Types of flowers and fruit setting based on style length

Except for ‘TN74128’ that contained almost the same percentages of the flower types, all the genotypes recorded greater percentages of LGs + MEs that are capable of fruit setting than SRTs (Table 2). Previous studies also revealed that among all types of eggplant flowers, LGs and MEs often occur in higher number than SRTs (Nagasawa et al., 2001; Pohl et al., 2019). However, the SRTs accounted for a considerable percentage of flowers on the plants (from about 20% to 45%).

A close relationship was established between fruit setting and style length in eggplant genotypes such that the LGs and MEs of all the genotypes investigated showed satisfactory fruit setting rates (42% to 76%), while only a small percentage (< 4%) of SRTs set fruits with most of their flowers aborted (Table 2). Pandit et al. (2010) and Pohl et al. (2019) also observed the higher percentage of fruit setting from LGs and MEs respect to non-reproductive flowers (SRTs).

The question that arises is: which differences between LGs and SRTs could have induced fruit setting in the former? To answer this question, certain features of the two types of flowers were studied in the two genotypes chosen based on their high SRT percentages.

 

 

Table 2.  Percentage of flowers based on style length and percentage of fruit setting from those flowers in 13 eggplant genotypes.

LG: long style flower. ME: medium style flower. SRT: short style flower. Values with the same letters between columns and rows for percentage of flowers and percentage of fruit setting have no significant difference using LSD test at 5% probability.

 

 

Features of LGs and SRTs

Morphological features

As expected, in both genotypes LGs exhibited not only substantially longer styles but also wider stigmas than SRT flowers did. Moreover, LGs recorded far greater pedicel diameters and lengths, as well as heavier flowers, pistils, ovaries, and stigmas (Table 3). In concordance with these results, Mohideen et al. (1977) found that higher fruit setting is associated with larger numbers of LGs, as they produce fleshy ovaries and thick pedicels. Kowalska (2006) reported lower stigma weights in SRTs and Salas et al. (2012) noted that SRTs had not only smaller ovaries but also smaller anthers and buds. Moreover, Sękara & Bieniasz (2008) observed that SRTs had smaller ovaries and stigmas.

 

 

Table 3.  Differences between long (LG) and short style flowers (SRT) of two eggplant genotypes in morphological features, nutrient concentrations and micromorphological traits.

Values with the same letters in each row for each genotype have no significant difference using LSD test at 5% probability. TTC: 2,3,5-triphenyl tetrazolium chloride

 

 

Nutrient concentrations

Given that special nutrients and nutritional elements are needed for pollen grains to germinate on the stigma surface and for pollen tubes to grow in the style tissue, the quantities of these nutrients in the stigma and style were determined. According to Table 3, LGs exhibited far greater protein, total sugar, and reducing sugar concentrations in their style and stigma tissues than did SRT ones in both genotypes. In both genotypes, K concentration in the style + stigma tissues of LGs was somewhat higher than that in SRTs, although this difference was not significant. Contrary to our expectations, greater amounts of Ca, Mg, and B were measured in SRTs than in LGs in both genotypes, although the Ca concentration in ‘TN74128’ and the Mg concentration in ‘TN74243’ were not significantly different between the two kinds of flowers (Table 3).

Rylski et al. (1984) and Handique & Sarma (1995) reported small quantities of total sugar and reducing sugar in underdeveloped stigmas of SRTs but larger amounts of protein, polysaccharides, and other nutrients in the stigmas of LGs due to their permeable tissues that make them more favorable to pollen grain absorption and germination. These findings are confirmed by Sękara & Bieniasz (2008), who reported lower sugar content in SRTs. The requirements for pollen germination vary from species to species. In addition to moisture, a carbohydrate source is needed for satisfactory pollen germination and tube growth (Dane et al., 2004). Carbohydrates function both as an energy source and a regulator of osmotic pressure (Fei & Nelson, 2003; Huang et al., 2004). One of the best carbon sources for pollen germination and tube growth is sucrose, followed by glucose, fructose, galactose, and lactose (Patel & Mankad, 2014).

