OPEN ACCESS
Journal of Multidisciplinary Applied Natural Science Vol.
5 No.
https://doi.
org/10.
47352/jmans.
Research Article Optimization of In Vitro Microcloning of Lagochilus inebrians Bunge Kumush Sultonova*.
Begali Alikulov.
Normat Xasanov.
Yulduz Abdullayeva.
Shahnoza Sharopova.
Baxtiyor Jabborov.
Hulkar Hotamova.
Mamadiyarova Dilshoda, and Boymurodov Khusniddin Received : November 6, 2024 Revised : April 1, 2025 Accepted : June 5, 2025 Online : July 26, 2025 Abstract In the following years, introduction .
n sit.
of rare and promising medicinal plants, determination of morphological and ecological adaptability to selected soil and climate conditions, and obtaining pathogen-free seedlings through microclonal .
n vitr.
reproduction in areas affected by environmental stress factors creation of plantations are gaining importance.
This research aimed at choosing the optimal conditions for obtaining pathogen-free seedlings of Lagochilus inebrians in vitro and acclimatization in arid The optimal amounts of added phytohormones were chosen to increase the efficiency of the nutrient media used in the microconal propagation of L.
inebrians, based on the possibility of acclimatization in arid conditions.
The obtained results serve to multiply L.
inebrians in vitro and create plantations in arid regions based on the obtained seedlings.
Keywords Lagochilus inebrians, explant, culture, phytohormones, in vitro, microclonal propagation, drought, ex vitro
INTRODUCTION
About 500,000 species of higher plants are known in the world.
The medicinal properties have been determined in 5% of them.
Today, 60% of pharmacological preparations are obtained from medicinal plants .
However, the raw material of medicinal plants is insufficient or their number is sharply reduced due to unplanned harvesting from nature .
Recently, breeding, preservation and use of medicinal plants on the basis of biotechnological methods are being considered as promising directions .
Accordingly, in order to supply the pharmaceutical industry with raw materials of medicinal plants, development of ways of their cultivation is one of the urgent issues.
In this regard, making recommendations for breeding and production of medicinal plants, including Lagochilus inebrians Bunge, is of great scientific and practical importance.
inebrians is an intoxicating plant locally known as Bozulbang which grows in hot climates PublisherAos Note:
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A 2025 by the author.
Licensee Pandawa Institute.
Metro.
Indonesia.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license .
ttps:// org/licenses/by/4.
0/).
According to its life form, it is considered a semi-shrub.
This unique type of plant grows in the foothills, in the mountains, on gravel and river tributaries, sometimes on the banks of canals and ditches, mainly in Samarkand.
Jizzakh.
Navoi.
Surkhandarya regions of Uzbekistan, and distributed in some other republics of Tajikistan and Turkmenistan in Central Asia .
inebrians is an annual plant with a main root reaching 20Ae60 cm in The length of the main root is 25Ae35 cm.
The stem is spiky, ascending, the base is rounded, four-sided, covered with hard glandular hairs.
The leaf is simple, divided into three to five parts, arranged oppositely on the stem and branches.
The length of green leaves is 2Ae3 cm.
The flowers are reddish in color and are arranged in a semi-ring on the stem and branches.
The fruit is 4 brown nuts and blooms in June-September.
The time to harvest the fruits of the L.
inebrians plant is July and August .
By studying the ontogeny of this plant in the conditions of Uzbekistan, it was determined that its lifespan is at least 25 years.
Selfpreservation of senororulations occurs only with A comparison of the ontogenetic structure of senororulations of L.
inebrians in different ecological and phytocenotic living conditions showed that all senororulations secretory gives the highest result in middle-aged generative Its leaves and flowers are mainly collected by the local population .
In its natural habitat.
inebrians grows only with seeds.
The seeds germinate in mid-February to early March.
Germination of seedlings does not exceed 26% .
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Table 1.
The effect of nutrient medium components on the recovery of shoots of L.
Inebrians.
Substance concentration .
g/L) Components Introducing explants into in vitro conditions (MS.
Multiplication (MS.
Rooting (MS.
NH4N3
Kinetin (C10H9N5O) KH24
l2A2H2
MgS4A7H2
MnS4A5H2
H3B3
ZnS4A7H20
uS4A5H20
N2M4A2H2
l2A6H2
FAS4A7H2
N2iDTaA2H2 Inositol Carantothenate Nicotinic acid Ryridoxine solution Thiamine solution Casein hydrolysate Sucrose Agar AuDifcoAy The chemical composition of L.
inebrians plant contains vitamin K, 0.
