Conditioning of Scots pine (Pinus sylvestris L.) sowing material

ABSTRACT Scots pine is the main forest forming species in Poland. Despite the great importance of natural reconditioning of forest trees, a substantial source of reforestation is the nursery production of seedling material. Pre-sowing seed conditioning, consisting of chemical and physical treatments, is commonly used to improve seed quality as well as to advance uniform development and increase plant efficiency. The influence of semiconductor laser irradiation wavelength on Scots pine seeds (Pinus sylvestris L.) was tested in both laboratory and pot experiments. Irradiation rates used in trials were (D3) three-, (D6) six- and (D9) nine-fold of the basic rate of 2.5·10−1 J·cm−2 with a control (D0) of non-irradiated seeds. Irradiation accelerated emergence and increased seedling numbers. The germination energy and capacity of tested Scots pine genotypes were reduced under higher (D6) doses of laser irradiation. Stimulation of morphological traits and increase in chlorophyll contents was observed under the lowest (D3) irradiation rate, while an increase in polyphenol contents occurred under the highest, nine-fold rate of irradiation.


Introduction
Scots pine (Pinus sylvestris L.) constitutes approximately 70% of forest stand areas in Poland. This species occurs throughout the country except in the Bieszczady (Migaszewski and Gałuszka 1997;Puchniarski 2008). It grows on a wide variety of soils; however, the optimal growth conditions are sandy soils with a layer of clay or marl underneath. Due to its low habitat requirements, it is a primary species for the reforestation of the lowest quality lands useless for agriculture. Scots pine is an acidophilic and photophilous tree, losing its needles rapidly in the shade. The deep root system adapts it to survival in severe drought conditions. Pine is a valued tree due to its wood, which is used in construction and carpentry as well as furniture, paper, cosmetic and pharmaceutical industries (thus so many pine monocultures for wood harvesting), with resin and volatile oils playing an important role in building climates and environments (Migaszewski and Gałuszka 1997;Androsiuk et al. 2011;Barzdajn et al. 2015;Kowalczyk and Wojda 2019).
One of the reforestation methods with species like pine, spruce, beech, linden, maple, sycamore, oak and fir is direct seeding. This method is cheaper than restoration by planting seedlings, but also, importantly, sowing the seeds coming from a particular area (in-situ) allows for obtaining plants better adapted to particular environmental conditions (Puchniarski 2008). One of the methods of genetic resource preservation ex-situ is the long-term storage of seeding material (seeds or parts of plants) in a Gene Bank. In Poland, the Forest Gene Bank has operated since 1995 and performs comprehensive seed evaluations (Rakowski and Jagodzińska 2001;Matras 2013). To meet the requirements of forest nurseries (coniferous trees fruit abundantly every few years) enormous amounts of tree and shrub seeds need to be stored. Seeds of different species are kept in storage rooms with proper humidity to decrease their vital activity. For pine and spruce, the humidity threshold is 3.5%. Another storage method is cryopreservation, which is the freezing of previously dried seeds at −10°C (Matras 2013). However, long-term storage of seeding material can cause a decrease in quality, due to changes in physiological state. Thus, seeds are subjected to specialised tests and conditioning (Copeland and Mc Donald 2001). Pre-sowing conditioning of seed materials includes seed soaking (stratification) or putting seeds with layers of moist sand or peat dust. These treatments shorten germination time and result in more homogenous germination. Chemical methods are also possible, such as the use of concentrated sulphuric acid, which causes damages in the hard seed cover and thus easier water penetration into the seed. However, chemicals may cause changes in the chemical composition of the seed itself or lead to soil contamination. Therefore, physical treatments may be preferred, such as microwave irradiation, or radiation from electric fields (Rybiński et al. 2003), magnetic fields (Martinez et al. 2000), visible light (Rybiński and Garczyński 2004), light-emitting diodes (Velasco 2020) and UV light (Hernandez-Aguilar et al. 2020).
Pre-sowing laser radiation conditioning is one of the methods used to stimulate the growth of crop plants (Dobrowolski and Różanowski 1998). Laser radiation modifies biochemical and physiological processes and affects enzyme systems, resulting in improved (e.g. faster) germination, evenness of emergence and better early development and yield of plants (Hernandez-Aguilar et al. 2016;Szajsner et al. 2017;Szajsner et al. 2019). There are few reports of laser stimulation of non-crop plants . This study aimed to determine the effect of pre-sowing irradiation of Scots pine seeds on the features determining the sowing value and the course of early development stages. The research was carried out on selected Scots pine genotypes using different doses of semiconductor laser radiation. The energy and germination capacity of seeds, morphological features of seedlings, as well as their fresh and dry weight, were assessed. To deepen the complexity of the analysis of pine genotypes response to pre-sowing laser stimulation, the content of photosynthetically active pigments was also determined: chlorophyll a, b, total and polyphenols.
