Breaking Seed Dormancy of Jaltomata procumbens (Cav.) J. L. Gentry Seeds with the Use of KNO₃

Abstract

Jaltomata procumbens (Cav.) J. L. Gentry presents seed dormancy mechanisms in its two populations, erect and decumbent, that make its germination and obtaining of plants difficult. Potassium nitrate (KNO₃) is used as a seed germination promoter by soaking in an osmotic solution. The objective was to break the dormancy of Jaltomata seeds by evaluating KNO₃. Treatments included: 2 populations (erect and decumbent), 2 soaking times (4 and 6 days) in 2 concentrations of KNO₃ solution (0.1% and 0.2%) and in distilled water used as a control. Days of germination including starting (DGS) and ending (DGE), energy period (EP), germination energy (GE), germination percentage (GP) and rate (GR) were measured. The erect population presented a more uniform germination and a lower number of DGS and DGE as well as 100% germination with the highest GR (2.56 seeds day⁻¹). No statistical differences were observed between KNO₃ and control; however, the positive effect of the KNO₃ and 4 days of soaking on the germinated seed was observed. The decumbent population exhibited a more variable germination; however, the same trend of the solution type and soaking days was observed, reaching 93.1 GP. Considering the economic and accessibility aspects of substances that promote germination on J. procumbens, distilled water imbibition and KNO₃ are recommended to accelerate the germination process.

1. Introduction

Jaltomata procumbens (Cav.) J. L. Gentry, of erect growth habit and blue-dark fruit color, is considered a semi-domesticated species and viable for future use. There are different populations in specific regions of Mexico; one includes the decumbent growth habit and green fruit at a mature stage. The first one is ruderal, on the edge or inside of crop fields; the second one is found inside of those sites. Both populations are used as edible fruit in Mexico [1].

The seed of some wild species, or during the domestication process, presents dormancy mechanisms that allow them to persist through time in unfavorable conditions. This is a limitation for growing a species since fast germination and growth are required [2]. Desirable characteristics are also important because these allow an early and uniform plant emergence, positively influencing yield, quality, and economic incomes [3].

In general, two kinds of dormancy exist: endogenous and exogenous. In the first one, some embryo characteristics prevent germination; in the second one, structural and chemical characteristics prevent the process. Derived from this, dormancy can be classified into five classes considering physical, morphological, and physiological aspects, and their combinations; with the non-deep physiological dormancy type present in most seeds [4]. To overcome this limitation, techniques to promote germination such as chemical and physical scarification, stratification, or their combinations are used.

The seed of Jaltomata procumbens populations presents dormancy mechanisms that limit its germination and ability to develop into plants. Some studies are reported to release dormancy on the erect population, where gibberellic acid and heat treatment were utilized [5]; however, these are still scarce, and high prices linked to break dormancy substances, thus making it difficult for its generalized usage [6]. Throughout the literature review, no information was found for the decumbent population.

Potassium nitrate is known as a germination promoter for a wide plant species variety; but in some species, it may also have the opposite effect [7]. It is applied through soaking, where the seed is hydrated under controlled conditions between water potential of a determined concentration solution and the water potential seed inside; therefore, osmotic solution activates the metabolism, preventing radicle emergence; after that, the seed is taken out from the solution and put it into an adequate environment, where the germination process is reactivated and accelerated [3,8]. The effectiveness of soaking with an osmotic solution depends on the solution water potential and treatment duration [2].

The effect of KNO₃ on seed germination could be due to oxidized forms of nitrogen such as NO₃ or NO [3]. It has been reported that the NO₃ ion reduces light requirements because it enhances the sensitivity of the seed to this factor [6]; however, its effect is not limited to photoblastic germination as it can also promote the germination for several species under dark conditions [7]. The response to nitrate appears to be via phosphorylation/dephosphorylation, for the kinase protein CIPK23, from nitrate transporter NRT1.1, while the light response is via phytochrome A [9]. Nitric oxide is recognized as a powerful agent for breaking seed dormancy in many species [10]. Its production is mediated by nitrite reductase (NiR) which reduces NO₂ to NO, using NAD(P)H as an electron donor. Its production could be beneficial at relatively low levels, functioning as a second messenger [11]. It could participate in either signaling or responses to environmental stress such as that generated by salinity [12]. Nitric oxide has different modes of action to promote germination such as reducing ABA sensitivity or accumulation, enhancing CYP707A2 gene expression or, through changes in the pentose phosphate pathway, promoting the increase in the glucose catabolism and thus promoting dormancy release [11].

