Kale seed priming with red seaweed biostimulant

Seed priming is a treatment that can contribute to improve seed physiological potential and increase its tolerance to abiotic stresses. Thus, this work evaluated the effect of kale seed priming with red seaweed biostimulant on physiological seed potential, seed health and tolerance to high temperature at germination. The experimental design was completely randomised, with a 2 x 4 factorial scheme. Treatments consisted of doses of 0, 0.25, 0.50 and 1.0 mL L­1 of red algae Solieria sp. biostimulant, and temperatures of 20 and 30°C. The biostimulant used was subjected to chromatographic analysis to detect bioactive compounds. Seed imbibition curves were used to determine priming duration procedure. Treatments effects were evaluated by seed health, germi­ nation, root and shoot length, and dry mass, under ideal (20°C) and stress (30°C) temperatures. The results were submitted to analysis of variance, Tukey’s test (temperatures) and regression (doses). The 22­h imbibition period is adequate for kale seed priming with Solieria sp. biostimulant. Kale seed prim­ ing with Solieria sp. does not interfere with seed health. The temperature of 30°C reduces kale seed germination index, as seedling root growth. The use of Solieria sp. biostimulant does not promote kale seed physiological potential.


Introduction
Kale (Brassica oleracea var. acephala) is a vegetable crop belonging to the Brassicaceae plant family, originating from the European continent, with excellent adaptation to mild temperatures. However, there is a demand for this vegetable throughout the year. Considering the tropical climate conditions on several countries, studies of the tolerance to high temperatures in kale production are necessary.
The use of seeds for kale seedlings production has been increasing in recent years, especially considering the increasing release of hybrid culti vars, which do not emit lateral shoots, preventing asexual propagation (Trani et al., 2015).
Seedlings production is one of the most important stages for horticul tural production systems, which requires highquality seeds. A key component of the performance of crop seeds is the complex trait of seed vigour. Crop yield and resourceuse efficiency depend on successful plant establishment in the field, and it is the vigour of seeds that defines their ability to germinate and establish seedlings rapidly, uniformly and robustly across diverse environmental conditions (Finch Savage and Bassel, 2016). One procedure that can be used to improve seed quality is physiological conditioning, also known as seed priming. Seed priming is a presowing treatment that partially hydrates seeds without allowing radicle emergence. Consequently, primed seeds demon strate rapid germination and improved germination rate and uniformity. Moreover, seed priming is often implicated in improving the stresstolerance of ger minating seeds (Chen and Arora, 2013). Some exam ples of success using this technique are verified in the literature, as exemplified in the use of seed priming in spinach seed treatment that resulted in high ger mination rates, seedling emergence, growth, maturi ty and yield; in this research the maximum seed vigour was obtained when seeds were treated with 6% concentration of Sargassum wightii (Brown Seaweed) (Takoliya et al., 2018).
Studying physiological and biochemical mecha nisms involved in heat stresstolerance in rice seeds, Hussain et al. (2016) found that the best performance and greater rice seedlings tolerance obtained from primed seeds, by different methods (hydropriming, osmopriming, redox priming, chemical priming, and hormonal priming) is associated with increased starch metabolism, high respiratory rate, lower peroxidation and increased capacity of the antioxidant defence sys tem. In addition, other experimental results have shown that the higher germination efficiency and vigour (seedling growth) occur due to the mobilisation of reserves and the activation of genes responsible for the synthesis of vital enzymes during the priming pro cedure (Lal et al., 2018).
Seaweeds are green, brown and red marine macroalgae. Extracts of brown seaweeds are widely used in horticulture crops, mainly for their plant growthpromoting effects and ameliorating effect on crop tolerance to abiotic stresses, such as salinity, extreme temperatures, nutrient deficiency and drought (Battacharyya et al., 2015). Red seaweeds (Rhodophyta) are sources of carrageenans, which are sulphated linear polysaccharides that represent major cellular constituents of this algae; Carrageenans improve plant growth by regulating various metabolic processes, such as cell division (Shukla et al., 2016).
Studying the effects of extracts and isolated molecules of two species of Gracilaria (Gracilariales, Rhodophyta) on early growth of lettuce, Torres et al. (2018) found a promoting effect of the aqueous extracts in lettuce root length. Nonetheless, there is a lack of studies testing red seaweeds for seed treat ment, especially in seed priming protocols.
Therefore, the objective of this work was to evalu ate the effect of kale seed priming with red algae biostimulant in seed performance under adequate conditions and thermal stress.