60Ae1.
97% lagoxilin, 0.
flavonoid glycosides, 44Ae77 mg% ascorbic acid, 6Ae 7% organic acids, 5Ae10 mg% carotene.
It contains 66Ae12.
42% tar, 2.
58Ae2.
78% slag and other substances, as well as calcium and iron salts.
inebrians leaf contains lagoxilin, 0.
03% essential oil, 11Ae14% digestive substances, organic acids, 7Ae 10 mg% carotene, and 77Ae100 mg% vitamin C.
From the tissues of L.
inebrians, the physiologically active compound diterrenoid, tetrahydric acid lagoxilin and its acetyl derivatives, which are very poorly soluble in water, are isolated and are used as the most important source in chemistry and pharmacology .
Based on the analysis of the above data, a promising medicinal plant was distributed in the territory of Uzbekistan is L.
inebrians Bunge.
The main goal of our research is to develop the biotechnology of microclonal propagation, preservation, and pathogen-free seedlings in vitro.
MATERIALS AND METHODS
Cultivation of Donor Plants In the studies, the mother plant L.
inebrians was grown in greenhouses under protected conditions.
The collection of planting material was carried out according to standard technology for growing flower crops .
The medium selected for growing L.
inebrians seedlings should have a saturated balanced nutrient composition .
The inebrians .
ield condition.
0A0.
5A0.
4A0.
reenhouse cultivatio.
0A0.
0A0.
Number of buds .
ieces/ex.
Regeneration rate (%) Number of buds .
ieces/ex.
Regeneration rate (%) Number of buds .
ieces/ex.
Regeneration rate (%) B5 5.
0 mkM Ba 2.
0 mkM NSK Table 3.
Regenerative performance of L.
Vrint BDS 5.
0 mkM Ba 2.
0 mkM NSK
NAA
Ba Concentration of growth regulators .
g/L) Option Table 2.
The effect of nutrient medium components on the recovery of shoots of L.
Multidiscip.
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substrate contained peat particles of 0Ae5 mm, dry matter was at least 10% of the substrate volume.
The moisture capacity was 78Ae82%, the air capacity was 8Ae12%, and the pH was 5.
To improve the structural properties, 15% of rellite was added to the peat .
In order to ensure rapid rooting of the rhizome and maintain high humidity, daily watering of the seedlings was carried out .
The rooted plant was planted in a larger container .
7Ae1.
0 L) with the same amount of substrate and nutrients as in the rooting stage, creating a ratio of N:P:K = 140:160:180 mg/L.
During the studies, the air temperature was maintained between 16 and 18 AC for the first 2Ae4 weeks, then the temperature was lowered from 8 to 10 AC to complete the vernalization period, which lasted 3Ae6 weeks.
During the cold season, regular but intermittent watering and periodic fertilization were carried out once a week with a complex fertilizer containing the main elements in the N:P:K ratio of 250:300:350.
At the end of the vegetative period, the plants were treated once with a stimulant in a ratio of 10:1 in 15 mL to form a bush.
The air temperature was gradually increased from 16 to 18 AC over 12Ae14 days.
The temperature for growing the donor plants was then increased from 18 to 22 A When the temperature reached the maximum value, the plants were illuminated with 9-10 KLK .
W/m.
lamps for 8Ae10 h/day for 14 days.
During the warm season, it was observed that the number of upper crusts increased twice a week.
Sterilization of Nutrient Medium and Planting Materials In accordance with the current recommendations, aseptic conditions were used to sterilize nutrient medium and planting materials.
Murashige and Skoog (MS) nutrient medium used in in vitro culture was autoclaved twice for 15 min at 0.
75 atm pressure and 117AC.
The initial explants for in vitro culture were sterilized in the following sterilization media: 70% ethanol.
5, 1, 2% of calcium hydroxide solution with an exposure time of 5 to 15 Stages of sterilization of plant material to obtain sterile explants: .
cleaning the plant from various contaminants.
holding in a soap solution for 30 min.
rinsing with running water.
sterilization with 70% ethanol for 5 min.
sterilization with 0.
5, 1, 2% calcium hypochlorite J.
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Figure 1.
Regeneration of L.
inebrians shoots on primary explant surface in BDS medium supplemented 0 M BAP and 2.
