Callus Induction, Proliferation, Enhanced Secondary Metabolites Production and Antioxidants Activity of Salvia moorcroftiana L. as Influenced by Combinations of Auxin, Cytokinin and Melatonin

Abstract:

Tissue culture technique is one of the best methods to reproduce salvia plant Therefore, the aim of this research was to enhance the in-vitro callus proliferation and production of secondary metabolites of S. moorcroftiana using different combinations of auxin, cytokinin and melatonin. Initially, callus induction was optimized using indole acetic acid (IAA), 2, 4-dichlorophenoxy acetic acid (2,4-D), and naphthalene acetic acid (NAA) applied at different concentrations in combination with 1 mg L^-1 of 6-benzylaminopurine (BAP). The results indicates that earliest days to callus induction (14.67 days) was occurred in the media fortified with 2, 4-D+BAP (2.0+1.0 mgL-1). Whereas the highest callus initiation (100%) was induced on MS medium incorporated with 2,4-D+BAP (1+1mgL^-1). Furthermore, maximum fresh weight was obtained when 2,4- D + BAP at the rate of (1+ 1mg L^-1) was incorporated and dry weight was attained when 2,4- D + BAP at the rate of (2+1 mg L^-1) was added to MS media. The maximum fresh and dry weight was obtained when melatonin at rate of 1.5 mg L^-1 was supplemented with MS media including 2,4-D + BAP (1+1mg L^-1), moreover the maximum DPPH scavenging activity, total phenolic and flavonoid content was noted when supplemented with melatonin at rate of 1.5 mg L^-1. In conclusion, among various concentrations of plant growth regulators, 2,4- D + BAP at the rate of (1+ 1mg L^-1) along with 1.5 g L^-1 melatonin was the best for callus growth and production of secondary metabolites of S. moorcroftiana.

Keywords:
Antioxidant activity; Growth regulators; Sage; Secondary Metabolites

HIGHLIGHTS

• Tissue culture technique is considered to be one of the best methods to reproduce salvia plant.

• Salvia is a medicinal plant with lots of chemical constituents.

• Salvia plays an important role in treating different fatal diseases.

• Maximum fresh and dry weights were obtained at 1.5 mg L^-1 melatonin.

• the highest DPPH scavenging activity, total phenolics and flavonoid content were obtained at 1.5 mg L^-1 melatonin.

INTRODUCTION

Sage (S. moorcroftiana) belongs to the largest genus of ornamental, aromatic and medicinal plants in the family Lamiaceae distributed over tropical regions [¹]. The plant is largely found in the Mediterranean Basin, Central and South America, South-East and Central Asia and is mostly cultivated for medicinal purposes. In Pakistan S. moorcroftiana is found in the northern areas of the Khyber Pakhtunkhwa province and Kashmir region at a height of about 5200ft [²]. S. moorcroftiana is a medicinal plant with lots of chemical constituents which play an important role in treating different fatal diseases. It contains chemical compounds which have anti-tumor, antibacterial, antifungal, antiviral and anti-inflammatory activity [³]. Salvia also contains several beneficial secondary metabolites like phenolic compounds, essential oils and terpenoids [⁴]. Sage's biological effects on human health are due to the anti-inflammatory, antibacterial, fungistatic, virostatic, astringent, eupeptic and antihydrotic properties of its components [³]. These can be used in the pharmaceutical and medical fields as a result of their hypoglycemic and antimutagenic actions and as such in the treatment of Alzheimer's Disease [⁵]. Sage is mixed into a combination of herbal preparations as an active ingredient and is useful for bronchitis treatment [⁵].

Propagation of common sage plants can be induced through cuttings and seeds, but conventional propagation methods are not practiced due to low seed germination, poor planting material and slow growth. However, the tissue culture technique is considered to be one of the best methods for reproducing salvia plants. Very limited information is available on the micro-propagation of this plant. Moreover, some other species of this genus have been tried for propagation in-vitro conditions such as hairy roots, callus and cell suspension [⁶]. Micropropagation of salvia species has been done successfully on various media such as Murashige and Skoog (MS), B5 vitamins and α- naphthalene acetic acid (NAA) for successful regeneration from explants taken from open field plants [⁷].

