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Ergasheva Farogat Sheralievna, postgraduate student, the Faculty of Natural Sciences Gulistan State University, Uzbekistan E-mail: [email protected] Sherbaeva Madina qodir qizi, student, the Faculty of Natural Sciences Gulistan State University, Uzbekistan Khushmatov Shunqor Sadullaevich, Institute of Biophysics and Biochemistry of National University of Uzbekistan, Laboratory of cell biophysics Kushiev Khabibjon Khojiboboevich, professor, the Faculty of Natural Sciences Gulistan State University, Uzbekistan
PRESERVING OF SAMPLES OF POMIGRANATE SORTS IN IN VITRO CONDITIONS
Abstract: The article presents the results of the slowdown in the growth of pomegranate varieties in in vitro conditions through increasing the concentration of sucrose in nutrient media. Increasing the concentration of sucrose from 30 g/l slows down the growth process of pomegranates. It was revealed that with the introduction of sucrose in the amount of 70 g/l into the nutrient medium, is possible to directly transplant the plants for 7 months while maintaining the viability of the plants. The reduction of sucrose in the nutrient medium from 70.0 g/l to 60.0 g/l remained from 7.1 to 42.85% of the plants. At concentrations of sucrose 70-90 g/l, clear inhibition of growth processes after storage for 5 months was noted, but the plants maintained their viability. In order to increase the duration of non-transportable storage of pomegranate plants in culture, it is necessary to combine the use of elevated concentrations of sucrose and cultivation of plants at a lower positive temperature for up to 1-2 years. It is noted that an increased concentration of sucrose (4-5%) in nutrient medium retards cell growth without causing a toxic effect, and therefore can be used to maintain cultures in a state of rest for a long period of time. Studies have established slowdown in growth process and the possibility of increasing the duration of direct cultivation to create a collection of pomegranate in vitro.
Keywords: pomegranate, sucrose, concentration, in vitro collection.
Introduction Conservation of plant collections is particularly impor-
Biotechnological methods are widely used in the world tant for future use, both for breeding purposes, the production for the long-term preservation of plant collections used in the of a healthy planting material, and for the preservation of the future for breeding purposes, the production of healthy plant- gene pool and plant biodiversity as a whole. And biotechno-ing material and for the preservation of the gene pool and logical methods are widely used for long-term preservation plant biodiversity. One of the methodological approaches to of plant collections.
the deposition of plants - is the content ofbiological objects in At the same time, special attention is paid to slowing down
a slow metabolism. It is known that various organic substances the growth and development of plants. One of the conditions with high osmotic activity are added to nutrient media to slow for slowing down growth is the use of osmotic [1, 60-62]. down the growth and preserve the invitational plant mate- To slow down the growth and preserve the invitational
rial. The researchers note that an increased concentration of plant material, various organic substances with high os-sucrose (4-5%) in the nutrient medium retards cell growth motic activity are added to the nutrient media: glucose, without causing a toxic effect, and therefore can be used to sucrose [2, 733-736] or sorbitol [3, 145-146]. Sucrose has keep cultures at rest for a long period of time. The goal of our inhibiting properties of plant development under in vitro study is to identify the effect of sucrose concentrations on the conditions. The inhibitory effect of sucrose is based on a growth processes of pomegranate mericloning for long-term change in the osmotic pressure of the fluid in the direction non-storage of plants in vitro collection. of exosmos.
While studying the growth and development of flax
[4, 64-66], winter wheat [5, 68], potatoes [6, 46; 7, 8-10; 8, 15-16], poplar [9, 28-30], strawberries [10, 75], grapes [11, 9-12] in vitro conditions, it was proved that the addition of sucrose in the redistribution of 4-5% in the nutrient medium, retards the growth of plant cells. Therefore, for the storage of biotechnological plant collections, sucrose is used as a retarding agent for the growth and development of plants [12, 202; 13, 65-66].
The purpose of this research is to determine the concentration of sucrose, affecting the growth and development of pomegranate for long-term direct storage in vitro conditions.
Materials and methods
As objects of the research, we have selected plants of 5 varieties of Punicagranatum L. In this research, both general biotechnological methods were used [14, 125] equally as methods developed in the laboratory of biotechnology and virology of plants of the NBS-NNC [15, 190-192]. Sterilization of plant material and its introduction to an isolated culture was carried out in laminar boxes.
