Evaluation of the synergistic efficiency of hormonal, biological and physical stimuli in the induction and growth of callus in lavender

Mohammed Abdulgafor Mohammed; Masara Dafer Alani; Ahmed O. Mashaan; Ali F. Almehemdi

doi:10.12688/f1000research.173637.1

Home Browse Evaluation of the synergistic efficiency of hormonal, biological and...

ALL Metrics

-

Views

-

Downloads

Get PDF

Get XML

Export

▬

✚

Research Article

Evaluation of the synergistic efficiency of hormonal, biological and physical stimuli in the induction and growth of callus in lavender

[version 1; peer review: 1 approved with reservations, 1 not approved]

Mohammed Abdulgafor Mohammed ¹, Masara Dafer Alani², Ahmed O. Mashaan¹, Ali F. Almehemdi³

PUBLISHED 18 Mar 2026

Author details Author details

¹ Department of Biotechnology, University of Anbar, Ramadi, 31001, Iraq
² Department of Studies and Planning, University of Anbar, Ramadi, 31001, Iraq
³ Center of Desert Studies, Department of conservation Agriculture, University of Anbar, Ramadi, 31001, Iraq

Mohammed Abdulgafor Mohammed
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration

Masara Dafer Alani
Roles: Data Curation, Formal Analysis, Investigation, Resources

Ahmed O. Mashaan
Roles: Funding Acquisition, Software, Validation, Visualization, Writing – Review & Editing

Ali F. Almehemdi
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Fallujah Multidisciplinary Science and Innovation gateway.

Abstract

Background

Lavender is a crucial source of secondary metabolites with medicinal and industrial importance. Its exploitation faces challenges due to the limited availability of raw materials and their sensitivity to climate. Tissue culture techniques, particularly callus culture, represent the most sustainable method for the continuous production of these active compounds.

Methods

This study focused on developing an effective protocol for inducing and stimulating callus cultures in lavender plants. In the growth phase, different concentrations of 2,4-D and benzyladenine were assessed. In the induction phase, different concentrations of the growth regulator 2, 4-D (0, 1, 2 and 3) mg L⁻¹ were tested under light and dark conditions. The experimental approach investigated three primary variables hormone concentration, environmental conditions and the type of nutrient medium. A comparative analysis was also completed between two nutrient media, MS and B5, supplemented with different Chitosan concentrations (0, 50, 100 and 200) mg. L⁻¹.

Results

The results indicated that the optimal concentration for callus induction was 1 mg L ⁻¹ of 2,4-D, with the highest callus formation rate 95% recorded under dark conditions. Among all treatments, the combination of 2, 4-D 1 mg L⁻¹ and BA 2 mg L⁻¹ was the most effective for sustainable callus growth. Regarding the nutrient medium, B5 was more efficient than the MS medium. For the biostimulants, Chitosan at 100 mg L⁻¹ recorded the highest efficiency in both fresh and dry callus weights.

Conclusions

This study underscores the necessity of integrating hormonal, nutritional and environmental factors for optimal callus production.

Keywords

Lavender, induction callus, biostimulants, hormonal, physical, biological.

Corresponding author: Mohammed Abdulgafor Mohammed

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2026 Mohammed MA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Mohammed MA, Dafer Alani M, Mashaan AO and Almehemdi AF. Evaluation of the synergistic efficiency of hormonal, biological and physical stimuli in the induction and growth of callus in lavender [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2026, 15:407 (https://doi.org/10.12688/f1000research.173637.1) First published: 18 Mar 2026, 15:407 (https://doi.org/10.12688/f1000research.173637.1) Latest published: 18 Mar 2026, 15:407 (https://doi.org/10.12688/f1000research.173637.1)

Introduction

Lavender (Lavandula angustifolia Mill.) is a member of the Lamiaceae family of flowering plants and is the most widely cultivated of the family, which also has over 39 other genera (Vârban et al., 2022, Kumar et al., 2025). The varied secondary metabolites in lavender provide a broad range of industrial uses in the pharmaceutical, cosmetic, and food industries (Crisan et al., 2023). The medicinal value of lavender has been well established over the centuries, considering the wide range of advanced herbal diseases and its other numerous pharmacological attributes, including antimicrobial, antifungal, antispasmodic and other active antioxidant (Hassiotis and Vlachonasios, 2025) as well as digestive function regulating (Firoozeei et al., 2021) properties. It is also used to treat gastrointestinal disorders, cardiovascular diseases, and respiratory and urinary disorders, anxiety, depression, and sleep disorders (Kumar et al., 2025), mood swings (Mehrabian et al., 2022), chronic pain (Pehlivan and Karadakovan, 2019), restless legs syndrome (Ghasemi et al., 2021), migraines (Yuan et al., 2021), and behavioural disorders in dementia patients. (Scuteri et al., 2019) as well as improving physical symptoms such as blood pressure and respiratory rate (Contrada et al., 2021).

There are difficulties in the plant based pharmaceutical industry related to the sourcing of raw materials attributable to the isolation of active compounds being arduous due to plants being low in active compounds or active compounds being affected by climatic conditions. Under these circumstances, the cultivation of plant tissues offers exciting possibilities, as it promotes the incessant active compounds synthesis irrespective of the season. This facilitates the overcoming of climatic and geographical limitations, hence aiding in the supply of growing the demand for medicinal and aromatic plants. This is one of the refined and improved strategies in the biotechnological domain that aims or is directed towards the amplified generation of secondary metabolites relevant for the industry (Ozyigit et al., 2023). This necessitates the adjustment and calibration of every single component in every single process of culture tissue namely, nutrient medium, the different and varied growth hormones, and the different types of stimulants to maximise biomass and secondary metabolites (Adil et al., 2025). The technology of callus culture is one of the sustainable methods for the valuable plant compounds and for the preservation of the habitat of the medicinal plants (Abed et al., 2020, Babich et al., 2020, Alrawi et al., 2022). Compounds can be derived from callus tissue in a lab setting without having to use whole plants, and this method can be commercially scaled by employing bioreactors for compound bioprocessing. in vitro callus tissue development depends on a number of factors; for example, a plant’s genetics, the plant organ type, and the composition of the nutrient medium and the growth regulators (Miri and Roughani, 2018). Furthermore, callus culture technology depends on the growth and sustained production and the qualitative and quantitative production of active compounds. The optimum environment must also be available. The importance of growth regulators during callus formation is significant because, in some cases, a tissue may be unresponsive or may even die if the medium’s hormone levels are set higher or lower than the tissue’s internal level (Yegorova et al., 2020). Previous research indicates that the callus formation is more pronounced when the nutrient medium is enhanced with growth regulators, specifically auxins and cytokinins (Jabr and Mohsen, 2024). The optimization of the hormonal milieu is only attained with the moderators acting synergistically within set ranges. Compared to using each of them separately (Tilkat and Onay, 2009). The balance between auxins and cytokinins in the nutrient medium is a key determinant that stimulates cell division and initiates callus formation. In addition, biostimulants such as chitosan can enhance biomass production and secondary metabolites in plant tissue cultures (Javed et al., 2025). It acts as a plant defence enhancer capable of activating various physiological and biochemical processes in different plant species (Iber et al., 2022). Chitosan is a biopolymer derived from chitin, which can be produced economically by recycling the exoskeletons of crustaceans. Given the notable lack of studies on callus induction in lavender, this study aims to evaluate the efficiency of biological and physical stimuli in inducing callus and promoting its growth in this plant.

Materials and methods

Plant materials

Lavender seeds were obtained from a specialised seed production company Al-Wadi seed Company located south of the capital Baghdad, Iraq. The seeds were stored in a laboratory at a temperature of 15°±2 C until use.