Plant growth regulators play a key role in different physiological processes during the growth and developmental stages of plants [⁸-¹³]. Different types of cytokinin including kinetin are known to be more efficient in the proliferation of shoots such as 6-benzylaminopurine “BAP”. Auxin is also essential for in vitro rooting of new growth with different concentrations in salvia [¹⁴]. The micropropagation and regeneration of adventitious shoots in S. moorcroftiana was higher when MS media was augmented with 6-benzylaminopurine (- 0.533 mg L^-1 BAP), naphthalene acetic acid (- 0.644 mg L^-1 NAA) and kinetin (1.12 mg L^-1). BAP could also be more effective in increasing axillary buds [¹⁵].

Apart from the auxin and cytokinin groups, certain other chemicals can be used for callus induction and proliferation. Melatonin (N-acetyl-5-methoxytryptamine) is plant growth regulator and rooting agent that plays an important role in plant development and protective role in plant stress [¹⁶]. Melatonin has been reported in different angiosperms and is mostly found in tomatoes, cucumbers, higher and medicinal plants [¹⁷]. Melatonin is considered to have scavenging properties which act on reactive oxygen and hydroxyl radicals [¹⁷-¹⁸] and produce cyclic 3-hydroxymelatonin having antioxidant properties. Melatonin has similar functions like auxin, thereby promoting cell development, organogenesis, and plant growth [¹⁹]. Higher plant survival rates, greater shoot, and root development, photosynthesis, improved stomatic morphologies, high amounts of sucrose and proline, lower concentrations of ROS/RNS, lipid membrane peroxidation, and cell damage were observed in melatonin-treated plants [¹⁷]. Application of melatonin can also improve the resistance to various stresses including drought, salinity, extreme temperature, radiation, and chemical stresses. There is very limited information available on the propagation of S. moorcroftiana both in in vitro and in vivo conditions. Therefore, this study was aimed at evaluating the effect of plant growth regulators and melatonin on callus induction, callus growth and secondary metabolites production in S. moorcroftiana.

MATERIALS AND METHODS

Experimental site

Two different experiments were carried out in the Laboratory of Plant Tissue Culture, Department of Horticulture, The University of Agriculture Peshawar to optimize the plant growth regulators (auxin and cytokinin) and melatonin for callus induction and proliferation of S. moorcroftiana.

Collection of explant material and preparation of cultured media

Moorcroftiana specie of the genus Salvia is native to the northern part of Khyber Pakhtunkhwa and Himalayan mountains of Pakistan, especially Kashmir. It grows between 5,000- and 9,000-feet elevation. This plant was introduced by botanists of Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex Peshawar and grown in natural environment in Medicinal Botanical Centre of PCSIR Peshawar-Pakistan.

Newly developed leaves were collected as explants from one year old plants of S. moorcroftiana, which are available in the Medicinal Botanical Centre of the Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex Peshawar (34° 02' N, 71° 37' E), Pakistan. The plants were identified and authenticated by Dr. Hina Fazal, PCSIR under accession number 1173PCSIR. In the first experiment, three plant growth regulators (PGRs) were optimized for callus induction from leaf explants. The auxins i.e., indole acetic acid (IAA), 2, 4-dichlorophenoxy acetic acid (2,4-D), and naphthalene acetic acid (NAA) were applied at the concentrations of 0.5, 1.0, 1.5 and 2.0 mg L^-1 in combination with 1 mg L^-1 of 6-benzylaminopurine (BAP) which were PGRs were supplemented to the media culture performed in a laminar flow unit in the culture room and then transferred to the growth room. Temperature was kept at 25±2^ºC with 16/8 h photoperiod. Leaf explants were thoroughly washed and sterilized with mercuric chloride (HgCl₂) to remove contaminants and cultured in MS-media culture (Murasheige and Skoog) having different concentrations of auxins (2,4-D, IAA, NAA) and cytokinin (BAP) (Table 1). Media without plant growth regulators were kept as control. The MS-media has 30 g L^-1 sucrose, 7-8 g L^-1 agar, and pH of 5.5-5.8 range was adjusted for the media.