The conditions of sterilization of original plant material were determined experimentally for each studied culture. At the same time it needs to be taken into account the origin of the explants as well. The concentrations and exposures of the sterilizing reagent are also tested. As sterilizing agents for the release of endogenous or exogenous infections, 1-2% solutions of sodium hypochlorite NaClO, 70% ethyl alcohol - C2H5OH,
0.08% solution of silver nitrate AgNO3 were used, that freed isolated explants from exogenous and endogenous infections. For cultivation of pomegranates, in vitro were used mineral base ofMurasige-Skooga nutrient media [16, 477-482].
Samples of pomegranate varieties were deposited after recovery by using the culture method of apical meristems with a relative explants size of 0.1-0.2 mm. In order to increase the regenerative capacity of the meristem during micro multiplication of obtained plants, there were used cloned micromultiplication of scheme and technology [12, 202-203].
Estimated plant viability at a frequency of once a month in the number of leaf and shoot tissue necrosis:
- visual death of plant - 0 points;
- necrosis of more than 50% of tissues plants - 1 point;
- necrosis of less than 50% of tissues - 2 points;
- plants without necrosis - 3 points.
In order to find the optimal condition for slowing the growth of pomegranate varieties Qizil anor, Qozoqi anor, Qaiym anor, Oq dona (tuya tish), Achchiq dona were studied in terms of the influence of sucrose at concentrations of 1090 g/l. The content of sucrose was in the amount of60-70 g/l in a nutrient medium.
Result and discussion
Discussions of the results of data on the state of growth and development of pomegranate plants of the Qozoqi anor variety at various concentrations of sucrose under in vitro conditions are given in the (table 1).
Table 1. - Viability of test-tube plants of grapes at various concentrations of sucrose, Qozoqi anor variety, 2017-2018.
Sucrose g/l Retention^ Roots Height, sm Leaf quantity, pc Growth rate. sm per day Polarity coefficient
Quantity, pc Length, sm Resogenous zone, см
1 2 3 4 5 6 7 8 9
Recording after 25 days of cultivation
10.0 100 4.6 1.8 8.3 1.9 2.5 0.08 4.4
20.0 96.4 4.1 2.2 9.0 1.8 2.3 0.07 5.0
30.0 100 4.8 2.6 11.0 2.2 2.5 0.09 5.0
40.0 96.4 4.0 2.8 11.2 1.8 2.3 0.07 6.2
50.0 96.4 4.0 2.9 11.2 2.2 2.3 0.09 5.1
60.0 82.1 3.7 3.0 11.1 1.8 2.2 0.07 6.2
Recording after 45 days of cultivation
10.0 100 5.0 2.8 14.0 4.0 5.6 0.09 3.5
20.0 96.4 4.9 2.9 14.2 4.0 5.7 0.09 3.6
30.0 100 4.9 3.7 18.1 5.0 6.8 0.1 3.6
40.0 96.4 4.7 4.0 18.8 4.6 6.4 0.1 4.1
50.0 96.4 4.6 3.8 17.5 4.8 6.3 0.1 3.6
60.0 82.1 4.4 4.1 18.0 4.7 6.4 0.1 3.8
1 2 3 4 5 6 7 8 9
Recording after 65 days of cultivation
10.0 100 5.1 3.0 15.3 6.2 8.5 0.09 2.5
20.0 96.4 5.5 3.3 18.2 6.2 8.7 0.09 2.9
30.0 100 5.1 4.3 21.9 7.6 10.0 0.11 2.9
40.0 96.4 5.3 4.4 23.3 7.4 9.5 0.11 3.1
50.0 96.4 5.1 4.3 21.9 7.8 9.4 0.12 2.8
60.0 82.1 4.5 4.8 21.6 7.2 9.3 0.11 3.0
Recording after 85 days of cultivation
10.0 100 5.3 2.2 11.7 8.4 10.3 0.11 1.4
20.0 96.4 4.1 3.1 13.0 8.4 10.6 0.11 1.5
30.0 100 4.4 4.3 18.9 9.8 12.0 0.12 1.9
40.0 92.8 4.2 3.6 15.8 9.4 11.8 0.12 1.7
50.0 92.8 4.0 3.8 15.2 10.0 11.7 0.13 1.5
60.0 78.6 3.7 4.1 15.6 8.7 11.4 0.11 1.8
It should be noted the high survival rate of micro cuttings and the safety of plants in the experiment. Only at the maximum concentration of sucrose the deterioration of the development of micro-cuttings was noted. During the entire cultivation period, the optimal development of plants occurred at a sucrose concentration of 30 g/l. At the minimum (10 g/l) and maximum (60.0 g/l) concentrations, root formation deteriorated, the length of the rhizogenic zone, the growth rate, and the decrease in the foliage of the shoots decreased. In general, the slowdown of growth processes was not significant; based on this, part of the plants were trans-
ferred to a culture room with a low temperature to extend the plant storage period.