Boron (B) also plays an important role in pollen germination and tube growth (Stephenson et al., 1994). By affecting H+-ATPase activity, B promotes pollen germination (Obermeyer & Blatt, 1995). The pollen tube wall is composed of callose, cellulose, and pectin, of which pectin is the major component (Li et al., 2002). Since B plays a direct role in pectin synthesis, it helps the development of the pollen tube membrane (Wang et al., 2003), increases sugar absorption and metabolism (Vasil, 1964), raises oxygen uptake (O’Kelley, 1957), and enhances the pollen response to Ca (Mascarenhas & Machlis, 1964). However, B can be toxic at a higher concentration or trigger deficiency symptoms (Pérez-Castro et al., 2012). Mondal & Ghanta (2012) reported that a low concentration of boric acid stimulated pollen germination and pollen tube growth in Solanum macranthum, but at higher concentrations had inhibitory effects. Moreover, Fang et al. (2016) showed that a high concentration of B inhibited pollen germination and tube growth in Malus domestica. Our findings confirm these results: the far higher B concentration in SRTs than in LGs observed in the present study might have given rise to its inhibitory effect on pollen germination in SRTs.

The results of many early studies indicated that Ca is necessary for pollen germination and pollen tube growth (Ge et al., 2007; Steinhorst & Kudla, 2013). Stephenson et al. (1994) emphasized the great reliance of pollen germination and tube growth on Ca content as it plays different roles in protein phosphorylation and enzyme activity of the pollen tube (Polya et al., 1986), in the rigidity of the pollen tube wall (Kwack, 1967), and in the permeability of the pollen tube membrane (Dickinson, 1967). It also helps the growth of the pollen tube tip by controlling vesicle movements toward the tip (Picton & Steer, 1983). In the present study, SRTs exhibited higher Ca concentration than did LGs. This is in agreement with the findings of Belho (1992), who reported that a low concentration of Ca in the culture medium was effective in stimulating pollen germination and pollen tube growth in Solanum khasianum and Solanum marginatum, while a high concentration inhibited pollen germination and reduced pollen tube elongation. K and Mg are also known to stimulate pollen germination and tube growth (Shivanna & Johri, 1985; Taylor & Hepler, 1997). These two ions in the culture medium serve as ions supporting the stimulatory effects of Ca (Brewbaker & Kwack, 1963). K and Mg have been reported to have stimulatory effects on pollen germination and tube growth in Solanum marginatum, but inhibitory effects in Solanum khasianum (Belho, 1992). Germination and the number of long pollen tubes decreased in the latter species with increasing pollen K and Mg contents (Belho, 1992). In accordance with these results, in the present study the higher amounts of Mg measured in SRTs had inhibitory effects, while K, with nearly the same amounts in LGs and SRTs, did not exhibit any inhibitory effects.

 

Micromorphological traits

Fig. 1 presents cross sections of LG and SRT styles of ‘TN74128’. These data shows that the surface area of the central canal is larger in LG (418 µm2 ) than in SRT (283 µm2 ). In addition, the number of vascular bundles in the LG style (6) is higher than in SRT (4). These canals are filled with pollen tube transmitting tissue. The nutritional role of this tissue has shown that pollen tubes nourish from products of cells comprising this tissue (Erbar, 2003). On the other hand, since vascular bundles also transport water, nutrient and organic molecules, increasing the number of vascular bundles in LGs can increase the growth of pollen tubes toward the ovules.

Pollen grain transfers male gametes to the female part of the flower playing an important role in the success rate of fruit setting. High crop yield typically depends on viable pollens. Pollen’s critical role has motivated further investigation of its features. According to Table 3, LGs in ‘TN74243’ recorded a greater pollen number than SRTs did, while this difference was not significant in ‘TN74128’. The polar axis and equatorial diameters of the pollens were measured to determine pollen shapes. In both genotypes, the polar axis of the pollen grains in LG was higher than that of SRT (Table 3). No significant differences were observed, however, in equatorial diameter between the two types of flowers. Fig. 2 presents the polar axis and equatorial diameter of pollen grains in two kinds of flowers in ‘TN74128’. Rylski et al. (1984) found no differences in pollen number, shape, size, or fertility between these two types of flowers in the studied varieties.