In the second experiment, the induced callus was exposed to various concentrations of melatonin (0, 0.5, 1.0, 1.5 and 2.0 mg L^-1) with the objective of enhancing callus growth and secondary metabolites production of S. moorcroftiana. Melatonin was added to the culture media before sterilization. Media were also kept saturated with an optimized level of PGRs from the previous experiment. Similarly, MS-media was having 30 g L^-1 sucrose and 7-8 g L^-1 agar with pH ranging from 5.5 to 5.8. The media were sterilized at 121 ℃ for 20 min. The culture was transferred to growth room at 25±2 ºC along with 16/8 hours of light and dark photoperiod respectively.

Table 1
Detail of PGRs treatments used in media culture

Treatments	Auxin (mg L-1)				Cytokinin (mg L-1)		Auxin to cytokinin ratio
	2, 4-D	NAA	IAA		BAP
Control	-	-	-		-		-
2,4-D0.5+BAP	0.5	-	-		1.0		0.5
2,4-D1+BAP	1.0	-	-		1.0		1
2,4-D1.5+BAP	1.5	-	-		1.0		1.5
2,4-D2+BAP	2.0	-	-		1.0		2
NAA0.5+BAP	-	0.5	-		1.0		0.5
NAA1+BAP	-	1.0	-		1.0		1
NAA1.5+BAP	-	1.5	-		1.0		1.5
NAA2+BAP	-	2.0	-		1.0		2
IAA0.5+BAP	-	-	0.5		1.0		0.5
IAA1+BAP	-	-	1.0		1.0		1
IAA1.5+BAP	-	-	1.5		1.0		1.5
IAA2+BAP	-	-	2.0		1.0		2

Studied attributes

The following attributes were studied during the research experiments

Days to callus induction

Explants cultured on media were under observation on a regular basis till callus emergence. The number of days was counted from the date of culturing till callus induction.

Percent callus induction

Data on callus induction (%) was recorded with the help of the following formula:

$$\text{Percent callus induction}=\text{No of explants that produced callus/No of explants}\times 100$$

Callus morphology

On full induction of callus for each treatment, callus texture and callus color were examined visually.

Callus fresh and dry weight

Fresh callus was detached from the media after 40 days and rinsed carefully, to remove the media. Callus was placed on tissue paper to drain extra water and then fresh weights were measured using a digital weighing balance. The callus was then kept in an oven at 40^ºC for a period of 48 h and the dry weight was calculated by using a digital balance.

Total phenolic content

The total phenolic of oven dried callus was determined according to the protocol by Singleton and Rossi [²⁰]. The methanol-based sample was primed by taking 1.5 mg of dried callus in 5 ml methanol. Then 40 µL of the sample was further diluted by adding 3.16 mL distilled water. Folin-Ciocalteu reagent (200 µL) and 600 µL sodium carbonate (20 µL) were incorporated into the mixture and centrifuged and incubated for 25 min. Gallic acid was used as a standard for plotting a standard curve.

Total flavonoid content

The total flavonoid content (TFC, mg g^-1 DW) was quantified as per the method used by Park and coauthors [²¹]. A methanol-based sample prepared for total phenolics was used. Rutin was used a as standard to calibrate the standard curve. The absorbance was recorded using a spectrophotometer.

DPPH free radical scavenging activity

DPPH-radical scavenging activity (DRSA) in treatments exposed to melatonin was quantified following the procedure explained by Ahmad and coauthors [²²]. The methanol solution of each sample (1 mL) was incorporated with DPPH-solution (2 mL). Samples were protected in dark conditions for 25 min and the absorbance reading was taken through a spectrophotometer. DRSA was calculated by the equation as under.

$$\mathrm{DRSA}(\mathrm{\%})=100\times (1-\mathrm{A}\mathrm{P}/\mathrm{A}\mathrm{D})$$

Where AP stands for the absorbance of shoots extract at 517 nm and AD for the DPPH solution without extract.

