{'fraction_non_alphanumeric': 0.02856440511307768, 'fraction_numerical': 0.01720747295968535, 'mean_word_length': 4.699355561782012, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
An estimation of the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn

The aim of the experiment was to determine the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn grown under controlled conditions. The experiment was to check whether an application of exogenous hormones during vegetative and generative stages of the plant had an effect on above-ground mass development, on nitrate reductase activity and on plastid pigments content. Experiment factor was synthetic auxin and cytokinin and the date of their application. Auxin was applied in the form of a synthetic indole-3-butyric acid, while cytokinin was sprayed as synthetic 6-benzylaminopurine. The control plants were treated with distilled water. Depending on the experimental variant, spraying was applied at the sixth true leaf stage and at the first flower bud stage. The research showed that the response of the alfalfa plants to the application of cytokinin and auxin was not uniform. It seems that the most effective was the application of a mixture of them both but only during the vegetative stage. Additionally, cytokinin caused an increase in plastid pigments content in alfalfa leaves. On the other hand, a mixture of auxin and cytokinin triggered the highest nitrate reductase activity in alfalfa roots and raised the ratio of total chlorophyll content to carotenoids. Synthetic auxin caused the decrease of the levels of most parameters compared to the control.

A characteristic feature of plant hormones is their cooperation with other hormones. One of the examples can be exogenous auxin and cytokinin, in particular in larger concentrations, both stimulating the biosynthesis of ethylene in tissues (Lorteau et al., 2001;Khan et al., 2002). It proves that a lot of processes which are attributed to the effect of those hormones can be in fact the reaction of the plant to a high concentration of ethylene. Another example can be a mutual influence of cytokinin and auxin, for example, in the stimulation of cambium activity and the vascular tissue formation. It results in a better supply of tissues in photosynthesis products and raises immunity to stress factors, like water stress (Aldesuquy, 2000).
According to many publications (Galoch et al., 1996;Nahar and Ikeda, 2002), plant hormones, to a large extent, affect flower differentiation. Noden et al. (1990) say that there is a relation between the level of endogenous cytokinins and the number of flowers and pods shed by the plants of the Fabaceae family. He continues saying that spraying those plants with synthetic cytokinin prevents these processes by balancing hormonal activity. It turns out that the level of endogenous auxin in budding flowers is too low, which may be the cause of shedding flowers but also the cause of shedding of developing fruits. Spraying plants with auxin can change these processes. It is worth noting that there are plant enzymes which dissolve natural auxin (Rylott and Smith, 1990). Resse et al. (1995) and Rodrigo et al. (1997) attribute to hormones an important role in the transport and distribution of nutrients. Hormones affect the rise in acceptors and the rise in activities of enzymes loading and unloading phloem. Hormones play the role of signal substances to convey information about need of an acceptor.
The aim of the experiment was to determine the effects of synthetic auxin and cytokinin and the time of their application on some morphological and physiological characteristics of Medicago x varia T. Martyn grown under controlled conditions. The experiment was to check whether an application of exogenous hormones during vegetative and generative stages of the plant had an effect on above-ground mass development, on nitrate reductase activity and on plastid pigments content (chlorophyll a and b as well as carotenoids).

The conditions of experiment establish
In March 2014, a pot experiment was conducted to grow Medicago x varia T. Martyn 'cv. Kometa' in a growing room at the Faculty of Natural Sciences of the Siedlce University of Natural Sciences and Humanities -Poland. The experiment conditions were: temperature 24 ± 2/16 ± 2°C; photoperiod 16/8 h; light intensity of 200 lmol m À2 s À1 achieved through the use of high-pressure sodium lamps; and humidity 40%. The experiment was completely randomised, with four replications with the control subject. The entire experiment was conducted in 40 pots, four pots for each variant and 3 plants in each pot. The experimental factor consisted of two growth regulators -synthetic auxin and synthetic cytokinin.
The pots were filled with 5 kg of soil each. The soil used in the experiment was composed of loamy medium sand, III soil valuation class, taken from the arable topsoil level. It was characterised with a very high content of assimilable phosphorus and magnesium, a high content of potassium, copper and zinc, and a medium content of boron, manganese and iron (Table 1).
Alfalfa seeds were sown in mid-March to a depth of 2-3 cm. After the seeds germinated, 3 representative plants in each pot were left for further research. During their growth, the alfalfa plants were sprayed with the growth regulators according to the methodology data presented in Table 2. Auxin was applied in the form of a synthetic indole-3-butyric acid (IBA -concentration of 30 mg dm À3 ), while cytokinin was sprayed as synthetic 6-benzylaminopurine (BAP -concentration of 30 mg dm À3 ). The control plants were treated with distilled water. The plants were sprayed until dip-off with 0.20 dm 3 of spray liquid per pot. Depending on the experimental variant, spraying was applied at the sixth true leaf stage (F 1 ) and at the first flower bud stage (F 2 ).
The experiment was replicated four times. When the flowers faded plants were harvested and the biomass was fractionated into that of roots, stalks, leaves and inflorescence.