Establishment of tissue culture

A nutrient medium consisting of half MS salts (Murashige and Skoog, 1962) and gibberellic acid at a concentration of 0.5 mg L ⁻¹ and 30 g L⁻¹ sucrose was prepared. The solution was then transferred to a graduated cylinder and the volume was made up to 1000 mL with sterile distilled water. The pH of the medium was adjusted to 5.7 ± 0.1 using NaOH or HCl solutions at a concentration of (0.1). 7 g. per litre of agar was added as a solidifying agent. The medium was mixed thoroughly and heated with stirring until it reached full boiling point (100° C) to ensure that the agar dissolved.

The medium was distributed into glass culture vessels 10 ml vessel. The containers were sealed with tight fitting lids and sterilised in an autoclave Model ABC at a temperature of 121° C and a pressure of 1.04 kg cm for 15 minutes. The sterilised medium was stored at 25° C until use. The seeds were sterilised according to the method used by the researcher, whereby the seeds were washed with running water to remove floating seeds and dust. The healthy seeds were transferred to the laminar air cabinet, then a 3% sodium hypochlorite sterilising agent was added to the seeds for 15 minutes with continuous stirring (Al-Alwani and Mohammed, 2022).

The seeds were rinsed with distilled water for 5 minutes and the process was repeated three times to ensure that the sterilising agent was removed. The sterilised seeds were planted on the prepared nutrient medium. The planted seeds were then transferred to the growth chamber at a temperature of 25° C and a light intensity of 1000 lux for 16 hours of light and 8 hours of darkness (Sekhi et al., 2025). After four weeks, seedlings ready for use were obtained for callus induction experiments. The chemicals listed in Table 1 were used in this study.

Table 1. Reagents used in the study, including the amount used, supplier information, and catalogue numbers.

Reagent item	Quantity	Supplier	Catalogue numbers
MS.	4.74 g. L⁻¹	Duchefa Biochemie	M0221
Gamborg B5 medium	3.92 g. L⁻¹	Duchefa Biochemie (Holland)	G0209
Dichlorophenoxyacetic acid	(0,1,2 and 3) mg. L⁻¹	Duchefa Biochemie (Holland)	D0911
Benzyladenine	BA (0,1 and 2) mg. L⁻¹	CDH (India)	022625
Sucrose	30 g. L⁻¹	CDH (India)	030299
Chitosan	(0,50,100, 200) mg. L⁻¹	CDH (India)	046555
Agar Agar	7 g. L⁻¹	Duchefa Biochemie (Holland)	P1001.1000

Callus induction

A nutrient medium based on full MS salts strength was prepared, with varying concentrations of the growth regulator dichlorophenoxyacetic acid (2,4-D) (0, 1, 2 and 3) mg L⁻¹. Then 30 g L⁻¹ of sucrose was added to the medium as a carbon source and 7 g L ⁻¹ of agar as a stabilising agent. The nutrient medium was then sterilised according to the standard protocol. After preparing the media, parts of the leaves were excised and then planted on the prepared nutrient media containing the specified concentrations of the growth regulator 2,4-D. The cultures were incubated in a growth chamber with controlled conditions at a constant temperature of 25 C. To investigate the effect of light on callus induction, the cultures were exposed to two different lighting regimes complete darkness and 1000 lux light intensity under a cyclic light regime 16 hours of light 8 hours of darkness. At the end of the four-week incubation period, data were collected by measuring Induction rate the percentage of tissue sections in which callus was formed, Fresh weight and Dry weight.

The nutrient medium was prepared mainly from full-MS salts strength at a concentration of 4.74 g L⁻¹, with the addition of 30 g L⁻¹ sucrose and 7 g L⁻¹ agar. This medium was then enriched with varying concentrations of auxin 2,4-D at levels of (1, 2, and 3) mg L⁻¹ combined with concentrations of cytokinin benzyladenine BA at levels of (0, 1 and 2) mg L⁻¹. After sterilising the media, callus pieces of a fixed weight 150 mg were transferred to it three days after preparation. The cultures were incubated in complete darkness at a constant temperature of 25° C for four weeks. At the end of the incubation period, data were collected by measuring the fresh weight and then the dry weight of the callus growth.

The role of nutrient components and chitosan in promoting callus growth

Two types of nutrient media MS and B5 were used, prepared with different concentrations of chitosan (0, 50, 100 and 200) mg L⁻¹, with the addition of 30 g L⁻¹ sucrose and 7 g L⁻¹ agar. Callus pieces weighing 150 mg were transferred to these sterile media containing different concentrations of chitosan. After a four-week incubation period, growth rates were determined by measuring the fresh and dry weights of the callus formed.

Statistical analysis

The data were statistically analysed using GenStat statistical software (Gen 12 ed) (Payne et al., 2009) according to a completely randomised design (C.R.D.), with ten replicates for each treatment. The treatments were arranged in a factorial arrangement. The means were compared on LSD test after partitioning the variance components via fisher's test by using probability ≤ 0.05 whereas, the callus pecentage was analysed using originPro 2025b (origin 2025).

Results and discussion

The effect of light conditions and 2,4-D concentrations on callus formation

Statistical analysis, as shown in Figure 1 revealed that the growth regulator 2,4-D had a significant effect on callus induction. It was found that the optimal concentration for callus formation from leaf tissue was 1 mg L⁻¹ of 2,4-D added to the nutrient medium, which reached 95%, while an increase in concentration above this level led to a significant decrease in the rate of callus formation.

Figure 1. Effect of light conditions and 2,4-D concentrations on callus formation.

X-axis (Abscissa): 2,4-D concentrations and illumination type: Dark and light, y-axis (ordinate): Callus percentage.

On the other hand, lighting had a relatively limited effect on callus induction compared to the effect of 2,4-D growth regulator concentration. The highest callus production values were recorded under complete darkness, and all auxin containing treatments outperformed their counterparts grown under lighting conditions ( Figure 1).

Effect of illumination and 2,4-D concentrations on callus fresh weight (g)

The results of statistical analysis according to the data shown in Table 2 showed a statistically significant difference in the fresh weight values of callus induced from lavender leaf tissue, where the fresh weight of callus differed according to concentration (0, 1, 2 and 3) mg L⁻¹ of 2,4-D added to the MS nutrient medium. The auxin concentration of 1 mg L⁻¹ showed the highest significant value for callus fresh weight, which reached 0.796 g. An increase in auxin concentration above this level led to a gradual decrease in the rate of callus formation, indicating that high auxin concentrations have an inhibitory effect on the callus formation process. It should be noted that the auxin free treatment 0 concentration did not show any visible or statistical response in callus formation, confirming the pivotal role of auxin in initiating induction and stimulating cell division in plant leaf tissues. As for the effect of lighting, the results showed a clear superiority of callus growth under conditions of complete darkness for all concentrations studied, with the average fresh weight values under these conditions showing a remarkable superiority of 0.557 g compared to their counterparts grown under lighting conditions, which reached 0.527 g. The effect of the light factor can be observed in Figure 2, where the callus appeared yellowish brown, while in the dark, the callus appeared white yellowish. This difference in response may be due to the inhibitory effect of light on callus induction and growth processes. It can be concluded that the process of callus induction from lavender leaf tissue requires a delicate balance between the optimal concentration of auxin and suitable environmental conditions, emphasising the complex interaction between hormonal and environmental factors in regulating growth and differentiation processes in plant cells. The results also show that the highest fresh weight of callus was achieved at a concentration of 1 mg L⁻¹ of regulator 2,4-D when grown under dark conditions, reaching 0.830 g. While no value was recorded for fresh weight of callus (0 g) in treatments free of auxin under dark or light conditions. This confirms the importance of auxin as a basic factor in callus formation.

