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Article

Dynamics of the Ocimum basilicum L. Germination under Seed Priming Assessed by an Updated BBCH Scale

by
Valentina Ancuța Stoian
1,
Ștefania Gâdea
1,
Roxana Vidican
2,
Dan Vârban
3,
Claudia Balint
4,
Anamaria Vâtcă
5,*,
Ancuța Rotaru
6,*,
Vlad Stoian
2 and
Sorin Vâtcă
1
1
Department of Plant Physiology, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
2
Department of Microbiology, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
3
Department of Crop Plant, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
4
Department of Engineering and Environmental Protection, Faculty of Agriculture, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
5
Department of Management and Economics, Faculty of Animal Science and Biotechnologies, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
6
Department of Applied Informatics and Mathematics, Faculty of Animal Science and Biotechnologies, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(11), 2694; https://doi.org/10.3390/agronomy12112694
Submission received: 6 October 2022 / Revised: 25 October 2022 / Accepted: 27 October 2022 / Published: 30 October 2022
(This article belongs to the Special Issue Effect of Agronomic Treatment on Seed Germination and Dormancy)
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Abstract

:
Germination of the medicinal and aromatic plant basil raises interesting questions due to its no seed periodicity and dormancy demand, and the seed priming could help to assure the permanent needs for this herb. The study aimed to provide an adapted BBCH (Biologische Bundesanstalt Bundessortenamt and CHemical industry) scale for the germination secondary stages of sweet basil Ocimum basilicum L. (var. MVSt). A standardized germination test was performed with hydropriming, electric field, and magnetic field as priming methods to assess the changes in the secondary stages of germination. The time range for each basil secondary stage was recorded and added to the adapted BBCH, to highlight the daily secondary stage changes. All the seed priming methods increased the germination capacity with 2–3% compared with the control, with the highest value for electropriming. The seed priming methods studied improved the germination and changed the pattern of secondary stages in the BBCH scale. The germination energy was set to more than 90% in all primed seeds, with a maximum of 91.75% in the magnetic field. The time needed for 50% germination of seeds was set to 6.5 days and 90% germinated seeds were recorded after 11.7 days. The 09a BBCH lasted for 9 days in control and hydropriming, 10 days in electric field conditions and 7 days for magnetoprimed seeds.

1. Introduction

Sweet basil (Ocimum basilicum L.) is an annual herb from the Lamiaceae family, Genus Ocimum, and represents the most widely used medicinal and aromatic plant [1]. Its unique properties from the strongly scented smell and spicy taste were used for different purposes such as food seasoning, ornamental, religious, and for homeopathic medicine [2,3,4]. Basil is distributed worldwide and comprises more than sixty cultivars [5]. Additionally, it is often called “king of the herbs” because of its economic expansion from a wide range of fields [6]. Traditionally, the consumption and uses of basil essential oil place together the leaves and flowers for an insecticide effect, medicinal remedies, and culinary flavoring purposes [4,7,8].
The main key stage in the plant’s future life strategy of growth and development is represented by its germination process. Because it has no seasonal periodicity, germination represents an interesting and problematic aromatic and medicinal weed [9]. The metabolic activity initiation of seed germination begins with the imbibition, biochemical processes, then embryo growth radicular emergence, and at the end, the emergence of aboveground plant components [9]. The first complex biological process that starts the engine of the future plant must gather several favorable conditions in a simultaneous manner [10].
Different methods of seed stimulation before the germination starts were tested for medicinal plants to compensate or replace some of these factors and to speed up the germination time [11,12,13,14]. These seed priming methods were also helpful to obtain more vigorous and resistant plants to diseases and pests. Another advantage was highlighted after priming techniques so that, in this way, different medicinal plants could thrive in unfavorable environmental conditions with a higher production of different chemical compounds with a maximum medicinal value [14,15,16,17,18]. Each technique has a specific procedure, most of them based on seed soaking, that implies different changes in seeds [19]: hydropriming refers to soaking the seeds in water followed by forced air-drying until initial weight; osmopriming presume the soaking of seeds in different osmotic solutions; hormopriming refers to soaking of seeds in different growth regulators; and biopriming use solutions with microorganisms placed on the seed surface before germination. Osmopriming strongly reduces the emergence time for Salvia przewalskii Maxim. [20], and it also increases with 14% of the germination rate of Physalis angulata L., Plantago ovata Forsk, Achillea millefolium L., and Momordica charantia L. [17,21,22,23]. Hormopriming significantly increases seed germination of soybean [24], Salvia officinalis L. [25], similar to Ocimum basilicum L. under salinity stress conditions with an increase in linalool and eugenol content percentages [14], which ensures the highest emergence percentages of Nigella sativa L. under drought [18] and under cadmium stress, and increases germination of Thymus vulgaris L. [12]. The beneficial effect of seed hydropriming was seen in the germination stage under optimal conditions for fennel and borage [26,27]. Another efficient technique for breaking fennel seed dormancy in addition to hydropriming was biopriming treatment [26]. Biopriming for 20 min to Dracocephalum Kotschy Boiss could enhance with more than 20% the germination stage [28]. Magnetic field was found to increase Salvia officinalis L. seedling length [25], alleviate salinity stress to Silybum marianum L. [13], and have a positive effect on medicinal properties to Hyssopul officinalis L. [16] and Momordia charantia L. [15]. A magnetopriming for Sinapsis alba L. seeds, exposed for 30 min, significantly increases the germination rate. Two new techniques for seed priming could be mentioned, respectively: laser for improving Salvia officinalis L. seed germination and the seedling growth and development [25], and cold plasma [29] tested for Catharanthus roseus L. with high efficiency on germination. For this last species, both hydro-, osmo- and hormopriming showed no beneficial priming effect and do not ensure a significant improvement of the germination process [29]. Another species, where these three priming treatments did not show any major advantages, is Matricaria camomilla L., the maximum germination being observed in the control treatment [30].
This context emphasizes two concepts: the first one states that the priming technique is species dependent and the second one targets future research—there is a need for identifying new priming procedures that will improve the germination in problematic plant species. Both concepts are necessary for science to deliver realistic solutions that can assure a maximum germination percentage.
In order to maintain the permanent need for dry and fresh basil, rapid production strategies and environmentally friendly organic techniques are needed. The general phenological principal growth stages are made up of ten in total, described in the BBCH scale by using numbers from 0 to 9 [31]. The germination process represents the first phenological principal stage and also the key stage because if it does not happen properly, subsequent stages could not be assessed. This scale was proposed as a general assesment instrument for the most cultivated crops, but it still remains general for the large majority of plants and needs to be updated. The first aim of the study was to adapt the general BBCH scale for basil germination, with an update of specific secondary stages. This process will fill the gaps and it is also necessary to present in detail every specific stage identified for basil on a daily basis. The second objective was to assess if hydropriming, electric, and magnetic field seed priming methods could be useful to improve basil germination energy and capacity. The third objective was to add a time range for each secondary stage in the updated BBCH scale for further comparison and standardization.