A positive effect of KNO₃ was reported in seeds of Helianthus annuus L., improving the germination parameters as germination index, mean germination time and time to 50% germination [13]; this also occurred in Datura stramonium seeds, applied alone or in combination with gibberellic acid, with the highest percentage of germination between 50 to 63% [14]. In Capsicum annnum var. Glabriusculum seeds, it was reported that osmopriming allows the efficient digestion of the endosperm by gibberellin induced enzymes, reducing the endosperm mechanical restrictions when being metabolized and, providing energy to start and maintain embryo growth [2]. In Solanum lycopersicum seeds a positive effect of KNO₃ on seed germination was reported, related with the activity of the enzyme nitrate reductase, which acted as a germination promoter [15]

The response to every technique used to break the dormancy depends on the species and dormancy type; then, it is necessary to develop a technique to break dormancy of Jaltomata procumbens erect and decumbent type. The objective of this research was to break seed dormancy of two J. procumbens populations (erect and decumbent) by evaluating potassium nitrate solution and two soaking times.

2. Materials and Methods

The experiment was carried out in a germination chamber (Model: ATTGPT-B; Serie: 143-958-201) at Botany Program of Postgraduate College, Campus Montecillo, Colegio de Postgraduados, State of Mexico from 29 May to 30 June 2019.

2.1. Genetic Material

Erect and decumbent populations of J. procumbens from Tlaxcala, Mexico, were evaluated (19°15′ N y 97°53′ W, at 2500 m of altitude); predominant temperate sub-humid climate C(w1) y C(w2); average annual temperature between 12 and 18 °C. Ripe fruit seeds collected in 2015 were used, which were given the following handling: extracted seeds were rinsed off with tap water; after that, the seeds were dried under shadow at room temperature; finally, they were stored in plastic jars of 30 mL capacity, with screw cap, and were maintained in refrigerated conditions at 4 °C.

2.2. Experiment Management

The treatments for both populations were 2 KNO₃ concentrations: 0.1 and 0.2%, and, in distilled water used as control; there were 2 soaking times: 4 and 6 days.

The seed with treatments was placed in transparent plastic cups of 24 mL capacity and maintained in a germination chamber with diurnal (12 h at 30 °C) and nocturnal (12 h at 20 °C) conditions. When necessary, KNO₃ solution and distilled water was used to keep seeds at soaking conditions.

After soaking time, the seeds were extracted and placed in a sieve (mesh number 22), and rinsed with distilled water during 10 s. After that, in petri dishes, seeds were distributed on moistened filter paper; finally, petri dishes were placed in the germination chamber under the aforementioned light and temperature conditions. Distilled water was applied to keep the paper moistened.

2.3. Experimental Design

The arrangement of treatments was a factorial design with the next factors and levels: 2 populations (erect and decumbent), 2 soaking times (4 and 6 days), 2 KNO₃ (0.1 y 0.2%), and distilled water used as control, providing 12 treatments in total. The treatments were distributed in a completely randomized design with three replications. The experimental unit was a petri dish with 25 seeds each, with 36 experimental units in total.

2.4. Variables Registered and Generated

For 33 days, germinated seeds were registered. Radicle emergence from the testa was used as biological germination criterion.

Days to germination starting (DSG) and ending (DEG) were determined. Furthermore, the following germination variables were calculated [16,17]:

Germination percentage (GP)

$$\mathrm{GP}=\frac{Number of germinated seeds}{Number of seeds set to germinate }\times 100$$

Energy period (EP). days after sowing (DAS) to reach 50% or more of germinated seeds.

Germination energy (GE). cumulative germination percentage once reached the energy period.

$$\mathrm{GE}=\frac{Cumulative daily total of germinated seeds }{Number of seeds set to germinate}\times 100$$

Germination rate (GR). number of germinated seeds every day.

$$\mathrm{GR}=\frac{Number of germinated seeds}{Number of days to first count }+\cdots +\frac{Number of germinated seeds}{Number of days to last count }$$

2.5. Statistical Analysis

Results of the germination percentage were analyzed with logistical regression using the Wald test. To evaluate the model, Forward and Stepwise tests were applied. For the germination variables, analysis of homogeneity of variances and normality were performed; those data that did not meet the statistical assumptions were transformed using square root. The data were analyzed with the SAS program (2008) 9.2 version statistical program [18]; performing a variance analysis, based on a completely random factorial model 2 × 3 × 2, and Tukey’s separation means test (p ≤ 0.05).