Materials and Methods
The experiment was carried out in a Seed and Phytopathology Laboratory, in two stages. In the ini tial stage, the imbibition curves were performed to determine the ideal time for priming. Kale seeds of the Brazilian cultivar Butter were used, and a Solieria sp. biostimulant, which has 7.5% Mn and 13% S; a density of 1.3 g cm 3 , and is rich in carrageenans. The experimental design used was completely ran domised, in a 2 x 4 factorial scheme (temperatures x doses), with four replications.
To determine the bioactive compounds present in the seaweed extract, a chromatographic analysis was performed, according to the methodology described below.

Methodology for the analysis of phenolic compounds by liquid chromatography high efficiency (HPLC)
The analyze were performed on HPLC equipment brand HP model 1100, Lichrospher RP18 column (5 µm) equipped with 210 nm UV detector and quater nary pump system. The reverse phase analysis con sisted of: solvent A MilliQ water with 1% phospho ric acid and solvent B Acetonitrile. The mobile phase pumping system was gradient, with 90% of solvent A from 0 to 5 min, 60% of A from 5 to 40 min and 90% of A from 45 to 50 min. The standard flow was main tained at 0.5 mL/min according to Morelli (2010). The samples were filtered through Nylon membranes of 0.45 µm pore diameter. The phenolic compounds were identified according to their order of elution and by comparing their retention time with those of their pure standards. Quantification was performed by the external standardization method, by correlat ing the area (mAU *s) of the compound peak to the standard curve performed with each standard evalu ated (gallic acid, epigallocatechin, catechin, epicate chin, epigallocatechin gallate, rutin, ferulic acid, naringin, hesperidin, myricetin, resveratrol, quercetin, apigenin and canferol). The result is expressed in µg/mL of extract.

Seed imbibition curves
Seed imbibition curves were constructed to define the priming period, performed using a method adapted from Ferreira et al. (2013). Briefly, four repli cates of ±0.1 g of seeds per treatment were soaked in solutions of 0, 0.25, 0.50 and 1.00 mL L 1 Solieria sp. biostimulant in a plastic box (11 x 11 x 3.5 cm). Each box contained 50 mL of solution in the bottom, and a metallic screen on the top, with the seeds placed between four sheets of germitest paper previously wet (at five times its weight). The boxes were placed in a germination chamber (BOD) at 20°C until protru sion of the primary root.
To determine the amount of solution absorbed, seeds were removed from the germination chamber and the gerbox, dried with paper towels and weighed on a digital analytical balance, with an accuracy of 0.001 g. After weighing, seeds were placed again in the gerbox and brought to the germination chamber. The evaluations were made within 60 min, and when protrusion of the primary root occurred, the process was interrupted, registering the corresponding peri od. Afterwards, the results obtained in the imbibition curves were analysed, and the appropriate period for priming was determined, which must be previous to the protrusion of the primary root (Bewley et al., 2013).

Seed priming
In the second research stage, Solieria sp. biostim ulant doses for seed priming were tested. The experi mental design was completely randomised in a 2 x 4 factorial scheme (temperatures x doses) with five replications. Seed priming occurs at 20°C for 22 h (defined in the previous stage), given the evaluated doses of 0, 0.25, 0.50 and 1.00 mL L 1 , according to the method described in the step of the seed imbibi tion curves. After priming, seeds were evaluated for health, germination (percentage and velocity index), root and shoot seedling length and dry seedling mass.

Seed health
Seed health was evaluated by the blotter test, with eight replicates of 25 seeds placed in plastic boxes (11 x 11 x 3.5 cm) containing three sheets of filter paper moistened with distilled water in a ratio of 2.5 times the dry paper weight. The seeds were incubated at 25°C for 7 days under a 12h photoperi od. After incubation, the seeds were examined indi vidually under a stereomicroscope and optical micro scope, counting the percentage of incidence, and the pathogens were identified based on their morpholog ical characteristics (MAPA, 2009 b).