2. Materials and Methods

The experimental design consisted of four seed treatments with an electric field, hydropriming, magnetic field, and control (Table 1) in four replications. The experiment was done in a growth chamber at 20 ± 2 °C, 40% humidity with full light assured by 20 neons × 18 W. A total number of 1600 seeds, 100 seeds/replication for each treatment, were placed on filter paper in Linhard germinators in controlled conditions. Made of white ceramics, the Linhard pots have a base diameter of 18 cm, with a deepness of 3 cm. The water was introduced at the pots reservoir bottom, then covered by a perforated thin glass on which the seeds were put on a filter paper. The germinator had four openings that facilitate gas exchange and the humidity was mantained by a top glass with 20 cm diameter which also allows light for the seed. Prior to germination assessment, the seeds were sterilized using a 50% bleach solution [32]. The standard germination test was performed according to the International Seed Testing Association (ISTA) [33].
The Ocimum basilicum L. seeds used for the experiment were represented by a Romanian local variety (MVSt) from Mihai Viteazu village, Cluj county (46°32′22 N, 23°44′33 E). Paper bags were used to store the seeds at 20 ± 2 °C for two weeks until the experimental start. The seeds were assessed every day for thirteen days (D1–D13) during germination time. All the secondary stages from germination were evaluated in each replication and the number of seeds were recorded. Representative images for each germination sub-stage assessed were captured using a stereomicroscope with an Optika camera of 8 MP resolution. For seeds assessment, the newest and most representative BBCH scale for growth and development was used [31], all seeds being assesed on 00–09 stages. In the secondary stage 06 BBCH, three extra phases were recorded, and also for the secondary stage 09 BBCH, two extra phases were assessed separately. All these supplementary phases were seen clearly during basil germination and were considered in assembling the newly updated BBCH scale.
The presented results are average results, percentages of germinated seeds from the total number of seeds per repetition, or from the total number of tested seeds. Data were compiled and analyzed separately and compared to the control and germination patterns that were presented as matrices. To express the general pattern of germination in all priming methods, all the percentages below 1 were not considered, being removed from the comparison with the control treatment and uncounted in each of the treatment germination patterns. Germination energy and capacity, respectively, and the number of days necessary for the germination of 25, 50, 75 and 90% seeds were selected as parameters for the analysis of differences induced by priming treatements on the entire seed pool [37]. All averages and their standard errors were analyzed with post hoc LSD test, with packages “psych” [38] and “agricolae” [39], under Rstudio version 1.4.1106 [40].