A laboratory experiment was carried out in an environmental chamber (SANYO, model MLR-351H). Scots pine seeds (moisture content: 4.55%) were treated with semiconductor laser beam irradiation (model CTL 1106MX, with a power of 200 mW and a wavelength of 670 nm).
The irradiated fragment of the seed surface was determined by the CTL 1202 S scanner, coupled with the laser. The seeds of Scots pine were preconditioned with laser light with previously fixed doses: C(D0), D3, D6 and D9. The basic radiation dose was 2.5·10 −1 J·cm −2 . The duration of individual exposure was 4.1 min. The control group C(D0) consisted of seeds without any dosages.
The germination energy and germination capacity were tested (after 7 and 14 days, respectively), moreover, the lengths of the radicle, hypocotyl and aboveground part of seedlings, the stem with cotyledons, were measured. As part of the experiment, fresh and dry matter amounts were determined.
Research with sowing material of pine treated with a laser beam (laboratory experiment) was also conducted in pots grown under a cover. The substrate in pots was peat moss with NPK fertiliser. After eight weeks, seedling parameters were assessed, including the number and length of roots and height of seedlings. Moreover, the photosynthetically active pigments in obtained plant material were tested: a, b and total chlorophyll as well as polyphenols (total phenolic compounds). Chlorophyll and polyphenol contents were tested using spectrophotometry by measuring the absorbance of prepared filtrates at wavelengths of 645 and 663 for chlorophylls and 765 for polyphenols (Lichtenthaler 1987).
The results of the research were statistically evaluated using the analysis of variance for a two-factor experiment, with the using STATISTICA 13.1 by Stat Soft Polska. The significance of differences was calculated at the level of α = 0.05 using the Duncan test.

Laboratory experiment
Research conducted in laboratory conditions was on the influence of semiconductor laser irradiation on germination and early development stages of seedlings. After the statistical analysis of data, reduction in values of germination energy and capacity was noted after using a six-fold dose of seed laser irradiation. Tested material was characterised by high germination parameters (86.4% germination energy and 91.6% germination capacity on average for genotypes) and was classified as an I quality group, and after irradiation, it was classified as a II quality group (78.5% and 88.1%, respectively Table 2). Quality classes for pine seeds according to thresholds and values for each class are (in %): cl. I at 91-95-100; cl. II at 81-85-81, cl. III at 70-75-80. Other doses did not influence germination energy and capacity.
Morphological features of seedlings: the length of radicle, hypocotyl and main stem with cotyledon, showed a diversified reaction from the laser beam. The length of radicles and stems were stimulated by the three-fold irradiation of the laser beam. The root increase was 11% and 9% for stems in comparison to the control seedlings from the seeds not irradiated by a laser beam, respectively (Table 2). Statistical analysis of the lengths of radicles, hypocotyls and stems with cotyledons showed interaction of genotypes with the laser beam doses used.
The genotypes A and C reacted with elongated roots: A after the D3 dose and C under all doses applied. Genotype C had the strongest root length stimulation, with more than 93% under the D3 dose, while genotype I decreased the length of its roots under the D6 dose. Other genotypes did not express any reaction on treating seeds with a laser beam ( Table 2). As for hypocotyl length, a positive reaction was observed in genotypes B and C. Genotype B reacted to the three-fold (D3) irradiation, while C form reacted to all doses of laser irradiation. Similarly to the length of the radicle, genotype I showed a reduction in the value of this parameter after the D6 irradiation treatment. Other genotypes did not react to seed irradiation (Table 2). Interaction for the length of stem with hypocotyl was similar for hypocotyl and expressed as stimulation in genotypes B (D3 dose, 34%) and C (all doses, from 44.5-80%), and genotype I reduced its length under all tested doses of a laser beam; other forms had no reaction (Table 3). Similar results (with the same methodological assumptions) were noted in sugar beet seeds treated with a semiconductor laser beam. Elongation of roots and hypocotyls was observed under three-fold irradiation (Prośba-Białczyk et al. 2013).