Germination test
The germination test was carried out at the ideal temperature for the species (20°C) and the stress temperature (30°C), separately. Four replicates of 50 seeds, already conditioned, were distributed on paper for germination ("germitest"), previously moistened with distilled water and kept in a germina tion chamber. The percentage of germination was evaluated on the fifth day (first count) and 10 days after sowing (DAS) (final count), according to the cri teria established in the Rules for Seed Analysis (MAPA, 2009 a).

Germination velocity index
The germination velocity index was determined in conjunction with the germination test, with daily counts, counting seeds with protrusion of 2 mm of primary root according to the protocol proposed by Matthews and Powell (2011).

Seedling length
Seedling length was determined at the end of the germination test (10 DAS), with 20 normal seedlings per experimental unit, from which the shoot length and root length were determined with a ruler gradu ated in centimetres (Nakagawa, 1999).

Seedlings dry mass
The same seedlings used to evaluate the length were separated in shoots and roots and dried in a forcedair circulation oven at 65°C for 72 h. Once dry, the samples were removed from the greenhouse and placed in a desiccator, and then weighed on a 0.001g precision scale (Nakagawa, 1999).

Statistical analysis
The results were submitted to analysis of vari ance. Tukey's test (p≤0.05) was used for the temper ature factor, and polynomial regression was realised for the dose factor. All analyses were performed using Sisvar software (Ferreira, 2011).

Results
Chromatographic analysis of the algae extract revealed the presence of gallic acid, at a concentra tion of 63.9 µg/L, as can be seen in Table 1.
Kale seed imbibition curves with red seaweed (Solieria sp.) biostimulant showed an increase in the wet mass accumulation up to 14 h, and the highest absorption peaks were obtained between 12 and 14 h. This period defines germination phase I (Fig. 1 a). Between 14 to 28 h of imbibition, the absorption was constant, without a significant increase in the control treatment, and this was denominated as phase II. After 28 h, phase III germination started. Using the doses of 0.25, 0.5 and 1.0 mL L 1 , the germination phase I occurred until 17, 13 and 17 h, respectively, and in all cases, the root protrusion occurred with 28 h of soaking ( Fig. 1 b, c and d).
For seed health, no difference was observed between treatments (Table 2). There was a low inci dence of fungi, such as Penicillium spp., Rhizopus spp. and Cladosporium spp., and absence of Fusarium spp. However, although no statistical difference was observed, 0.37% of Trichoderma spp. was detected in the treatment with 0.25 mL of red algae.
Regarding the germination seed performance, dif ferences between temperatures, without dose effects, were observed for the percentage and veloci ty index (Table 3).
There was a significant difference in seedling root length between the temperatures used, with a pro  4.6 a 3.5 a 3.5 a 4.8 a nounced reduction in root size when the seeds were exposed to 30°C. However, the doses of biostimulant evaluated in this study did not differ and were not able to attenuate the effects of thermal stress (Table  3). In relation to shoot seedling growth, the opposite response was observed, with higher mean lengths at 30°C compared with 20°C (Fig. 2). In examining the effect of the biostimulant doses, at 30°C, there was growth increment until the dose of 0.25 mL L 1 , after which, the growth declined (Fig. 2b).
In regards to the accumulation of dry mass in roots and shoots of seedlings, no effects of biostimu lant doses were observed (Table 3). For the tempera ture, a reduction in root length and increase in shoot length was verified in the control, which reinforces the hypothesis of the compensatory effect, related to plasticity.
some Trichoderma strains are used to control phy topathogens and promote plant growth due to their versatility of action, such as parasitism, antibiosis and competition, as well as acting as inductors of plant resistance against diseases. Melo et al. (2017) observed progressive increases in phytoalexin con centration in soybean and sorghum seeds treated with increasing algae doses, demonstrating a high cor relation between dose used and the amount of phy toalexin produced. Phytoalexins are compounds that have been studied a few years ago, which present mainly antimicrobial activity (Arruda et al., 2016).
The highest phytopathogenic fungi observed was Alternaria spp. (Table 2), however no differences between treatments were found. This fungus is a pathogen that can be transmitted by seeds and inter feres with the germination and development of seedlings (TorresCórtes et al., 2019).
Regarding the germination seed performance, dif ferences between temperatures, without dose effects, were observed for the percentage and veloci ty index (Table 3). In general, germination capacity was reduced at 30°C, indicating an abiotic stress effect in this process. Temperature exerts a marked effect on germination because it influences the per formance of enzymes involved in the mobilisation of reserves, as well as enzymes associated with hor monal regulation.
In Arabidopsis, a model species in studies of physi ology and genetics, and of the same botanical family of kale, it was verified that when seeds are exposed to high temperatures during germination, there is a stimulus to biosynthesis of abscisic acid (ABA) and, consequently, a repression of gibberellin synthesis (Toh et al., 2008). The balance between ABA and gib berellins is responsible for the occurrence of germi nation or maintenance of dormancy (Kucera et al., 2005). Furthermore, it should be noted that gib berellins stimulate the synthesis and production of hydrolases, especially alphaamylase, resulting in seed germination (Miransari and Smith, 2014).
However, the inhibitory effects of ABA on seed germination include the impedance of radicle expan sion and endosperm weakening, as well as increased expression of transcription factors, which may adversely affect the seed germination process (Graeber et al., 2010).
The absence of the effect of red algae on germina tion might indicate that this response is associated with the composition of the biostimulant and to the moment of application. One of the main compounds

