermal time to germination (θ T). Tb values were determined by extrapolating plots of mean germination rate (GR) versus temperature (T) to the intercept on the abscissa.
lowest and Aroma 2 the highest Tb (Fig. 3, Table 1). ermal time did not vary statistically among cultivars, except for ILL63-X, which germinated more slowly at a given temperature (Table 1). ILL63-X germinated the slowest of all cultivars, indicating that GR declined with seed aging, while its Tb was more stable and similar to other cultivars, including the related ILL. ILL and ILL63X were similar genetically, but ILL63-X seeds were stored since 2006 while ILL were commercial new crop seeds. e older ILL63-X seeds had a similar Tb but lower percentage germination, and germinated more slowly compared to ILL (Table 1, Figs. 1 and 3). Tb did not change with relatively long-term storage even though vigor and viability decreased. We observed little variation in θ T and Tm among basil cultivars except for ILL63-X, which had a greater θ T and lower Tm. e variation shown by ILL63-X was likely the product of seed aging and not genetics, since ILL63-X was the only cultivar subjected to longterm storage in this study (Table 1). erefore, Tb did not appear to be a genetically controlled constant in basil seed but is independent of other seed quality indicators. Tb did not change with the storage-related aging that reduced the viability and vigor of ILL63-X.
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An accurate graphic determination of Tm was complicated by the abrupt decline in germination percentages above 40 °C for most cultivars. Tm was estimated by comparing the temperatures that inhibited germination by 50% (Fig. 1). ANOVA revealed no cultivar differences in Tm, which ranged from 44.1 °C for Nufar to 40.4 °C for ILL63-X, although most cultivars were clustered between 44.3–44.6 °C (Table 1). To did not differ significantly among cultivars and ranged from 32–42 °C, with a mean range for all cultivars of 34.5–39 °C (Table 1). Only limited information is available about effects of temperature on basil seed germination from previous studies. Basil seed was reported to germinate over a temperature range of 13–30 °C, with the majority of seeds germinating aer 4 d, with an optimum of around 25 °C (Ramin, 2006). In another study, sweet basil germinated rapidly between 21–30 °C to over 80% aer 4 d. At all other temperatures, either the percentage or the speed of germination was reduced (Putievsky, 1983). is study suggests that basil seed germinates reliably over a much wider range of temperatures (Table 1, Fig. 1). e thermogradient table was well suited for characterizing temperature responses since multiple replicates of several cultivars could be simultaneously tested over a wide range of temperatures. However, since 6 different cultivars were tested, the experiment was divided into two tests. is approach required that seeds of some cultivars be stored for longer periods than others before testing. Aging differences between the two testing groups likely had minimal effects on results since all seeds were stored in sealed containers at low moisture content (9% DWB) and temperature (4 °C). Also, there were no discernible differences in the germination performance of the early and late testing groups. Basil is a warm-season, frost-sensitive crop and germinated faster and to greater percentages at higher temperatures (Figs. 1 and 2). It was interesting that the base temperatures were relatively low, close to 10 °C for most cultivars, and lower than the previously reported 13 °C (Ramin, 2006). Variations in Tb have long been debated among seed scientists (Ellis and Butcher, 1988; Welbaum and Bradford, 1991). For many species, germination rate increases linearly with temperature above Tb (Ellis and Butcher, 1988). e ceiling temperature (Tm) is the maximum temperature where germination occurs and oen well beyond the linear range of the germination rate versus temperature plots (cf. Figs. 2 and 3). Because of this, Tm and other temperatures beyond the linear range were excluded from calculations of Tb (Fig. 3). Earlier work proposed that in the absence of dormancy, Tb is genotypically rather than developmentally controlled (Ellis and Butcher, 1988). However, in this study, sweet basil exhibited significant variation in Tb among cultivars (Fig. 3, Table 1). Variation in Tb is not unique to basil. A number of other studies have shown that Tb may vary with environment and hormonal conditions (Welbaum et al., 1990). Storage reduced estimated Tb for germination of both 40 and 60 DAA (days aer anthesis) seeds by approximately 5 °C (Welbaum and Bradford, 1991). Variations in seed maturity and production environment were not investigated for basil seeds, but may affect Tb. e current study contradicts the proposal that Tb is fixed for a given species because there were significant statistical