3. Results

3.1. Germination Stage Description and Completion According to the BBCH Scale for Basil

The transition from seed to seedling or all the germination secondary stages were observed and monitored during basil development (Figure 1). All the described germination secondary stages of basil seeds served to complete the extended BBCH scale [31] (Table 2).
The 00 secondary stage for basil was set as dry seed in the extended general BBCH scale (Figure 1, Table 2). From this secondary stage when breaking dormancy happens, the seed will end the inactivity or extremely low metabolic activity period. In the present study, all the seeds that showed inactivity were considered, which did not swell nor produce mucilage. The basil seeds are oval-shaped ellipsoidal and quite small, ranging between 2–2.5 mm in length and 1–1.4 mm in width. The seed surface is porous with small blisters that look like dots and the seed coat color is dark brown.
The 01 germination secondary stage represents the initiation of the seed imbibition. Hereby, it is considered that all the seeds are partially covered with gelatinous mucilage for around (30)50–80% around the pericarp (Figure 1, Table 2). This mucilage is present within minutes after hydration and has different roles. First, it represents a source of nutrients for embryo growth, conserves water, and it keeps the seed hydrated for the establishment of the seed-soil contact [9]. It usually appears to seeds as an adaptation to survive drought conditions [41]. In contact with water, the seeds produce a dense layer of mucilage gelatinous that can keep the seeds in the liquid solution if they are placed in a test tube upside down. The seed volume is higher, and the seeds started to begin their biochemical transformation. After imbibition, the seeds changed their dimensions between 2.4–2.8 mm in length and 1.4–1.5 mm in width. This secondary stage is important because many seeds could be lost in the soil bank without continuing further development. Basil seeds in this sub-stage were observed for 2–4 days.
The 03 secondary stage is the first one representing the best beginning of the vegetative stage of the future plants (Figure 1, Table 2). It includes all the seeds that are doubled in volume, swelled and covered totally with mucilage. After the imbibition process, it is considered that the enzymes are activated to break down the starch into sugars (hydrolysis) for assuring the embryo nutrition needs. The well-imbibed seeds had between 2.7–2.9 mm in length and 1.7–2.2 mm in width. This secondary stage lasted between 4–7 days.
The 05 secondary stage is represented by the emergence of radicle from the seed coat. The seed evaluation can present some difficulties because the seed must be moved, and sometimes even turned to be able to observe this secondary stage (Figure 1, Table 2). The seed tegument does not crack identically in all seeds; therefore, it requires increased attention to be able to identify the emergent radicle. This stage lasted between 4–6 days.
The 06 secondary stage phases a, b and c are clearly differentiated, and their evolution is different from one day to the next (Figure 1, Table 2). In the first phase (a), a brief elongation of the radicle is happening, and therefore it is more visible than in the secondary stage 05. Then, in the next phase (b), tiny hairy roots appear on the radicle. In phase c, the radicle continues its straight growth from the hairy roots zone. The phase 06a of this secondary stage lasted between 3–8 days, the phase 06b between 3–7, and the phase 06c between 2–4.
In the 07 secondary stage, the appearance of the arc between the radicle and the hypocotyl was seen (Figure 1, Table 2). Besides the elongated radicle, in this secondary stage, the green base of the cotyledons can be observed. The hypocotyl arc changes its color to green and also thickens. Here, the photomorphogenesis begins, and the embryo grows toward the light source. The cotyledons begin to break and release from the seed coat. This secondary stage lasted between 3–5 days.
The 08 secondary stage could be characterized by the growth in the purpose of cotyledons elongation, whereby they try to reach the soil surface (Figure 1, Table 2). Usually, this process takes time, and it is directly related with the substrate principal parameters and fertility. A percentage between 30–80% of the cotyledons is visible and the folded cotyledons are still kept under the seed coat. The hypocotyl continues to elongate. This sub-stage lasted between 5–8 days.
The 09 secondary stage phases a and b between which there is a clear difference were evaluated separately (Figure 1, Table 2). Phase a is represented by the seedling with semi-expanded and smaller cotyledons. This represents the embryo’s complete development stage and from here it is considered a young seedling. Phase b is characterized by all seedlings with both fully developed cotyledons, with an intensely green leaf surface, these being visibly larger than in the previous phase. This sub-stage for each phase lasted between 8–9 days. From this secondary stage, the thick cotyledons serve as food reserves, and they shrivel and fall after the nutritive substances are consumed by the seedling.

3.2. Dynamic of Seed Germination Development Due to Specific Priming Treatments

3.2.1. Seed Development in the Control Treatment

Inactive seeds 00 BBCH which did not germinate existed until the end of the tested period. The number of these seeds was within the average range of 10–16% and decreased over time by 2% from the next day until the end of the germination assessment. In only two days, the 01 BBCH was seen with the highest values between 38–50% on D1 and an immediate drop (34%) in number the next day (Figure 2). Both 03 and 05 BBCH were visible within the first five days. A total of 36–48% of the seeds on D1 were in the 03 BBCH, slightly similar to D2, when a decrease of only 2% was visible for this stage. On D4 and D5, the seeds in this phase were present within 2/4 and 1/, respectively, from the replications. On D2, a percentage range between 24–34% was registered with the seeds that exhibit an emerged radicle (05 BBCH). After two days (D3), the seeds from this phase decreased by 26%, present in 2/4 of the replications. Further stages 06a–06b BBCH and 06c–07 BBCH show the same trend and begin on D2 and D3, respectively. The phases 06a and 06b BBCH were observed only from the second day with a close percentage of 26% for 06a and 24% for 06b. The seeds stayed six days in the 06b BBCH with no extra seeds on D6, compared with 4 days in the case of 06a BBCH and a lack of new seeds on D4. The following secondary stage, 06c BBCH, was observed for only two days, on D3 when a range of 10–14% seeds entered in this stage and a two times lower value on D4. On D3, the 07 BBCH was dominant with a percent between 30–46% of the seeds, the number decreased two times until the next day (D4) and five times until D5. The most advanced seeds from D3 were in 08 BBCH, within a 6–28% range. On D4, the number tripled, but was followed by a daily decrease. On D7 and D8, only 3/4 of tested replications presented seeds in the 08 BBCH. In the following two days, D9–10, the seeds in this secondary stage were found only in half of the replications. The 09 BBCH comprises nine days in total. A number of 24 seeds were in 09a BBCH on the fourth day already. On the next day, the number decreased three times, next with 12% and the last drastic reduction was on day 7 with 42%. From D7 until D11, the seeds in this phase occupy a 3/4 share of the replications. In the following days, this share decreased by one part per day. The last secondary stage is characterized by a total time frame of eight days from the sixth day (70%) from the germination beginning until the last assessed day (94%). The value of the seeds in this stage increased daily by 14%, 6%, and 2% (from D9 until D12).

