Using pyrimidinecarboxylic acids as growth stimulants for Rhododen-dron

: Synthesised organic compounds of pyrimidi-necarboxylic acids are characterised by high biological activity, even when their concentrations are low. These compounds, when applied to the seeds of Rhododendron , a genus of woody plants, with concentrations of 0.01, 0.05, and 0.1 %, stimulate the growth of the plants. The effect is more obvious 3 months after the start of the experiment, rather than 7 months after. Thus, Rhododendron ledebourii Pojark. seedlings grew by 13.3-33.5 %, and Rhododendron smirnowii Trautv. seedlings grew by 29.6-48.1 %. Rhododendron ledebourii and Rhododendron smirnowii seedlings demonstrated similar direct correlations: when the concentration of 2-benzylamino-4-methyl-pyrimidine-5-car-boxylic acid and 4-methyl-2-morpholin-4-pyrimidine-5-car-boxylic acid (for Rhododendron ledebourii seeds only) rose from 0.01 to 0.1 %, the height of the seedlings increased. It is suggested using 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid, 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid, and 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid at concentrations of 0.01, 0.05, and 0.1 % as growth stimulants for the species of Rhododendron genus .


INTRODUCTION
Over the past few years, the cultivation of plants has been facing a number of challenges, including a dramatic increase in temperatures, lack of soil water, and the need for more resistant and hardy plants, which grow even under the harshest conditions.It is therefore vital to minimise the time required for the growth of planting material, which can be done using growth and germination stimulants, including synthesised chemical compounds Pentelkina, 2003;Vasin et al., 2008Vasin et al., , 2009;;Ostroshenko and Ostroshenko, 2011;Schuchka, 2006;Baranova, 2013a;Khodaei-Joghan et al., 2018;Nesterkina et al., 2019) and other original modes (Shibaeva et al., 2018).
It has been noted high biological activity of pyridines and pyrimidines.For example, 1, 4-dihydropyridines have received large attention because of their fundamental role in different biological processes (Goldmann and Stoltefuss, 1991;Litvinov, 1998;Balalaie et al., 2008).It has been reported about the wide biological activity of dihydropyridine derivatives (Ghoneim and Assy, 2015).But a promising area of the study, related to the growing need for the development of effective and safe drugs, is the synthesis of new heterocyclic systems, containing a pyrimidine fragment.Pyrimidine fragments and pyrimidine base are included in DNA.So pyrimidine derivatives show different biological activity.There is another mode for applying chemical compounds, containing pyrimidine fragments, for instance, as mutagens.
The composition of diethyleneimide-2-amidopyrimidyl phosphoric acid (phosphazine, phosphomid, syn.phosphemide -phospemidum) includes two ethylene imine groups connected to phosphorus and a pyrimidine base.Ethyleneimine causes mutations, the pyrimidine base is included in the chromosome during DNA synthesis, determining the specificity of the effect (Weisfeld, 2015).Laboratory seed germination and morphometric parameters of seedlings (length of roots and shoots, number of leaves) were studied after seed treatment with heterocyclic sulfur-containing compounds on the example of woody plants (Vostrikova et al., 2020) and agricultural crop (Vostrikova et al., 2021).As the composition of the phosphemide mutagen, pyrimidine-carboxylic acids contain a pyrimidine base.Sulfur (in the composition of alkylating compounds: diethyl-and dimethylsulfonate, ethylmethanesulfonate) enhances biological activity.It is known that mutagens in low concentration have stimulating effect for plant objects.A necessary working step is the determination of concentration ranges (Bome et al., 2017).
The effect of pyrimidine carboxylic acids on seed germination and plant height was studied using another annual flower -spreading marigold (Tagetes patula L.) (Vostrikova et al., 2012;Kalaev et al., 2013a).However, the effect of pyrimidinecarboxylic acids on the growth of other plants has not been studied yet.
Up to the present time, there have also been no studies considering the results of application of synthesised chemical compounds of pyrimidinecarboxylic acids to the seeds of woody plants, which grow much slower than annual plants.It is therefore of great importance to conduct a longitudinal study and measure the height of ornamental woody plants over long time intervals (eg. 3 and 7 months after the application of the growth regulator) in order to determine, whether the growth stimulating effect lasts or deteriorates over time.
Two ornamental woody plants, namely Rhododendron ledebourii Pojark.and Rhododendron smirnowii Trautv.were used.The long history of studying these species (Moiseeva et al., 2012b;Baranova, 2013b) at the B.M. Kozo-Polyansky Botanical Garden of Voronezh State University has demonstrated, that Rhododendron ledebourii is a winter-hardy, drought-resistant, and fruitbearing shrub.Rhododendron smirnowii is also quite winter-hardy, though less drought-resistant.It also grows slower than Rhododendron ledebourii (Alexandrova, 2003;Vostrikova, 2011).It has been reported about the antioxidant activity in Rhododendron leaves, connections between this characteristic in maternal plant and the cytogenetic structures in the seedlings of the Rhododendron species (Vostrikova et al., 2022).Cytogenetic polymorphism of seed progeny of introduced plants has been studied on the example of Rhododendron ledebourii (Burmenko et al., 2018).It has been revealed the high seeds quality.So the representatives of the Rhododendron genus are valuable resource plant.
The aim of our research was to study the effect from pre-sowing seed treatment of the following woody plants: Rhododendron ledebourii and Rhododendron smirnowii by pyrimidine carboxylic acids.