Plants, that were transferred in the culture room with a lower temperature were maintained for 6 months (Table 2). In versions with minimal sucrose concentrations in surviving plants, the viability was 2 points. When the concentration of sucrose is 40 g/l, pomegranate plants are characterized by satisfactory viability - 1.6 points and shoot aging in single plants. With increased sucrose concentrations of 50-60 g/l, the viability and the number of surviving plants decreased, but green leaves still existed, shoots matured.
Table 2. - The condition of plants after storage for 6 months in the cultural room with a low temperature, variety of pomegranate sort of Qozoqi anor, 2018
Sucrose, Kept, Height, Leaves, pc Reten- Band, pc
g/l pc.% CM Green Yellow Dried tion Green Dry Yielded
10 3/100 14.0 14.6 3.4 - 2.0 3 - -
20 3/100 15.3 20.3 - 1.6 2.0 3 - -
30 2/66.7 10.3 4.0 - 5.5 1.0 1 1 -
40 6/100 14.5 9.4 5.3 6.0 1.6 1 2 3
50 3/50 10.0 5.3 - 8.0 1.0 - 1 2
60 6/66.6 9.8 3.6 - 11.5 1.0 1 3 2
The presence of plants for 3 months at a temperature of plants remained in a satisfactory condition, their cultivation
25-27 °C on a nutrient medium with a high content of sucrose, continued. Similar results were obtained in experiments on the
followed by transfer to conditions of low temperature ensured study elevated concentrations ofsucrose (4-6%) on varieties of
their safety without a decrease in viability for 9 months. Single pomegranate Qayim anori, Achchiq dona.
Table 3. - Viability of test-tube plants of grapes at various concentrations of sucrose, Oq dona (Tuya tish) sort, 2012-2013
Sucrose g/l Kept,% Roots Heght, sm Leaf quantity, pc Growth rate. Cm per day Polarity coefficient
Quantity, pc Length, sm Resogenous zone, sm
1 2 3 4 5 6 7 8 9
Recording after 30 days of cultivation
10.0 100 5.7 3.3 19.2 3.6 2.3 0.12 5.3
1 2 3 4 5 6 7 8 9
50.0 100 4.9 3.5 17.1 2.6 1.8 0.09 6.5
60.0 96.5 4.5 3.1 14.1 2.1 1.5 0.07 6.9
70.0 100 4.1 3.0 12.3 2.4 1.3 0.09 5.1
80.0 96.5 3.0 2.7 8.1 2.0 1.3 0.07 4.0
90.0 100 3.2 2.0 6.5 1.8 2.0 0.06 4.0
Recordin g after 60 days of cultivation
10.0 100 5.5 3.4 18.7 7.0 4.7 0.11 2.7
50.0 82.2 4.8 3.9 18.8 4.7 4.5 0.08 4.1
60.0 92.9 4.7 3.8 18.0 4.3 3.5 0.07 4.2
70.0 89.3 4.2 3.9 16.4 4.1 3.5 0.06 4.0
80.0 71.6 3.0 3.5 11.0 2.9 2.5 0.05 3.8
90.0 71.4 3.5 2.5 9.0 2.9 2.3 0.05 3.1
Recordin g after 90 days of cultivation
10.0 100 5.8 3.6 20.8 10.5 6.6 0.11 2.0
50.0 82.1 5.2 4.5 22.8 7.2 6.5 0.08 3.1
60.0 67.9 5.0 4.4 21.5 6.6 6.2 0.07 3.3
70.0 85.7 5.0 4.9 24.5 6.3 5.8 0.07 3.8
80.0 39.3 5.0 4.8 24.0 4.7 4.2 0.05 5.1
90.0 25.0 5.4 4.1 21.8 3.8 3.4 0.04 5.0
In setting the experiment on the Oq dona variety (tuya tish), higher concentrations of sucrose were included in the study: 50, 60, 70, 80, 90 g/l. From the analysis of the data given in table. 3, it follows that the optimal development of plants occurs when the sucrose content in the nutrient medium is 10.0 g/l. At elevated concentrations of sucrose, the decrease in the safety of plants and the intensity of growth processes was observed. Reducing the growth rate of plants and the intensity of growth processes are in this situation a positive phenomenon, since they ensure the minimization of growth for the deposit of plants in the collection.