3.2.2. Electric Field Effect as Priming Method upon Seed Germination Secondary Stages

Inactive seeds 00 BBCH which did not germinate existed until the end of the tested period (Figure 2). On D1, the number of these seeds was within the average range of 10–19%. This percentage interval decreased over time by 7% until D4 and then by 10% from D5 until the end of the germination assessment. On D2, D5, D7, and D8 were observed seeds that developed from 00 BBCH to others. The 01 BBCH, 03 BBCH, and 05 BBCH were visible in the first four days. The seeds partially covered with mucilage (01 BBCH) were only observed in the first four days. The higher number of seeds was on D2 in a range between 7–29%. On D3, the seed in this secondary stage (01 BBCH) were missing from 1/4 of the total tested replications. Within the following day, in 2/4 of the replications, seeds passed this secondary stage, then over the following days, all seeds evolved to the next stages. The seeds completely soaked with water, 03 BBCH, were visible until D7, with no seeds in this stage on D8 (4/4) and only one seed on D9. The seeds missing this secondary stage were on D3, and D5 in 1/4 of the total replications, and on D4, D6, and D7 for 2/4 of the replications. This secondary stage was prevalent in the first two days due to the high number of seeds found. On D1, the seeds in 03 BBCH were in the highest range of 49–71%, but this percentage was diminished by 23% on D2. From D3, the number of seeds in this secondary stage presented a daily reduction. The emergence of radicle (05 BBCH) was observed during 10 days with the highest number of the seeds 14–32% on D2. A percent between 32–55 of the total seeds found in the 03 and 05 BBCH needed two days to move to further stages. The radicle elongation (06a BBCH) and root hair appearance (06b BBCH) were observed only from D2 with the highest percentage of 19% for 06a BBCH and 24% for 06b BBCH on D3. Both secondary stages last similarly to the previous one until D10. An average range percentage between 19–44% from the seeds reached 06c BBCH only on D3, then they quickly passed (33%) to the following stages. This phase ended completely on D9 with the remark that on D7 not a single seed was in this stage. The 07 BBCH and 08 BBCH were observed from D3 until D10. The highest seeds recorded percentage was 64% (07 BBCH) on D3 but on D8, no seeds were seen in this phase. In the following two days, 20–33% of seeds passed this phase. The 08 BBCH registered the highest value of 72% on day 5 with a sudden reduction of 34% on the following day. From D6 and D7, in 3/4 of the replications there were observed seeds in this phase, and from D8 until D10, only in 1/4 of the replications. A total of three seeds were in the 09a BBCH on D3 already. This number increased for each D4, D5 by 2 times until D6, after which a decrease was observed with around 1.2 times lower rates of seeds in the 09a BBCH. D11 and D12 of the experiment registered only one seed in this phase, which took two days to jump into the last secondary stage. The 09b BBCH is characterized by a total time frame of nine days, starting in D5 (10%) from the germination beginning until the last assessed day (95%). Here, the percentage range increased as the days passed. Similar percentages were registered on D7 and D8, then with a 2% increase on D9 and D10. On D11, the highest percentage was reached, and the plants began to grow and further develop the first true leaves.

3.2.3. Hydropriming Effect upon Germination Secondary Stages

On the first day, the number of inactive seeds (00 BBCH) which did not germinate was present until the end of the tested period (Figure 2). These seeds were within the average range of 10–12%, the lowest range within all the tested treatments, and decreased over time by 1% until D5 and then again by 1% until D7, with the same percent of decrease until D11 and again until the end of the experiment. The secondary stages 01–06b BBCH are increasing in terms of the days’ number and the seeds can be recorded in a progressive development (Figure 2). The seeds from the 01 BBCH were observed on D1 in a range between 10–19%, with a decrease of 9% on the next day and 4% on D3. The seeds from the 03 BBCH were between 51–58% on D1, the highest percentage registered. The percentage of 03 BBCH seeds decreased by 21–26% on D2 and D3. The seeds from 05 BBCH recorded the same percentage as those from the 01 BBCH. This time, the number increased on D2 by 8%, then a decrease of 18% was observed on D3. The seeds in this stage are present in 1/4 of the total number of replications on D5 and D7 and 2/4 on D6 and D8. The following phase, 06a BBCH, had the highest range of germinated seeds between 5–20% on D2 with a slight decrease of 7% on D3. Until the end of the experiment, from D9–11, no seeds were observed in this phase. The seeds shared from the total replications was 3/4 on D6 and 2/4 on D7 and D12. On D2 and D3, 5–24% of the seeds were already in 06b BBCH. The next day a decrease in the seed percent of 21% was observed and the lower values were maintained until D8. The share of seed present was 1/4 on D9, 2/4 on D5, D7, D8, D13, and 3/4 on D4. The seeds in 06c BBCH and 07 BBCH began to appear on D2. The higher ranges were between 2–15% on D2 for 06c BBCH and between 18–31% on D4 for 07 BBCH. The seeds share from the 06c BBCH decreased from 3/4 on D4 to 1/4 on D9. All the gaps observed in the seed development were grouped in 1/4 share of presence from the total tested replications. The seeds observed in the 08 BBCH were comprised in the highest range between 17–27% on D3. In the following three days, 3% of the seeds advanced to this phase each day. From D7 until D10, the number of seeds in this stage were within the range of 4–9 with a seed presence share of 3/4 on D8 and 1/4 on D10. One seed evolved quickly on D2 to 09a BBCH. The highest number of seeds in this stage was recorded on D5, represented by a rate between 58–65%. The values dropped by 28% after two days. The seeds in this secondary stage were observed in all the replications for the period of 9 days, which was the duration of this phase. The last assessed stage, 09b BBCH, began on D5 for a number between 4–7 seeds. The seed number from the following days increased progressively with 24% on D6 and with 50% on D7. From D8, the 09b BBCH seed percentage increased only with 2–3 units until the last day of the assessment.