Statistical analysis

Data was analyzed using the statistical package (STATISTIX 8.1, Inc, Tallahassee FL, USA). The differences between means were calculated by using LSD (least significant difference) at 5% level of significance [²³].

RESULTS AND DISCUSSION

Optimization of type and concentration of auxins for callus induction of S. moorcroftiana from leaf explant

During the experiment the Murashige and Skoog (MS) media was supplemented with different concentrations (0.5, 1, 1.5 and 2 mg L^-1) of auxins, i.e., indole acetic acid (IAA), 2, 4-dichlorophenoxy acetic acid (2,4-D), and naphthalene acetic acid (NAA) in combination with 1 mg L^-1 of 6-benzylaminopurine (BAP). The results of this experiment are as follows.

Days to callus induction

The data regarding days to callus induction indicates a significant effect of auxin on callus induction (Table 2). The earliest callus induction (14.7 d) occurred in the medium augmented with 2 mg L^-1 2,4-D followed by 1 mg L^-1 2,4-D. While the media fortified with 2 mg L^-1 IAA and 0.5 mg L^-1 NAA takes the longest time of 24 d and 22 d, followed by 22 d by 1.5 mg L^-1 BAP.

The physiological activity of auxin within the plant tissue makes the difference in callus formation and morphology [²⁴]. Furthermore, mutual action of auxins and cytokinin control the process of cell division. Auxins play a vital role in the stimulation of proteins in the cell cycle related to the cdc2/cdk2 class of cyclin-dependent kinases. Furthermore, the applications of auxins with cytokinin synergistically influence an increase in the activity of cdc2/cdk2 like kinase [²⁵] producing more callus. Auxins to cytokinin optimum ratio are essential for maximum callus induction [²⁶]. The present results are in close conformity with those of Hesami and Daneshvar [²⁷] who observed compact callus of Ficus religiosa in NAA and IBA treatment as compared to friable callus obtained in MS media supplemented with 2, 4-D. Huang and Staden [²⁸] also investigated days to callus induction in S. chamelaeagnea, and reported minimum days to callus induction on 2, 4-D and BAP medium.

Table 2
Effect of plant growth regulators for percent and days to callus induction of S. moorcroftiana

Treatments	Days to callus induction	% Callus induction
Control	-
2,4-D0.5+BAP	21.13±0.6bcd	77.4f
2,4-D1+BAP	18.81±0.5f	100.0a
2,4-D1.5+BAP	16.58±0.5g	99.8a
2,4-D2+BAP	14.67±0.4h	82.5c
NAA0.5+BAP	21.69±0.7bc	77.3f
NAA1+BAP	18.01±0.3f	66.8i
NAA1.5+BAP	20.24±0.5de	71.8g
NAA2+BAP	23.33±0.3a	96.4b
IAA0.5+BAP	20.91±0.5cd	70.8h
IAA1+BAP	19.11±0.4ef	79.4e
IAA1.5+BAP	22.14±0.4b	80.7d
IAA2+BAP	24.24±0.3a	61.0j
ANOVA	***	***

Percent callus induction

Statistical analysis revealed a significant mean difference between auxins and their concentrations. Overall, 2,4-D showed higher callus induction than NAA and IAA. The highest percentage (100%) of callus induction was observed in media fortified with 1 mg L^-1 2,4-D + BAP. Statistically similar results (99.8%) were observed with 1.5 mg L^-1 2, 4-D, followed by 2 mgL^-1 NAA (96%). The lowest callus induction was at 2 mg L^-1 IAA (61%) and 1 mg L^-1 NAA (67%) (Table 2).