Table 2. Effect of illumination and 2,4-D concentrations on callus fresh weight (g).

Light conditions	2,4-D (mg L⁻¹)
Light conditions	0	1	2	3	Mean
Dark	0.000	0.830	0.752	0.647	0.557
Light	0.000	0.762	0.723	0.624	0.527
LSD_0.05	0.014				0.007
Mean	0.000	0.796	0.737	0.635
LSD_0.05	0.010

Figure 2. Effect of illumination (light (A) and dark (B)) on the growth of callus induced from lavender leaves, after four weeks.

Effect of illumination and 2,4-D on callus dry weight of callus (g)

Effect of auxin on dry weight of callus the results of statistical analysis shown in Table 3. Indicate a clear variation in biological response when treating lavender Lavandula spp. leaf tissues with different concentrations of the growth regulator 2,4-D. A concentration of 1 mg L⁻¹ of 2, 4-D recorded the highest significant dry weight of callus 0.064 g. An increase in concentration to 2 mg L⁻¹ resulted in a significant decrease in dry weight 0.058 g, while a concentration of 3 mg L⁻¹ recorded a decrease of 0.046 g. The auxin free treatment did not show any visible or statistical response in callus formation, confirming the pivotal role of auxin in the process of induction and initiation of cell division.

Table 3. Effect of light conditions and 2,4-D concentrations on callus dry weight (g).

Light conditions	2,4-D (mg L⁻¹)				Mean
Light conditions	0	1	2	3	Mean
Dark	0.000	0.068	0.059	0.048	0.044
Light	0.000	0.061	0.057	0.044	0.040
LSD_0.05	0.002				0.001
Mean	0.000	0.064	0.058	0.046
LSD_0.05	0.001

Statistical analysis showed a significant interaction between auxin concentration and lighting conditions. Treatments under lighting conditions recorded an average dry weight of 0.040 g, while treatments in complete darkness significantly exceeded this with an average dry weight of 0.044 g.

Effect of 2,4-D and BA on fresh weight of callus (g)

The results in Table 4, Showed that adding synthetic auxin of type 2,4-D at different concentrations to the nutrient medium had a significant statistically significant effect on the fresh weight of the callus formed. A clear response to the concentrations added to the nutrient medium was observed, with the low concentration 1 mg L⁻¹ outperforming all other treatments, recording the highest significant value for fresh weight of 0.920 g. With an increase in hormone concentration to 2 and then 3 mg L⁻¹, a gradual and significant decline in growth efficiency was observed, with the fresh weight of callus decreasing to 0.811 g and 0.632 g, respectively. This decrease indicates the possibility of an inhibitory effect of high concentrations of auxin on the process of development and growth.

Table 4. Effect of 2,4-D and BA on fresh weight (g) of callus.

BA (mg L⁻¹)	2,4-D (mg. L⁻¹)			Mean
BA (mg L⁻¹)	1	2	3	Mean
0	0.838	0.728	0.628	0.731
1	0.880	0.843	0.636	0.786
2	1.042	0.863	0.634	0.846
LSD_0.05	0.018			0.010
Mean	0.920	0.811	0.632
LSD_0.05	0.010

On the other hand, the results also showed statistically significant differences between the different concentrations of cytokinin benzyladenine (BA) added to the nutrient medium. The effect of BA was characterised by a positive linear relationship, where the growth rate of callus measured by fresh weight increased significantly with increasing hormone concentration. Growth peaked at a concentration of 2 mg L⁻¹, achieving the highest fresh weight value of 0.846 g. In contrast, the treatment containing a concentration of 0 mg L⁻¹ free of cytokinin recorded the lowest fresh weight value of 0.731 g, highlighting the vital importance of cytokinin in supporting cell division and callus formation. The most significant scientific importance of this study lies in evaluating the complementary and reciprocal effects of these two hormones when used together. The results showed a clear superiority of some combinations over others. The addition of a combination of auxin 2,4-D at a concentration of 1 mg L⁻¹ in conjunction with cytokinin BA at a concentration of 2 mg L⁻¹ resulted in the highest total value of 1.041 g. This result reflects a synergistic effect between the two hormones, each enhancing the effect of the other, leading to optimal stimulation of growth and development processes. In contrast, the combination of auxin 2,4-D at a concentration of 3 mg. L⁻¹ with cytokinin at a concentration of 0 mg. L⁻¹ recorded the lowest fresh weight value, which was 0.628 g. This poor performance confirms the negative effects of high auxin concentrations in the absence of a balancing factor such as cytokinin, supporting the hypothesis that the balance between these two hormones, rather than the presence of only one, is the decisive factor in directing plant cell growth and differentiation in tissue cultures.

Effect of 2,4-D and BA on dry weight of callus (g)

The results in Table 5 showed a clear response when auxin was added at different concentrations to the nutrient medium. The concentration of 1 mg L⁻¹ significantly outperformed all other treatments, recording the highest dry weight of callus at 0.064 g. This result indicates that this concentration is optimal for stimulating cell division and hypertrophy, which positively affects biomass accumulation. However, a gradual and significant decline in growth efficiency was observed with increasing auxin concentration. Dry weight decreased to 0.060 g at a concentration of 2 mg L⁻¹ and to 0.044 g at a concentration of 3 mg L⁻¹. This decrease indicates that higher concentrations of auxin may have an inhibitory or toxic effect on cells, hindering their growth and limiting callus formation, which is common in plant responses to growth regulators when the optimum limit is exceeded.

Table 5. Effect of 2,4-D and BA on dry weight (g) of callus.

BA (mg L⁻¹)	2,4-D (mg L⁻¹)			Mean
BA (mg L⁻¹)	1	2	3	Mean
0	0.065	0.055	0.041	0.054
1	0.062	0.060	0.042	0.055
2	0.070	0.065	0.050	0.062
L.S.D_0.05	0.002			0.001
Mean	0.065	0.060	0.044
L.S.D_0.05	0.001

The results showed statistically significant differences in the dry weight of callus when different concentrations of cytokinin were added. The effect of cytokinin was characterised by a positive linear relationship, with the dry weight of induced callus increasing significantly with increasing hormone concentration. This effect peaked at a concentration of 2 mg L⁻¹, achieving the highest dry weight value of 0.062 g. This confirms the vital role of cytokinin in regulating cell division and differentiation, which is a complementary and important factor in the callus induction process.

The greatest scientific value of this study lies in assessing the combined and complementary effects of auxin and cytokinin hormones, as it is known that the balance between them is the main driver of plant cell growth. In this context, the results showed a significant advantage for the combination of auxin at a concentration of 1 mg. L⁻¹ with cytokinin at a concentration of 2 mg. L⁻¹. This combination achieved the highest dry weight value for callus 0.070 g. This outstanding performance is attributed to the synergistic effect between the two hormones, where auxin at its optimal concentration 1 mg. L⁻¹ reduces cell elongation, while cytokinin at its optimal concentration 2 mg L⁻¹ promotes cell division and prevents differentiation, which together leads to optimal cell mass enlargement and higher dry matter accumulation.

In contrast, the combination with the highest concentration of auxin 3 mg. L⁻¹ and a complete absence of cytokinin 0 mg L⁻¹ recorded the lowest dry weight value, at 0.041 g. These results confirm that the success of callus induction and its maximum growth measured by dry weight depend critically on the selection of the optimal concentration of each plant regulator separately and more importantly, on the selection of the optimal combination of the two. The interaction between auxin and cytokinin is what directs the final physiological response of plant tissues.