3.2.4. Magnetic Field Effect as Priming Seed Method upon Germination Secondary Stages

On the first day, the number of inactive seeds (00 BBCH) which did not germinate was present until the end of the tested period (Figure 2). These seeds were within the average range of 10–16%, and decreased over time by 1% until the next day and then again by 3% until the end of the experiment. Equal time was recorded for the 01 and 03 BBCH for the first four days. On D1, the seeds were between 34–40% in 01 BBCH with a decrease of 13% on the next day, followed by a 24% lower seed number from D3 and D4. The phase of seed complete imbibition (03 BBCH) was within the range of 45–52% on D1. A total of 11% of seeds passed into the next secondary stages on D2, around 21% of the seeds changed stage on D3, and with 17% (from D3 seeds), fewer seeds were observed on D4. A similar number of seeds were observed in 05 and 06a BBCH and partially 06b BBCH. The seeds were seen in these sub-stages on D2 of evaluation at a value of around 20% from the total. From D4, the seeds were seen only in a share of 1/4 (05 BBCH), 2/4 (06a BBCH), and 3/4 (06b BBCH) from the replications. The seeds with radicle present (05 BBCH) were seen in a lower number range of only 1–5% (D3–10) until the end of the experiment, with a day (D9) without any seeds in this secondary stage. In the case of the 06a BBCH, no seed was observed for 4 days (D6–9), then one appeared on D10. Phase 06b BBCH ended on D6. The next group of secondary stages were 06c BBCH and 07 BBCH following the same pattern as the previous group and lasting the same number of 9 days. The seeds from the 06c BBCH were between 11–35% on D3, with a decrease of 20% on D4. From D5 until D11, only 1 or 2 seeds were observed in this germination phase with a presence share of 2/4 on D5 and 1/4 on D6–11. The 07 BBCH started on D3 with a seed percent between 22–53%. The next day, a decrease of 31% of the seed abundance was seen. From D5, the seeds in this secondary stage decreased by 19% and kept the value until D11. Four days without any seeds in this stage between D7 and D10 was noted, and a seed share of 2/4 on the D5, and 1/4 on D6 and D11. Another group of secondary stages 08 and 09a BBCH was formed because of the seed presence that lasted 11 days and they appeared on D3. However, the percentage distribution differs greatly between them. The seeds found in 08 BBCH were between 5–15% on D3, with a 27% increase on the next day and another 1% on D5. From D6, the seeds decreased by 16% and followed by a 21% decrease on D7. On D10, the seed number in the 08 BBCH increased by 1%, followed by another increase on D12 with the same percentage. On D6 and D7, the seed share in 08 BBCH was 3/4 from total replications, then the share was reduced to 2/4 between D8–10 and D12, and 1/4 on D11 and D13. The seeds found in 09a BBCH were 3% on D3 with a 16% increase on the following day, 41% on D5, and another 21% on D6. From D7, the seeds decreased by 23%, by 10% on D8, and by 2–3% on D9 and D10. On D11, the seed number in the 09 BBCH increased by 2%, followed by a decrease of 4% and another increase on D13 by 1%. On D9–10 and D13, the seed share was 2/4 and between D11–12, the share was reduced to 1/4 from total treatments.

3.3. Changes in Seed Germination Pattern Due to Priming Treatments

The basil seeds secondary stages changed over time depending on the priming treatments applied (Table 3, Supplementary Materials—Tables S1 and S4). The electric field shows multiple differences from the control treatment. A 20% increase in the number of completely swelled seeds (03 BBCH) was observed compared to the control treatment, where this percent was represented by seeds in 01 BBCH. On D2, the seeds from 01 BBCH and 03 BBCH were at 12%, with much more in the electric priming treatments. On the same day, the number of seeds from 05–06b BBCH was much lower than the control. Less than 5% of seeds, in 01–05 BBCH (D3), show increases over control, with a peak at 06c BBCH when almost 15% of seeds exceed the control. At the opposite, both 07 and 08 BBCH have a more than 10% reduction in seeds. A similar trend is visible on D4, when the seeds in 07 BBCH exceed the control with more than 17%, while the difference for seeds in 08 BBCH is set to 28%. During D5, the last high difference between the two treatments was seen, with a more than 20% reduction in seeds in the 09a BBCH for the electric treatment. This phase also exhibited a negative difference on D6, but this overcomes the control during D7–10 with a range of 2.5–8.75% of seeds. In the final growing phase (09b BBCH), the electric treatment presents a faster achievement on D5, when more than 5% of seeds exceed the control. For the same growth phase, the interval between D7–9 presents an opposite phenomenon; in this period, the seeds from the control exceed the seeds from the electric treatment. The last days of the experiment, D11–13, shows another turn, with the electropriming exceeding the control with more than 3%.
The hydropriming method was more efficient compared to the control, on D1 there were already seeds in 06b BBCH (Table 3). Around 15% of the seeds on D1 were completely imbibed (03 BBCH), with most of the seeds from the control treatment in 01 BBCH. In D2, most of the seeds from 03 BBCH jumped two stages into 06c BBCH. In D3, the electric field helped the seeds to develop up to 09a BBCH with a peak percentage on 08 BBCH compared to the control. The seeds from 07 BBCH were reduced with around 20% compared with the control. On D4, all the secondary stages recorded higher seed percentages except for 07 BBCH, whose highest percent was lower in electric treatment compared with the control. On the next day, the differences compared to control were lower. The seed percentage was lower in 08 BBCH and higher in the last assessed phase 09b BBCH. The period between D6–10 recorded an opposite phenomenon, where in this period the seeds exceeding the control were in phase 09a BBCH. Then, until the end of the experiment, the seeds in the last phase overcame the control by between 1–4% (Table 3, Supplementary Materials—Tables S2 and S4).
The application of a magnetic field on basil seeds produces only a reduced number of cases where the differences are higher compared to the control seed pool (Table 3, Supplementary Materials—Tables S3 and S4). The inactive seed maintains its value below −2%, which suggests a lower geminating potential of this priming method, even if on D1 of the experiment the value recorded was higher. There is a rapid change observed in the first secondary stage (01 BBCH), when the first day presents a higher value in the control treatment, followed by a change of up to 11% after 24 h. An opposite trend is visible in the 03 BBCH, with more than 8% higher seeds in this stage compared to the control, but a 5% decrease during D2 and D3. The 05 BBCH has a start below the control, maintained in this trend during the first two days, and overcomes the control only in D3. A similar trend was observed for 06a BBCH, starting with D2. Days D3 and D4 present the higher positive impact of a magnetic field for 06b and 06c BBCH, both of them having a higher number of seeds compared to the control. Starting with the 07 BBCH, a high positive/negative difference change was visible between the two treatments. The stage 07 BBCH exhibits lower values than the control, followed after 24 h by an increase of more than 11% difference. The next secondary stage, 08 BBCH, starts with over 5% less seeds than the control, a difference that goes up to five times higher in favor of the control after 24 h. The interesting phenomenon is that the difference was recovered and even directed toward magnetic treatment, which overcame the control by more than 10% on day 5.
After this point, the difference between the two treatments decreased. The 09a BBCH presents a variable trend, as observed in the 07 and 08 BBCH, but the most interesting aspect is that in the final secondary stage, the magnetic field overcame the control by 2–8% during the entire length of this phase (Table 3).
Overall, the use of priming on basil seeds induced a change in the number of seeds in different BBCH stages recorded on each day. The analysis of germination energy and capacity, respectively, and the time for 25–90% seeds germinated revealed the lack of significant differences between the applied treatments (Table S5). In terms of germination capacity, the 90.5% recorded in control was exceeded by all treatments with 2–3% and the electropriming was the most effective. Germination energy reveals the magnetopriming as the most effective treatment for basil seeds, with almost 2% higher than in control. For this parameter, all priming methods exceed 90% of germinated seeds. The time needed for 25% seeds to germinate was set as 3.3 days for all treatments, whereas for 50% of seeds to geminate, 6.5 days were necessary. For 75% to 90% of total seeds to germinate, only two days are necessary, all treatments required 11.7 days to reach at least 90% germinated seeds.