The de-differentiation process from active proliferated and meristematic cells tends to produce callus that leads to thicker, stiffer, swollen and visible tissues of the explant. Unorganized masses of cells generally termed callus are produced by actively divided cells on the surface of an explant. In the present study 1.5 mg L^-1 2,4-D produced the highest callus which encouraged most parts of the explant which shows the efficiency of 2,4-D to produce more callus as compared to IAA and NAA. As a common auxin, 2,4-D produces active proliferation from the dedifferentiation of explant cells [²⁹]. Furthermore, growth regulator concentrations especially auxin and cytokinin in the culture medium are critical to controlling growth and morphogenesis. Generally, high concentrations of auxins and low cytokinin's in the medium promote abundant cell proliferation with the formation of callus [³⁰]. The provision of exogenous auxin and cytokinin influence callus in various plant species. Generally, an equal ratio of auxin and cytokinin stimulates callus induction, while a high ratio of cytokinin-to-auxin or auxin-to-cytokinin induces shoot and root regeneration, respectively [²⁶]. An excellent callus induction was observed at 3.5 and 3.0 mg L^-1 2, 4-D after a culture period of 4-5 weeks [³¹]. The present results are in close conformity with Mastuti and coauthors [³²] who reported more callus induction and growth in Physalis angulata when growth medium was fortified with 2,4-D and Kinetin as compared to IAA and NAA. Further, Kintzios and Skoula [³³] also reported almost similar results, who investigated that a higher rate of callus induction was obtained when an intermediate auxin and cytokinin concentration were used for S. officinalis.

Callus fresh and dry weight

The fresh and dry weight of the callus was significantly influenced by the auxins. The media supplemented with 2,4-D produced higher fresh and dry weight of callus as compared to NAA and IAA. The highest callus fresh weight (1.89 g L^-1) was observed at 1 mg L^-1 2,4-D and dry weight (1.13 g L^-1) at 2 mg L^-1 2,4-D. Media supplemented with 0.5 and 1 mg L^-1 of IAA developed callus with the lowest fresh weight of 0.72 and 0.7 g which is statistically at par with 0.5 mg L^-1 NAA (0.78 g L^-1). The minimum dry weight was observed at 0.5 mg L^-1 IAA, followed by 2 mg L^-1 NAA. This indicated that 2,4-D was more suitable regarding the callus weight (Table 3 and Figure 1).

Table 3
Effect of plant growth regulators for callus fresh weight, dry weight, callus color and texture of S. moorcroftiana

Treatments	Callus fresh weight (g L-1)	Callus dry weight (g L-1)	Callus color	Texture
Control			-		-
2,4-D0.5+BAP	1.02±0.01d	0.45±0.01e	whitish-brown		compact
2,4-D1+BAP	1.89±0.02a	0.86±0.01c	green		compact
2,4-D1.5+BAP	1.68±0.02b	0.94±0.02b	whitish		friable
2,4-D2+BAP	1.62±0.01b	1.13±0.01a	whitish		friable
NAA0.5+BAP	0.78±0.01e	0.45±0.01e	whitish-brown		friable
NAA1+BAP	0.98±0.01d	0.85±0.02c	whitish-green		Friable
NAA1.5+BAP	1.26±0.01c	0.34±0.01f	green		Compact
NAA2+BAP	1.53±0.02b	0.24±0.01g	green		Compact
IAA0.5+BAP	0.72±0.02e	0.15±0.01h	green		Compact
IAA1+BAP	0.70±0.03e	0.30±0.01fg	green		Compact
IAA1.5+BAP	1.13±0.03cd	0.60±0.01d	green		Compact
IAA2+BAP	1.24±0.04c	0.93±0.03b	green		Compact
ANOVA	***	***	***	***