Analysis of morphological and physiological traits
The following features were measured in the experiment: the number of stomata (pieces mm À2 ), the length of stomata (lm), the breadth of stomata (lm), the length of stomatal pores (lm), the stalk length [cm], the number of shoots per plant (pieces), the leaf number per shoot (pieces) and the diameter of root collars (mm). Moreover, the total mass of one plant was determined (g DM), the mass of a shoot (g DM), the mass of leaves per shoot (g DM), the mass of inflorescence per shoot (g DM) and the mass of the root system (g DM).
Measurements of stomata were taken after the harvest, choosing randomly ten leaves from each plant. It was done with the microscope Olipmus CX 41 and with the DP -soft program for image processing and analysis. The raw material for the research was the upper epidermis taken from the middle part of the leaf blade (Braune et al., 1975). All measurements were taken with the images magnified 400 times.
After withering of the plants, the collection and fractionation of biomass for roots, leaves, stems and inflorescences was conducted. In stems, leaves and roots nitrate reductase activity was determined with the in vitro method according to Jaworski (1971). Moreover, plastid pigment content was marked in leaves. Chlorophyll a and b were marked with the method by Arnon et al. (1956) as modified by Lichtenthaler and Wellburn (1983), and the content of carotenoids -using the method by Hager and Mayer-Berthenrath (1966). Plant material for the marking was collected from each plant at the full flowering stage (50% of open flowers). As for the pigments, the optical density of the obtained supernatants was determined with the Marcel Mini spectrophotometer with wavelengths: 440, 465 and 663 nm. Next, the results were calculated according to the following formulas: where: E -extinction at a particular wavelength; v -amount of 80% acetone [cm 3 ] used for extraction; w -sample weight [g].
Tukey's test was used to find means that were significantly different from each other, at the significance level of NIR 0.05.

Stomatal parameters and plant biometrics
The results of the research (Table 3) shows that application of synthetic hormones to the alfalfa plants did not affect the number and length of stomata in the leaf epidermis. However, it was noted that application of synthetic auxin and cytokinin had an impact on the breadth of stomata, increasing it by 33% on average. On the other hand, statistically significant growth in the length of stomata pores was noted when synthetic cytokinin was applied (by 20.6% on average) and the mixture of auxin and cytokinin caused the same result (a growth by 13.7% on average). Analysis of growth and development stages of the plants clearly indicated that application of hormones during bud formation did not affect development of stomata in the leaf epidermis of the alfalfa plant. The data presented in Tables 4 and 5 show that after the application of synthetic auxin and cytokinin and the mixture of the two hormones the growth and development of particular parts of the alfalfa plants varied considerably. Auxin, when applied during the stage of sixth true leaves, caused the highest growth in the length of stalks, in the diameter of the root collar and the highest growth of dry matter. Cytokinin applied during the vegetative stage increased the number of leaves but made them grow smaller, which resulted in a smaller mass compared to other experiment objects. Because cytokinin caused shortening of the stalks, the mass of stalks was smaller too. In turn, the mixture of auxin and cytokinin diminished the mass of inflorescence. The same mixture when applied during the vegetative stage caused an increase in the number of shoots (on average Auxin 21.7% compared to the control plot) and an increase in the mass of well developed shoots (on average 47.8% compared to the control plot).