4. Discussion

The marketplace requires stable and secure information about seed testing [42]. Without germination tests, healthy and vigorous seedlings could not be produced. Seed testing represents the science of assessing the seeds’ quality for determining their value [41] for setting up a business in the case of basil. The basil herb needs, for an increased production, vigorous seeds to provide stable and secure products for the market and consumption [41]. The complex process of germination determines physical changes in the seeds which intensify the embryo metabolism and enable the activities of the enzymes. The imbibition represents the starting point of the seed metabolism and intensifies the chemical reactions for the development of the embryo [43].
The need to test and use priming techniques can be argued by the qualitative threats to the soil level as a result of intensive climate changes. Seed priming could influence germination, seed quality, and also seedling performance. As a warm season aromatic and medicinal plant, the basil seedling establishment is difficult during cold soil, and the reason for how temperature impacts seed germination was studied [9,10,41,44]. Usually, for the first and final count, days 2–3 and 6–7 were considered [10]. More than 80% of seeds germinated on day 4 at a temperature range of between 21–30 °C in another experiment [45].
In the control treatment, the seeds were already in the first day with 44% at the beginning of imbibition and 41% completely imbibed. The basil seed mucilage has beneficial effects on seed germination [46]. Water content from the seeds increased four times because of the mucilage presence. Additionally, the oxygen entry into seeds was changed [41]. The seed coat produces a gelatinous mass very quickly [47]. This aspect was observed during hydropriming when we rotate a test tube upside down and the seeds did not fall within the test tube.
Soaking the seeds in water for up to 48 h, and drying with forced air prior to sowing, represents the hydropriming technique. Several advantages target the low-cost of this method and the environmentally friendly character [48,49,50]. During this process, the seeds could gain resistance to unfavorable climatic growth conditions and improved water uptake [51]. This improvement represents a weak aspect of the method, the water uptake being uncontrolled, and promotes uneven imbibition [52], unequal emergence of seedlings and a reduction in germination speed [53]. Based on the observations in the current experiment, hydropriming is the most successful priming methods for the beginning of the germination process. Almost 84% of seeds required less than 24 h to pass the imbibition process. Almost 20% of seeds were in the 05 and 06a/b secondary stages after this period. Half of the secondary stages recorded for this priming method required eight days to be completed, the 84% percent of seeds that complete the germination stage is achieved in day 10, and the maximum recorded 92% is recorded after another 1 day. This repartition of seeds in multiple germination stages indicates an unbalanced reaction of seeds to this priming method. Hydropriming could be considered “time-consuming” and can cause a problem when the seeds stick together during the process, thus causing difficulty in sowing. Depending on the species, this method can sustain the entire interval of effects—positive, neutral and negative—acting for a higher germination, followed by a rapid emergence of roots and shoots, especially for plants cultivated in different thermal regimes or salinity stress [54,55,56]. Both metabolic and biochemical activities are activated in seeds subjected to hydropriming; when this process is stopped by dehydration, the seeds gain a stress memory which improve the response to future stresses and also present an osmotic stability and a reduction in the time required for imbibition prior to the germination stage [57].
Generally, seeds exposed to an electric field show an increased biological activity, with higher values of respiration and water absorption [58]. The application of this priming method stimulates the appearance of dipole–dipole interactions in seeds, a change in the fluxes of polar molecules through the plasma membrane, but do not significantly affect the metabolic activity [59,60]. A synthesis of electropriming results shows positive effects on increasing germination rate and physiological changes, an increase in protein content and formation of reducing sugars, while no effect was observed on plant hormones, surface modification and permeability, with no biochemical changes, biotic stress removal or photosynthetic pigment alterations [58,61,62]. The results obtained in the current experiment revealed that more than 65% of electro-primed seeds exceeded the imbibition stage up to 03 and 05 secondary stages within the first 24 h. The general trend of the seed full germination process shows 1–3 days’ longer periods of time allocated for the completion of each secondary stage, but with a continuous passage of seeds from one stage to another. The end of the germination stage for this treatment occurred after 11 days from the beginning of the experiment, with almost 94% of seeds in the 09b phase. Several authors reported a more than 10% increase in germination, a slightly reduced mean germination time and increases in antioxidant enzymes activity [63,64] and up to 100% seed germination after electro-priming with greater seedlings [65].
Magnetic fields applied to seeds, as a priming method, may interact with ionic currents in the membrane of an embryo cell and induce a change in osmotic pressure and ionic fluxes that influence the osmoregulation potential [66]. Overall, magnetic fields stimulate the plant and root growth at higher rates and activate the protein formation and are a viable priming treatment for non-standard seeds [67]. Depending on the plant species, the efficiency of this technique varies largely from no effect up to increases in germination and early/late development of plants and even an inhibition of germination [68]. The experiment on basil seeds revealed that magnetopriming reduced the overall length of 05–07 secondary stages to only 3 days. A maximum 2% increase in the germination is visible compared to the control, but this still shows a lack of homogeneity in seeds that pass from a secondary stage to another. Only the final secondary stages of germination (phases 09a and 09b) showed a homogenous passage of seeds from 09a to 09b. This phenomenon sustains the capacity of magnetopriming technique to ensure at least 91% of seeds in the final phase of germination after 14 days. This method has the advantage of being low-cost, short-duration and non-invasive, and came in return with an increase in the tolerance to biotic and abiotic stresses [48,69] and have a prolonged effect, up to the end of the vegetation period of the plants.