MATERIALS AND METHODS
The research was conducted at the B.M. Kozo-Pol-yansky Botanical Garden of Voronezh State University (geographic coordinates: 39°22' N, 51°40' E; 168.2 metres above sea level).
Prior to the sprouting process, the seeds of Rh. ledebourii and Rh.smirnowii were soaked in a water suspension of the above listed compounds with concentrations of 0.01 %, 0.05 %, and 0.1 % for 18 hours.The control group consisted of the same type of seeds soaked in tap water solution of a commonly used growth stimulator, Epibrassinolide (commercial fraction Epin Extra produced by NNPP NEST M, Russia), with the concentration of 0.05 % (in accordance with the instruction).In case of each of the studied concentrations of the acids, as well as the control group, the experiment was conducted three times using a set of 100 seeds.After soaking, the rhododendron seeds were placed in Petri dishes containing blotting paper, and germinated in the laboratory conditions at a constant temperature 22 о С.On the 21st day, the sprouts were planted in containers filled with highmoor peat and then kept in a greenhouse.The height of the seedlings of Rhododendron ledebourii was measured with a ruler, 3 and 7 months after the start of the experiment.Since Rhododendron smirnowii grows slower, the height of its seedlings was measured 7 months after the start of the experiment.
Sprouts are formed during the early stage of plant ontogenesis, which starts after the germination stage, i.e. when the seed coat develops, and finishes, when the first leaf of the hypocotyledonous stem (the shoot rising from the plumule) develops (Korovkin, 2007).After the first true leaves appear, young plants are considered seedlings (Korovkin, 2007).
The results were statistically processed using the STADIA software package.The procedures of data grouping and processing were described by A. P. Kulaichev (2006).The mean values were compared using Student's t-test.Coefficient of variation (Cv) was counted according to G. F. Lakin (1990).If Cv was below 10 %, it meant that the degree of variation was low, with Cv between 10 and 25 % it was medium, and when Cv was over 25 % -the degree of variation was high (Lakin, 1990).Average values of seedlings (plants) height were compared using Student's t-test.The seed germination in control and experimental variants were compared using Z-test for equality of frequencies.To estimate the influence of various concentrations of the chemical compounds on the height of the plants, one-way analysis of variance was used.The power of influence was calculated according to Snedecor (in %).

RESULTS AND DISCUSSION
The standard growth stimulant hasn't shown influence of the seed treatment on the height of Rhododendron ledebourii seedlings.It hasn't been revealed the significant difference between Сontrol group and Epin group.That means special stimulant impact for sowing qualities in Rhododendron seeds or specific seeds reaction for the treatment.The seeds germination wasn't strongly increased (Table 1-3).But there were significant differences between control and experimental groups in this parameter (Table 1-3).
Within 7 month after the start of the experiment all the studied compounds at any concentration proved to be effective for Rhododendron ledebourii (Table 2).
Within 7 month after the start of the experiment, the height of the Rhododendron smirnowii seedlings also increased (Table 5).The parameter "height of the seedlings" varied greatly for the control group of Rhododendron ledebourii 3 months after the start of the experiment.This is indicated by Cv = 44 %.For the experimental groups, Cv was medium (Table 1), which indicated that the reactions of individual seedlings to the compounds used were similar.7 month after the start of the experiment, the degree of variation was medium in the control group, and even low in the experimental group.This might mean that older seedlings adapt better to experimental conditions.Changes of the seedlings growth are illustrated in Fig. 1-3.
For the seedlings of Rhododendron smirnowii, a medium coefficient of variation was observed (Table 3).Because of the slow growth and the small size, differences between experimental and control groups of Rhododendron smirnowii are not significant 3 month after the start of the experiment.Designations: Cv -variation coefficient * -differences with the control group are significant (p < 0.05) ** -differences with the control group are significant (p < 0.01) *** -differences with the control group are significant (p < 0.001) 1 -differences with the Epin group are significant (p < 0.05) 2 -differences with the Epin group are significant (p < 0.01) 3 -differences with the Epin group are significant (p < 0.01)