3. Results

3.1. Effects Analysis on the Germination

Population (Pop), solution (Solu) and soaking time (Soak) affected the germination probability (p ≤ 0.05). The erect population registered 100% of germination and the decumbent 93.1%. The Pop × Solu and Pop × Soak interactions indicate that each type of solution and soaking time had a different effect within each population; for Solu × Soak, however, the soaking time affected germination differently within each type of solution. Finally, the effect of the day corresponding to the total evaluation period shows the probability that germination increases with time (

Table 1. Effects analysis on the germination of two J. procumbens populations. Colegio de Postgraduados, Campus Montecillo, Texcoco, State of Mexico. Data taken between 29 May and 30 June 2019.

Effect	Degrees of Freedom	Wald Chi-Squared	Pr > ChiSq
Population	1	437.3	<0.0001
Solution	1	35.4	<0.0001
Soaking	1	91.9	<0.0001
Pop × Solu	1	11.4	0.0007
Pop × Soak	1	24.4	<0.0001
Solu × Soak	1	39.5	<0.0001
Day	1	5796.4	<0.0001

).

The erect population showed a more uniform germination pattern, the combinations of 4 soaking days with KNO₃ at 0.1 or 0.2% stood out whereby the germination started earlier with the highest number of seeds, reaching on average at 12 DAS 100% of germination (Figure 1). On the contrary, the decumbent population showed a heterogeneous pattern, where KNO₃ at 0.2% with 4 soaking days started the germination process earlier; however, the control and 4 soaking days finished this process first. In addition, it registered a germination interval of 90.7 to 98.7%.

3.2. Germination Model

When the model was evaluated, the erect population, the solution, the soaking and their combinations each had an effect on the probability of germination as the days of the evaluation period increased (p ≤ 0.05). Based on the estimators of these parameters (

Table 2. Analysis of maximum likelihood estimates on the germination of two J. procumbens populations. Population (Pob): erect (E); solution (Solu); soaking (Soak). Colegio de Postgraduados, Campus Montecillo, Texcoco, State of Mexico. Data taken between 29 May and 30 June 2019.

Parameter	Estimator	Standard Error	Wald Chi-Squared	Pr > ChiSq
Intercept	−4.90	0.20	619.16	<0.0001
Population E	2.62	0.13	437.33	<0.0001
Solution	−0.86	0.14	35.37	<0.0001
Soaking	−0.35	0.04	91.91	<0.0001
Population E × Solution	0.10	0.03	11.37	0.0007
Population E × Soaking	−0.11	0.02	24.37	<0.0001
Solution × Soaking	0.18	0.03	39.45	<0.0001
Day	0.47	0.01	5796.41	<0.0001

) the probability of germination was obtained, taking into account each of the treatments and time from sowing. As a result, the same trend was obtained where, with increasing the total evaluation time, there is a probability that germination percentage will increase; the erect population, with the three types of solution and both soaking times, presents the highest probability of starting and ending the germination process earlier than the decumbent. However, in both populations, there is a 100% germination probability: outstanding out the control with four soaking days with the best effect on germination probability (Figure 2).

3.3. Germination Variables

For calculated germination variables, the population had an effect on all variables, while soaking influenced EP and GR (p ≤ 0.05). The highest coefficient of variation was detected in the percentage of germination energy, at a percentage of 14.23%.

In the Pop × Soak interaction there was a statistical difference in GR, indicating that for this variable, the effect of soaking time was not the same within each population.

The erect population presented a superior response in all variables (

Table 3. Mean values of germination variables of two J. procumbens populations. Solution: 0.1, 0.2% of KNO₃ and control; soaking: 4 days (4 d) and 6 days (6 d). Colegio de Postgraduados, campus Montecillo, Texcoco, State of Mexico. Data taken between 29 May and 30 June 2019.

	Days to Start Germination	Days to End Germination	Energy Period	Germination Energy	Germination Rate
Population
Erect	7.7 bꬷ	13.0 b	9.8 b	64.0 a	2.6 a
Decumbent	12.7 a	25.6 a	17.9 a	57.1 b	1.3 b
Solution (%)
0.1	10.1 a	19.0 a	13.7 a	58.3 a	1.9 a
0.2	10.0 a	18.2 a	13.2 a	60.7 a	2.0 a
Control	10.1 a	19.2 a	13.8 a	62.7 a	1.9 a
Soaking (days)
4 days	9.6 a	18.2 a	13.1 b	59.3 a	2.0 a
6 days	10.4 a	19.5 a	14.0 a	61.8 a	1.9 b

^ꬷ Different letters indicate statistical difference (Tukey, p ≤ 0.05).