Effect of nutrient medium components and chitosan on callus fresh weight (g)

The results of statistical analysis, shown in Table 6, revealed a clear variation in the components of different nutrient media in stimulating callus growth. The comparison showed that the nutrient medium B5 significantly outperformed the other media, recording the highest average fresh weight of callus induced from leaf tissue, with a value of 2.197 g. In contrast, the MS medium showed the lowest fresh weight growth rate for the same type of callus, with a value not exceeding 1.233 g.

Table 6. Effect of nutrient medium components and chitosan on callus fresh weight(g).

Medium components	chitosan (mg L ⁻¹)				Mean
Medium components	0	50	100	200	Mean
MS	1.000	1.206	1.904	0.824	1.233
B5	1.892	2.374	2.392	2.131	2.197
L.S.D_0.05	0.060				0.030
Mean	1.446	1.790	2.148	1.477
L.S.D_0.05	0.042

As for the results related to the analysis of the effect of chitosan concentration, presented in Table 6. They showed statistically significant differences between the four concentrations studied (0, 50, 100 and 200) mg L⁻¹ was observed that all tested concentrations of chitosan significantly outperformed the control group ( Figure 3), confirming the positive effect of this substance in stimulating soft weight gain in callus. This effect followed a clear pattern of proportionality, as the rate of soft weight gain in calves increased significantly with increasing chitosan concentration, reaching its highest value at a concentration of 100 mg L⁻¹, with a peak of 2.148 g. Performance reversed significantly when the highest concentration 200 mg L⁻¹, where the fresh weight of the callus decreased compared to the optimal concentration 100 mg L⁻¹, indicating that the positive effect of chitosan is at its maximum at a specific concentration, and that increasing the concentration beyond this limit may lead to adverse results.

Figure 3. Effect of chitosan concentrations: CHTS₀: without chitosan, CHTS₅₀: 50 mg chitosan per liter, CHTS₁₀₀:100 mg chitosan per liter and CHTS₂₀₀: 200 mg chitosan per liter on callus growth of lavender leaves, after four weeks.

Effect of nutrient medium components and chitosan on dry weight of callus (g)

The results of statistical analysis, as shown in Table 7, revealed a clear variation in the performance of nutrient medium components in terms of their effect on the dry weight of callus. The results showed a marked superiority of the B5 diet in achieving the highest average dry weight of callus induced from leaf tissue, recording a value of 0.103 g. In contrast, the nutrient medium MS recorded the lowest dry weight, with a value of 0.076 g. indicating that the composition of the nutrient medium B5 was more suitable and effective in promoting growth and dry matter accumulation in callus compared to the medium MS.

Table 7. Effect of nutrient medium components and chitosan on dry weight of callus (g).

Medium components	Chitosan (mg L⁻¹)				Mean
Medium components	0	50	100	200	Mean
MS	0.068	0.071	0.087	0.081	0.076
B5	0.101	0.103	0.106	0.102	0.103
L.S.D_0.05	0.002				0.001
Mean	0.084	0.087	0.096	0.091
L.S.D_0.05	0.001

As for the results shown in Table 7, Statistical analysis revealed statistically significant differences between the different concentrations of chitosan included in the study (0, 50, 100 and 200) mg L⁻¹. It was observed that all concentrations treated with chitosan significantly outperformed the control group, to which no concentration of the substance was added 0 mg L⁻¹. This superiority confirms the stimulating and positive effect of chitosan on callus growth.

The results also revealed a positive correlation between increased chitosan concentration and the dry weight of induced callus, with values increasing steadily with higher concentrations, peaking at a concentration of 100 mg L⁻¹. The average dry weight of the callus reached its highest value, amounting to 0.096 g. A decline in this positive effect was also observed when using the highest concentration 200 mg L⁻¹, where the average dry weight decreased to 0.091 g. This decrease indicates that the stimulating effect of chitosan follows an optimum pattern, achieving maximum growth response, and that exceeding this concentration may have an inhibitory or less effective effect on the accumulation of dry matter in callus.

Discussion

Growth regulators, especially auxins, are key factors in stimulating callus formation. The addition of auxin to the nutrient medium elicited a response in the leaf tissues of lavender plants, stimulating callus formation. The added auxin induces fundamental changes in the plant cells in contact with the medium and stimulates the initiation of cell division. During this stimulation phase, constructive processes such as protein synthesis and DNA replication are activated, followed by a series of changes in cellular activity that culminate in cell division and the formation of a callus mass covering most parts of the plant fragment.

The response of plant tissue varies depending on the concentration of auxin in the nutrient medium. An increase in concentration promotes callus formation until the optimum concentration of 1 mg. L⁻¹ is reached, which achieved the highest callus formation rate 95%, in addition to an increase in fresh and dry weight. Conversely, exceeding this optimal concentration, as observed at concentrations of 2 and 3 mg. L⁻¹, leads to adverse effects that inhibit growth. The experiment also showed that light was an inhibitory factor for callus growth induced from lavender leaves compared to dark conditions, which were more conducive to growth. The reason for the reduced callus growth under light can be attributed to the reduced effectiveness of auxin.

It is important to note that continuing to supplement the nutrient medium with auxin at the optimal concentration in order to increase callus production may inhibit growth due to auxin accumulation, necessitating the addition of cytokinin. Cytokinin plays an important role in sustaining callus growth. and previous studies indicate that the interaction between auxins and cytokinins at different concentrations is a decisive factor in achieving optimal hormonal balance (Jabr and Mohsen, 2024; Tilkat and Onay, 2009). In this regard, the treatment consisting of 1 mg L⁻¹ auxin and 2 mg L⁻¹ benzyladenine BA recorded the highest fresh and dry weight values, indicating that it is a suitable combination for sustained callus growth. This can be explained by the ability of cytokinins, especially benzyladenine, to promote nutrient uptake, stimulate cell division and inhibit protein degradation, which supports the division process when the appropriate hormonal balance is achieved. Cytokinins also promote the synthesis of RNA, proteins, and enzymes within the cell. Overall, the increase in soft and dry weight of callus reflects changes in cellular components such as carbohydrates, proteins and amino acids associated with the process of division and growth (Bulya et al., 2023). Therefore, the balance between auxins and cytokinins in the nutrient medium remains a key determinant of continued cell division and callus growth. In addition to growth regulators, the type of nutrient medium played an important role in fresh and dry weight increase, with nutrient medium B5 outperforming MS medium, which is attributed to the difference in the composition of the two media.

Bioactive chitosan was also used as a callus growth stimulator, and the results were consistent with previous studies on the role of chitosan in stimulating growth (Pirbalouti et al., 2017; Byczyńska, 2018; El-Khateeb et al., 2018; Atteya et al., 2023). chitosan treatments recorded significant increases in growth indicators and a positive correlation was observed between chitosan concentration up to a certain level and callus growth, with the highest fresh and dry weight values recorded at a concentration of 100 mg. L⁻¹. This stimulation is attributed to the biological properties of chitosan, which enhances the absorption of key nutrients such as nitrogen and phosphorus, thereby supporting metabolic processes and the accumulation of organic matter. This was reflected in an increase in fresh weight due to higher water and organic matter content and dry weight due to the accumulation of proteins and carbohydrates, resulting in improved biomass of the callus. Stimulation must be limited and sensitive to concentration; at a concentration of 200 mg L⁻¹, growth indicators decreased significantly, and this decrease can be explained physiologically by ionic disturbance and inhibition of nutrient absorption.