The weight of seedlings, both fresh and dry, showed stimulation after all doses of a laser beam (Table 2). Statistical analysis for dry weight revealed the interaction of genotypes with doses of laser beam irradiation. Among the ten tested genotypes, stimulation was noted in six of them: B, C, E, G, H and I. The most frequently observed reaction to stimulation was under the D3 dose treatment (genotypes B, E, G) and the D9 dose (B, H, I). Genotypes D and J did not react to presowing laser beam irradiation in any of the tested traits (Table 3). In the research on the conditioning of sugar beet seeds by Prośba-Białczyk et al. (2013), after irradiation by different doses of a laser beam, an increased dry weight of leaves and roots was observed.

Pot experiment
In a pot experiment (conducted under a cover), similar to the laboratory experiments, dose D3 was the most effective. Three-fold irradiation stimulated all three tested morphological traits of pine seedlings. The heights of seedlings increased by more than 7%, the length of radicles increased by 17%, and root numbers increased on average from 1.2 pc to 1.8 pc. At higher doses (D6 and D9), the heights of seedlings reacted to reducing the value of this trait, and nine-fold irradiation, the strongest dose, resulted in a reduction in root length (Table 4). Statistical analysis showed the diverse reaction of tested genotypes of pine to laser irradiation. The weakest dose (D3) stimulated the heights of seedlings in five tested genotypes: B, D, G, H and I (9.15%; 20.28%; 3.77%; 19.68% and 10.40%, respectively). In three genotypes, the elongation of seedlings was noted from nine-fold irradiation in form H and I and from treatment D6 for genotype D. Reduction was caused the most frequently by the strongest dose, nine-fold irradiation, in forms A, C, D, F, G. Genotype E did not show any reaction to pre-sowing irradiation. The root number was stimulated by the weakest three-fold irradiation dose. An increase in values of this trait was observed in half of the tested genotypes: A, D, E, F and I. However, forms B, G, H and J were not sensitive to laser irradiation. The root length reaction to laser irradiation of seeds occurred only in three genotypes. In two of those, elongation was under the D3 dose, while form G reduced root length after doses D6 and D9 (Table 5). Chlorophylls, the most important assimilate pigments, are organic compounds playing a crucial role in the photosynthesis process, especially in the light phase. They can absorb solar irradiation and change solar energy into chemical energy (Kopcewicz and Lewak 2002). Chlorophyll a is the only pigment with the capacity for photochemical reactions (Biczak et al. 2016). In many research studies on stress, including oxidation stress (Percival and Sheriffs 2002;Wang et al. 2009;Zhang et al. 2013;Liu et al. 2014;Gengmao et al. 2015), there has been shown as one of its symptoms a change in chlorophyll levels (Carter and Knapp 2001;Zarco-Tejada et al. 2004;Boquera Ma et al. 2010). Some authors (Islam et al. 2014) give information that chlorophyll content is an indicator of leaf health and is correlated with plant growth. Plants have developed mechanisms that allow them to maximise the use of available light. Photophilous plants grow in full light, and photophobic plants grow in shaded areas. Photophilic plants have more chlorophyll a, and photophobic plants have more chlorophyll b (Kopcewicz and Lewak 2002). Research on the content of assimilating pigments in pine needles shows a wide diversity in their contents according to the age of needles and trees (Wężyk et al. 2003). An increase was observed with the age of needles while the aging of the trees affects a decrease in the value of this trait. With the increase of tree age, a decrease in the ratio between chlorophyll a and b was also noted. Chlorophyll contents also depend on the place of origin of the tree stands. In the pine needles from Puszcza Niepołomicka, the chlorophyll a content was three-fold lower than in pines from the region of Olkusz and Ojcowski National Park, where chlorophyll b was four-fold lower (Wężyk et al. 2003).