0 M NAA .
days ol.
solutions for 7Ae10 min.
re-washing with distilled water 3Ae5 times.
Selection of Nutrient Medium and Growth Regulators for In vitro Propagation of L.
Explants The optimal time for grafting from donor plants was during the period of active growth.
Plant sample explants were grown on MS medium .
ucrose 30 g/L, agar 6 g/L.
BAP 2.
0 mg/L.
NAC 1 mg/L).
Morphogenetic activity was assessed by the number of explants formed during the observation period .
Ae21 day.
The composition of the modified MS medium was changed stepwise during the studies (Table .
In vitro cultivation of explants was carried out in MS nutrient media with two different growth regulators: BAP diazonium from 2 to 6 mg/L and NSK from 0.
01 to 0.
2 mg/L.
In the cultivation of explants, we used different ratios of BAR and NAA concentrations as growth regulators (Table .
In addition, the effect of different concentrations of BAP, kinetin and thidiazuron (TDZ) .
1 and 0.
M) on the viability of cultures, shoot regeneration and stem number increase was also studied.
At the initial stage of in vitro cultivation, cultures were fed with an agar medium supplemented with 5.
BAP and 2.
0 M NAA according to the BDS and B5 recipe in addition to MS.
In vitro culture of explants, ascorbic acid medium solution was used to reduce the negative effects of phenolic compounds released during the cultivation process .
After pretreatment, sterile axillary buds were dissected into 1 mm samples in a Retry dish under laminar flow box conditions.
The isolated explants were transferred to a nutrient medium in 1 cm diameter flasks for in vitro cultivation .
Microcloning and Rooting of Explants Microcloning was carried out in several stages.
The rhizomes grown from the flasks were transferred to a nutrient medium with a reduced concentration of growth regulators in laboratory flasks for propagation.
One transplant was carried out for 27Ae38 days.
The living environment was the same as that in the cultivation of explants.
Subsequently, the conglomerates that had grown and sprouted were divided into more sprouts under sterile conditions and planted for rooting.
For rooting, shoots with 3Ae4 leaves, 1.
5Ae2.
0 cm long, were cut from the base and placed in a A MS hormone-free medium with a 2-fold reduced nutrient concentration and a fixed amount of sucrose .
g/L).
In the studied samples, the rooting indicators, average rooting frequency, 100% rooting frequency of plants planted for rooting or rooting in hormonal and non-hormonal media were observed, and in the presence of auxins in various concentrations - NAA.
IAA.
IBA - on A BDS and A MS mineral bases.
The most effective concentration of the agent for stimulating rhizogenesis in L.
inebrians was determined.
Root formation began on average after 14 days.
Rooted regenerated plants were ready for planting in vitro in 21Ae28 days.
To gradually acclimate the regenerated plants to harsh in vitro conditions with high humidity and high infectious background, a small hole was drilled in the tops of the flasks.
Note: p O 0.
Au-Au Ae no information Control .
ormone-free mediu.
Ba 0.
Ba 0.
Ba 5.
Ba 5.
0 NAA 2.
Ba 5.
0 NAA 5.
TDZ 0.
TDZ 0.
TDZ 5.
TDZ 5.
0 NAA 2.
KinAtin 0.
Ba 5.
0 IAA 5.
Ba 0.
4 NAA 3.
2 IAA 2.
Growth regulators Regeneration rate (%) 6A0.
8A0.
8A0.
1A0.
3A0.
BDS
Random shoots .
/exr.
5A0.
Regeneration rate (%) Mineral base 1A0.
Random shoots .
/exr.
6A0.
8A0.
8A0.
0A0.
4A0.
1A0.
5A0.
3A0.
7A0.
0A0.
Table 4.
Effect of nutrient medium mineral content and growth regulators on regeneration of L.
inebrians buds in in vitro cultures.
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which was gradually enlarged over 3Ae5 days, and then the tops were completely removed and For 7Ae14 days, the plants were grown in pre-sterilized, moistened peat granules, where the living environment was maintained at a temperature of 23A1 AC and 100% humidity.
Starting from the beginning of intensive growth, the regenerants were placed in normal conditions.
The period from planting the explant to receiving the adapted regenerants was an average of 75 days.
RESULTS AND DISCUSSIONS
Getting Input Materials In the initial stage of in vitro culture, agar nutrient medium supplemented with 2.
0 M NAA
0 M BAP was used for the cultures of BDS and B5 resert.
The use of nutrient media containing plant growth regulators induced a regeneration process in the tissues of L.
inebrians primary explants compared to media without control hormones .