Discussion and Conclusions
Regarding results of chromatographic analysis, which revealed the presence of gallic acid, it is impor tant to consider that gallic acid is a secondary metabolite present in most plants and algaes (Cotas et al., 2020). Considered one of the major phenolic acids, gallic acid (or gallate) is a benzoic acid of great importance for the formation of a socalled gala totaninhydrolyzable tannins group formed by a unit of sugar and a variable number of phenol acid mole cule. This metabolite is known to exhibit a range of bioactivities including antioxidant and antimicrobial (Fernandes and Salgado, 2016).
Considering results of seed health, is important to mention that according to Machado et al. (2012), found in red algae is carrageenans, sulphated linear polysaccharides. Recent research has uncovered the biological activity of carrageenans and their oligomeric forms as plant growth promoters and elic itors of defence responses (Shukla et al., 2016).
However, carrageenans have shown a relatively greater effect when applied to plants at adult devel opmental stages. For example, according to Gonzales et al. (2013), oligocarrageenans obtained by depoly merisation of red algae carrageenans increase the growth of tobacco plants by increasing photosynthe sis and nitrogen assimilation, as well as stimulate the growth of 3yearold Eucalyptus globulus plants.
Regarding results of seedling root length (Table 3) is worth mention that temperature stress has a detri mental effect on plant metabolism by interrupting cell homeostasis, and the direct result of cellular changes is increased accumulation of toxic compounds in cells, including reactive oxygen species (Essemine et al., 2010). According to Tsukagoshi (2016), reactive oxy gen species regulate the activity of the root meris tems and root development. Therefore, this mecha nism is probably involved in the reduced growth of the kale seedling root accompanying the elevation of temperature, as verified in this work.
However, in relation to shoot seedling growth, the opposite response was observed (Fig. 2). This response might be explained by a mechanism of plas ticity, in an attempt to balance the growth of the seedling, due to the reduction in root size. HernandezHerrera et al. (2014) found that there was a stimulatory effect of algae extracts on the length of plumule in tomato seedlings.
In regards to the accumulation of dry mass in roots and shoots of seedlings, no effects of biostimu lant doses were observed (Table 3). As mentioned by Mašková and Herben (2018), the ratio of biomass partition between roots and shoots is essential for the ability of plants to compensate for the limited resources in the environment and thereby to survive and succeed in competition. Allocation plasticity is an important process for seedlings, and this is one of the most vulnerable phases of the breeding cycle, for most species. In this context, a rapid allocation response may have a direct impact on its survival (Lloret et al., 1999).
Regarding the biostimulant doses evaluated, it is possible that there were no effects on the accumula tion of seedling dry mass because this process is more related to the issue of mobilisation and parti tion of the reserves, with little influence exerted by the compounds present in algae. However, consider ing that to date, there are no scientific studies to prove the direct effect of algae biostimulants in the process of mobilising seed reserves and partitioning of biomass in seedlings, additional studies in this area are necessary.
In conclusion, the 22h imbibition period is ade quate for kale seed priming with Solieria sp. biostim ulant. Kale seed priming with Solieria sp. biostimulant does not interfere with seed sanity. The temperature of 30°C reduces velocity germination index and kale seedling growth, obtained from seeds primed with Solieria sp. biostimulant. The use of Solieria sp. bios timulant does not promote improvements in kale seed physiological potential under the conditions in which this research was carried out.