2025 Volume 94 Issue 4 Pages 483-490
Although Basella alba and B. rubra have been used as annual leafy vegetables, ornamental plants, in dyes and in medicine for a long time in many countries, factors affecting seed germination in these species remain unclear. Therefore, in this study the effects of scarification treatment, temperature and light on seed germination were investigated in B. alba and B. rubra. In darkness, the germination percentage of scarified seeds was increased compared to non-scarified seeds in both species, suggesting that they show physical dormancy. Thermal optima for germination in B. alba and B. rubra were 25°C under dark conditions. In addition, light (white light) irradiation to seeds promoted germination, although they were able to germinate even if they were kept in darkness. It was also clarified in both species that the light quality of red (R) and far-red lights (FR) affected seed germination. Red light irradiation promoted seed germination, but far-red irradiation inhibited it. After far-red light irradiation for four days, the promotive effect of a red light on seed germination was reversed by a far-red light given immediately afterwards. Successive irradiation with R/FR/R promoted seed germination, but the subsequent FR irradiation cancelled the promotive effect of red light. Such reversible effects of red and fa-red light are typical in phytochrome-mediated seed germination in other species. Our results indicated that seeds of B. alba and B. rubra exhibit physical dormancy and light sensitive traits that are controlled by phytochrome(s), possibly phytochrome B.
Basella, which belongs to the family Basellaceae, widely distributes from the tropical and subtropical regions mostly in America, Africa, Madagascar and south India to New Guinea, and it is native to tropical Asia, probably India or Indonesia (Deshmukh and Gaikwad, 2014; Reddy et al., 2014; Renfrew and Sanderson, 2005; Saroj et al., 2012). Among Basella, B. alba and B. rubra are well-known as Indian spinach, Malabar spinach, Ceylon spinach or vine spinach (Adhikari et al., 2012; Deshmukh and Gaikwad, 2014; Kumar et al., 2013; Palada and Crossman, 1999; Roy et al., 2010). These species, B. alba and B. rubra, are classified according to leaf characteristics and stem color (Adegoke and Ojo, 2017; Palada and Crossman, 1999). The former has dark green oval or nearly round leaves with green stems, whereas the latter has round oval leaves and red stems. They are an extremely vigorous plant with that run and climb, and the vines typically grow to a length of 3 to 5 meters (Adhikari et al., 2012; Deshmukh and Gaikwad, 2014; Harold, 1963; Kumar et al., 2013).
Indian spinach has been cultivated for long time as annual leafy vegetables (substitutes for spinach in the tropics and subtropics), ornamental plants, in dyes and in medicine in almost all parts of India, tropical Asia and Africa (Adhikari et al., 2012; Deshmukh and Gaikwad, 2014; Harold, 1963; Reddy et al., 2014), because they are very nutritious, contain functionally important phytoconstituents or coloring compounds for dyes (Kumar et al., 2015; Murakami et al., 2001; Oloyede et al., 2013; Premalatha and Rajgopal, 2005). In Japan, B. alba and B. rubra are mainly used as a vegetable and as an ornamental plant, respectively, although both B. alba and B. rubra are edible. In other countries, however, B. rubra is preferred by some consumers for consumption due to its delicious taste (Roy et al., 2010).
The seeds of B. alba are generally sown between late April and mid-May in agricultural practice. Subsequently, the apical bud of main stem is removed after the emergence of 5 to 6 leaves, and then lateral shoots approximately 15 cm in length are harvested, leaving two leaves. These lateral shoots are harvested from late June to October.
Indian spinach seeds are well known to exhibit low germination and subsequent slow emergence. Miura et al. (1997) improved the emergence rate in B. alba seeds by post-sown priming with a potting mixture (soil moisture content) under dark conditions, and the emergence rate increased up to approximately 85% by controlling the soil moisture level and the duration of soil moisture retention. Although the effect of soil moisture content on emergence has been confirmed in B. alba, the relationships between other environmental conditions and seed germination have not been sufficiently investigated.
Seed germination is a complex physiological process influenced by a variety of environmental factors such as temperature, light and soil moisture (Baskin and Baskin, 1988; Finch‐Savage and Leubner‐Metzger, 2006; Ghorbani et al., 1999). The primary environmental factor regulating seed dormancy and germination is temperature, and light and moisture are of secondary importance (Baskin and Baskin, 1988).
