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DOI: http://dx.doi.org/10.20534/ESR-16-9.10-21-24
Safarova Nigora Karimdjonovna, E-mail: nigorask@mail.ru Safarov Karimdjon Safarovich, Tashkent Botanical Garden of the Institute of the Gene Pool of Plants and Animals, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan
E-mail: ksafarov@mail.ru
Optimization of nutrient medium composition for the Cultivation of Ostrowskia magnifica Regel in Vitro Conditions
Abstract: This article presents the results of the experiment on the cultivation of Ostrowscia magnifica Regel in the Tashkent Botanical garden. Peculiarities of cultivation, the effect of different nutrient medium on the growth and development of regenerated plants in vitro are studied.
Keywords: in vitro culture, callus, organogenesis, phytohormones, nutrient medium.
1. Introduction
In recent years there is a trend of downward in biological diversity. Many species of plants and animals become rare and in some areas are met no longer. Therefore, the conservation of biological diversity is one of the most important issues in the world. Along with traditional methods of plant conservation ex situ, the use of bio-technological methods of reproduction acquire greater importance. Besides such methods of biotechnology, as microclonal propagation, callus culture in vitro, embryoculture and other methods are used.
2. Materials and methods
The technology of clonal propagation in vitro at the laboratory level developed in the world for more than 2400 species of plants; however, this method is more often used for plants propagated with difficulty by conventional methods, as well as for solving problems, one of which is the conservation of rare and endangered species of plants.
Currently a topical area is the preservation and reproduction of rare and hard-clonal plant species [1]. Ostrowskia magnifica Regel, distinct monotype native, rare relic of Central Asia, included in the Red Data Book of the Republic of Uzbekistan [2]. It is a perennial grassy plant, its height up to 170 cm, common in Tashkent and Ter-mez provinces of our Republic: the Western Tien-Shan: the Ugam river basin; the Hissar range: the basin of the river Tupalang and Shargun. The total number does not exceed 30 thousand individuals. It is propagated by seeds.
The study of biology, morphology and reproduction of Ostro-vskia in the Tashkent Botanical garden is engaged by M. I. Roza-nowskaya [3]. According to the author, the best time of sowing of Ostrowskia magnifica seeds — autumn and the end of February. In both cases, the shoots appear in early April.
Picking flowers, digging up tubers and low seed production are the reasons for the changes in the population and habitat of this species. And so the question of the introduction in culture in vitro of Ostrowskia magnifica Regel is highly relevant.
In the scientific literature, studies on the introduction in culture in vitro Ostrowskia magnifica Regel are absent.
The purpose of this study was optimization of nutrient medium composition for the growth and development of Ostrowskia magnifica Regel plant in vitro.
Materials and methods of research. It is known that primary explant should be completely freed from all microorganisms (bacteria, fungi, mycoplasmas, etc.) and its continued existence in vitro requires the maintenance of absolute asepsis, as fungal and bacterial infection inhibits cell growth and leads to the death of culture [4].
Ostrowskia magnifica seeds were stratified in the freezer, at temperatures below — 4 °C for 14 days.
In the first stage of clonal micropropagation is necessary to obtain a sterile and well-growing culture. Great importance has the choice of sterilizing solution and the method of disinfection from which depends the explants infection, their physiological state and level of viability [1].
It is found that the most effective method of sterilization of Os-trovskia seeds is the following method: initial treatment in 0.06% of AgNO3 solution (20 min), then in 70% of ethanol solution (1 min) and subsequent washing in sterile water 3 times (10 min each).
In this arrangement, the step of seeds sterilizing the seeds retained high viability. After sterilization the seeds were sown in agar-agar (0.6 percent), unhormonical nutrient medium prepared on tracing medium of T. Murashige and F. Skoog (MS) [5], as this medium is the most suitable for microclonal propagation of many herbaceous plants.
The culture was kept at temperature of 23±2 °C, relative humidity 80-85%, light white fluorescent lamps with 16-hour pho-toperiod.
With this method of sterilization and germ cultivation of Ostrowskia magnifica appeared after 17-18 days, true leaves were formed after 10 days. Six weeks after germination Ostrovskia seal hypocotyls was observed, leaves started to turn yellow and formed callus tissue.
It is known that cell culture of each plant species and even different organs and tissues of the same species requires a specific nutrient medium composition. Moreover, for the initiation of callus, maintenance its growth, for the induction of organogenesis have in each of these cases. It is necessary to change the composition of the medium in each of these cases.
The callus cells can grow indefinitely in vitro, and periodically transplanting them to fresh nutrient medium, but often there are phenomena, which undesirable in micro-propagation of plants, namely: the change in ploidy of the cultured cells; structural chromosome rearrangements and accumulation of gene mutations; loss of morphoge-netic potential of cultured cells. Prolonged cultivation of callus cells exacerbates these changes, so the period ofunorganized growth in the micropropagation should be reduced to a minimum [6, 11].