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differences in base temperatures calculated for diverse cultivars of O. basilicum. ese differences were likely not caused by dormancy since all seeds germinated rapidly at most temperatures. e calculation of Tb was based on graphic interpolation to a germination rate of zero, so subtle differences in slope could affect base temperature calculations. However, regression lines used to calculate Tb had six or more points and were largely immune to the influences of aberrant data points; each value was the average of four replications. erefore, significant variation in Tb exists among basil cultivars so that it may be possible through breeding to decrease Tb and improve low temperature germination performance. At low temperatures, germination was very slow, so field planting near Tb is not recommended due to slow germination and the possibility of pathogenic attack that could reduce field emergence. REFERENCES Almeida I., D.S. Alviano, D.P. Vieira, P.B. Alves, A.F. Blank, A. Lopes, C.S. Alviano and M.D.S. Rosa. 2007. Antigiardial activity of Ocimum basilicum essential oil. Parasitology Res. 101: 443–452. AOSA. 2015. Rules for testing seeds, Vol. 1. Principles and procedures. Assoc. Offic. Seed Anal., Washington, D.C. Bozin, B., N. Mimica-Dukic, N. Simin and G. Anackov. 2006. Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem. 54: 1822–1828. Chiang, L.C, L.T. Ng, P.W. Cheng, W. Chiang and C.C. Lin. 2005. Antiviral activities of extracts and selected pure constituents of Ocimum basilicum. Clin. Exp. Pharmacol. Physiol. 32: 811–816. Duke, J.A. 2008. Basil as the Holy Hindu highness. Altern. Complementary erap. 14: 5–8. Elias, S.G., L.O. Copeland, M.B. McDonald and R.Z. Baalbaki. 2012. Seed testing: principles and practices. Michigan State University Press, East Lansing. Ellis, R.H. and P.D. Butcher. 1988. e effects of priming and ‘natural’ differences in quality amongst onion seed lots on response of the rate of germination to temperature and the identification of the characteristics under genotypic control. J. Exp. Bot. 39: 935–950. Gummerson, R.J. 1986. e effect of constant temperatures and osmotic potentials on the germination of sugar-beet. J. Exp. Bot. 37: 729–741. Jett, L.W. and G.E. Welbaum. 1996. Effects of matric and osmotic priming treatments on broccoli seed germination. J. Am. Soc. Hortic. Sci. 121: 423–429. Medany, M.A. and A.K. Hegazy. 2007. Prediction of seed germination and seedling growth of four crop plants as affected by root zone temperature. World J. Agric. Sci. 3: 714–720. Nerson, H. and H.S. Paris. 1988. Effect of fruit age, fermentation and storage on germination of cucurbit seeds. Sci. Hortic. 35: 15–26. Orozco-Segovia, A., L. Gonzalez-Zertuche, A. Mendoza and S. Orozco. 1996. A mathematical model that uses Gaussian distribution to analyze the germination of Manfreda brachystachya (Agavaceae) in a thermogradient. Physiol. Plant. 98: 431–438. Putievsky, E. 1983. Temperature and daylength influence on the growth and germination of sweet basil and oregano. J. Hortic. Sci. 58: 583–587.
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Ramin, A.A. 2006. Effects of salinity and temperature on germination and seedling establishment of sweet basil (Ocimum basilicum L.). J. Herbs Spices Med. Plants. 11: 81–90. Schwember, A.R. and K.J. Bradford. 2005. Drying rates following priming affect temperature sensitivity of germination and longevity of lettuce seeds. Hortsci. 40: 778–781. Simon, J.E., 1998. Basil. New Crop FactSHEET. Purdue University. Retrieved from http:// www.hort.purdue.edu/newcrop/cropfactsheets/basil.html (verified 20 April 2016). Umar, A., G. Imam, W. Yimin, P. Kerim, I. Tohti, B. Berke and N. Moore. 2010. Antihypertensive effects of Ocimum basilicum L. (OBL) on blood pressure in renovascular hypertensive rats. Hypertens. Res. 33: 727–730. Welbaum, G.E. and K.J. Bradford. 1991. Water relations of seed development and germination in muskmelon (Cucumis melo L.). VI. Influence of priming on germination responses to temperature and water potential during seed development. J. Exp. Bot. 42: 393–399. Welbaum, G.E., T. Tissaoui and K.J. Bradford. 1990. Water relations of seed development and germination in muskmelon (Cucumis melo L.). III. Sensitivity of germination to water potential and abscisic acid during development. Plant Physiol. 92: 1029–1037.
