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Original article
203
IN VITRO MICROPROPAGATION OF AJUGA TURKESTANICA (REGEL) BRIQ.: A
PATHWAY TO PATHOGEN-FREE SEEDLINGS
Kushiev Kh.Kh.,
Mannabova M.M.
Research Institute Agrobiotechnologies and
Biochemistry of Gulistan State University
Email: mmannabova@gmail.com
ABSTRACT:
In recent years, the application of in vitro techniques has become a cornerstone in
the conservation and propagation of plant biodiversity. This study focuses on the
micropropagation of Ajuga Turkestanica, a medicinal plant species, under in vitro conditions.
The research aimed to identify the optimal nutrient media and growth regulators for the efficient
propagation of pathogen-free seedlings. The results demonstrated that the B5 nutrient medium
supplemented with 0.1 µM BAP (6-Benzylaminopurine) was the most effective for the
proliferation of Ajuga Turkestanica. Additionally, the BDS medium enriched with 5.0 µM BAP
and 2.0 µM NAA (Naphthalene Acetic Acid) showed high efficiency in promoting shoot
regeneration. The study also highlighted the importance of cytokinins, particularly BAP, over
other growth regulators like TDZ (Thidiazuron) and Kinetin in the multiplication phase.
Keywords:
Ajuga Turkestanica, in vitro, micropropagation, callus, MS medium, phytohormones
INTRODUCTION
The rapid decline in biodiversity due to ecological changes, anthropogenic activities, and
overexploitation of natural resources has necessitated the development of advanced conservation
strategies [1]. Among these, in vitro micropropagation has emerged as a powerful tool for the
preservation and propagation of endangered plant species [2]. This technique not only allows for
the rapid multiplication of plants but also ensures the production of pathogen-free seedlings,
which is crucial for the restoration of natural populations [3].
The genus Ajuga, particularly Ajuga Turkestanica, is of significant interest due to its medicinal
properties. However, the natural populations of this species are under threat due to
overharvesting and habitat destruction [4]. Therefore, the development of efficient in vitro
propagation protocols is essential for its conservation and sustainable utilization [5].
This study aims to explore the morphogenetic responses of Ajuga Turkestanica to various growth
regulators and nutrient media, with the ultimate goal of establishing an optimal protocol for its
micropropagation.
MATERIALS AND METHODS
Plant Material and Sterilization
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The study utilized Ajuga Turkestanica explants collected from greenhouse-grown plants. The
explants, consisting of shoot tips and nodal segments, were carefully excised and washed under
running tap water for 30 minutes to remove surface contaminants. For sterilization, the explants
were treated with 70% ethanol for 30 seconds, followed by immersion in a solution of 0.1%
mercuric chloride (HgCl₂) and 0.1% Tween 80 for 30 minutes. After sterilization, the explants
were rinsed three times with sterile distilled water to remove any traces of the sterilizing agents.
This protocol resulted in a high survival rate of 87-96%, with minimal contamination observed
during the initial culture phase [6].
Nutrient Media and Growth Regulators
The explants were cultured on two primary nutrient media: MS (Murashige and Skoog) [7] and
VM (Van der Meer) [8]. Both media were supplemented with different concentrations of growth
regulators, including BAP (6-Benzylaminopurine), Kinetin, and NAA (Naphthalene Acetic
Acid). The pH of the media was adjusted to 5.8 using 1N NaOH or HCl before autoclaving at
1210C for 15 minutes. The media were solidified with 0.8% agar (w/v) and dispensed into sterile
culture vessels.
Experimental Design
The study was designed to evaluate the effects of different growth regulator combinations on the
morphogenetic potential of Ajuga Turkestanica. A total of 10 different media formulations were
prepared, each with varying concentrations of BAP (0.1–5.0 µM) and NAA (0.02–2.0 µM). The
explants were cultured in these media under controlled environmental conditions, with a 16-hour
photoperiod provided by cool-white fluorescent lamps (40 µmol m-2 s-1) and a temperature
maintained at 25±20C. Each treatment consisted of 50 explants, replicated three times to ensure
statistical reliability [9].
Data Collection and Analysis
The regeneration frequency (percentage of explants forming shoots), shoot proliferation (number
of shoots per explant), and callus formation (percentage of explants forming callus) were
recorded over a period of 8 weeks. Data were analyzed using one-way ANOVA, and mean
values were compared using Duncan’s Multiple Range Test (DMRT) at a significance level of
p<0.05. Statistical analyses were performed using SPSS software (version 25.0) [10].
RESULTS
The results of the study are presented in detail, with a focus on the effects of different growth
regulators and nutrient media on the shoot regeneration, callus formation, and rooting of Ajuga
Turkestanica. The findings are supported by tables and figures to provide a clear and thorough
explanation of the outcomes.
