Seed priming enhances seed germination and plant growth in four neglected cultivars of Capsicum annuum L.

Plant material

Capsicum annuum L. (Solanaceae) is an annual plant native of the South America, arrived in Europe, together with tomato, in the 16th century. It has an elongated floral axis with 1 flower per node. The flowers have a white perianth and the fruit (with a variable color from ivory to purple, but in most varieties from yellow to red) is a berry about fifteen centimeters long, with numerous discoidal seeds with a diameter of 3–5 mm. The fruits of C. annum are known for their antioxidant properties, thanks to the presence of vitamin C and beta-carotene, which help protect cells from oxidative damage, furthermore, they are rich in vitamin A, potassium, and fiber.

C. annuum is widespread in Campania, and its fruits are considered one of the main vegetables grown in the region. Campania is famous for its traditional cultivars of peppers, including “sassaniello” and “papaccella”. For the present study we examined four ancient and neglected cultivars (i.e., cultivated by few households, or on small areas; Orobiyi et al., 2017): sassaniello giallo (SY), sassaniello rosso (SR) (i.e., yellow and red sassaniello), papaccella liscia gialla (PY), and papaccella liscia rossa (PR) (i.e., smooth yellow and red papaccella). Seeds were provided by the cooperative company Arca 2010, a germplasm bank of Campania region.

Priming treatments

Seeds provided by Arca 2010 were obtained from the ripe fruit, dried in the sun, and stored 1 month at +4 °C until they were used. The experiments were carried out in the period October 2021–January 2022. One gram seeds (about 120 seeds) for each cultivar were subjected to each priming treatments: halopriming with KNO₃ at three different concentrations (2%, 4%, 6%) and for three different times (24, 48, 96 h); hydropriming for 24, 48, 96 h; acid priming (100 mM HCl) for 5, 10 and 15 min, for a total of 15 priming treatments; further 0.5 g of untreated seeds were tested as control. We chose these acid priming and halopriming with KNO₃ according to literature reports (Agoncillo, 2018; Kumar, Chaurasia & Marmat Sandip, 2021; Quintero et al., 2018; Venkatasubramanian & Umarani, 2010). For each treatment seeds were submerged in water or aqueous solution for the selected times. Then, seeds were dried in oven at +30 °C, until constant weight.

Seed vitality assessment

Seed vitality was evaluated by 2,3,5-triphenyl-tetrazolium chloride (TTC) test after priming and re-drying of the seeds. The tetrazolium test indirectly verifies the respiratory activity in the cells of the seed tissues. The test is based on the activity of dehydrogenase enzymes, and particularly malate dehydrogenase; this latter by oxidation of its substrate, determines the release of electrons and hydrogen ions, which reduce the TTC in living tissues. The reduced form of the TTC-salt is a red-colored, stable, non-diffusible substance called triphenyl-formazan (França-Neto & Krzyzanowski, 2022). Specifically, 20 seeds for each cultivar and treatment were put in Petri dishes on wet paper added of 5 mL distilled water at +20 °C for 18 h. Seeds were then cut along the embryonic axis and immersed in 1% tetrazolium chloride solution (in phosphate buffer) for 6 h at +30 °C, in the dark (Kusumawardana, Pujiasmanto & Pardono, 2018). Seeds were observed under a stereomicroscope at 40x magnification.

Germination and growth

For each germination test 30 seeds were used, and the experiments were carried out in triplicate. After the priming treatment and drying, the seeds were germinated in Petri dishes on wet filter paper in the dark at +20 ± 1 °C, until the seedling development. During this period the following parameters were calculated: the germination percentages (GP) at 5, 10 and 15 days (i.e., the percentage of germinated seeds with 2 mm emerged radicle); the mean germination time (MGT); T₅₀ index, i.e., the time (days) necessary for 50% of viable seeds to germinate; the simple vigor index (SVI); the germination development index (DGI).

The mean germination time (MGT) was calculated using the following formula (Ellis & Roberts, 1979):

$$\mathrm{M}\mathrm{G}\mathrm{T}={\displaystyle \frac{\sum Dn}{\sum n}}$$where n is the number of seeds germinated on day D and D is the number of days counted from the start of germination at each count. The denominator is the summation of the seeds germinated at each count; therefore, it is equal to the total number of germinated seeds.

The parameter T₅₀ is the time needed for 50% of the final germination percentage (FGP), and together with MGT allows to estimate the germination speed.

The simple vigor index (SVI) (Tu et al., 2022; Wang et al., 2021).

$$\mathrm{S}\mathrm{V}\mathrm{I}=FW\times FGP.$$

Equal to the product of the total fresh weight of the seedlings (FW) for the final percentage of germination (FGP).

The development germination index (DGI) was calculated following the formula (Quintero et al., 2018):

$$100\times \left({\displaystyle \frac{FGP\left(t\right)}{FGP\left(0\right)}}\right)\times \left({\displaystyle \frac{PRL\left(t\right)}{PRL\left(0\right)}}\right).$$

Equal to the percentage product between FGP observed in primed seeds compared to control seeds, and primary radicle length observed in primed seeds compared to control seeds.

