THE INFLUENCE OF PHYSIOLOGICALLY ACTIVE COMPOUNDS ON GROWTH AND DEVELOPMENT POTATOES (Solanum tuberosum L.) IN THE CONDITIONS IN VITRO

ABSTRACT

The following article is devoted to the study of influence of some phytohormones (PH) of glycyrrhizic acid (GA) (indole–3–acetic acid (IAA); indole–3-butyric acid (IBA); 1- naphthaleneacetic acid (NAA) and kinetin) on supramolecular complex in order to determine the state of high yield due to the plant growth exacerbation and the development of ‘Santy’ potato species as well as the development of vegetative organs. The increased activity of amylolytic ferments like α– and β–amylase was determined having been compared to the controlled ones. Also, the enzyme activity index value was determined on the fifth day and it equaled to 85,4±3,2 (GA:IAA) per day; 90,3±3,3 (GA:NAA); 110,7 (GA:IBA)±4,3 and 100,0±2,6 (GA:Kinetin) mg. With respect to control in 10^-5 M concentration of GA-IBA supramolecular complex, it was found that the callus tissue of potato develops 1, 2 times, in 10^-7 M concentration of nutrition the vegetative organs were 1, 8 times higher.

АННОТАЦИЯ

Данная статья посвящена изучению влияния некоторых фитогормонов (ФГ) глицирризиновой кислоты (ГК) (индол-3-уксусная кислота (ИУК); индол-3-масляная кислота (ИМК); нафталин-1-уксусная кислота (НУК) и кинетин) на супрамолекулярный комплекс с целью определения состояния высокой урожайности на основе влияния обострения роста и развития растений картофеля сорта «Санти. При этом влияние супрамолекулярного комплекса на суммарные амилолитические ферменты определялось повышением активности α- и β-амилаз по сравнению с контрольными. Установлено, что значение индекса активности ферментов на пятые сутки составляет 85,4±3,2 (ОА: ИУК) в сутки; 90,3±3,3 (GA:NAA); 110,7 (ГА:ИФА)±4,3 и 100,0±2,6 (ГА:Кинетин) мг. Относительно контроля в 10^-5 М концентрации надмолекулярного комплекса ГА-ИФА установлено, что каллусная ткань картофеля развивается в 1,2 раза, в 10-7 М концентрации питания вегетативные органы в 1,8 раза выше.

Keywords: potato, in vitro, glycyrhisic acid, amylolytic ferments, supramolecular complexes. 

Ключевые слова: картофель, in vitro, глициризиновая кислота, амилолитические ферменты, надмолекулярные комплексы.

Introduction

Potato is one of the most important crops in the world and therefore the subject of constant interest and numerous studies including those classified as plant biotechnology. In the rapidly developing field of plant biotechnology, the use of plant in vitro culture techniques is the common, integration feature. In vitro culture techniques enable us to excise from plants and under controlled, axenic laboratory conditions culture their cells, tissues, organs or even the whole plants. These techniques are also a starting point for many practical applications that we will briefly present and discuss.

The first report on the establishment of potato in vitro cultures by Stewart and Caplin [1] was made more than half a century ago. Since then, hundreds of research articles have been published and some of them can not be easily obtained today.

Phytohormones are considered the most important regulators in the process of plant growth and development [2-5], the basis of this effect is associated with the process of bio-regulation of plant activity.

The intensity of biochemical/physiological processes occurring during the development of plants is directly related to the activity of the ferment complexes [6]. In particular, amylolytic ferments have been identified as important ones in the initial development process (micro phenological phase) of the plant [7].

Amylolytic ferments activity is regulated under the influence of endogenous phytohormones and it is noted that adaptation mechanisms occur directly through the activity of ferments at the cellular level when the plant organism is exposed to a variety of stress factors [8].

The purpose of this research is to investigate the effect supramolecular complexes of glycyrrhizic acid (GA) and a number of phytohormones (IAA, IBA, NAA, kinetin) on α – and β–amylase ferment activity in the development of potato in in vitro conditions.

Material and methods

In the research there were used a variety of potatoes such as Santa in order to analyze biological activity of GA:PH (IAA, NAA, IBA and kinetin) supramolecular complexes.

We used nutritious environment Murashige-Skoog [9] according to the following scheme for the cultivation of potato varieties tissues in in vitro:

1. There was prepared nutritious environment Murasige-Skug which contained 7000 mg/l agar, 30000 mg/l sucrose, 2 mg/l GA-IBA -4-5-6-7-8 cm, 2 mg/l glycine, 160 mm KNO₃ in order to reproduce it in in vitro conditions.

2. There were used vegetative bodies of regenerated plant of potato varieties in nutritious environment. Bodies of regenerated plant were kept in +23...+25°С temperature with light for 16 hours in in vitro conditions. The light was 5000-7000 LK in this condition. In the chosen nutritious environment, there were grown vegetative bodies by 10-12 repetition. Bodies of the regenerated plant were grown during 25-55 days in vitro.

In the composition of the extract of potato tubers during the experiments, there was determined the activity of amylolytic (α–, β–amylase) ferments by the spectrophotometric method [10,11].