Fig. 1:
The height of the seedling of Rhododendron ledebourii (in mm) seedlings after the seed were treated with 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid; 1 -seedlings (21 days), 2 -plants 3 months, 3 -plants 7 months Designations: Cv -variation coefficient * -differences with the control group are significant (p < 0.05) ** -differences with the control group are significant (p < 0.01) *** -differences with the control group are significant (p < 0.001) 1 -differences with the Epin group are significant (p < 0.05) 2 -differences with the Epin group are significant (p < 0.01) 3 -differences with the Epin group are significant (p < 0.01)  The growth ratio of Rhododendron ledebourii seedlings (in the experimental group as compared to the control group) after the seeds were treated with organic compounds of pyrimidinecarboxylic acids, demonstrates that the stimulating effect is most obvious 3 month after the start of the experiment.However, some treatment do not effect growth (Table 1).7 month after the start of the experiment, the stimulating effect deteriorates, but all the compounds at any concentration studied in this paper still have a positive effect (Table 2).
It was shown significant influence of the treatment of seeds with the synthesised organic compounds (as a factor) on the height of the Rhododendron seedlings.The power of influence of the treatment of seeds with the synthesised organic compounds on the height of the seedlings was evaluated using the one-way analysis of variance (Table 4-6).4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid, 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid, and 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid with concentrations of 0.01, 0.05, and 0.1 % resulted in the increase in the height of Cv -variation coefficient * -differences with the control group are significant (p < 0.05) ** -differences with the control group are significant (p < 0.01) *** -differences with the control group are significant (p < 0.001) 1 -differences with the Epin group are significant (p < 0.05) 2 -differences with the Epin group are significant (p < 0.01) 3 -differences with the Epin group are significant (p < 0.01) Table 3:  Rhododendron ledebourii seedlings.A direct correlation can be observed: the higher the concentration of 2-benzylamino-4-methyl-pyrimidine-5-carboxylic acid (within the range between 0.01 and 0.1 %), the higher the seedlings of Rhododendron ledebourii and Rhododendron smirnowii are.The same correlation is observed for Rhododendron ledebourii seedlings, when 4-methyl-2-morpholin-4-pyrimidine-5-carboxylic acid is applied.When Rhododendron ledebourii seeds were treated with 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid at the concentration between 0.01 and 0.1 %, a negative correlation was observed: the lower the concentration, the higher the seedlings.
We can thus say, that the studied synthesised organic compounds of pyrimidinecarboxylic acids are characterised by high biological activity and stimulate the growth of ornamental woody plants when their concentrations are low.The stimulating effect is most obvious 3 months after the seed treatment (the seedlings are 55.6-116.7 % higher than the seedlings in the control group).7 month after the start of the experiment the stimulating effect starts deteriorating.Compounds of pyrimidinecarboxylic acids increase the height of Rhododendron ledebourii seedlings by 13.3-33.5%, and the height of Rhododendron smirnowii seedlings by 29.6-48.1 %.Apparently, pyrimidinecarboxylic acids are more biologically active than other chemical substances, as their chemical structure is similar to the structure of a natural growth stimulator -indole acetic acid (heteroauxin).It can be assumed that auxin activity was kept better within 3 month, than during 7 month after the start of the experiment.
Earlier research studied the stimulating effect of 0.01-0.05% 4-methyl-2-piperidin-1-yl-pyrimidine-5-carboxylic acid on the germination and growth of the seedlings of spreading marigold: with the concentration of 0.03-0.05%, the height of the plants increased (differences with the control group are reliable, p < 0.001).However, other compounds of pyrimidine-5-carboxylic acids at the studied concentrations demonstrated stronger stimulating effects (Vostrikova et al., 2012;Kalaev et al., 2013 a).
Obtained results are consistent with earlier studies by R. G. Gafurov and co-workers on carbon N-and Obenzyl-containing compounds that have bright auxin activity, which is ensured by the presence of a benzyl group at the nitrogen or oxygen atom (Gafurov and Makhmu-    2003,2005).These compounds contain effector fragments that together determine the stress protective activity, namely, the quaternary ammonium and benzyl groups and the hydroxyethyl group -an analog of the benzoxyethyl group (Budykina et al., 2005;Timeyko et al., 2005).Tested substance (2-benzylamino-4-methylpyrimidine-5-carboxylic acid) contains similar fragments, so it also shows bright auxin activity.Based on the literature data (Budykina et al., 2005;Timeyko et al., 2005) and the results of our research, it can be assumed that used compound has the stress-protective activity for valuable ornamental plants of Rhododendron species.

Table 1 .
The seedlings height of Rhododendron ledebourii seedlings 3 months after the seed were treated with the studied synthe-

Table 2 :
The height of Rh. ledebourii seedlings 7 months after the seed were treated with the studied synthesized organic compounds

Table 4 :
The power of influence (in %) of the seed treatment on the height of Rhododendron ledebourii seedlings on the 3 month after the start of the experiment

Table 5 :
The power of influence (in %) of the seed treatment on the height of Rhododendron ledebourii seedlings on the 7 month after the start of the experiment

Table 6 :
The power of influence (in %) of the seed treatment on the height of Rh. smirnowii seedlings on the 7 month after the start of the experiment