Temperature changes the activity of enzymes in seeds and affects both the capacity and rate of germination. Germination rates generally increase with a rise of temperature to an optimum temperature, but temperatures above the optimum lead to a sharp decrease in germination rates. (Bewley et al., 2013). In addition, optical temperature ranges for germination differ according to species.
Light is another important environmental factor that affects seed germination in many plant species (Shinomura, 1997). Red light (R) or white light promote germination in positive photoblastic seeds, whereas their light qualities suppress germination in negative photoblastic seeds. Light-insensitive seeds, however, germinate under both dark and white light conditions.
The objectives of this study were to evaluate factors influencing seed germination in B. alba and B. rubra. The effects of scarification, different temperatures and different light qualities on seed germination were investigated. In addition, the role of phytochrome(s) in seed germination of these species was discussed.
Seeds of B. alba and B. rubra (Tohoku seed CO., LTD., Tochigi, Japan) were used in all experiments. They were immersed for about one hour in distilled water for imbibition. Twenty-five seeds were placed inside a 9 cm diameter Petri dish with filter paper moistened with 5 ml of distilled water, and then Petri dishes were sealed with vinyl tape to prevent drying. Germination trials consisted of three Petri dishes per treatment. Germination was considered as emergence of the radicle. Germination percentages (number of germinated seeds/number of used seeds) were calculated for each Petri dish.
Effect of scarification treatment on seed germinationSeeds were subjected to scarification or non-scarification treatment using sand paper, and 25 seeds of each were sown in a Petri dish after imbibition for about one hour. The Petri dishes were placed in the dark at 25°C in a growth chamber. The dark condition was generated by wrapping each Petri dish in aluminum foil. Germination percentages were determined 12 days after sowing.
Effect of temperature on seed germinationThe Petri dishes were arranged in growth chambers at 20°C, 25°C or 30°C under a dark condition. The number of germinated seeds was counted 12 days after sowing.
Effect of light conditions on seed germinationScarified seeds were imbibed with distilled water for about one hour, and then grown under dark or light conditions in a growth chamber. Light and dark conditions were generated by continuous irradiation from a white florescent tube (FL40SS·EX-N/37; Panasonic, Osaka, Japan) with a light intensity of 80 to 85 μmol·m−2·s−1 and by wrapping each Petri dish in aluminum foil, respectively. Germination percentages were determined 12 days after sowing.
Effect of different light qualities on seed germinationScarification treatment was carried out in all seeds. The seeds were immersed in distilled water for one hour and grown in a controlled room at 25°C with or without irradiation of different lights. For different light quality treatments, white light, blue light, red light and far-red light were generated from a white fluorescent tube (FPL27EX-N; Panasonic), a red LED (Sanyo Electric Co., Ltd., Osaka, Japan), a blue LED (Sanyo Electric Co., Ltd.) and a far-red LED (Sanyo Electric Co., Ltd.), respectively. Light intensity with white light, red light and blue light was 15 ± 1 μmol·m−2·s−1, and that with far-red was 20 ± 2 μmol·m−2·s−1. Germination percentages were determined 14 days after sowing.
Effect of successive red and far-red light irradiation treatments on seed germinationScarification-treated seeds were incubated at 25°C for four days under far-red light irradiation, and then light treatment was given in five different ways: 1) no irradiation (Control), 2) irradiation with red light (R), 3) irradiation with far-red light followed by red light (R/FR), 4) successive irradiation with red light, far-red light and red light (R/FR/R), and 5) successive irradiation with red light, far-red light, red light and far-red light (R/FR/R/FR). Each irradiation was carried out for 10 min. After finishing each treatment, seeds were placed at 25°C under dark conditions by wrapping each Petri dish in aluminum foil for 10 days. Germination was checked 14 days after sowing.
Measurements of light quality and light quantityLight quantity was measured with a Li-Cor LI-1000 (Li-Cor, Inc., Lincoln, Nebraska, USA) quantum sensor. The wavelength of the light source was determined by a USB2000+ spectrometer (Ocean Optics, Dunedin, Florida, USA).
Statistical analysisGeneralized Linear Models (GLM) with a quasibinomial distribution and logit link function were used to compare the final germination percentages among treatments for the two species: B. alba and B. rubra. The respective effects of scarification, temperature or light in the two species were tested using two-way analysis of variance (ANOVA), followed by Tukey’s test. All statistical analyses were performed with the statistical programming language R 3.3.3 (R Development Core Team, 2017) [R Development Core Team (2017) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org].