To determine the activity of amylolytic ferments contained in the  potato tubers, which sprouted in the experiments, were scraped from the pieces of the flourishing tubers and brought to a homogenous state in the solution of NaCl (1%) in the mortar with separated biomass (1 g), in the solution of the homogenated NaCl was extracted at a temperature of +4°C (90 min.), at the next stage 4 000 rot/min. speed for 10 minutes during centrifugation, the formed supernatant is added 3 ml of starch solution (5,5%) to the composition of the incubation environment at the next stage 3 ml acetate buffer (рН=5,5), at a temperature of +40°C (in a water bath) saved for 30 minutes. The reaction was stopped by adding 2 ml 1М HCl to the composition of the mixture, starch glucose in the medium was determined using iodine solution (0,5 ml of the mixture was injected into 50 ml of the tube, 30 ml of distilled water was poured and 1 ml of 0,1 M HCl, 5 drops of KJ solution (0,3%), optic density was analyzed in wave length with 595 nm density value. Amylolytic activity in the incubation environment were noted in the presence of 1 ml of ferment solution, amount of hydrolyzed starch amount dissolved during 30 minutes, that is (hydro. mg. starch/30 min. ×g. ferment extract) [12-15].

The activity of amylolytic ferments was determined in 2, 7 and 10–day standard method in the process of budding of potato tubers.

Method of determining α–amylase activity. In the experiments, when analyzing the activity of the α–amylase ferment, the mass of the potato tuber, which develops by budding, was brought to a homogeny state in the acetate buffer environment (0,05 М; рН=5,5) and centrifugated for 10 minutes during the 4 000 rotation/minute speed. Incubation medium (5 ml) contains 2 mg of acetate powder for β–amylase neutralization with the addition of a supernatant by heating for 15 minutes at a temperature of +70°C, and then quickly cooled [16,17].

Results and Discussion

In the table below there are presented the experimental results obtained on the effect of GA and IAA, NAA, IBA, kinetin supramolecular complex on the parameters of budding and development of ‘Santa’ potato variety in laboratory conditions (Table 1).

From the results obtained (Table 1), it can be seen that the length of the stem of ‘Santa’ variety(cm), the length of the root (cm) in laboratory conditions compared to the control under the influence of supramolecular complex (100 µМ) GA:PH (IAA, NAA, IBA and kinetin), there was a significant increase in the length of the stem (cm).

The following picture 1 shows the experimental results obtained on the effect of GA:phytohormone supramolecular complex on the total amylolytic activity value in the process of budding and development of  potato tubers.

Table 1.

Indicators of effects during 10 days after usage of GA:phytohormones (IAA, NAA, IBA and kinetin) to the indicators of budding and development of Santa variety of potato in laboratory conditions (M±m)

Note:* – the level of statistical reliability with respect to control is р<0,05, ** –р<0,01 (n=3–4).

Thus, in the experiments, the value of total amylolytic activity was determined in the tuber composition of the budding-development process of the ‘Santa’  potato variety in laboratory conditions in the control variant. At this point, it can be noted that in the process of budding-development, the total amylolytic activity in the tuber composition was noted increase in 1-6 days, maximum on day 6 and decrease from day 7 (Fig. 1).

Figure 1. The influence dynamics of GA: phytohormones (IAA, NAA, IBA and kinetin) supramolecular complexes on α–amylase activity in the process of budding and development of ‘Santa’ potato variety in laboratory conditions (1-10 days)

In the experiments, it was found that in the incubation conditions of GA:IAA (4:1), GA:NAA (4:1), GA:IBA (5:1) and GA:Kinetin supramolecular complex in the concentration of 100 µМ, the overall amilolytic activity value of the tuber composition of  potato in the process of budding-development of the ‘Santa’ variety was significantly increased compared to the control. In particular, there were determined that the maximum value of this indicator is 70±5,0 (GA:IAA); 75±4,1 (GA:NAA); 95±3,4 (GA:IBA) and 80,0±2,2 (GA:Kinetin) (hydro. mg starch×30 min./g) on day 5 (Fig. 1).

In order to draw a more complete conclusion about the stimulating properties of supramolecular complex of GA formed by IBA, we observed the effect of its above-mentioned solutions on the development of the vegetative organs of potato in in vitro conditions.

To do this, on the basis of Murashige-Skoog nutrient, there were prepared nutrients with concentration of 10^-4, 10^-5,10^-6 and 10^-7 М by adding complexes. This nutrient was used in obtaining calluses from the vegetative tissues of potato.

Based on the obtained results, it can be noted that under the influence of the applied different concentrated solutions of supramolecular complex of GA formed by IBA, the tissues of potato organs gave positive indicators in in vitro conditions.

In the 10^-4 and 10^-5 M concentration of GA without adding IBA phytohormone there were observed higher results for 1,0 and 1,2 times in  potato, and 1.6 and 1.8 times in nutrients with concentration 10^-6 and 10^-7 М of GA-IBA complex rather than in the control ones.

We observed the influence of glycyrrhizic acid salts on the growth and development of potato. Based on the data obtained from the results of the observation, it can be noted that on the example of GA’s IBA with supramolecular complex, there were observed 12, 3-12, 5% higher than control in the development of potato in complex stimulants formed by phytohormones of the auxin group.

Similar indicators were observed in the biomass of tubers, along with the observation of potato at all stages of growth and development.

Hence, it can be concluded that supramolecular complex formed by GA with IBA, is characterized by the quality of a physiologically active substance that accelerates plant growth and development.

Conclusion

Under the influence of GA with phytohormones of GA:IAA (4:1), GA:NAA (4:1), GA:IBA (5:1) and GA:Kinetin supramolecular complexes, there was an increase in total amylolytic activity in the content of  potato tubers. The ferment activity index value is 70±5,0 (GA:IAA); 75±4,1 (GA:NAA); 95±3,4 (GA:IBA) and 80,0±2,2 (GA:Kinetin) mg made up on day 5.

It was determined that the supramolecular complex, formed by GA with IBA, has the property of a physiologically active substance that accelerates plant growth and development.

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