) and showed uniform and faster germination than the decumbent population; recorded fewer DSG (7.7 days) and DEG (13.0 days). This concentrated the germination process in an average period of 5.3 days, which derived in fewer DAS (9.8 days) to reach 50% or more of germinated seed, being earlier in 82.5%, with an RG of 2.6 seeds day⁻¹. These characteristics are important to use a species for agronomical purposes, to obtain homogenous seedlings in size in the shortest possible time; furthermore, costs are reduced by shortening the time designated to this production stage [3].

There were no statistical differences in the solution type. However, a positive effect of KNO₃ at 0.2% was observed on DSG, DEG, and EP with the lowest values; in the same way, this effect was registered for GR, showing the highest number of germinated seeds per day (Table 3).

The soaking time produced statistical differences on EP and GR. In both variables, 4 days favored them with a difference of 6.6 and 9.1%, respectively. For DSG and DEG, although there were no statistical differences, the same trend with that time was registered; on contrary, with six days, the highest EG was obtained but without being statistically different with respect to four days of soaking (Table 3).

3.4. Effect of Population and Solution on Germination

The Pop × Solu combination had an effect between populations on DSG, EP, and GR. Within populations, it was observed that the minor number of DSG was favored, with the control and KNO₃ at 0.2% on erect and decumbent populations (Figure 3). For the last population, the same KNO₃ concentration favored the minor number of DEG; while, for the first, the lowest value was obtained with 0.1% of KNO₃ (Supplementary Material, Figure S1).

With KNO₃ at 0.2%, the lowest EP in both populations was registered, where the erect population stands out; with a difference of 84.3% lower (Figure 3). This same KNO₃ concentration was favorable on the erect population for obtaining the highest GR with 260.3%, in comparison with the decumbent that registered its highest GR with the control.

3.5. Effect of Population and Soaking on Seed Germination

For Population × Soaking combination, the lowest DAS to start germination was observed with four soaking days, without being statically different from six days within populations. In this sense, with 4 soaking days, the erect population stood out with 71.8% lower DSG than the decumbent population (Figure 4).

In EP and GR, with the combination of erect population and 4 soaking days, better values were obtained and exhibited a statistical difference to the other combinations. In this way, this population stood out with 96.3% in EP and with 103.8% in GR with regard to decumbent and 6 days combination, presenting the highest EP and the lowest GR.

4. Discussion

Potassium nitrate alone and in combination with soaking time had a different effect on each population. This indicates that, even between populations of the same species, there was a differential response to treatments to break dormancy; in addition to the observation between species, [8] and [3] reported that the response to a soaking treatment with an osmotic solution depends on the species, in addition to the solution type used, and the soaking time. KNO₃ at 0.2% and 4 soaking days registered the lowest number of DSG; however, it was not statistically different to the other combinations (Figure 5). This same combination presented the lowest EP value, statistically different to the control and 6 soaking days; with a difference between both of 15.0%.

On the other hand, a positive effect of the control on the germination process was also observed in both populations; registering the control and four soaking days the highest GR, but without being statistically different to the other combinations, with exception to the control and six soaking days that presented the lowest value.

This could be attributed to the elimination through soaking of substances such as tannins presented in the seed, recognized as germination inhibitors. One of these substances in the fruit of both populations was reported, with a content of 1.3 and 1.2 g of floroglucin/100 g of dry sample [19]. Tannins reduce the gibberellins activity through protein precipitation, resulting in an inhibition of the growth caused by gibberellin, also inhibiting enzymes related to the synthesis of sugars as glucose phosphate isomerase, aldolase and, glucose-6-phosphate-dehydrogenase and thus, the inhibition of radicle emergence [20,21]. The positive effect of tannins elimination using distilled water was reported in seeds of Phaseolus aureus L., reducing its content among 18 to 35% by the leaching out of the polyphenols through the used water [22]. Thus, in this work, this situation could be presented, registering similar results to the treatments with KNO₃, without statistical differences. This response was similar to that reported in the germination of Foeniculum vulgare Mill., where no significant behavior was found when was applied KNO₃ compared with distilled water [23].

However, the greatest positive trend in the germination process was with KNO₃. This can be explained by the KNO₃ osmopriming effect, through which, the metabolism is activated without radicle emerging, but when seed is removed from the solution and under suitable conditions the process is accelerated [3,8]; the phenomenon that was observed when registering the greatest initial number of germinated seeds, the highest GR and the lowest EP. A similar response was observed in Capsicum chinense Jacq., where with KNO₃ osmopriming, a radicle emergence was not observed during the KNO₃ osmopriming compared with distilled water, but a greater seedling emergence was registered [24].