Seeds began to germinate within three days after imbibition under dark conditions in both species irrespective of scarification treatment (Fig. S1). Subsequently, the germination percentage increased more rapidly in scarification treatment compared to that in non-scarification treatment in both species. As a result, the germination rate in scarification treatment was higher than that in non-scarification at 12 days after imbibition. As expected, a two-way ANOVA of germination with a generalized linear model (GLM) indicated significant effects of scarification (F = 45.23, P < 0.001) (Table 1). When the seeds of both B. alba and B. rubra were imbibed without scarification in darkness, approximately 43–53% of the seeds germinated (Fig. 1). On the other hand, scarified seeds exhibited a high germination rate over 67% in both species. It was demonstrated from these results that scarification treatment improves germination rates in these species.
Analyses of variance (ANOVA) of the effects of each treatment and their interaction on seed germination.
Effect of scarification treatment on seed germination. Scarified or non-scarified B. alba and B. rubra seeds were placed under dark conditions at 25°C in a growth chamber for 12 days.
Germination in seeds of B. alba occurred three days after imbibition at 20°C or two days at 25°C and 30°C under dark conditions (Fig. S2). Thereafter, germinated seeds increased more quickly at 25°C and 30°C than at 20°C. Twelve days after imbibition, the germination rate at 30°C was, however, equal to that at 20°C because it increased slowly from six days at 30°C. Effect of temperature on seed germination in B. rubra was slightly different to that in B. alba. When seeds of B. rubra were kept under dark conditions, they germinated in two or three days in all temperature treatments. The increases in the germination rates at 25°C and 30°C were rapid, although the germination rate increased rather slowly at 20°C. After 12 days, imbibed seeds at 25°C and 30°C had higher germination rates than those at 20°C. Two-way ANOVA indicated that there was a significant effect of temperature (F = 8.91, P < 0.01) and interaction between species and temperatures (F = 6.62, P < 0.05) on the germination rate (Table 1). Final percentages of germination in each temperature in both species are shown in Figure 2. The highest rate of germination (65%) was observed at 25°C in B. alba, while it was approximately 50% at 20°C and 30°C. In B. rubra, 31% of seeds germinated at 20°C. In contrast, the rate of germination showed higher values at 25°C and 30°C, for which the rates were 69% and 64%, respectively. These results thus indicated that the highest germination rate was obtained at 25°C in B. alba and at 25°C or 30°C in B. rubra.
Effect of temperature on seed germination. Scarified B. alba and B. rubra seeds were placed in growth chambers for 12 days at 20°C, 25°C or 30°C under dark conditions. The different letters indicate significant difference (P < 0.05) using Turkey’s HSD test.
In B. alba, seeds began to germinate two days after imbibition independent of the light condition (Fig. S3). Subsequently, the germination rate under light conditions increased rapidly compared to that under dark conditions. These results in B. alba were similar to those in B. rubra. Two-way ANOVA analysis confirmed that light irradiation (F = 37.79, P < 0.001) affected seed germination (Table 1). When the seeds of both species were imbibed in the darkness, approximately 70% of seeds germinated (Fig. 3). On the other hand, white light irradiation resulted in a high germination rate in B. alba and B. rubra, with rates of 89% and 97%, respectively. These results demonstrated that white light irradiation promotes seed germination in these species.
Effect of light conditions on seed germination. Scarified B. alba and B. rubra seeds were grown at 25°C under dark or light conditions in a growth chamber. Values are expressed with mean ± SE.
Seed germination was observed at two days after imbibition under dark conditions in B. alba, and then the rate of germinated seeds increased slowly for 14 days (Fig. S4). Continuous blue light irradiation exhibited a similar germination pattern to the darkness condition. The increase in the number of germinated seeds was rapid under continuous white light or red light irradiation, although germination occurred at two days after imbibition as in the dark condition. Far-red light irradiation resulted in late germination, and only a few germinated seeds 14 days after imbibition. While seeds began to germinate at three days after imbibition under darkness and blue light-irradiation in B. rubra, germination occurred at two days after imbibition under white and red light-irradiation.
Effects of light quality on the final germination rate were independent of the species used. The ANOVA analysis for the germination rate at 14 days after imbibition indicated significant differences among light qualities (F = 237.09, P < 0.001), although a significant, but quite small, interaction between species and light quality (F = 0.0447, P < 0.05) (Table 1) was also observed. While 65% of seeds germinated in darkness in B. alba, continuous white and red light irradiation promoted germination at a higher rate over 84% (Fig. 4). On the other hand, an inhibition effect of far-red light irradiation on seed germination was observed, and the rate of germinated seeds was only 7%. Under blue light irradiation, there were no positive and negative effects on seed germination. The results were similar to those in B. rubra. It was thus clarified that irradiation with white and red lights promotes seed germination, but that with far-red light inhibits seed germination. It was also indicated that there are no effects of blue light irradiation on seed germination.