In our research on the effect of laser beam irradiation on pine seeds, conducted under a cover, there was noted a significant stimulating effect of irradiation on chlorophyll contents in seedlings. The use of three-fold irradiation increased in chlorophyll a, b and total chlorophyll (by 21.9%, 96.7% and 34.2%, respectively). Six-fold irradiation only stimulated chlorophyll a and total chlorophyll, while the D9 dose did not make significant changes in tested pigments (Table 6). Statistical analysis showed an interaction between the genotype and laser the irradiation dose for chlorophyll a and b and total chlorophyll. The stimulation of chlorophyll a in seedlings was achieved after the D3 dose used for irradiation in the genotypes A, D and G. The use of the D9 dose also affected the increase in chlorophyll a in the genotypes A, C and D, with a reduction in genotypes B, H and F. Forms B and H responded favourably to treatment D6. Genotypes E, F and I did not show any reaction on presowing irradiation with a semiconductor laser beam. For chlorophyll b, the best result was achieved after irradiation with the D3 dose. An increase in the content of this pigment was noted in four genotypes: A, D, E and G. Other doses stimulated chlorophyll contents in individual genotypes: dose D6 for genotype B and dose D9 for genotype C. A reduction in the value of this trait was present only in two cases; genotype A reacted negatively to the D6 and D9 doses, and genotype J reacted negatively to the D6 dose. The interaction for total chlorophyll showed a diversified reaction of genotypes to laser irradiation. Six of the ten tested forms showed a stimulating effect. Genotypes A, B, D and G reacted with an increase in the content of total chlorophyll from treatment D3, B and H increased with six-fold irradiation, and another two increased with the strongest dose. A reduction was observed only in two genotypes: H after D3 and D9 doses and J with the D6 and D9 doses (Table 7). Changes in chlorophyll contents in the leaves of sugar beet seedlings under the use of a laser beam were tested by Prośba-Białczyk et al. Polyphenols, plant organic compounds being natural antioxidants, are present in many different parts of the plant: stems, leaves and fruits (Clemens 2001;Hall 2002;Źrobek-Sokolnik et al. 2007;Kováčik et al. 2011). Among the ability to deactivate free radicals, they have antibacterial, insecticide and anti-inflammatory properties (Huang et al. 2005;Farah et al. 2008;Qi et al. 2012). Moreover, the activation of the antioxidant system is one of the most important mechanisms of plant tolerance to toxic substances in the environment, like heavy metals (Clemens 2001;Hall 2002). Statistical analysis of the results for polyphenol contents showed significant stimulation effects for the nine-fold irradiation of seeds with semiconductor laser irradiation; values of this trait raised by more than 15%. In the case of Scots pine, an increase in values of these antioxidants can have a significant meaning, as this species, in comparison to other coniferous trees, is more sensitive to changes in the environment (Pukacka and Pukacki 2000). At the same time, dose D9 did not affect any changes in chlorophyll contents, while the other doses D3 and D6 did not cause changes in polyphenol contents ( Table 6). The interaction of genotypes with doses showed that in five among ten tested forms, stimulation was caused by the D9 dose: B, D, E, F and I. Stimulation was observed also in form C under three-fold irradiation. On the other hand, a reduction of this trait value was noted in genotypes A, H and J after the D3 dose in addition to E and H under D6 and G and J under nine-fold irradiation (Table 8).
In Figure 1(A-J), the dynamics of Scots pine emergence during eight weeks are presented. The comparison of seedlings developed from non-irradiated control seeds with the seeds pre-treated with a semiconductor laser beam showed that stimulation during the pot experiment was observed in six among ten tested genotypes. The best effect was in the case of form D after three-fold irradiation, for control seeds emergence occurred 26 days after using irradiation at the level of 82 pc (irradiated 90 pc). An increase in emergence was also noted after D3 in genotypes F, G and J. The sixfold irradiation of seeds caused an increase in seed emergence in five genotypes: E, F, G and J. An unfavourable effect, decrease in emergence, was noted only in four forms: B, C, H and I.

Summary
The research on the influence of different doses of laser on the biotimulation of seeds of selected genotypes of Scots pine allowed for conclusions. Under laboratory conditions,  genotypes B and C were the most susceptible to irradiation with laser rays, showing stimulation of all the examined traits. In the pot experiment, as in the laboratory experiment, the D3 dose was the most effective cause of stimulation of the studied features, with nine times irradiation causing the most frequent reduction in these values. The use of an optimal dose of a semiconductor laser beam (usually triple irradiation) for pre-sowing biostimulation of pine seeds resulted in an increase in the content of chlorophyll in seedlings, which may increase the intensity of photosynthetic activity and plant biomass and phenolic compounds in pine seedlings, which can be interpreted as a defense response of plants to the stress factor. Observation of seed emergence in the pot experiment showed accelerated emergence and an increase in the number of seedlings under the conditions of pre-sowing laser stimulation. The germination energy and germination capacity of the studied pine genotypes were reduced (negative effects) with the use of higher (D6) doses of laser radiation.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by WrocÅaw University of Environmental and Life Sciences, Poland.