Observations on the state of plants in the collection
the plants dry out, the number of green, yellowed plants and the number of dry leaves decrease, and the plants' vitality decreases. At elevated concentrations of sucrose, a decrease in the intensity of growth processes is most pronounced and is expressed primarily in the slowing down of plant growth. When cultivated for 220 days, this process is aggravated and the drying of plants and the decrease in their viability at sucrose concentrations above 70.0 g/l were observed. It should be noted that in this experiment the maturation of plants occurred. At a concentration of 50 g/l-9 plants, at a concentration of 60 g/l - 3 plants, 70 g/l - 6 plants and 90 g/l -1 plant matured.
(Table 4) showed that when they were stored for 200 days,
Table 4. - Indices of plant viability in direct cultivation on a nutrient medium with various concentrations of sucrose, Oq dona (Tuya Tish) grape variety, 2018.
Sucrose g/l Kept,% Plant height, см Leaves, quantity Survivability, rates
Green Yellow Dry
1 2 3 4 5 6 7
Cultivation during 214 days (07.11.13.)
10.0 26/92.8 15.9 7.8 2.7 3.4 1.8
50.0 20/71.4 11.3 5.9 2.9 4.0 1.8
60.0 12/42.8 11.0 7.7 3.0 2.5 1.8
70.0 11/39.2 100 5.6 2.5 5.3 1.5
80.0 8/28.5 5.0 3.1 2.0 4.5 1.4
90.0 2/7.1 5.5 3.0 2.5 5.0 1.4
Cultivation during 260 days (23.12.13.)
10.0 26/92.8 - 3.5 6.0 4.4 1.0
50.0 19/67.8 - 4.5 3.8 4.5 1.1
60.0 12/42.8 - 4.1 5.0 4.1 1.2
1 2 3 4 5 6 7
70.0 8/28.5 - 4.0 4.8 4.6 1.2
80.0 6/21.0 - 2.1 2.3 4.9 1.0
90.0 2/7.1 - 2.0 3.0 5.5 1.0
The storage period ofOq dona variety (tuya tish) on a nutrient medium with different sucrose contents was 7 months. The greatest number of plants was preserved at a sucrose content of10 g/l - 9 pieces, 4 plants each were preserved at concentrations of 50 and 60 g/l, individual plants were preserved at high concentrations of sucrose - 70-90 g/l.
With the introduction of sucrose in the amount of 70 g/l into the nutrient medium, the possibility of direct cultivation of plants of the pomegranate varieties Bedona, Ulfi, Qayim anor, Pushti Gulosha (clone 2-3) and Qoraqayin was established in 9 months. At the same time, their aging occurs and viable plants remain in storage.
Thus, for most of the studied varieties, the possibility of direct storage of plants up to 90 and even 240 days on a nutrient medium with a sucrose content of 60.0 g/l has been proved.
Cultivation of pomegranate plants continued under standard conditions for 240 days. During this period, 8 plants from a variant with a sucrose concentration of 5.0 g/l.4 plants from a variant with a concentration of 20.0 g/l, and 7 pomegranate plants from a variant with a concentration of 60.0 g/l were preserved viable. The results of our research on the use of elevated concentrations for long-term storage of pomegranate plants have been confirmed, moreover, the possibility of using for these purposes lower concentrations of sucrose has been revealed.
Conclusion
With an increase in sucrose concentration in nutrient
media above 30 g/l, the growth processes of granate plants slow down: root formation, root length and rhizogenic zone length, plant growth and leafiness of plants decrease: Installed opportunity of introduction into the composition of the nutrient medium sucrose in the amount of 70 g/l possible direct cultivation of plants for 7 months with preserved viability of plants. A decrease in the sucrose content in the nutrient medium from 70.0 g/l to 60.0 g/l increases the depositing time up to 360 days.
From storage, from 7.1 to 42.85% of plants remained, depending on the variety and condition of the plants before putting them for storage. At sucrose concentrations of 70-90 g/l, a clear inhibition of growth processes after storage for 5 months was noted, but the plants maintained their viability. However, when deposited for 8-10 months in these variants of the experiment, a sharp decrease in viability and intensive drying of the plants was observed. It is not possible to recommend these concentrations of sucrose for the deposition of in vitro pomegranate. In order to increase the duration of non-transportable storage of pomegranate plants in culture, it is necessary to combine the use of elevated concentrations of sucrose and cultivation of plants at a low positive temperature for up to 1-2 years.
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