Conclusion

This study evaluated the effects different concentrations of 2,4-D have on tissue culture callus induction of lavender leaf explants. From the results, all of the hormone concentrations caused callus formation to begin, but the MS medium with 1 mg L⁻¹ of 2,4-D was the most efficient and most effective in terms of the rate of callus formation. The addition of the growth regulator combination 2,4-D with BA. Significantly improved the capacity of callus formation. it was evident that the synergistic effect between the two hormone groups was responsible for the significant increase in callus yield. With respect to the biostimulant chitosan, it was concluded that chitosan addition to the culture medium enhanced the growth of the formed callus.

Data and software availability

All raw data files underlying the results of this study are available on OSF: Raw data for lavender study, https://doi.org/10.17605/OSF.IO/AMV4Z (Almehemdi 2025).

This project contains the following underlying data:

• Lavenderdata1.xlsx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

References

Abed AS, Majeed DM, Mohammed MA, et al.: In vitro influences of sugar nutrition and light condition on accumulation of some phytochemicals in Verbascum thapsus L. culture. Plant Archives. 2020; 20(1): 1126–1130.
Adil M, Khalil AK, Younas Z, et al.: Impact of media type on callus morphology and standardization of Withania somnifera cell culture for enhanced secondary metabolite production. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2025; 53(3): 14542.
Al-Alwani AAM, Mohammed MA: In vitro production of active compounds in the callus of the chia plant (Salvia hispanica). SABRAO Journal of Breeding and Genetics. 2022; 54(5): 1113–1124.
Almehemdi AF: raw data for lavender study.2025, November 26. osf.io/xz3wg. Publisher Full Text
Alrawi MS, Mohammed MA, Ismail EN, et al.: Stimulation and production of some active compounds in the chia plant with melatonin in vitro. Agron. Res. 2022; 20(S1): 899–912.
Atteya AKG, Abdel-Maksoud BA, Yahia A, et al.: Impact of chitosan on growth, chemical components and essential oil yield of Lavandula officinalis plants. Journal of Agricultural & Environmental Sciences (Damanhour University). 2023; 22(2): 264–288.
Babich O, Sukhikh S, Pungin A, et al.: Modern trends in the in vitro production and use of callus, suspension cells and root cultures of medicinal plants. Molecules. 2020; 25: 5805.
Bulya TE, Glukhareva TV, Kovaleva EG: Obtaining cell cultures of medicinal plants. Recent Research and Advances in Soilless Culture. IntechOpen; 2023.
Byczyńska A: Chitosan improves growth and bulb yield of pineapple lily (Eucomis bicolor Baker), an ornamental and medicinal plant. World Scientific News. 2018; 110(9): 159–171.
Contrada M, Cerasa A, Tonin P, et al.: Aromatherapy in stroke patients: Is it time to begin?. Front. Behav. Neurosci. 2021; 15: 749353.
Crisan I, Ona A, Vârban D, et al.: Current trends for lavender (Lavandula angustifolia Mill.) crops and products with emphasis on essential oil quality. Plants. 2023; 12: 357.
El-Khateeb MA, El-Madaawy AE, Saber AA: Growth and quality of Spathiphyllum wallisii L. as affected by foliar sprays of algae, chitosan, atonik and humic acid. Biosci. Res. 2018; 15(2): 618–627.
Firoozeei TS, Feizi A, Rezaeizadeh H, et al.: Complementary Therapies in Medicine.2021; 59: 102679.
Ghasemi M, Rejeh N, Bahrami T, et al.: Aromatherapy massage vs. foot reflexology on severity of restless legs syndrome in female hemodialysis patients. Geriatrics. 2021; 6: 99.
Hassiotis CN, Vlachonasios KE: How biological and environmental factors affect the quality of lavender essential oils. Physiologia. 2025; 5: 11.
Iber BT, Kasan NA, Torsabo D, et al.: A review of various sources of chitin and chitosan in nature. Journal of Renewable Materials. 2022; 10: 1097.
Jabr RM, Mohsen MM: Effect of growth regulators and BA on lavender callus induction in vitro. Euphrates Journal of Agricultural Science. 2024; 16(2): 484–491.
Javed MU, Mahmood T, Khan UA, et al.: Chitosan-mediated elicitation of secondary metabolism in Rhazya stricta and in-silico exploration of phytochemicals as potential drug candidates against H1299-NSCLC cell lines. Ind. Crop. Prod. 2025; 224: 120180.
Kumar D, Samota MK, Roy S, et al.: Lavender essential oils as natural food protectants: myth or viable substitute?. Sustainable Food Technology. 2025; 3: 354–374. Publisher Full Text
Mehrabian S, Tirgari B, Forouzi MA, et al.: Effect of aromatherapy massage on depression and anxiety in elderly adults: a randomized controlled trial. International Journal of Therapeutic Massage & Bodywork. 2022; 15: 37–45. PubMed Abstract | Publisher Full Text
Miri SM, Roughani A: Factors affecting tissue culture success in ornamental crops: I. medium composition. 2nd International and 3rd National Congress on Flower and Ornamental Plants, Mahallat, Iran. 2018.
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 1962; 15(3): 473–497. Publisher Full Text
OriginLab Corporation: OriginPro 2025b [Computer software]. Northampton, MA: OriginLab Corporation; 2025.
Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, et al.: Production of secondary metabolites using tissue-culture–based biotechnological applications. Front. Plant Sci. 2023; 14: 1132555.
Payne RW, Murray DA, Harding SA, et al.: GenStat for Windows. Hemel Hempstead: VSN International; 12th ed.2009.
Pehlivan S, Karadakovan A: Effects of aromatherapy massage on pain, functional state and quality of life in an elderly individual with knee osteoarthritis. Jpn. J. Nurs. Sci. 2019; 16: 450–458. PubMed Abstract | Publisher Full Text
Pirbalouti AG, Malekpoor F, Salimi A, et al.: Exogenous application of chitosan on biochemical and physiological characteristics, phenolic content and antioxidant activity of Ocimum ciliatum and Ocimum basilicum under reduced irrigation. Sci. Hortic. 2017; 217: 114–122.
Scuteri D, Rombolà L, Morrone LA, et al.: Neuropharmacology of neuropsychiatric symptoms of dementia and role of pain: essential oil of bergamot as a novel therapeutic approach. Int. J. Mol. Sci. 2019; 20: 3327.
Sekhi YS, Abdulkareem AA, Almehemdi AF, et al.: Optimizing micropropagation and growth enhancement of Lavandula angustifolia using two cytokinins. Anbar Journal of Agricultural Sciences. 2025; 23(1): 667–678. Publisher Full Text
Tilkat E, Onay A: Direct shoot organogenesis from in vitro–derived leaf explants of pistachio. In Vitro Cell. Dev. Biol. - Plant. 2009; 45: 92–98.
Vârban R, Vidican R, Ona AD, et al.: Modelling plant morphometric parameters as predictors for successful cultivation of medicinal Agastache species. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2022; 50: 12638.
Yegorova N, Kruglova N, Galin I, et al.: Induction of morphogenesis in callus culture of Lavandula angustifolia Mill. BIO Web of Conferences. 2020; 24: 00098.
Yuan R, Zhang D, Yang J, et al.: Review of aromatherapy essential oils and their mechanisms of action against migraines. J. Ethnopharmacol. 2021; 265: 113326.