In this study.
inebrians segments were found to have the least regenerative performance among other species studied (Table .
The samples growing in field conditions, after 51 Ae56 days, the first changes on the surface of the buds, i.
, a slight growth of excretory tissues, were In addition, it was noted that the regeneration of random shoots from the appearance of the tumor on the surface of the explant to the formation of primary vegetative organs lasted 12Ae17 days on average .
It should be noted that the development of callus tissue did not occur during the initial stages of cultures in vitro.
bud regeneration proceeded directly through the process of organogenesis.
Regeneration of buds was noted in the intact part of the tissue above the surface of the nutrient medium, but their formation occurred in the part closer to the original cut surface (Fig.
At the same time, the process of regeneration occurred based on the accidental division of buds into segments, that is, tissue damage.
Similar results caused by the positive effect of tissue damage during the regeneration process are explained by the activation of cell division .
In the course of research, stratification of buds in cold conditions .
A2 A), i.
, the influence of regeneration processes in the tissues of primary explants, was studied .
In the case of L.
inebrians taken from shoots growing in greenhouse conditions, it was noted that morphogenic reactions occurred in 20Ae25 days with prior storage of the tissue at the right Regeneration began after 5Ae7 days in samples that were not subjected to stratification in cold conditions.
In both cases, the average number of additional tissues was the same .
4Ae2.
6 units/ However, the absence of the initial suppression of cold differentiation resulted in the arrest of the growth of the primary vegetative organs formed in the secondary transition and the delay in the formation of subsequent shoots in the true seedling The formation of buds was not observed in the microclones obtained from the stratified plant tissue in cold conditions.
Effect of Nutrient Medium Mineral Content and Growth Regulators on the Morphogenesis of L.
Regeneration of L.
inebrians microbuds proceeded synchronously during the visual stress.
On 35Ae40 days of culture, the diameter of Figure 2.
The influence of the mineral content of the nutrient medium and growth regulators on the morphogenesis of L.
inebrians (A Ae shoot regeneration on the surface of morphogenic callus.
BDS medium supplemented with 0.
5 AAM BAP, 31 days of cultivation.
B Ae grown explants shoots formed in tissues.
B5 medium supplemented with 0.
1 M BAP, day .
Ba 5.
Ba 5.
0 NAA 2.
Ba 10.
0 NAA 2.
TDZ 0.
TDZ 0.
TDZ 5.
TDZ 10.
0 HUK 2.
KinAtin 0.
Ba 2.
2 2,4-D 0,5
Ba 0.
4 NAA 3.
2 IAA 2.
Note: p O 0.
A-A Ae no information 5A0.
Ba 0.
0A0.
3A0.
5A0.
4A0.
3A1.
5A0.
0A0.
5A0.
3A1.
Ba 0.
3A0.
Number of rows .
ieces/ex.
Regeneration speed (%) BDS Control .
ormone-free mediu.
Growth regulators Regeneration speed (%) Mineral base 5A0.
5A0.
5A1.
2A0.
5A1.
7A0.
0A0.
5A0.
5A0.
1A1.
4A0.
Number of rows .
ieces/ex.
Table 5.
Effect of mineral composition of nutrient medium and growth regulators on the regeneration of L.
inebrians shoots in vitro.
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Table 6.
Effects of growth regulators on the regeneration of L.
inebrians shoots in the in vitro cultures on B5 medium.
Growth regulators Control .
ormone-free mediu.
Ba 5.
0 NAA 2.
Regeneration speed (%) Number of buds .
ieces/e.
5A0.
0A0.
Ba 0.
4 NAA 3.
2 IAA 2.
0A0.
Figure 3.
inebrians tissues with developed absorptive leaves on BDS medium supplemented with 0.
M BAP 3.
2 M NAA 2.
3 M IAA .
days ol.
microbuds reached 3.
0Ae4.
5 mm.
They were transferred to a new hormone-free medium for further growth.
It was not possible to determine the most important factors affecting the number of shoots formed in the actual field of vision .
However.
BDS and B5 mediums were characterized by a similar rate of regeneration of L.
microplants, and a decrease was noted in the MS Taking this into account, further work was carried out using BDS and B5 mineral bases.
Similar results were obtained in the cultivation of other studied species.
In the control samples, in BDS medium without hormones .
egeneration Ae 7%, the number of shoots Ae 4.
1A0.