Where FGP (t) is the final germination percentage of primed seeds, FGP (0) is the final germination percentage observed in control seeds, PRL (t) is the primary root length of treated seeds, and PRL (0) is the length of the primary root of control seeds. Of course, being expressed in comparison to control, DGI is equal to 1 (or 100%) in the control; DGI > 1 (100%) indicates that priming treatment improve germination performance, and vice versa.

The seedlings were grown in seedbeds on vermiculite, at the temperature of 25/20 °C (day/night), relative humidity (RH) of 60–75% (day/night) and a photoperiod of 16 h of light per day with a photosynthetic photon flux density (PPFD) at the top of the canopy of 180–190 μmol photons m⁻² s⁻¹. Seedlings were watered with Murashige Skoog salt liquid medium diluted with water 1:1. At 40 days of culture, the following parameters were measured: total fresh and dry weight of the plants, the weight of the shoots and roots, the length of the roots and the height of the plants.

Morphological observations

Morphological traits considered in previous studies (e.g., Kumar, Chaurasia & Marmat Sandip, 2021; Venkatasubramanian & Umarani, 2010), such as root length and fresh weight, shoot length and fresh weight, total fresh weight, total dry weight, and leaf length (second leaf stage), were measured in 10 plants per species and treatment at 40 days culture. The shoot height was measured from the collar to the end of the highest leaf; the root length was measured from the collar to the longest tip of the primary root. The dry weight was measured after oven treatment at +60 °C until constant weight.

Total antioxidants

Total antioxidants were estimated in the leaves of the four cultivars showing the best germination and growth performance (i.e., Hydropriming for 24 h, KNO₃ 4% for 48 h, and KNO₃ 6% for 96 h), and in relative controls, by the ferric reducing antioxidant power (FRAP) assay (Sorrentino et al., 2023). In brief, fresh leaf samples (250 mg), were homogenized and resuspended with a 60:40 (v/v) methanol/water solution; then, the suspension was centrifuged at 14,000 rpm for 15 min at 4 °C; once recovered, the extract was added to 300 mM acetate buffer containing 10 mM tripyridyltriazine (TPTZ) in 40 mM HCl (1:1.6), and 12 mM FeCl₃ (1:16), with a final concentration 1:20 (extract: buffer) and pH 3.6. After 1 h incubation at 4 °C, the absorbance at 593 nm was measured with a spectrophotometer (UV-VIS Cary 100; Agilent Technologies, Palo Alto, CA, USA) using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as standard. The total antioxidant capacity was expressed as μmol Trolox equivalents for mg of fresh tissue.

Protein content

Protein content was estimated in the primed seeds of the four cultivars showing the best germination and growth performance (i.e., Hydropriming for 24 h, KNO₃ 4% for 48 h, and KNO₃ 6% for 96 h), in addition to untreated seeds as control. For protein extraction seeds (300 mg) were pulverized and suspended in a solution containing 10% tri-chloro-acetic acid (TCA) and 80% methanol in 100 mM ammonium acetate. The suspension was centrifuged at 13,000 rpm at +4 °C for 3 min at each addition; finally, the pellet was recovered and dried in oven at +50 °C for 10 min. Then phenol and SDS buffer were added for pellet resuspension. After this step, samples were centrifuged for 10 min at 13,000 rpm and room temperature, to separate the phenol phase containing protein fraction. After adding 80% methanol in ammonium acetate samples were stored overnight at −20 °C. Afterwords, samples were centrifuged at 13,000 rpm for 10 min and the pellet obtained was washed with 100% methanol and 80% acetone two times. Finally, the protein pellet was suspended in a buffer containing a protease inhibitor cocktail (Sigma) and phenyl-methyl-sulfonyl fluoride (PMSF) in 0.1% ethanol. The protein content was determined according to the Bradford method (1976), based on the binding between protein and Coomassie Brilliant Blue G-250, measuring absorption at 595 nm, which is proportional to the protein content.

Data analysis

The data were processed using Microsoft Excel and IBM SPSS for Windows (IBM Corp. Released 2020, Version 27.0. Armonk, NY, USA). As for the percentage data, the analysis was performed after arcsine transformation. Generalized linear models (GLMs) were applied to MGT, SVI, DGI, T₅₀ and morphological data (considered as dependent variables) to test the main and the interactive effects of cultivar (fixed effect with four levels: PR, PY, SR and SY), treatments (fixed effect with five levels: halopriming with three different concentrations −2%, 4%, 6%, hydropriming and control), time (fixed effect with three levels: 24, 48, 96 h). For the priming treatments showing the best germination and growth performance, the differences between the means of total antioxidant and protein content were tested by ANOVA. As post hoc test, the Tukey’s HSD was used with a significant level for p < 0.05.