Nutrient medium for cultivation contains mineral salts, carbohydrates, vitamins, growth regulators, amino acids. The basis of all nutrient media for cultivation of isolated plant tissues is a mixture of mineral salts, represented as macro-and micronutrients. In most media includes vitamins. Necessary components of any nutrient
Fig.2. The callus cells are transferred into organogenesis
Fig.3. Formation of the flower on the apex of the shoot
Fig.4. Temp of growth is very good
medium are plant hormones — compounds that are involved in the regulation of physiological processes in plants. For the growth and differentiation of plant cells auxins and cytokinins are required.
Callus tissue of Ostrovskia from unhormonical medium was transplanted to the MS medium with addition of hormones IAA/BAP in the ratio of 0.25/0.02 mg/l. In this medium callus cells began to grow (increase in size), has gained a certain critical mass and gave rise to the process of organogenesis (Fig.1-4). Callus cells are very differ in growth rate, texture, color, ability to turn green in light and other properties. Cell colonies on agar medium can be compact and solid, but also loose, that in removing the callus break into individual pieces.
The last type of callus in the liquid medium very easily separates single cells and gives rise to suspension culture. Noted that the compact callus can give rise to friable tissues, but not vice versa [7; 8].The consistency of the callus to a large extent depends on the composition of the medium. Anatomical structure offriable callus is characterized by many organized centers of meristematic activity, separated by large undifferentiated cells.
Dense callus cells are less differentiated and contain many large vacuolation cells [9; 10].
Callus cells of Ostrovskia magnifica are brown, friable and hydration, splits into individual pieces in extraction (Fig.1).
Differences in the chemical composition: total amount of polysaccharides of the cell wall is higher in dense callus, but the percentage of cellulose in comparison with pectin substances and hemicelluloses is reduced [11].
Fugitive growing callus cells, characterized by monotonous reproduction and growth, owing to the processes of secondary differentiation can form tissues (histogenesis), organs (organogenesis) and the bud like structures — embryos (embryogenesis, or somatic embryogenesis) [12].
Initially, the callus grows, gains a certain critical mass, and then proceeds to morphogenesis (Fig.2-3). However, only single cells change their program development. This suggests that the transition to morphogenesis should be strictly appointed combination of external factors action primarily the inductor, in the condition of cells determinancy, i. e., with its willingness to realize their hereditary properties under the action of this inductor.
Apparently, the main reason that causes the differentiation of cells and their transition to the histogenesis followed by the formation of organogenic structures, can be considered the action of phytohormones.
Phytohormones are necessary component of any nutritional medium, as they participate in the regulation of physiological processes in plants. It is important to emphasize that the ratio of auxin and cytokinin in the nutrient medium determines the type of morphogenesis. This pattern was first discovered by Skoog and Miller in 1957, and then confirmed by numerous experiments.
Type of formed meristem depends on the ratio between auxin and cytokinins, but their specific involvement in different stages of morphogenesis remains unclear [12].
The combinations of these two preparations are used for induction of shoots formation. In experiments on the introduction to the culture and the microclonal reproduction of Ostrowskia magnifica several variants of culture media were used.
3. Results and discussion
During the conducted research it was found that the optimum media for shoots and further growth of Ostrovskia cells are: MS medium with the addition of 0.2 mg/l of BAP and 2.0 mg/l of IAA; MS medium with the addition of 2.0 mg/l of BAP and
4.0 mg/l of IAA; MS medium with the addition of 2.0 mg/l of BAP. For the formation of callus and growth of these cells MS medium is an an optimal medium without the addition of phy-tohormones and MS medium with 1.0 mg/l of IAA and 0.5 mg/l of kinetin (table).
Callus cells — the main object during long-term cultivation in vitro. The famous scientist-botanist N. Krenke 50 years ago, part of his works devoted to studying the role of callus tissue in the whole plant. He tied it up with the phases, which passes callus tissue:
• The first phase — the rapid growth of callus cells, protecting the injury site;
• The second phase — the storage of nutrients in callus cells;
Table 1. - Nutrient media for seeds cultivation
• Third, the most important phase — the regeneration of callus tissue of lost organs (roots and shoots).
All these three phases were observed in the Ostrovskia magnifica.
From the hormonal MS medium (IAA 0.25 mg/0.02 mg of BAP per liter of medium) explant of Ostrovskia was transplanted to the medium IAA 2.0 mg/0.2 mg of BAP per liter and in a month the beginnings of white flowers with 5-6 petals are formed on the apex of the shoot (Fig.3).