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 18 Mar 2026

Author details Author details

¹ Department of Biotechnology, University of Anbar, Ramadi, 31001, Iraq
² Department of Studies and Planning, University of Anbar, Ramadi, 31001, Iraq
³ Center of Desert Studies, Department of conservation Agriculture, University of Anbar, Ramadi, 31001, Iraq

Mohammed Abdulgafor Mohammed
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Project Administration

Masara Dafer Alani
Roles: Data Curation, Formal Analysis, Investigation, Resources

Ahmed O. Mashaan
Roles: Funding Acquisition, Software, Validation, Visualization, Writing – Review & Editing

Ali F. Almehemdi
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (1)

version 1

Published: 18 Mar 2026, 15:407

https://doi.org/10.12688/f1000research.173637.1

Copyright

© 2026 Mohammed MA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

0

SEE MORE DETAILS

CITE

how to cite this article

Mohammed MA, Dafer Alani M, Mashaan AO and Almehemdi AF. Evaluation of the synergistic efficiency of hormonal, biological and physical stimuli in the induction and growth of callus in lavender [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2026, 15:407 (https://doi.org/10.12688/f1000research.173637.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Version 1

VERSION 1

PUBLISHED 18 Mar 2026

Views

21

Reviewer Report 08 May 2026

Tassanai Jaruwattanaphan, Kasetsart University, Bangkok, Thailand

Not Approved

https://doi.org/10.5256/f1000research.191471.r476658

This manuscript investigates the effects of 2,4-D, BA, light conditions, culture medium (MS versus B5), and chitosan on callus induction and growth in lavender. The main topic of the study is relevant to the field of plant biotechnology, and the ... Continue reading

This manuscript investigates the effects of 2,4-D, BA, light conditions, culture medium (MS versus B5), and chitosan on callus induction and growth in lavender. The main topic of the study is relevant to the field of plant biotechnology, and the presented results, especially positive effects of 1 mg L⁻¹ 2,4-D in dark conditions, along with 2 mg L⁻¹ BA, B5 medium, and 100 mg L⁻¹ chitosan on callus production, are considered acceptable. However, the manuscript needs major revision to be considered scientifically accurate and acceptable for indexing.

Is the study design appropriate and is the work technically sound? Partly

The three experimental stages are not clearly related as the settings adjusted in phase 1 were not explicitly taken to phases 2 and 3. Thus, it is impossible to evaluate the alleged synergistic effects. The explant description is also not detailed enough, as key characteristics such as leaf age, location and size are not included.
Furthermore, the callus development experiment lacks a zero-auxin control and the chitosan experiment does not state the plant growth regulators in the basal media.
The manuscript refers to Lavandula angustifolia Mill. in the Introduction but uses Lavandula spp. in the Results. This inconsistency is taxonomically unacceptable and must be corrected throughout.
The authors could define sequential experimental design and possibly include a basic workflow diagram illustrating how each phase was related.

Are sufficient details of the methods and analysis provided to allow replication by others? Partly

Methodological information is partially adequate for reproduction. Basic culture conditions are presented; however, numerous important aspects are omitted.
Inadequate description of explant preparation (leaf age, location, size and developmental stage are not provided)
There is no characterization of chitosan; degree of deacetylation (DD%) and molecular weight should be given since they have a substantial influence on biological activity.
While the sterilizing approach (3% NaOCl for 15 min) may be too severe, there is no information on contamination or explant survival rates. No mention of strategies to reduce oxidative browning either.
The use of GA₃ in germination media is not supported or referred to; however, it may affect explant physiology and subsequent induction of callus.
The physiological condition and origin of the 150 mg callus inoculum employed in later phases, such as treatment source, callus age, and subculture history, are unknown.

Is the statistical analysis and its interpretation appropriate? Partly

The statistical approach (CRD analyzed by ANOVA with LSD at p ≤ 0.05) is generally appropriate, and the use of ten replicates per treatment is commendable.
However, ANOVA outputs are not reported. Tables include only treatment means and LSD₀.₀₅ values, without F-values or p-values for main effects and interactions.
Pairwise comparison letters are absent from all tables, making treatment differences difficult to interpret.
Figure 1 contains a major scaling error, as the Y-axis exceeds 100% despite representing callus formation percentage.
Error bars are not defined (SD or SE), and the large variation observed in some treatments is not discussed.

Are the conclusions drawn adequately supported by the results? Partly

Overall, indicated trends are generally consistent with provided data; however, several findings are misleading or inadequately supported.
The notion of “synergistic efficiency” has no justification. Most factors were tested in isolation in the studies, and no real cross-factor interaction analysis was performed.
Physiologically, the concept of auxin action is incorrect. Auxin is mostly related to cell division and dedifferentiation in callus induction, not to decreased cell elongation.
The reason given for the advantage of B5 medium is too superficial and should mention precise compositional differences between B5 and MS media.
The introduction highlights the synthesis of secondary metabolites, but no metabolite analysis was done; hence, the data do not support these assertions.
Only biomass measurements were taken to determine callus quality, and essential features such as texture, color, viability, and oxidative browning were not investigated.

Comments on Figures

Figure 1 contains a critical Y-axis error (scale to 200%) that must be corrected. Error bar definitions must be stated, and the source of high variability acknowledged.
Figure 2 is of insufficient quality for publication. The image is low resolution, includes distracting background elements, does not specify which 2,4-D treatment is shown, and lacks a scale bar.
Figure 3 does not specify whether images are from MS or B5 medium.
No photographic documentation is provided for the 2,4-D + BA experiment.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant germplasm conservation, plant molecular genetics, plant tissue culture, and cytogenetics.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

Report a concern

Respond or Comment

Views

29

Reviewer Report 27 Apr 2026

Duha Mysire Majeed, Plant Biotechnology, Al-Nahrain University / Biotechnology Research center, Baghdad, Iraq

Approved with Reservations

https://doi.org/10.5256/f1000research.191471.r476656

This study investigates the combined effects of hormonal, environmental, and biological factors on callus induction and growth in lavender. The topic is relevant and of interest in plant tissue culture; however, several aspects require improvement to enhance the scientific quality ... Continue reading

This study investigates the combined effects of hormonal, environmental, and biological factors on callus induction and growth in lavender. The topic is relevant and of interest in plant tissue culture; however, several aspects require improvement to enhance the scientific quality of the manuscript.

Major comments:

The manuscript requires substantial language editing to improve clarity and scientific writing style. Several sentences are repetitive and lack precision.
The novelty of the study is limited, as it mainly focuses on optimization using well-established factors such as 2,4-D, BA, nutrient media, and chitosan. The authors should better highlight the originality of their contribution.
The experimental design is not sufficiently detailed. The authors should clearly describe the explant type, treatment structure, and factorial design to ensure reproducibility.
The statistical analysis needs to be strengthened. The manuscript lacks detailed ANOVA results, p-values, and a clear explanation of statistical significance.
The discussion section should be improved by providing deeper interpretation of the results and better comparison with recent literature.

Minor comments:

Ensure consistency in units and formatting (e.g., mg L⁻¹).
Improve figure quality and labeling.
Correct grammatical and typographical errors.

Conclusion:
The conclusions are generally supported by the results but are somewhat overstated and should be moderated.

Recommendation: Major Revision

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: I have previously collaborated with one of the authors, Mohammed A. Mohammed, as a co-author on a published research article in 2020. This collaboration was limited to that publication and there is no ongoing collaboration related to the current manuscript. I have had no direct contact with the authors regarding this article, and the manuscript was not discussed with them. I confirm that I am able to assess this work impartially.