Nitrate reductase activity in different parts of plants
The research ( Table 6) has shown that nitrate reductase activity in the control subject plants was 32.1 lmol NO 2 À g À1 fresh weight in leaves, 8.40 lmol NO 2 À g À1 fresh weight in stems, and 4.20 lmol NO 2 À g À1 fresh weight in roots. Nitrate reductase activity in leaves and stems increased or decreased, depending on the combination of hormones and the stage at which spraying was applied. A similar effect of auxin on nitrate reductase activity was also observed in the fresh weight of alfalfa stems. On the other hand, irrespective of the hormone type and the spraying number and stage, nitrate reductase activity in roots was higher compared to the control. The highest activity (66.7%) was observed for the plants sprayed only once with a mixture of auxin and cytokinin at the first flower bud stage -F 2 .

Plastid pigments content in leaf blades
The study (Table 7) showed that spraying the plants with hormones triggered an increase in the chlorophyll a content in alfalfa leaf blades. Additionally, application of cytokinin only at the sixth true leaf stage (F 1 ) or only at the first flower bud stage (F 2 ) resulted in a substantial increase in the content of chlorophyll a, about 147% and 132% respectively, in comparison to the control. The weakest effect (from 6.64% to 23.9% relative to the control) was observed after auxin spraying. It should be noted that it led to a decrease in the content of chlorophyll b. The sharpest drop (48.3%) compared to the control was observed in the plants sprayed once with auxin at the sixth true leaf stage -F 1 . As for the total chlorophyll (368-384 mg 100 g À1 fresh weight), the best results were obtained after applying cytokinin at both stages of alfalfa development. Similarly, cytokinin spraying resulted in the largest increase in carotenoid content (Table 8). Spraying the plants with this hormone at the sixth true leaf stage (F 1 ) and at the first flower bud stage (F 2 ) caused a 107% rise in carote-  noid content compared to the control. Application of cytokinin at the F 1 stage contributed to a 95.3% increase, whereas when applied at the F 2 stage, it led to a 74.5% change in the value of this particular characteristic. Auxin spraying had an influence on the decline in carotenoids, but only in the variant with a single spraying at the F 1 stage. The use of auxin only at the first flower bud stage improved pigment content in the plant material by 5.11%. The most dramatic drops (6.80%) in carotenoids were observed for a mixture of auxin and cytokinin used only at the sixth true leaf stage (F 1 ). The highest ratio of total chlorophyll to carotenoids (13.3 on average) was found for the plant material treated with a mixture of auxin and cytokinin, regardless of the number of sprayings and their stage. In contrast, values lower than the control were achieved with auxin.