The choice of a nutrient medium is defined by a type of a plant which is introduced into culture, as well as by tasks of experiment.
We in the experiments on introduction in culture and microclo-nal propagation of Ostrowskia magnifica used some versions of the nutrient media are shown below in table 1. of Ostrowskia magnifica Regel in vitro conditions
Options MS BAP IAA Kin Rate of progress
1 MS - - - In 16-18 days the seeds sprouted, except for cotyledons, four true leaves appeared; the hypocotyls increased in volume and formed a callus.
2 MS 0.2 0.1 - The nutrient medium for shoots. From the callus cells shoots with leaves formed. The temp of development is fast.
3 MS 0.02 0.25 - The formed callus cells in unhormonal medium with a fast temp of rising, increasing in volume, give rise to the process of organogenesis.
4 MS - 1 0.5 The leaves of transplanted explants, rapidly yellowing, again formed callus. But if to this nutrient medium to replant the callus cells, then they will continue to grow, develop and move to the organogenesis.
5 MS 0.2 2.0 - The optimal nutrient medium, in which regenerated plants grow well, unidentified items are formed, very similar to the flower.
6 MS 0.08 1 - The transplanted explants' leaves turn yellow, wither, but from the base of the petiole formation of leaves starts again. The pace of development is weak.
7 MS 4.0 4.0 - The transplanted explants grow well. The formation of callus cells wasn't observed.
8 MS 0.05 0.5 Shoot formation wasn't observed, the leaves are formed from callus cells, but they quickly start to lose their green color.
9 MS 2,0 - - An optimal nutrient medium for faster growth of seedlings.
10 MS 4.0 2.0 Transplanted explants grow well.
11 MS 2,0 4,0 From the callus cells shoots with leaves are formed. Growth rate is fast.
With the purpose of taking root of sprouts the plantlets of Ostrowskia were transplanted in MS medium with supplementation of phytohormones in combination with 2 mg/l of IAA + 1 mg/l of BAP. Fresh medium was watery than before prepared media, because into 1 litre of the main MS medium we added agar-agar not 6 g per liter, and 5 g per liter. After six weeks in Ostrovskia we have observed the formation of roots. The plants have 8-9 leaflets. Regenerated plants developed normally.
As previously noted, the type of forming meristem depends on the ratio between auxin and cytokinins, however, a range of plants not always change in balance of phytohormones to regenerate buds or roots. On the interaction of hormones in regulation of differentiation of buds or roots can be affected by other factors, such as the content of sugars and phosphates, a source of nitrogen and other components of the medium.
Thus, the concept of hormonal regulation of differentiation and morphogenesis in vitro cannot be considered as universal. First, the phytohormones in regulation of differentiation and morphogenesis do not have specificity. The same hormones regulate both cell division with undifferentiated growth of callus and cell division associated with differentiation.
4. Conclusions
As a result of conducted research we can draw the following conclusions:
1) Ostrowskia magnífica seeds for germination require stratification at temperatures below — 4 °C for 14-16 days.
2) For sterilization and preservation ofviability Ostrovskia magnifica one can use the following method: initial treatment in 0.06% of AgNO3 solution for 20-25 min, followed by washing in 70% of ethanol for 1-2 min, in sterile water several times for 10 minutes each.
3) It is better to use sterile jars with a volume of200-250 ml for the cultivation of plant material.
4) Optimal conditions for the cultivation of Ostrowskia magnifica is 16-hour light regime and temperature — 25 °C.
5) For an introduction to the culture and micropropagation of Ostrovskia magnifica the following nutrient media fit: MS with 0.25 mg/l IAA + 0.02 mg/l BAP, MS 2 mg/l IAA + 0.2 mg/l BAP, MS 4.0 mg/l IAA+2.0 mg/l BAP.
6) For the formation of callus and growth of cells the optimal medium is MS medium without hormones and MS medium with addition of 1.0 mg/l IAA and 0.5 mg/l kinetin.
At the moment there is no universal technology of in vitro cultivation, which would be suitable for all plants. For each plant species the development of specific techniques for formation ofplants-regenerants in tissue culture are required. The conducted research of the initial stages of the cultivation of Ostrowskia magnifica Regel will allow to continue development of micropropagation technology for solving problems of preservation of the given species.
Acknowledgments
The work is done within the "Introduction assessment and biology of seed multiplication of promising new species of ornamental and medicinal plants" project (№ F.5 — F A-O-12154, Academy of Sciences of Uzbekistan).
References:
1. Zaripova A. A. Initial stages of micropropagation of peony lateral buds. Vestnik OGU - № 6. - Ufa, - 2009. - P. 140-142.