Seed vitality and germination

The observation of control and primed seeds after tetrazolium test showed red-colored seed tissues in all control and treated seeds apart from those seeds subjected to acid priming. Here, regardless the treatment times, tissues appeared white or poorly colored both in the embryo and in the endosperm (Fig. S1). Due to this result, acid priming was excluded from further analyses.

The result of the GLM (Table S1) applied to MGT showed a significant interaction between cultivars, treatments and times. All priming treatments significantly decreased the MGT compared to control, with a reduction overall cultivar higher under KNO₃ 4% and 6% for 48 and 96 h, respectively.

The MGT under the different treatments and times grouped for each CV is illustrated in Fig. 1. In control seeds the MGT was higher in papaccella compared to sassaniello. Specifically, in PR the highest decrease of MGT was observed with KNO₃ 4% for 48 h and KNO₃ 6% for 96 h; in PY the highest reduction of MGT was recorded in seeds primed by KNO₃ 4% for 48 h; in SR the highest reduction of MGT occurred under KNO₃ 4% for 48 h and KNO₃ 6% for 96 h treatments, whereas in SG only by KNO₃ 4% for 48 h.

Morphological observation

The results of the GLMs (Table S1) applied to morphological data highlighted a significant interaction between cultivars, treatments and times. Morphological traits measured at 40 days culture (Table 1) showed that all plant characters were affected by seed priming treatments. Specifically, in SR the most promising treatments were KNO₃ 2% for 48–96 h and KNO₃ 6% for 96 h; the first significantly increased root length and weight and total weights; the latter significantly increased all measured traits. In SY instead, hydropriming for 24 h, and KNO₃ 6% for 48 and 96 h significantly increased all selected traits. In PR and PY most traits significantly increased in plants grown from seeds subjected to KNO₃ 4% for 48 h and KNO₃ 6% for 48 and 96 h.

In general, all seed priming boosted plant development, inducing a significant increase in biomass production (i.e., total weight). The different contribution of root and shoot weights to total weight is reported as well (Table 1). In all cultivars priming treatments affected both hypogeal and epigeal weight, with a more pronounced effect in plants from seeds primed by KNO₃, compared to hydropriming. Specifically, by comparing the total weight measured in control plants and in plants from seeds primed with KNO₃ 6% for 96 h, a significant increase was found in the latter in all four cultivars, highlighting that this treatment seems overall the most reliable.

Indexes related to germination performance

The results of the GLMs (Table S1) applied to the indexes related to germination indicated a significant interaction between the cultivars, treatments and times. Indexes related to germination performance (DGI, T₅₀ and SVI) are reported in Table 2 for all cultivars and treatments. In SR DGI and SVI provided the best values under hydropriming 24 h, KNO₃ 2% for 24–48 h and KNO₃ 6% for 96 h priming treatments; T₅₀ instead, was about halved by KNO₃ 4% for 24–48 h and KNO₃ 6% for 96 h. A similar trend was observed in SY. In both PR and PY the priming by KNO₃ 6% for 96 h provided the best performance, since it significantly increased DGI and SVI and strongly reduced T₅₀.

Cultivar-specific differences were observed in DGI. This index reached the highest values in PY/PR; specifically, in PR and PY, DGI under KNO₃ 6% for 96 h, was about twice the control 179.0 ± 32.6% and 216.5 ± 35.1%, respectively. In Sassaniello instead the highest values for DGI were >160%, and were observed in SR under hydropriming 24 h, KNO₃ 2% for 24–48 h and KNO₃ 6% for 96 h, while in SY, under hydropriming 24 h and KNO₃ 6% for 96 h. Also, in SY, PR, and PY a reduction in DGI of about 20% compared to control was sometimes observed due to a reduced root length (see Table 1).

Antioxidant efficacy

For antioxidant evaluation, only priming treatments providing the best germination and growth performance at each individual cultivar were considered and compared to control. The selected treatments were common to all four cultivars; they were hydropriming for 24 h, KNO₃ 4% for 48 h and KNO₃ 6% for 96 h (Fig. 2). In PR control antioxidant content in leaf tissue was lower (about halved) than PY, and no significant difference was observed between the antioxidant concentration in leaf from primed seeds in comparison with control. In PY instead, KNO₃ 6% for 96 h significantly increased antioxidants in leaf tissue. In SR both hydropriming and even more KNO₃ 6% for 96 h significantly increased antioxidant concentration compared to control. In SY all priming treatments significantly increased the concentration of antioxidant in the leaves, with KNO₃ 6% > KNO₃ 4% > H.

Protein content

In SR seeds, all priming treatments significantly stimulated the production of protein in comparison with control, with hydropriming which approximately tripled it (Fig. 3). Similarly, in SY all priming treatments increased protein content, but in this case KNO₃ 6% for 96 h showed the best performance, with a protein content almost quadrupled compared to control. In PY and PR all treatments significantly increased protein content in seeds, with a similar trend in both cultivars: i.e., KNO₃ 6% > Hydropriming > KNO₃ 4%