The positive effect on germination of KNO₃-soaking seeds could be due to the influence of the oxidized forms of nitrogen as NO₃ or NO [3]. Nitrate induces the genetic expression such as genes involved in the nitrate assimilation (nitrate and nitrite reductase); nitrate transport (CHL1/NRT1.1); energy production and metabolism of carbon (glucose-6-phosphate dehydrogenase); GARP-like transcription factors; genes involved in hormonal metabolism and signaling [7]. Nitric oxide is recognized as a potent agent to break dormancy on seeds [10] working through the influence on ABA accumulation and sensitivity, CYP707A2 gene expression or, pentose phosphate pathway [11]. In Arabidopsis seeds, after six hours of soaking with NO₃ the CYP707A2 gene induction has been observed, which encodes an abscisic acid 8′-hydroxylase, the main catabolic enzyme of abscisic acid in imbibed seeds, presenting an accelerated decrease in this hormone recognized as germination inhibitor [7]. The effect of NO has been reported on Paulownia elongata seeds in combination with light conditions or cold stratification, where the accumulation of endogenous NO in the early stage of absorption can promote the break dormancy [25].

Furthermore, the NO₃ seems to be related to phytochromes, functioning as sensors to different factors as the light, and which have an important role in the germination process [9]; therefore, nitrate could stimulate the accumulation of cGMP [10], a molecule recognized as a key signaling in many process in plants including seed germination through direct effect on the phytochrome signal transduction [26], which promotes phytochrome responses [10]. The response to light presents two types of action where the first one, very-low-fluence response (VLFR), is mediated by phytochrome-A, and the second one, low-fluence response (LFR), is mediated by phytochrome-B [27]. In Arabidopsis, the greatest phytochrome-A expression seems to be linked to the nitrate presence in combination with a long exposure to darkness and low temperatures, improving the germination process [9]. In this research, in addition to nitrate, low temperature (20 °C) and darkness conditions were provided, which could have generated a response similar to that observed in Arabidopsis where the expression of DOG1, PHYA and CIPK23 genes act as temperature, light and nitrate sensors, respectively, recognizing the appropriate conditions to release the seed dormancy through the enhanced expression of GA3ox1 [9] increasing a positive trend in the germination process.

The trend of the results obtained with KNO₃ treatment were similar to those reported in Brachiaria humidicola, where it was registered a favorable response with KNO₃ in GP, because the nitrate influences the break seed dormancy by its action in the pentose phosphate pathway [28]. In the same way, in the Citrus limonia Osbeck seeds stored up to 3 months, preconditioned with KNO₃ for up to 9 days, it was reported a trend in the improvement of the germination, getting results for up to 85 GP with 84.5% of emergence; concluding, that osmopriming seems to reverse the storage effect in the seed, improving germination percentage, which indicates a metabolic recovery [29].

Results obtained for the erect population in germination percentage (100%) and germination period (13.0 DAS on average) were superior to those registered by [30], who, with unfermented fruits, 40 days of storage of the seed and 20 °C germination temperature, obtained 61.0% of germination to 27 DAS. Moreover, in those reported by [5], who evaluated gibberellic acid, with a concentration of 250 mg L⁻¹ on seed emergence, a registering of up to 87% of germination in a period of 25.5 days was observed. However, these authors carried out the evaluation in substrate (moss and perlite).

For the decumbent population, the germination period (25.63 days after sowing) was between the data reported by [5,30]; also, an improvement in the germination process was observed, registering a high percentage of 93.13% of germination. In this population, in a study about morphological differences and infertility, 36.2% germination was observed in 2 months [31].

5. Conclusions

Seed dormancy can be broken in J. procumbens populations (erect and decumbent) with KNO₃ in combination with 4 days of seed soaking.

Differential responses to treatments between and within populations were observed; however, the erect population stood out with the best data for germination variables.

No statistical differences between KNO₃ solution and the control were obtained; however, the greatest positive trend with KNO₃ in the germination process was observed.

It is necessary to determine the influence of factors such as temperature and light on the germination process of J. procumbens seeds, and the expression of genes linked to these factors.

Considering economic and accessibility aspects on the of use substances that promote germination on J. procumbens, distilled water imbibition and KNO₃ are recommended to accelerate the germination process.