Reviewer Expertise: Plant Biotechnology, Plant Tissue Culture, Molecular Biology, Plant Genetic Engineering

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Respond or Comment

Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 18 Mar 2026

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2
Version 1 18 Mar 26	read	read

Duha Mysire Majeed, Al-Nahrain University / Biotechnology Research center, Baghdad, Iraq
Tassanai Jaruwattanaphan, Kasetsart University, Bangkok, Thailand

Comments on this article

All Comments(0)

Add a comment

Sign up for content alerts

Browse by related subjects

Back to all reports

Reviewer Report

21 Views

08 May 2026 | for Version 1

Tassanai Jaruwattanaphan, Kasetsart University, Bangkok, Thailand

21 Views Cite this report Responses(0)

Not Approved

This manuscript investigates the effects of 2,4-D, BA, light conditions, culture medium (MS versus B5), and chitosan on callus induction and growth in lavender. The main topic of the study is relevant to the field of plant biotechnology, and the presented results, especially positive effects of 1 mg L⁻¹ 2,4-D in dark conditions, along with 2 mg L⁻¹ BA, B5 medium, and 100 mg L⁻¹ chitosan on callus production, are considered acceptable. However, the manuscript needs major revision to be considered scientifically accurate and acceptable for indexing.

Is the study design appropriate and is the work technically sound? Partly

The three experimental stages are not clearly related as the settings adjusted in phase 1 were not explicitly taken to phases 2 and 3. Thus, it is impossible to evaluate the alleged synergistic effects. The explant description is also not detailed enough, as key characteristics such as leaf age, location and size are not included.
Furthermore, the callus development experiment lacks a zero-auxin control and the chitosan experiment does not state the plant growth regulators in the basal media.
The manuscript refers to Lavandula angustifolia Mill. in the Introduction but uses Lavandula spp. in the Results. This inconsistency is taxonomically unacceptable and must be corrected throughout.
The authors could define sequential experimental design and possibly include a basic workflow diagram illustrating how each phase was related.

Are sufficient details of the methods and analysis provided to allow replication by others? Partly

Methodological information is partially adequate for reproduction. Basic culture conditions are presented; however, numerous important aspects are omitted.
Inadequate description of explant preparation (leaf age, location, size and developmental stage are not provided)
There is no characterization of chitosan; degree of deacetylation (DD%) and molecular weight should be given since they have a substantial influence on biological activity.
While the sterilizing approach (3% NaOCl for 15 min) may be too severe, there is no information on contamination or explant survival rates. No mention of strategies to reduce oxidative browning either.
The use of GA₃ in germination media is not supported or referred to; however, it may affect explant physiology and subsequent induction of callus.
The physiological condition and origin of the 150 mg callus inoculum employed in later phases, such as treatment source, callus age, and subculture history, are unknown.

Is the statistical analysis and its interpretation appropriate? Partly

The statistical approach (CRD analyzed by ANOVA with LSD at p ≤ 0.05) is generally appropriate, and the use of ten replicates per treatment is commendable.
However, ANOVA outputs are not reported. Tables include only treatment means and LSD₀.₀₅ values, without F-values or p-values for main effects and interactions.
Pairwise comparison letters are absent from all tables, making treatment differences difficult to interpret.
Figure 1 contains a major scaling error, as the Y-axis exceeds 100% despite representing callus formation percentage.
Error bars are not defined (SD or SE), and the large variation observed in some treatments is not discussed.

Are the conclusions drawn adequately supported by the results? Partly

Overall, indicated trends are generally consistent with provided data; however, several findings are misleading or inadequately supported.
The notion of “synergistic efficiency” has no justification. Most factors were tested in isolation in the studies, and no real cross-factor interaction analysis was performed.
Physiologically, the concept of auxin action is incorrect. Auxin is mostly related to cell division and dedifferentiation in callus induction, not to decreased cell elongation.
The reason given for the advantage of B5 medium is too superficial and should mention precise compositional differences between B5 and MS media.
The introduction highlights the synthesis of secondary metabolites, but no metabolite analysis was done; hence, the data do not support these assertions.
Only biomass measurements were taken to determine callus quality, and essential features such as texture, color, viability, and oxidative browning were not investigated.

Comments on Figures

Figure 1 contains a critical Y-axis error (scale to 200%) that must be corrected. Error bar definitions must be stated, and the source of high variability acknowledged.
Figure 2 is of insufficient quality for publication. The image is low resolution, includes distracting background elements, does not specify which 2,4-D treatment is shown, and lacks a scale bar.
Figure 3 does not specify whether images are from MS or B5 medium.
No photographic documentation is provided for the 2,4-D + BA experiment.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Plant germplasm conservation, plant molecular genetics, plant tissue culture, and cytogenetics.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

29 Views

27 Apr 2026 | for Version 1

Duha Mysire Majeed, Plant Biotechnology, Al-Nahrain University / Biotechnology Research center, Baghdad, Iraq

29 Views Cite this report Responses(0)

Approved With Reservations

This study investigates the combined effects of hormonal, environmental, and biological factors on callus induction and growth in lavender. The topic is relevant and of interest in plant tissue culture; however, several aspects require improvement to enhance the scientific quality of the manuscript.

Major comments:

The manuscript requires substantial language editing to improve clarity and scientific writing style. Several sentences are repetitive and lack precision.
The novelty of the study is limited, as it mainly focuses on optimization using well-established factors such as 2,4-D, BA, nutrient media, and chitosan. The authors should better highlight the originality of their contribution.
The experimental design is not sufficiently detailed. The authors should clearly describe the explant type, treatment structure, and factorial design to ensure reproducibility.
The statistical analysis needs to be strengthened. The manuscript lacks detailed ANOVA results, p-values, and a clear explanation of statistical significance.
The discussion section should be improved by providing deeper interpretation of the results and better comparison with recent literature.

Minor comments:

Ensure consistency in units and formatting (e.g., mg L⁻¹).
Improve figure quality and labeling.
Correct grammatical and typographical errors.

Conclusion:
The conclusions are generally supported by the results but are somewhat overstated and should be moderated.

Recommendation: Major Revision

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

I have previously collaborated with one of the authors, Mohammed A. Mohammed, as a co-author on a published research article in 2020. This collaboration was limited to that publication and there is no ongoing collaboration related to the current manuscript. I have had no direct contact with the authors regarding this article, and the manuscript was not discussed with them. I confirm that I am able to assess this work impartially.

Reviewer Expertise

Plant Biotechnology, Plant Tissue Culture, Molecular Biology, Plant Genetic Engineering

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

Responses (0)

[1] Abed AS, Majeed DM, Mohammed MA, et al.: In vitro influences of sugar nutrition and light condition on accumulation of some phytochemicals in Verbascum thapsus L. culture. Plant Archives. 2020; 20(1): 1126–1130.

[2] Adil M, Khalil AK, Younas Z, et al.: Impact of media type on callus morphology and standardization of Withania somnifera cell culture for enhanced secondary metabolite production. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2025; 53(3): 14542.

[3] Al-Alwani AAM, Mohammed MA: In vitro production of active compounds in the callus of the chia plant (Salvia hispanica). SABRAO Journal of Breeding and Genetics. 2022; 54(5): 1113–1124.

[4] Almehemdi AF: raw data for lavender study.2025, November 26. osf.io/xz3wg. Publisher Full Text

[5] Alrawi MS, Mohammed MA, Ismail EN, et al.: Stimulation and production of some active compounds in the chia plant with melatonin in vitro. Agron. Res. 2022; 20(S1): 899–912.

[6] Atteya AKG, Abdel-Maksoud BA, Yahia A, et al.: Impact of chitosan on growth, chemical components and essential oil yield of Lavandula officinalis plants. Journal of Agricultural & Environmental Sciences (Damanhour University). 2023; 22(2): 264–288.