2 units/ex.
during a long cultivation period .
, high pictures of shoot growth and development were It was found that the addition of exogenous growth regulators to the nutrient medium in the growth medium in vitro slightly increased the activity of inebrians bud formation, and the regeneration activity in some samples was higher than in the control medium (Table .
Addition of the growth regulators 5.
0 M BAP
0 M NAA to BDS medium resulted in the formation of additional shoots on day 12Ae15 of cultivation, compared with 22Ae25 days on BDS medium without hormones did.
The average number of buds in each explant was 4.
6A0.
4, the regeneration rate was 56.
Higher growth and development rates were also observed in BDS medium supplemented with 10.
0 M BAR and 2.
M NAA.
The regeneration rate was 66.
2% and 4.
A 0.
8 random shoots were formed per explant.
The use of rast concentrations .
1 and 0.
5 M) of BAP.
Kinetin and TDZ resulted in the formation of yellow, morphogenic callus, regardless of the mineral composition of the medium.
At the same time, the rate of formation of morphogenic callus did not exceed 38.
The formation of buds on the surface of the callus was observed only after 5 weeks of cultivation, which occurred much later than in medium supplemented with cytokinins and From these shoots, new shoots emerged, that is, indirectly, organogenesis continued.
This was especially evident in BDS medium supplemented 5 AAM TDZ.
Although analysis of variance revealed no significant effect of environmental factors on the number of microbuds that developed, addition of 5.
0 M BAP and 2.
0 M NAA to BDS nutrient medium increased regeneration compared to control and medium supplemented with J.
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cytokinins alone.
It was found that it accelerates almost twice.
Addition of 0.
1 M BAR to the medium of B5 resert resulted in the growth of explant tissue and the highest number of explants on its surface with a frequency of 56.
5% .
0 A 1.
units/led to the formation of exr.
This made it possible to consider this selected environment as optimal in the formation of buds (Figure .
At the same time, kinetin .
5 M) had the most accurate morphogenic activity among all used During the study, the influence of the mineral content of the nutrient medium (B5 and BDS) on the regeneration activity of L.
buds was determined.
At the same time, the effect of growth regulators on the number of shoots formed was analyzed (Table .
Analyzing the results, it was found that the highest concentration of BAP and TDZ .
1 and 0.
AAM) was more effective.
The combination of high concentrations of BAP .
0 M) and NAA .
M) reduced the visual factor by 19% with a regeneration frequency of 1.
3A0.
5 per explant.
This combination of growth regulators was effective in the microarray of L.
inebrians, and the obtained research results were analyzed as follows.
Regeneration of L.
inebrians Buds The development of L.
inebrians larvae continued directly in the process of organogenesis.
Cultivation in medium supplemented with growth regulators improved shoot formation and regeneration rate increased from 50.
0 to 75.
compared to control.
The use of nutrient medium B5 containing 5.
0 M BAP and 2.
0 M NAA was considered the most effective.
In this environment, the maximum speed of regeneration and the number of additional shoots per explant were obtained (Table .
It has been noted that the response of plant development in the in vitro conditions when using mineral media of different composition varies in different plants .
The combination of 5.
BAP and 2.
0 M NAA and BDS of the mineral base was considered the most effective for L.
inebrians microblooming.
Such a combination of growth regulators in nutrient medium B5 led to the acceleration of shoot formation in L.
When 0.
4 M BAP was combined with 2 M NAA and 2.
3 M IAA, a high frequency of inebrians hemogenesis was observed in nutrient medium B5.
Both direct and indirect organogenesis were observed during cultivation of L.
Only indirect organogenesis was observed in L.
in all types of nutrient media.
This can be explained by species characteristics.
The first visible changes on the surface of L.
inebrians explants were observed in 25Ae31 days in nutrient medium supplemented with 5.
0 M BAP and 2.
0 M NAA.
After 2.
5Ae5.
0 weeks in the food medium, all the microbuds of the studied species formed in the explants began to form in the form of 2Ae3 leaves (Figure .
Formation of the Root System of L.
Regenerants and Adaptation to Ex Vitro Conditions The formation of the root system of L.
in vitro was observed mainly in nutrient medium with added auxin and partly without hormones.
Seedlings obtained under in vitro conditions were Figure 4.
Transplantation of L.
inebrians from in vitro conditions to growth.
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Figure 5.
Rooting rate in L.
inebrians explants obtained in vitro, in % (A-laboratory conditions: 1-1/2BDS.