5. Conclusions

The results reveal the beneficial effect of seed priming during the secondary stages of germination for basil. The highest number of total percent of germinated seeds was given by the electric field priming method. Hydropriming is placed in the middle of all priming treatments studied with higher seeds stimulation in the first four days of the germination stage. Apparently, the magnetic field does not have an important impact on the total germinated seeds; however, it represents the only priming treatment that provides more seeds in the last secondary phase 09b compared with the control and also with one day earlier. Magnetopriming ensures the lowest duration of middle germination secondary stages. In contrast, at least 1–2 supplementary days are necessary for the seeds primed with an electric field to complete the germination secondary stages. Hydropriming determine unequal seed germination and up to 8 days for seeds to complete a secondary stage. All tested seed priming treatments act differently upon the basil seeds germination. The obtained results were integrated in the general BBCH scale, as an update and adaption for the basil germination principal stage and were completed with the outlook and time frame of the process.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12112694/s1, Table S1. Electric field as seed priming method and germination percentages in each secondary stages every day for basil; Table S2. Magnetic field as seed priming method and germination percentages in each secondary stages every day for basil; Table S3. Hydropriming as seed priming method and germination percentages in each secondary stages every day for basil; Table S4. Control treatment and germination percentages in each secondary stages every day for basil; Table S5. Differences induced by tretment methods on seed germination parameters.

Author Contributions

Conceptualization, V.A.S., S.V., D.V. and V.S.; methodology, V.A.S., C.B. and D.V.; software, V.S. and C.B.; validation, C.B., D.V. and Ș.G.; formal analysis, S.V. and V.S.; investigation, D.V., R.V. and A.V.; resources, Ș.G., S.V., V.S. and D.V.; data curation, V.A.S., C.B., R.V. and V.S.; writing—original draft preparation, V.A.S., S.V., V.S., R.V. and C.B.; writing—review and editing, V.A.S., S.V., V.S., D.V., A.V., Ș.G. and A.R.; visualization, V.A.S., R.V., V.S. and C.B.; supervision, V.A.S., S.V. and Ș.G.; project administration, V.A.S., V.S., A.V. and A.R.; funding acquisition, A.V. and A.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available on request from the first author and corresponding authors.