[7] Babich O, Sukhikh S, Pungin A, et al.: Modern trends in the in vitro production and use of callus, suspension cells and root cultures of medicinal plants. Molecules. 2020; 25: 5805.

[8] Bulya TE, Glukhareva TV, Kovaleva EG: Obtaining cell cultures of medicinal plants. Recent Research and Advances in Soilless Culture. IntechOpen; 2023.

[9] Byczyńska A: Chitosan improves growth and bulb yield of pineapple lily (Eucomis bicolor Baker), an ornamental and medicinal plant. World Scientific News. 2018; 110(9): 159–171.

[10] Contrada M, Cerasa A, Tonin P, et al.: Aromatherapy in stroke patients: Is it time to begin?. Front. Behav. Neurosci. 2021; 15: 749353.

[11] Crisan I, Ona A, Vârban D, et al.: Current trends for lavender (Lavandula angustifolia Mill.) crops and products with emphasis on essential oil quality. Plants. 2023; 12: 357.

[12] El-Khateeb MA, El-Madaawy AE, Saber AA: Growth and quality of Spathiphyllum wallisii L. as affected by foliar sprays of algae, chitosan, atonik and humic acid. Biosci. Res. 2018; 15(2): 618–627.

[13] Firoozeei TS, Feizi A, Rezaeizadeh H, et al.: Complementary Therapies in Medicine.2021; 59: 102679.

[14] Ghasemi M, Rejeh N, Bahrami T, et al.: Aromatherapy massage vs. foot reflexology on severity of restless legs syndrome in female hemodialysis patients. Geriatrics. 2021; 6: 99.

[15] Hassiotis CN, Vlachonasios KE: How biological and environmental factors affect the quality of lavender essential oils. Physiologia. 2025; 5: 11.

[16] Iber BT, Kasan NA, Torsabo D, et al.: A review of various sources of chitin and chitosan in nature. Journal of Renewable Materials. 2022; 10: 1097.

[17] Jabr RM, Mohsen MM: Effect of growth regulators and BA on lavender callus induction in vitro. Euphrates Journal of Agricultural Science. 2024; 16(2): 484–491.

[18] Javed MU, Mahmood T, Khan UA, et al.: Chitosan-mediated elicitation of secondary metabolism in Rhazya stricta and in-silico exploration of phytochemicals as potential drug candidates against H1299-NSCLC cell lines. Ind. Crop. Prod. 2025; 224: 120180.

[19] Kumar D, Samota MK, Roy S, et al.: Lavender essential oils as natural food protectants: myth or viable substitute?. Sustainable Food Technology. 2025; 3: 354–374. Publisher Full Text

[20] Mehrabian S, Tirgari B, Forouzi MA, et al.: Effect of aromatherapy massage on depression and anxiety in elderly adults: a randomized controlled trial. International Journal of Therapeutic Massage & Bodywork. 2022; 15: 37–45. PubMed Abstract | Publisher Full Text

[21] Miri SM, Roughani A: Factors affecting tissue culture success in ornamental crops: I. medium composition. 2nd International and 3rd National Congress on Flower and Ornamental Plants, Mahallat, Iran. 2018.

[22] Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 1962; 15(3): 473–497. Publisher Full Text

[23] OriginLab Corporation: OriginPro 2025b [Computer software]. Northampton, MA: OriginLab Corporation; 2025.

[24] Ozyigit II, Dogan I, Hocaoglu-Ozyigit A, et al.: Production of secondary metabolites using tissue-culture–based biotechnological applications. Front. Plant Sci. 2023; 14: 1132555.

[25] Payne RW, Murray DA, Harding SA, et al.: GenStat for Windows. Hemel Hempstead: VSN International; 12th ed.2009.

[26] Pehlivan S, Karadakovan A: Effects of aromatherapy massage on pain, functional state and quality of life in an elderly individual with knee osteoarthritis. Jpn. J. Nurs. Sci. 2019; 16: 450–458. PubMed Abstract | Publisher Full Text

[27] Pirbalouti AG, Malekpoor F, Salimi A, et al.: Exogenous application of chitosan on biochemical and physiological characteristics, phenolic content and antioxidant activity of Ocimum ciliatum and Ocimum basilicum under reduced irrigation. Sci. Hortic. 2017; 217: 114–122.

[28] Scuteri D, Rombolà L, Morrone LA, et al.: Neuropharmacology of neuropsychiatric symptoms of dementia and role of pain: essential oil of bergamot as a novel therapeutic approach. Int. J. Mol. Sci. 2019; 20: 3327.

[29] Sekhi YS, Abdulkareem AA, Almehemdi AF, et al.: Optimizing micropropagation and growth enhancement of Lavandula angustifolia using two cytokinins. Anbar Journal of Agricultural Sciences. 2025; 23(1): 667–678. Publisher Full Text

[30] Tilkat E, Onay A: Direct shoot organogenesis from in vitro–derived leaf explants of pistachio. In Vitro Cell. Dev. Biol. - Plant. 2009; 45: 92–98.

[31] Vârban R, Vidican R, Ona AD, et al.: Modelling plant morphometric parameters as predictors for successful cultivation of medicinal Agastache species. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2022; 50: 12638.

[32] Yegorova N, Kruglova N, Galin I, et al.: Induction of morphogenesis in callus culture of Lavandula angustifolia Mill. BIO Web of Conferences. 2020; 24: 00098.

[33] Yuan R, Zhang D, Yang J, et al.: Review of aromatherapy essential oils and their mechanisms of action against migraines. J. Ethnopharmacol. 2021; 265: 113326.

Evaluation of the synergistic efficiency of hormonal, biological and physical stimuli in the induction and growth of callus in lavender

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Plant materials

Establishment of tissue culture

Table 1. Reagents used in the study, including the amount used, supplier information, and catalogue numbers.

Callus induction

The role of nutrient components and chitosan in promoting callus growth

Statistical analysis

Results and discussion

The effect of light conditions and 2,4-D concentrations on callus formation

Figure 1. Effect of light conditions and 2,4-D concentrations on callus formation.

Effect of illumination and 2,4-D concentrations on callus fresh weight (g)

Table 2. Effect of illumination and 2,4-D concentrations on callus fresh weight (g).

Figure 2. Effect of illumination (light (A) and dark (B)) on the growth of callus induced from lavender leaves, after four weeks.

Effect of illumination and 2,4-D on callus dry weight of callus (g)

Table 3. Effect of light conditions and 2,4-D concentrations on callus dry weight (g).

Effect of 2,4-D and BA on fresh weight of callus (g)

Table 4. Effect of 2,4-D and BA on fresh weight (g) of callus.

Effect of 2,4-D and BA on dry weight of callus (g)

Table 5. Effect of 2,4-D and BA on dry weight (g) of callus.

Effect of nutrient medium components and chitosan on callus fresh weight (g)

Table 6. Effect of nutrient medium components and chitosan on callus fresh weight(g).

Figure 3. Effect of chitosan concentrations: CHTS0: without chitosan, CHTS50: 50 mg chitosan per liter, CHTS100:100 mg chitosan per liter and CHTS200: 200 mg chitosan per liter on callus growth of lavender leaves, after four weeks.

Effect of nutrient medium components and chitosan on dry weight of callus (g)

Table 7. Effect of nutrient medium components and chitosan on dry weight of callus (g).

Discussion

Conclusion

Data and software availability

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated

Figure 3. Effect of chitosan concentrations: CHTS₀: without chitosan, CHTS₅₀: 50 mg chitosan per liter, CHTS₁₀₀:100 mg chitosan per liter and CHTS₂₀₀: 200 mg chitosan per liter on callus growth of lavender leaves, after four weeks.