2-1/2BDS NSK5.
3-1/2BDS NSK1.
4-1/2MS IMK5.
5-1/2BSK5.
B - in greenhouse conditions: 11/2BDS.
2-1/2BDS NSK5.
3-1/2BDS NSK1.
4-1/2MS IMK.
Figure 6.
Effect of sucrose concentration on the formation of microflora in L.
inebrians in laboratory (A), greenhouse (B), and natural (C) conditions, in piece .
= .
transplanted one by one.
Therefore, the plant obtained in vitro was planted on the substrate in the The top of the substrate is a simple substrate, wood shavings were placed on the surface layer (Fig.
It was found that L.
inebrians callus tissue proliferation under in vitro conditions is optimally realized in the combination of BAP .
mg/L) NAA .
mg/L) in MS nutrient medium.
In the combination of BAP .
mg/L) NAA .
1 mg/L) GA3 .
5 mg/L), the intensity of root formation was relatively high.
It should be noted that the plants in the studied samples took root easily in both hormonal and non-hormonal media .
Average rooting rate in all tested samples was A 100% rooting in L.
inebrians microplants was observed only in the presence of auxins in A BDS and A MS mineral bases at a concentration of 0 M - NAA.
IAA.
IBA.
The most effective medium for inducing rhizogenesis in L.
was A B5 supplemented with 5.
0 AAM NAA (Figure 5.
Tables 7 and .
The type of auxins used did not affect rhizogenesis activity.
It can only be shown that the presence of 5.
0 M NAA.
IAA or IMK in the nutrient medium induced root formation compared to medium without hormones and medium containing NAA .
5 M).
Table 9 shows the growth and development of the studied samples of inebrians in two temperature regimes.
According to the obtained data, the length of the roots is 1.
5Ae 5 times shorter at a temperature of 23A2 A.
the same time, other growth indicators did not depend on the temperature regime.
Samples grown in a sterile environment under laboratory conditions Samples grown in greenhouse conditions Samples grown in natural conditions The place where plant samples were taken Table 8.
Growth and development of regenerated L.
inebrians plants.
Samples grown in natural conditions Samples grown in greenhouse conditions The place where plant samples were taken Samples grown in a sterile environment under laboratory conditions Number of roots 7 A 20A2 A 5A0.
5A0.
4A0.
0A1.
5A0.
0A1.
1A1.
0A1.
A MS IAA 5.
0 mkM A B5 NAA 5.
0 mkM 4A2.
5A2.
0A1.
2A2.
4A1.
0A1.
Root length .
Root length .
7 A 20A2 A 4A1.
5A2.
5A0.
5A1.
5A2.
5A2.
4A1.
A MS IAA 5.
0 mkM 5A0.
1A1.
3A1.
A BDS NAA 5.
0 mkM A MS IMA5.
0 mkM
A BDS
Number of roots Nutrient medium Table 7.
Indications of growth and development of L.
inebrians regenerated plants .
A).
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Another phenomenon of interest in microplant cultivation is the formation of stolons in L.
The development of stolons at the rooting stage was recorded only in 3 microcuttings grown on A B5 medium with high sucrose content, but all of them had the same morphology.
At the top of the formed stolon there is a bud of regeneration - a special vegetative part, which is formed from the maternal specimens and can later replace it.
Adventitious roots also begin to form at the base of a special new vegetative part.
Thus, under in vitro conditions.
inebrians microplants underground organs in accordance with those grown in natural conditions.
From the above observations, we can conclude that differences in the level of underground organs affect rooting.
The size of roots obtained in vitro indicates the successful adaptation of regenerated plants to nonsterile conditions in the future .
To evaluate the effect of sucrose concentration on the growth and development of plants during rooting, we used 30.
, 40.
We obtained 50.
0 and 70.
0 g/L of In the research, it was found that the increase in the concentration of sucrose .
0 g/L ga.
led to the acceleration of root growth in L.
inebrians plants (Figure .
To adapt to non-sterile conditions, vegetative parts of at least 5.
0Ae5.
5 cm in size with a developed root system were selected.
In this case, a mixture of sphagnum moss, peat and sand .
, crushed coconut fiber was used as a substrate .
The regenerants of the studied samples were kept in laboratory and greenhouse conditions at a temperature of 23A2 A and during the corresponding light period, the development of new leaves did not take place and caused a slowdown in A similar result was observed in the transition step from the used substrate and root In this case, a resting period was determined at a temperature of 23A2 A.