Pollen viability was determined using the two chemicals TTC and IKI. The IKI test failed to distinguish viable from non-viable pollen grains, as they all turned black in this test. TTC, however, showed significant differences between the two types of flowers (Fig. 3a). In both ‘TN74128’ and ‘TN74243’, 60.67% and 59% of the LG pollen grains were found to be viable, while only 39.5% and 35% of the SRT ones were identified as viable, respectively (Table 3). The in vitro pollen germination method was also conducted to find differences in pollen germination between LGs and SRTs (Fig. 3b). This germination was insignificant after 2, 4, 6, and 8 hr; about 60% of the pollen grains in both LGs and SRTs germinated after 8 hr in both genotypes (data not shown). Pollen tube growth was also examined after 8 hr to find significantly longer tubes in LGs than those in SRTs in both genotypes (Table 3). Simple and fast, this test method (in vitro pollen germination) is most commonly used for determining pollen grain viability. The IKI test overestimated pollen viability as it identified all the pollen grains from both LGs and SRTs as viable. Compared with the in vitro pollen germination test, the TTC test also underestimated pollen viability in SRTs as it identified as viable only 37% of their pollen grains. Both tests, however, showed LG pollen viability percentages to be identical. Shivanna & Rangaswamy (2012) found that the IKI test would not be useful for assessing pollen viability, since such non-vital stains as iodine in potassium iodide (IKI), acetocarmine, and aniline blue in lactophenol import color to both fresh and dead pollen grains alike. Hence, all the pollen grains in this study turned black after using IKI. As for the tetrazolium test, despite the satisfactory results reported in many species, no positive correlation has been established between the tetrazolium and in vitro pollen germination test results in some other species (Dafni et al., 2005). Furthermore, a graduated color (from very bright to deep red) reportedly developed in pollen grains stained in this method, such that setting a boundary point to distinguish between viable and non-viable pollen grains would be quite subjective (Shivanna & Rangaswamy, 2012).

 

 

Figure 1.  Cross section of style tissue in short style flower (a), and long style flower (b) of eggplant ‘TN74128’. Scale bar 5.0 µm

 

 

Figure 2.  Polar axis and equatorial diameter of the pollen grain in long style flower (a), and short style flower (b) of eggplant ‘TN74128’

 

 

Figure 3.  Using triphenyl tetrazolium chloride (TTC) for pollen viability: red color shows viable pollen and the bright one shows non-viable pollen (a), and pollen germination and pollen tube growth from in vitro pollen germination test (b). Scale bar 5.0 µm

 

 

Effect of NAA and Spd on flowering and initial fruit setting

As seen in Table 4, in both genotypes application of 1.0 and 1.5 mM Spd led to significant increases in the percentage of LG+ME in both solitary and inflorescence flowers compared to the control. Although treatment of 20 mg L-1 NAA had the same effect on increasing the percent of LG+ME, its difference was not significant compared to the control. Moreover, in ‘TN74128’ results obtained from higher concentrations of NAA (30 and 40 mg L-1) in both solitary and inflorescence type were very similar to the control and differences were not significant. On the other hand, in ‘TN74243’, although 30 and 40 mg L-1 NAA decreased the percentage of LG + ME and increased the percentage of SRT in both solitary and inflorescence type, however they didn’t show a significant difference to the control.

In ‘TN74128’, although 1.5 mM Spd and 20 mg L-1 NAA increased the percentage of initial fruit setting from solitary flowers, they did not show a significant difference with the control, while these treatments significantly increased the percentage of initial fruit setting from inflorescence flowers. Both of these treatments also increased the percentage of the total initial fruit set, but just 20 mg L-1 NAA was significant compared to the control (Table 5). This pattern is also present in ‘TN74243’ in how both 1.5 mM Spd and 20 mg L-1 NAA caused an increase of the percentage of initial fruit set from not only solitary type but also inflorescence type, as well as total initial fruit set. However, only 1.5 mM Spd showed a significant difference when compared with the control (Table 5).

In accordance with our results, Ravestijn (1983) found that growth regulators were able to convert SRTs into LGs and MEs, allowing these flowers to set fruits. This author also reported that the best results were obtained with the application of auxin and GA3 among the various growth regulators used to increase fruit setting rates in eggplant (Ravestijn, 1983). Moniruzzaman et al. (2015) also investigated the effects of GA3 and NAA, their results showing that all the tested concentrations of the two growth regulators increased LG and ME percentages in eggplants, with the highest value obtained for NAA applied at a concentration of 40 mg L-1. Moreover, Hoque et al. (2018) also showed that using 40 mg L-1 NAA increased the percentage of LGs and MEs and fruit setting rate of eggplants. Although no published report was found on the effects of polyamines on heterostylous species, the association of polyamines with flower sexuality or fertility has been reported. Rastogi & Sawhney (1990b) found that the male sterile mutants in tomato plants contained more polyamines than normal plants. Application of putrescine (Put) or Spd at concentrations 1× 10-3 and 1× 10-4 mol L-1 has also shown significant increases in the number of female flowers in walnut tress (Jizhong et al., 2004).