Effect of different light qualities on seed germination. Scarified B. alba and B. rubra seeds were grown at 25°C for 14 days under white light, blue light, red light or far-red light irradiation. Values are expressed as mean ± SE. The different letters indicate significant difference (P < 0.05) using Turkey’s HSD test.
Successive red and far-red light treatment had a much more significant impact on germination rates at 14 days after imbibition among successive light treatments (F = 49.55, P > 0.001) than species (F = 10.49, P > 0.01) (Table 1). When seeds were placed in darkness after far-red irradiation for four days, they showed a low germination rate (16%) in B. alba (Fig. 5). Irradiation with red light for 10 minutes, however, increased the germination rate up to 55%. In contrast, the effect of red light was abolished with subsequent far-red irradiation for 10 minutes, and the germination rate was 12%. Although irradiation with R light given immediately after R/FR treatment restored the germination rate up to 59%, irradiation with FR light given immediately after R/FR/R treatment again cancelled the effect of the R light irradiation leading to a 15% germination rate. Similar responses were observed in successive light treatments for B. rubra. These results demonstrated that far-red light irradiation reversibly cancelled the promotive effect of red light irradiation in germination of B. alba and rubra.
Effect of successive red and far-red light irradiation on seed germination. Scarification-treated B. alba and B. rubra seeds were incubated at 25°C for four days under far-red light irradiation, and then light treatment was delivered as follows: no irradiation, irradiation of red light, irradiation of far-red light followed by red light, successive irradiation of red light, far-red light and red light and successive irradiation of red light, far-red light, red light and far-red light. Subsequently, they were placed at 25°C under dark conditions for 10 days. Values are expressed as mean ± SE. The different letters indicate significant difference (P < 0.05) using Turkey’s HSD test.
Basella alba and B. rubra seeds are protected by a hard seed coat, and the seed coat becomes harder as the fruit and seeds attain maturity (Kumar and Giridhar, 2021; Labhane et al., 2014). When seeds with hard seed or fruit coats need to be germinated efficiently, mechanical or chemical scarification treatments are employed to break physical dormancy, which is caused by water-impermeable layers of palisade cells in the seed or fruit coat (Baskin and Baskin, 2004; Jayasuriya et al., 2008; Orozco-segovia et al., 2007). In the present study, about 20–30% of the of B. alba and B. rubra seeds could break physical dormancy because the germination percentages in non-treated and treated seeds were 40–50% and 70%, respectively.
In a temperature range from 20°C to 25°C, higher temperatures resulted in an increase in the germination percentage in both B. alba and B. rubra, while temperatures more than 25°C caused a slight decrease only in B. alba. This indicated that germination in B. alba and B. rubra is optimal at 25°C and 25°C to 30°C, respectively. The fact that there is an optimal temperature for germination has been noted for various species (Benvenuti et al., 2001). There was no significant difference in germination rates between 25°C and 30°C in B. rubra, but there were different germination patterns at 30°C between the two species. It has been shown that a species’ geographic ranges are significantly associated with the range of temperature in which it can germinate, and species with a wider range of germination temperatures had larger geographic ranges (Brandle et al., 2003; Grime et al., 1981). Therefore, this result may be attributed to the adaptive differences associated with their geographic ranges, although the geographic distribution of each species remains unclear.