Effect of Growth Regulators on Shoot Regeneration
The results indicated that the B5 medium supplemented with 0.1 µM BAP was the most effective
for shoot regeneration, with a regeneration frequency of 56.5% and an average of 5.0 ± 1.5
shoots per explant (Table 1). The BDS medium enriched with 5.0 µM BAP and 2.0 µM NAA
also showed high efficiency, with a regeneration frequency of 66.2% and an average of 4.0±0.8
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shoots per explant. In contrast, the MS and VM media showed lower regeneration frequencies
and fewer shoots per explant, indicating that the B5 and BDS media were more suitable for the
micropropagation of Ajuga Turkestanica.
Table 1. Effect of Different Growth Regulator Combinations on Shoot Regeneration
MediumBAP (µM)NAA (µM)Regeneration Frequency (%) Average Shoots per Explant
B5
0.1
0.02
56.5
5.0±1.5
BDS
5.0
2.0
66.2
4.0±0.8
MS
2.0
0.1
49.4
2.9±0.7
VM
2.0
0.1
41.8
2.9±0.5
Callus Formation and Indirect Regeneration
The study also observed callus formation in response to low concentrations of BAP (0.5 µM) and
TDZ (0.5 µM). However, the regeneration frequency from callus was relatively low, with only
28% of the callus-derived shoots developing into viable plantlets (Table 2). This suggests that
direct organogenesis is more efficient for the micropropagation of Ajuga Turkestanica compared
to indirect regeneration via callus.
Table 2. Callus Formation and Indirect Regeneration
Growth RegulatorConcentration (µM) Callus Formation (%) Regeneration Frequency (%)
BAP
0.5
37.0
32.0
TDZ
0.5
38.0
28.0
Rooting and Acclimatization
For rooting, the regenerated shoots were transferred to a hormone-free BDS medium, where they
developed roots within 4-5 weeks. The rooted plantlets were then acclimatized in a greenhouse
environment. The acclimatization process involved transferring the plantlets to a substrate
composed of a mixture of sand and peat (1:1 v/v) under high humidity conditions (80-90%) for
the first two weeks, followed by gradual exposure to ambient conditions. The survival rate of the
acclimatized plantlets was 85%, indicating successful adaptation to ex vitro conditions.
Discussion
The results demonstrate that the B5 medium supplemented with 0.1 µM BAP is highly effective
for shoot regeneration in Ajuga Turkestanica, with a high regeneration frequency and a
significant number of shoots per explant. The BDS medium enriched with 5.0 µM BAP and 2.0
µM NAA also showed promising results, particularly in terms of regeneration frequency. These
findings are consistent with previous studies that have highlighted the importance of cytokinins,
particularly BAP, in promoting shoot proliferation in in vitro cultures [11, 12].
Callus formation was observed in response to low concentrations of BAP and TDZ, but the
regeneration frequency from callus was relatively low. This suggests that direct organogenesis is
a more efficient pathway for the micropropagation of Ajuga Turkestanica, as it bypasses the
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callus phase and directly induces shoot formation from explants. This is in line with studies on
other medicinal plants, where direct regeneration has been shown to be more reliable and
efficient [13, 14].
The successful rooting and acclimatization of the regenerated plantlets further validate the
effectiveness of the in vitro propagation protocol. The high survival rate of 85% indicates that
the plantlets were well-adapted to ex vitro conditions, which is crucial for the large-scale
production and conservation of Ajuga Turkestanica.
Conclusion
In recent years, the application of in vitro techniques has become a cornerstone in the
conservation and propagation of plant biodiversity. This study focuses on the micropropagation
of Ajuga Turkestanica, a medicinal plant species of significant interest due to its therapeutic
properties, under in vitro conditions. The research aimed to identify the optimal nutrient media
and growth regulators for the efficient propagation of pathogen-free seedlings.
The results demonstrated that the B5 nutrient medium supplemented with 0.1 µM BAP (6-
Benzylaminopurine) was the most effective for shoot proliferation, achieving a regeneration
frequency of 56.5% and producing an average of 5.0 ± 1.5 shoots per explant. Additionally, the
BDS medium enriched with 5.0 µM BAP and 2.0 µM NAA (Naphthalene Acetic Acid) showed
high efficiency in promoting shoot regeneration, with a regeneration frequency of 66.2% and an
average of 4.0±0.8 shoots per explant.
The study also highlighted the importance of cytokinins, particularly BAP, over other growth
regulators like TDZ (Thidiazuron) and Kinetin in the multiplication phase. Callus formation was
observed in response to low concentrations of BAP and TDZ, but the regeneration frequency
from callus was relatively low (28%), indicating that direct organogenesis is a more efficient
pathway for micropropagation.
For rooting, the regenerated shoots were transferred to a hormone-free BDS medium, where they
developed roots within 4-5 weeks. The rooted plantlets were successfully acclimatized in a
greenhouse, with a survival rate of 85%.
This study establishes an efficient protocol for the in vitro micropropagation of Ajuga
Turkestanica, ensuring the rapid production of pathogen-free seedlings and providing a reliable
method for the conservation and sustainable utilization of this valuable medicinal plant.
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