Acknowledgments

On behalf of all authors we would like to thank Horia Radu Criveanu from the Biophysics Department for providing the devices used in this experiment. Additionally, special thanks to Vasile Stoian and Dan Vârban for assuring the materials used.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 02694 g001 550
Figure 1. Basil germination stage completed according to BBCH scale for growth and development (00—dry/inactive seed, 01—beginning of seed imbibition, 03—seed imbibition complete, 05—radicle emerged from seed, 06a—radicle elongation, no root hair, 06b—radicle elongation with root hair present, 06c—radicle elongation further the root hair zone, 07—hypocotyl with cotyledons, 08—hypocotyl with cotyledons outside seed coat, 09a—emergence, folded cotyledons, 09b—emergence, unfolded cotyledons).
Figure 1. Basil germination stage completed according to BBCH scale for growth and development (00—dry/inactive seed, 01—beginning of seed imbibition, 03—seed imbibition complete, 05—radicle emerged from seed, 06a—radicle elongation, no root hair, 06b—radicle elongation with root hair present, 06c—radicle elongation further the root hair zone, 07—hypocotyl with cotyledons, 08—hypocotyl with cotyledons outside seed coat, 09a—emergence, folded cotyledons, 09b—emergence, unfolded cotyledons).
Agronomy 12 02694 g001
Agronomy 12 02694 g002 550
Figure 2. Percentage of basil seeds germination stage according to BBCH scale for growth and development with all secondary stages: 01—beginning of seed imbibition, 03—seed imbibition complete, 05—radicle emerged from seed, 06a—radicle elongation, no root hair, 06b—radicle elongation with root hair present, 06c—radicle elongation further the root hair zone, 07—hypocotil with cotyledons, 08—hypocotil with cotyledons outside seed coat, 09a—emergence, folded cotyledons, 09b—emergence, unfolded cotyledons. C—control; E—electropriming; H—hydropriming; M—magnetopriming.
Figure 2. Percentage of basil seeds germination stage according to BBCH scale for growth and development with all secondary stages: 01—beginning of seed imbibition, 03—seed imbibition complete, 05—radicle emerged from seed, 06a—radicle elongation, no root hair, 06b—radicle elongation with root hair present, 06c—radicle elongation further the root hair zone, 07—hypocotil with cotyledons, 08—hypocotil with cotyledons outside seed coat, 09a—emergence, folded cotyledons, 09b—emergence, unfolded cotyledons. C—control; E—electropriming; H—hydropriming; M—magnetopriming.
Agronomy 12 02694 g002
Table
Table 1. Experimental design and seed priming procedures.
Table 1. Experimental design and seed priming procedures.
TreatmentSpecificationProcedure
CControlDry seeds directly placed on germinator
EElectric fieldThe homogenous electric field 158 V/m was created between the plates (d = 15.8 cm) of a capacitor with 25 V with an exposure time of 20 min [34]
HHydroprimingBatches of 100 seeds were soaked in 5 mL distilled water at 20 ± 2 °C for 48 h, then dried for 20 min with forced air (electric fan) [35]
MMagnetic fieldExposed 20 min in a pair of Helmholtz coils with a uniform magnetic field intensity of 0.22 × 10−3 T [36]
Table
Table 2. Description of the phenological growth stages of basil (Ocimum basilicum L.) according to the updated BBCH scale.
Table 2. Description of the phenological growth stages of basil (Ocimum basilicum L.) according to the updated BBCH scale.
BBCH Code—Two DigitDescription According to BBCH General Extended Scale [35] Additional Phases and Description for Basil Period
(Day’s Range)
Principal growth stage 0: germination
00Dry seed Inactive seed/Seminal dormancy1–13
01Beginning of seed imbibition Beginning of seed imbibition
+ 30–80% of the seed is covered with gelatinous mucilage		2–4
03Seed imbibition complete Seed imbibition complete
+ the seed is completely covered with gelatinous mucilage		4–7
05Radicle emerged from seed Radicle emerged from seed
+ a clear crack is visible on seed		4–6
06Elongation of radicle, formation of root hair and/or lateral roots06aElongation of the radicle3–8
06bRoot hair zone formation3–7
06cRadicle continues its straight growth from root hair zone2–4
07Hypocotyl with cotyledons or shoot breaking through seed coat Hypocotyl with cotyledons breaking through seed coat
+ the hypocotyl arc appearance		3–5
08Hypocotyl with cotyledons growing towards soil surface Hypocotyl with cotyledons growing towards soil surface
+ 30–80% cotyledons surface visible still kept under seed coat		5–8
09Emergence: cotyledons break through soil surface09aEmergence: cotyledons break through soil surface
+ folded cotyledons		8–9
09b+completely unfolded cotyledons5–13
Table
Table 3. The effect of seed priming methods on the pattern germination compared with control treatment.
Table 3. The effect of seed priming methods on the pattern germination compared with control treatment.
Secondary
Stages		Days
12345678910111213
E–C
002.25 −1.75−2−2.25−2.5−3−3−3−3−3
01−2211.7552.75
0319.7512.251.513.51.251
05 −7.52.5 1.251
06a −5.75−4.53.25
06b −101.754−1 −1−1
06c 14.7531.5
07 −10.2517.254.751.251.25
08 −11.75−28.256.5 −1−2.5−2.25
09a 1.25−2.5−20.5−4.257.758.7582.5−2.25−1.25
09b 5.51−6.5−4.5−4.5 5.254.253
H–C
00 −1.5−1.5−1.5−1.5−2.25−2.25
01−282.752.25
0314.5−1.53.75
059−92.751.75
06a41−1.2531.5 11.5
06b1.25−23 2
06c 8.25−5.51.251
07 1−19.2513.75−1.25
08 10.75−39−6.75−1
09a 3.7518.25 9.7562.53.752.5−2.5−1.5
09b 5.5−11−7.75−3.25−3.25−1.75431.5
M–C
002 −1−1−1−2.25−1.5−1.5−1.5−2.5−1.75−1.75−1.75
01−6.511.5
038.5 3.25
05−4−91.5−1 1 1
06a −2.25−61.25
06b 2.51.25 −1−1
06c 9.7511.251
07 −6.59.5
08 −5−25.7510.753.25−2.25−2.5−2 1
09a 4−16−9.5 1 −1.75−1.25−1.25
09b 77.523.252.752.751.752
Note: E—electropriming; H—hydropriming; M—magnetopriming; C—control. Values in the table represent percentages (%). Color differences legend: blue: <−10%; green: >−10%, <10%; red: >10%.
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Stoian, V.A.; Gâdea, Ș.; Vidican, R.; Vârban, D.; Balint, C.; Vâtcă, A.; Rotaru, A.; Stoian, V.; Vâtcă, S. Dynamics of the Ocimum basilicum L. Germination under Seed Priming Assessed by an Updated BBCH Scale. Agronomy 2022, 12, 2694. https://doi.org/10.3390/agronomy12112694

AMA Style

Stoian VA, Gâdea Ș, Vidican R, Vârban D, Balint C, Vâtcă A, Rotaru A, Stoian V, Vâtcă S. Dynamics of the Ocimum basilicum L. Germination under Seed Priming Assessed by an Updated BBCH Scale. Agronomy. 2022; 12(11):2694. https://doi.org/10.3390/agronomy12112694

Chicago/Turabian Style

Stoian, Valentina Ancuța, Ștefania Gâdea, Roxana Vidican, Dan Vârban, Claudia Balint, Anamaria Vâtcă, Ancuța Rotaru, Vlad Stoian, and Sorin Vâtcă. 2022. "Dynamics of the Ocimum basilicum L. Germination under Seed Priming Assessed by an Updated BBCH Scale" Agronomy 12, no. 11: 2694. https://doi.org/10.3390/agronomy12112694

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