B. alba and B. rubra seeds were light-sensitive for germination, and white light irradiation promoted germination. The fact that B. alba and B. rubra seeds were positively photoblastic was a surprising finding because it has been previously recognized that their seeds were negatively sensitive to light. Actually, to date B. alba seeds have always been sown in darkness (Miura et al., 1997). In this study, the white light treatment resulted in a germination rate of over 90%, which was approximately 5% higher than the method reported by Miura et al. (1997). In addition, as a method to improve the germination and emergence rates, white light treatment is an easy approach compared to the soil moisture adjustment reported by Miura et al. (1997). They were able to germinate seeds with a certain moisture content even if they were kept under dark conditions. Some varieties of tomato, lettuce, and Arabidopsis can germinate readily in the dark as well, but they are responsive to light (Hendricks et al., 1959). Subsequently, the fact that Pfr, an active form of phytochrome, pre-exists in the seeds of tomato varieties as well as lettuce, cucumber, and Arabidopsis was confirmed (Mancinelli and Borthwick, 1964; Mancinelli and Tolkowsky, 1968; Mancinelli et al., 1966, 1967; Yaniv et al., 1967). In addition, it was found that the light environment in which maternal parents were grown determined the dark germination level in Arabidopsis (Hayes and Klein, 1974; McCullough and Shropshire, 1970). Harvested seeds from maternal plants exposed to incandescent lamp light (rich in far-red light) during seed development exhibited a low germination percentage in the dark, while seeds from maternal plants grown under cool white fluorescent lamp light (deficient in far-red light) resulted in a high germination percentage in the dark. Germination was not observed under dark conditions in seeds from maternal plants grown under incandescent lamp light alone. In addition, it was indicated that light quality was perceived by the developing seed itself, and that phytochrome may persist in the Pfr form for long periods (9 months) in dry seeds. Therefore, it seems that Pfr, which is photoconverted by the environmental light spectrum during embryogenesis in maternal plants, may be already present in Basella seeds, and germination can be induced even under dark conditions when seeds are imbibed.
The spectral sensitivity to seeds of the two species was also observed in the present experiment. While red light irradiation to seeds exhibited a promotive effect the same as white light irradiation, far-red light irradiation inhibited germination. Further, subsequent far-red light after red irradiation cancelled the promotive effect on seed germination. These results indicate that B. alba and B. rubra seeds have a photo-reversible response, in agreement with the results for lettuce, Lepidium, tomato, and other many species (Borthwick et al., 1952; Frankland and Taylorson, 1983; Furuya, 1968; Mancinelli et al., 1966; Sage, 1992; Toole et al., 1955, 1956), and that they are under phytochrome regulation. However, neither a promotive or inhibitory effect of blue light was found based on the result that the germination ratio under blue light irradiation was similar to that under darkness, although a promotive or inhibitory effect of blue light has been observed in some species such as Arabidopsis and lettuce (Borthwick et al., 1952; Evenari et al., 1957; Koller et al., 1964).
Phytochrome-mediated responses have been classified into three modes of action according to photoreversibility, fluence requirement and presence of reciprocity (Franklin and Whitelam, 2004; Seo et al., 2009; Sullivan and Deng, 2003). Low fluence responses (LFR), which are saturated at low light intensities, exhibit the classical R/FR reversibility response. Very low fluence responses (VLFR), which are very low fluence response with reciprocity, do not show an R/FR reversibility response. High irradiance responses (HIR), which are characterized by a dependence on the intensity of light (fluence rate), are induced by prolonged high irradiance or very frequent pulses of light without reciprocity or an R/FR reversibility response. Shinomura et al. (1996) examined action spectra for the PhyB response mediated thorough LFR and the PhyA response mediated thorough VLFR in Arabidopsis seed germination, and showed that phyB was photoreversibly regulated by low fluence at wavelengths of 540–690 nm (red light) and 695–780 nm (far-red light) and that phyA can capture light of wavelengths from 300 to 780 nm. The inhibitory effect of prolonged white, far-red or blue illumination, which are mediated by the HIR response, on seed germination is well known in Citrullus colocynthis, Cucumis sativus, C. anguria and C. lanatus (Koller et al., 1963; Loy and Evensen, 1979; McDonough, 1967; Noronha et al., 1978; Thanos, 1984; Thanos and Mitrakos, 1992; Yaniv et al., 1967). It has also been reported in Arabidopsis that the HIR response of hypocotyl elongation was mediated by PhyA, and PhyA perceived both FR light (690–750 nm) and UV-A to blue light (320–500 nm) (Shinomura et al., 2000).
Therefore, the result in the present study, in which Basella seeds exhibited an R/FR reversible germination response, suggests that seed germination was mediated by LFR through phyB. On the other hand, if phyA-mediated VLFR or HIR is involved in Basella seed gemination, a promotive or inhibitory effect of blue light on seed germination should be observed. Therefore, it seems that neither VLFR or HIR regulate seed germination in Basella.
In conclusion, we revealed that B. alba and B. rubra seeds are light-sensitive with physical dormancy and germination is promoted by scarification treatment, an optimum temperature (25°C) and light irradiation. Reversible responses to exposure to red/far-red light suggest that germination of the two species is regulated by phytochrome B. Additionally, white light treatment is expected to further improve germination and emergence rates in these species when they are sown.