 

 

Table 4.  Differences between long (LG) and short style flowers (SRT) of two eggplant genotypes in morphological features, nutrient concentrations and micromorphological traits.

LG: long style flower. ME: medium style flower. SRT: short style flower. In each row, values with at least one same letter do not have a significant difference at the 5% probability level according to the LSD test.

 

 

Table 5.  Percentage of initial fruit set of two types of eggplant flowers (solitary and inflorescence), final fruit set, fruit dropping and final yield of two eggplant genotypes affected by spermidine (Spd) and 1-naphthaleneacetic acid (NAA).

In each row, values with at least one same letter do not have a significant difference at the 5% probability level according to the LSD test.

 

 

Effect of NAA and Spd on final fruit set and final yield

Although application of 1.0 and 1.5 mM Spd increased the percentage of LG + ME, and 1.5 mM Spd along with 20 mg L-1 NAA generally increased the percentage of the initial fruit set, to our surprise, results revealed that final yields and final fruit setting percentage at none of the Spd and 20 mg L-1 NAA concentrations used were significantly different from those recorded by the control treatment in both genotypes (Table 5). This table shows that the percentage of fruit dropping is higher in these treatments compared to the control. This fruit abscission in terms of using NAA may be due to the thinning effect of NAA, or in terms of using both NAA and Spd may be explained by this hypothesis that the plant has a self-regulating mechanism that adjusts excessive plant load due to excessive SRTs by abscising this kind of flower. While the plants that have a high number of LGs turn these kinds of flowers to fruits, they finally adjust their excessive load by abscising some of these fruits before ripening. Our results are consistent with Singh et al. (2002) and Sarker et al. (2011), who reported that NAA did not affect yield in tomatoes and eggplants, respectively. Khezri et al. (2010) also showed that, unlike Put, spermine (Spm) increased pistachio yield, while Spd had no effect on pistachio yield (although it reduced fruit abscission). However, other studies found that foliar spraying of NAA on eggplants increased the fruit number and yield of this crop (Singh, 2010; Moniruzzaman et al., 2015; Hoque et al., 2018). Akhtar et al. (1997) also investigated the effects of NAA at concentrations of 25, 50, 75, and 100 mg L-1 on tomato plants observing that while the yield reached its highest level at a concentration of 25 mg L-1, it gradually decreased with an increasing NAA concentration. Using Put and Spd sprays on date flower clusters, Tavakoli & Rahemi (2014) found that both polyamines increased yield and reduced fruit abscission relative to the values recorded by their control. Based on the results of this study and others, it may be concluded that the timing of polyamine application and the concentrations used are important factors. Moreover, no definitive mechanism can be claimed for polyamines since different effects have been reported for different types of polyamines on different plant species under different treatment conditions. The effects of polyamines on flower development thus await further research. Although this present study has several before-mentioned strengths, an important limitation that should be addressed is that among all three kinds of polyamines, we studied just Spd that we found more effective on flowering, while in regard to the different effects of three kinds of polyamines, further research should be done with all of them.

In general, smaller stigmas in SRTs lead to less pollen grain absorption. Moreover, the low nutrition (protein, total sugar and reducing sugar) of the stigma surface reduces the possibility for pollen germination. Although these types of flowers showed greater Ca, Mg, and B concentrations, the high concentrations of these nutritional elements may have had inhibitory effects on pollen germination and pollen tube growth. SRTs also showed shorter pollen tubes and lower pollen germination percentages, as vital parameters for fertilization and fruit setting. Thus, the decreased pollen germination percentage and pollen tube growth might underlie the failure of SRTs to set fruits. Further study is required, however, to shed more light on the causes underlying this failure. On the other hand, although 1 and 1.5 mM Spd increased LG and ME percentages, and 1.5 mM Spd along with 20 mg L-1 NAA increased the total initial fruit setting, they did not lead to any significant increases in the final yield and final fruit set when compared with the control. A possible explanation for this result might be that the plant has a self-regulating state. This means that when it has more SRTs it adjusts its load by abscising this kind of flower, while when it has less SRTs but more LGs, it adjusts its load by abscising its fruits before turning to the harvestable fruits. Although we could increase the LG to SRT proportion and initial fruit set by using plant growth regulators in this study, an important but unanswered question is how we can protect these initial fruits to increase final yield and final fruit set. Therefore, further work is needed to determine how to prevent fruit abscission.

 

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