2. The Red Data Book of the Republic of Uzbekistan. - Vol.1. Plants the Fungi, - Tashkent, Chinor ENK. - P. 319.
3. Rozanowskaya M. I. Ostrovsky majestic - Ostrowskia magnifica R G L./Introduction and acclimatization of plants. - Vol. 4. - Tashkent, - 1966. -P. 137-143.
4. Butenko R. G. Cell biology of higher plants in vitro and biotechnology based on them. - M.: FBK-PRESS, - 1999. - 160 p.
5. Murashige T., Skoog F. A revised medium for repid growth and bioassays with tobacco tissue culture//Physiol.plant, - 1962. - Vol.15, -No. 3. - P. 473-497.
6. Kalinin F. L., Sarnatskaya V. V., Polishchuk V. E. Tissue culture methods in physiology and biochemistry of plants. Kiev: Naukova Dumka, - 1980. - 488 p.
7. Reinert, J. and P. R. White, - 1956. The cultivation in vitro of tumor tissues and normal tissues of Picea glauca. Physiol. Plant., - 9: 117-189.
8. Torrey, J. G. and Shigemura, Y. (1957). Growth and controlled morphogenesis in pea root callus tissue grown in liquid media. Am. J. Bot. - 44, - 334-344.
9. Blakely, L. M., and F. C. Steward. Growth induction in cultures of Haplopappus gracilis Amer. J. Bot. - 48, - 351 - (1961).
10. GRANT ME, and FULLER KW. - 1968. Tissue culture of root cells ofVicia faba. Journal of Experimental Botany - 19: - 667-680.
11. Kataeva N. B., Butenko R. G. Clonal micropropagation of plants. - Moscow: Nauka, - 1983. - P. 22.
12. Valikhanova G. J. Plant biotechnology. - Almaty, «Konjic», - 1996. - 272 p.
DOI: http://dx.doi.org/10.20534/ESR-16-9.10-24-26
Urazbaev Ismatulla Ummatovich, Gulistan State University, Department of Soil Sciences, Docent E-mail: ismatullaurazboev@gmail.com
Saline resistance of vegetable and melon crops and valuation factors on the degree of soil salinity
Abstract: The article is devoted to the discussion of results obtained from the valuation of saline soils in Golodnaya steppe of Mirzachul oasis, correlation between the crop capacity of vegetable melon crops and salinity degree of soil. As a result of the research there were developed valuation factors for cultivated crops such as potato, onion, tomato and cabbage. Keywords: Golodnaya steppe, valuation, salinity, vegetable crop, crop capacity.
Introduction. Practical works on soil evaluation carried out in the Republic revealed that the transition in conditions of market economy requires effective use of every hectare of land. In order to achieve this there is a need in developing bonitation methods which take into account natural, economical and agricultural conditions in the process of evaluating soils used in producing vegetable and melon crops.
At present, soil valuation and assessment of soil's fertility in scoring units in the Republic of Uzbekistan are carried out by the Institute of Soil Sciences and Agricultural Chemistry and regional branches of the land Registry "Uzdavyerloyiha". It is generally known that the data obtained from soil valuation procedures is widely employed by agriculture planning institutions and tax inspections with the aim of planning the productivity of vegetable and melon crops and identifying the amount of tax on land.
Objective of the study was to develop valuation factors on the degree of soil salinity for the vegetable crops such as potato, tomato, onion and cabbage.
Material of the research. The research was carried out in Golodnaya steppe in developing methodological fundamentals of bonitation of soil found in the Steppe and used for growing main vegetable crops. Thus, irrigated meadow soils of Golodnaya steppe was taken as an object of the present study.
In 2010-2013 field studies were carried out in 20 key areas and 200 areas were chosen for calculation of crop capacity. Areas in dimension of10X10 meters were located in such a way that they could cover all main soil varieties. Calculation of crop capacity was carried out employing the method developed by Dospehov [1, 117-290]. Then the statistical data processing obtained from crop capacity calculation was done in order to compute average yields and search of correlation dependencies between certain properties of soil and productivity of vegetable crops.
Soils of Golodnaya steppe are saline in diverse levels where content of chloride sulfate type of salinity prevails. With the increase of salinity, the approach of soil horizons with the content of Mg and Na to the surface occurs.
Results of the research. Results of analysis of aqueous extracts obtained from studied soil types revealed (Table 1) that amount of solid residue in the upper horizons is insignificant. In non-saline soils content of water soluble salts is equal to 0.1% whereas in mid-saline soils the content is equal to 1%. Weak saline soils have intermediate position. Up to 1% of salt is concentrated in morphologically observed soil horizon. Formation of the horizon is due to the maneuvering of soil from overlying strata and possibly due to the capillary pulling up from the bottom salt solutions due to evaporation during subsurface soil desiccation after spring rain.