Discussion
Controlling gas exchange and, at the same time, photosynthesis, stomata have an impact on plant growth and development (Jones, 1998). Klamkowski et al. (2008) say that a higher leaf stomatal density is related to a more dynamic gas exchange. Thus, the number of stomata is related to stomatal conductance, which means that a higher number results in a higher rate of photosynthesis and transpiration (Klamkowski et al., 2008). In our study application of synthetic hormones to the alfalfa plants did not affect the number and length of stomata in the leaf epidermis. Also analysis of growth and development stages of the plants clearly indicated that application of hormones during bud formation did not affect development of stomata in the leaf epidermis of the alfalfa plant. Hormones regulate plant growth and development and, at the same time, the intake of nutrients. Thus, hormones are endogenous substances regulating distribution and accumulation of nutrients (Panwar et al., 1990;Nowak and Ciec´ko, 1991;Nowak et al., 1997;Czapla et al., 2003). Nowak and Ciec´ko (1991) suggest that a higher biomass growth and a higher concentration of some minerals in the above-ground parts of plants after application of synthetic hormones are caused by a bigger growth of the root system, in particular because of the lengthening of the root hair zone (Svenson, 1991;Meuwly and Pilet, 1991;Ali et al., 2008). In consequence of this lengthening the intake of ground water and intake of nutrients are both higher. Reinhardt et al. (2000)  -Means in lines marked with the same small letters do not differ significantly -Means in columns marked with the same capital letters do not differ significantly K -control, A -auxin (indole-3-butyric acid), C -cytokinin (6-benzylaminopurine), AC -auxin + cytokinin, F1 -sixth true leaf stage, F2 -first flower bud stage.  and Friml (2003) say that auxin is the key factor here since it functions as a signal informing about physiological processes in cells and their growing demand for nutrients. Moreover, cytokinin and auxin stimulate the activity of cambium and forming of phloem, making it possible for different kinds of nutrients to be delivered to all parts of a plant (Reinhardt et al., 2000;Cho et al., 2002;Friml, 2003). In our study auxin, when applied during the stage of sixth true leaves, caused the highest growth in the length of stalks, in the diameter of the root collar and the highest growth of dry matter. Cytokinin applied during the vegetative stage increased the number of leaves but made them grow smaller. Rylott and Smith (1990), Weijers and Ju¨rgens (2004) say that treating plants with synthetic auxin may contribute to the growing competition between vegetative and reproductive organs. Research done by Pandey et al. (2003) confirmed that. They used indoliloacetic acid, IAA, on cotton plants and noted a considerable growth of inflorescence. In turn, Nagel et al. (2001) suggest that application of synthetic cytokinin resulted in better vascularity of tissues and, in consequence, a better transport of photosynthetic products form vegetative to reproductive organs. At the same time the concentration of photosynthetic products in vessels of plants tested was higher. After the research on the pigeon pea with BAP, 6-benzylaminopurine, applied, Barclay and McDavid (1998) noted that its pods grew much thicker and as a result their mass was higher. In our study nitrate reductase activity in leaves and stems increased or decreased, depending on the combination of hormones and the stage at which spraying was applied. A similar effect of auxin on nitrate reductase activity was also observed in the fresh weight of alfalfa stems. Irrespective of the hormone type and the spraying number and stage, nitrate reductase activity in roots was higher compared to the control. The lowest nitrate reductase activity in the tissues of alfalfa roots compared to the leaf and stem tissues was also observed by other authors Ilieva, 2007, 2011;Ilieva and Vasileva, 2013). It should be pointed out, however, that the obtained values of the activity of the examined enzyme were within the limits described by the available scientific literature. In our study nitrate reductase activity in leaves and stems increased or decreased, depending on the combination of hormones and the stage at which spraying was applied. A similar effect of auxin on nitrate reductase activity was also observed in the fresh weight of alfalfa stems. Irrespective of the hormone type and the spraying number and stage, nitrate reductase activity in roots was higher compared to the control. Fu et al. (2000) reported that when plants from the Fabaceae family age, chlorophyll and carotenoid content in tissues decreases. The use of exogenous cytokinin may increase the content of chlorophyll in senescent leaf tissues, because it slows down chlorophyll degradation and delays the aging process. In their studies on the influence of BAP on cabbage, Costa et al. (2005) found that it slows the degradation of the total chlorophyll compared to the control plants. Authors also focused on determining the activity of enzymes taking part in chlorophyll degradation, such as chlorophyllase and magnesium dechelatase. It turned out that there was a significant drop in the activity of these enzymes in the plants sprayed with BAP compared to the control plants. Using two types of synthetic auxins -IBA and NAA (1-naphthaleneacetic acid) -and mixtures thereof (all in a concentration of 20 mg dm À3 ), Czapla et al. (2003) observed the largest effects when using IBA. On  the other hand, Nahar and Ikeda (2002) sprayed soybean plants with auxin -ethyl-5-chloro-3-indazolyl acid -and found an average of 23% increase in the analysed features compared to the control. The positive effect of plant hormones on plants was also reported by Barclay and McDavid (1998) and Czapla et al. (2003).

Conclusions
No matter what the time of synthetic hormone application was, the alfalfa responded with broader stomata whereas the length of the stomatal pore was the highest in objects with synthetic cytokinin applied and with a mixture of cytokinin and auxin applied. The highest biomass of the above-ground parts was noted for plants treated with the mixture of auxin and cytokinin. The plants also responded with the highest number and length of shoots. Statistical analysis showed that the time of spray affected the length and number of shoots, the diameter of the collar root and the mass of inflorescence. The value of those features was higher for plants sprayed at the stage of sixth true leaves (vegetative stage). It seems that the most effective was the application of a mixture of them both but only during the vegetative stage. Treating the plants with auxin alone decreased the activity of this enzyme in stems and roots compared to the control. Spraying the plants with exogenous auxin and cytokinin contributed to an increase in chlorophyll content in alfalfa leaves. The content of chlorophyll b and a + b in the plant material was very diverse. The highest concentration of carotenoids was found in the alfalfa leaves sprayed twice with cytokinin. Auxin decreased their content. Spraying alfalfa with a mixture of auxin and cytokinin had the most visible effects only on the ratio of total chlorophyll to carotenoids, as the ratio value increased by 65%.