The use of sphagnum moss as a substrate is suitable only as an initial step in adaptation.
This is due to the need to re-transplant these regenerants to a more diverse substrate when growing in a greenhouse or in open ground .
In this substrate, an increase in the length of the roots and the appearance of root hairs were observed, but the process of leaf regrowth was not noted after 4Ae5 months of work.
It was more effective to use a mixture of peat and sand, as well as crushed greens and sand.
At the same time, adaptation to room conditions by alternation of high and low temperatures was carried out on the basis of adding growth-promoting hormones to transplanting of regenerated plants.
Thus, keeping the planted plants in the substrate for 4Ae6 weeks in a dark place at a temperature of 5A2 A and transferring them to a temperature regime of 23A2 A, inducing them from the dormant period and accelerating the assimilation of the leaves came.
was developed only in samples with a developed root system.
It should be noted that when grown in room conditions, even after cold stratification, the leaf quickly died and a second dormancy occurred.
This can be explained by the relatively high temperature and dryness of the air.
This adaptation made it possible to eliminate the secondary dormancy period by transferring the planted plants to the greenhouse.
Acclimatization of regenerated plants in a greenhouse .
old storag.
was carried out by another research group .
in the case of cold air temperature (October-Decembe.
In both options of the substrate, the growth of the first leaf was recorded in February-March.
The average air temperature in the cold part of the greenhouse during this period was 8.
5A1.
4 A.
Activation of growth processes of regenerative plants at positive Table 9.
Efficiency of adaptation of L.
inebrians regenerated plants to ex vitro conditions.
Adaptation (%) Options Peat and sand .
Coconut fiber and sand .
Samples grown in a sterile environment under laboratory conditions Samples grown in greenhouse conditions Samples grown in natural conditions J.
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Figure 7.
Microclonal breeding of L.
inebrians in vitro and adaptation to species conditions (A-B - a callus formed from excrement.
C - formation of a cyst.
D - rooting process.
E Ae air conditioning.
F-ready temperatures .
n the greenhous.
was observed, regrowth when the temperature exceeds 5 A .
In this.
inebrians samples showed a normal seasonal rhythm of development.
At the same time, the synchronous sprouting of tissues is characteristic of all studied samples.
The degree of adaptation of plants was evaluated by the number of shoots with ex vitro formed leaves (Table .
According to the obtained data, the optimal substrate for adaptation of the regenerative plants of the studied samples of L.
inebrians growing in different conditions is a mixture of coconut fiber and sand .
: .
This made it possible to develop 7% hashish in plants adapted to a temperate climate with well-developed leaves and root The type of substrate also affected the level of development of leaf organs of plants .
Thus, when a mixture of peat and sand .
was used, the presence of the first leaf in the studied samples of L.
inebrians was recorded after 4 months of cultivation, and the presence of one leaf in each L.
inebrians tissue was recorded.
At the same time, the percentage of tissues successfully adapted with absorbent leaves did not exceed 41.
The vegetation period was 3 months from the growth of the first leaf to the death of the last one.
At the end of the specified period.
inebrians specimens growing in natural, laboratory, and greenhouse conditions were characterized by one large and one or two human tissues.
When a mixture of peanut flour and sand .
was used, the adaptation efficiency of all studied samples growing under different conditions varied from 71.
9 to Depending on the sample, regrowth of the first assimilative leaf was noted 5Ae9 weeks after transfer of in vitro rooted L.
inebrians tissues to non -sterile conditions .
old part of the greenhous.
The regenerants grown in the temperature regime of 5A1.
4 A helped the development of 1Ae3 assimilatory leaves, depending on the samples grown in different conditions.
The cultivation of plants in the next step biotechnological method was carried out in the experimental area of the At the same time, 3.
5Ae4.
0 months have passed after the first leaf appears.
During this period, the effect of cold stratification .
A) on rooting from shoots in vitro was studied.
The favorable temperature conditions had a positive effect on the development of ex vitro shoots of the The growth of the first leaf in L.
was observed in 48Ae56 days after the start of adaptation in a mixture of coconut fiber and sand.
Regrowth occurred twice as slowly after 89Ae94 days in samples without Rast temperature Therefore, temperatures are transferred to ex vitro conditions, it helps to eliminate the dormant state of vegetative organs and their germination.
The rest period is necessary for the normal development of geophytes, without it during the annual cycle, the growth of J.
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tissues slows down and the development of generative organs does not take place.
Choosing the optimal duration of the period with the right temperature helps the rapid development of leaves and roots .
Thus, the method of cultivation at a positive temperature .
A) is the most effective in terms of rooting and tissue formation, as it allows to obtain renewed plants with a developed root system .
5Ae8.
0 per shoot, 0 roo.
At the same time, the use of nutrient medium supplemented with auxins .
0 AAM NAA or IBA) promotes the formation of a more developed root system in L.
Changes in the concentration of sucrose in the nutrient medium have almost no effect on the growth and development of micro-plants during the rooting stage.
The results of our research showed that plant development at the right temperature not only promotes more intensive rhizogenesis, but also promotes faster development of regenerating plants in ex vitro conditions.
At the same time, the adaptation of regenerant L.
inebrians plants grown in different conditions showed that the most optimal substrate was a mixture of walnut meal and sand .
, with the growth and development of the plant under the selected different conditions.
A 82.
increase in adaptability.
A network of in vitro samples of L.
inebrians under different conditions was created based on the microclonal display systems developed in the research.
At the same time, the collection includes more than 10,000 micro-plants in the state of active growth and more than 1,000 samples of them in the state of slow We have developed a schematic system related to microclonal display, maintenance and development of L.
The scheme includes the selection of primary explants, cultivation and preservation of tissues in vitro, rooting, adaptation to different climatic conditions and subsequent ex vitro conditions (Figure .
CONCLUSIONS
In vitro microcloning of L.
inebrians, the intensity of the processes .
allus tissue formation and proliferation.
shoot formation, root formatio.
explant, explant sterilization method, depends on factors such as combinations of phytohormones used in the nutrient medium.
In the option where leaf axil buds and leaf fragments were used as initial explants.
MS medium containing a combination of BAP .
mg/L) NAA .
4 mg/L), shoots and leaf fragments located at the tip of the stem when lakshas are used, the intensity of callus tissue formation is high in the combination of BAP .
Ae4 mg/L) NAA .
4 mg/L) in MS nutrient The obtained information provides the possibility of effective reproduction of L.
plant and serves to organize plantations of this AUTHOR INFORMATION Corresponding Author Kumush Sultonova Ai Department of Genetics and Biotechnology.
Samarkand State University.
Samarkand-140104 (Uzbekista.
org/0009-0006-5246-5821 Email: sultonovakumush1990@gmail.
Authors Begali Alikulov Ai Department of Genetics and Biotechnology.
Samarkand State University.
Samarkand-140104 (Uzbekista.
org/0000-0002-0875-2210 Normat Xasanov Ai Department of Genetics and Biotechnology.
Samarkand State University.
Samarkand-140104 (Uzbekista.
org/0009-0001-7662-830X Yulduz Abdullayeva Ai Department of Biotechnology.
Samarkand State Veterinary Medicine.
Animal Husbandry and Biotechnology University.
Mirzo Ulugbek-77 (Uzbekista.
org/0000-0002-6112-6665 Shahnoza Sharopova Ai Department of Biology.
Bukhara State University.
Bukhara705018 (Uzbekista.
org/0000-0002-6112-6665 Baxtiyor Jabborov Ai Department of Biology.
Bukhara State University.
Bukhara-705018 (Uzbekista.
org/0009-0004-4361-9556 Hulkar Hotamova Ai Department of Preclinical Subjects.
Zarmed University.
Bukhara-200100 (Uzbekista.
org/0009-0006-2001-6925 Mamadiyarova Dilshoda Ai Department of Physiology.
Samarkand State Medical J.
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Appl.
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Sci.
University.
Samarkand-140104 (Uzbekista.
org/0000-0001-9596-9401 Boymurodov Khusniddin Ai Department of Biotechnology.
Samarkand State Veterinary Medicine.
Animal Husbandry and Biotechnology University.
Mirzo Ulugbek-77 (Uzbekista.
org/0009-0008-1778-6253 Author Contributions .
and M.
the experiments.
, and B.
and K.
statistically analyzed results.
All authors wrote the draft of the manuscript.
and N.
conducted the critical revision of the manuscript.
and B.
worked out the concept and design, supervised and funded the All authors read and approved the final manuscript.
Conflicts of Interest The authors declare no conflict of interest.
ACKNOWLEDGEMENT
The authors would like to thank the Molecular Biotechnology Laboratory of Samarkand State University named after Sharof Rashidov.
REFERENCES