An important approach to callus induction and growth is to determine the optimum amount of growth regulators especially auxin and cytokinin in the culture medium. The proper amount of auxin and cytokinin concentration should be optimized for more biomass and secondary metabolites production [³⁴]. Further, callus growth generally depends on the explant nature and also the auxin and cytokinin amalgamation [³⁵] which may be the fact that exogenously applied PGR’s increase the synthesis of endogenous cytokinin and auxin which results in increment in callus growth and mass especially fresh and dry weight as for callus induction and growth, auxins and cytokinins are widely used [³⁶]. The increase in callus biomass (fresh and dry) might also be due to the increase in cell division, elongation, differentiation in vascular tissues, root formation and rhizo-genesis, auxiliary shoot growth inhibition and embryogenesis by auxins and cytokinin [³⁷]. Furthermore, reprogrammed differentiated cells due to methylated DNA than usual by auxin also start cell division and elongation thereby increasing the fresh and dry biomass [²⁹]. The present results are in close conformity with Rehman coauthors [³⁸] who reported that callus biomass was significantly increased with the application of 2,4-D furthermore the addition of BAP enhanced the role of 2,4-D which resulted in increased biomass of Caralluma tuberculata callus. The results are supported by Blinstrubiene and coauthors [³⁹] who observed that media when augmented with BAP and 2,4-D significantly increased the callus fresh weight of Stevia rebaudiana. These results are in agreement with Aghaei and coauthors [⁴⁰] on Pistacia atlantica, Wang and Bao [⁴¹] on Viola wittrockiana and Manisha and Rajesh [⁴²] on Tecomella undulata. Moreover, these results are also in accordance with Andre and coauthors [⁴³] who reported that a significant increase in callus weight was observed when treated with 2,4-D and BAP. Similar results were also observed by Rahayu and coauthors [⁴⁴] on Centella asiatica who found that the addition of auxin to the media significantly increased callus biomass. These results are also in conformity with Fatima and coauthors [³⁵] on Digitalis lanata who reported that an increase in 2, 4-D hormone was related to an increase in dry weight.

Figure 1
Effect of plant growth regulators for callus fresh weight, dry weight, callus color and texture of S. moorcroftiana. W1: Control, W2: 2,4-D_0.5+BAP, W3: 2,4-D₁+BAP, W4: 2,4-D_1.5+BAP, W5: 2,4-D₂+BAP, W6: NAA_0.5+BAP, W7: NAA₁+BAP, W8: NAA_1.5+BAP, W9: NAA₂+BAP, W10: IAA_0.5+BAP, W11: IAA₁+BAP, W12: IAA_1.5+BAP, W13: IAA₂+BAP.

Callus morphology

Callus of whitish brown color and compact texture were observed in media fortified with a combination of 2,4 D (0.5 mg L^-1). On the other hand, callus with the same whitish brown color but having afriable texture were observed in media supplemented with NAA (0.5 mg L^-1). The calluses of green color and compact texture were found in MS media augmented with 2,4 D (1 mg L^-1), NAA (1.5 mg L^-1), NAA (2 mg L^-1), IAA (0.5 mg L^-1), IAA (1 mg L^-1), IAA (1.5 mg L^-1) and IAA (2 mg L^-1). A callus of whitish green color with a friable texture was observed in media containing growth hormones 2,4-D (1.5 mg L^-1) and 2,4-D (2 mg L^-1). One of the media amongst all supplemented with NAA (1 mg L^-1) developed callus of whitish green color with the friable texture (Table 3, Figure 2 and 3).

Figure 2
Callus proliferation under various levels of Melatonin. A) Control, B) 0.5, C) 1, D) 1.5, and E) 2 mg L^-1 of Melatonin keeping 2,4-D + BA

Figure 3
Sampling and culturing of nodal cuttings for multiplication under laminar flow unit, callus formation and plants formation after successful multiplication through in-vitro propagation.

Usually, a single explant comprises of callus having various strains which considerably affect the amount of consistency, color and morphogenetic competence of the callus [²⁹]. The color and texture of the callus can be altered by using different concentrations and types of PGR’s (alone and in combination) present in MS media [⁴⁵]. Application of exogenous PGR’s also affects the internal concentration of enzymes and plant hormones. Endogenous plant hormones are synthesized with the help of these exogenous plant hormones [⁴⁶] thereby affecting callus color, texture and nature. Auxin is dynamic for callus growth and development; it causes various distinct effects with the help of diverse levels used in the culture medium [⁴⁷]. It is considered that auxin promotes physiological modification, which mainly starts the process of cell division and differentiation. The manipulation of endogenous cytokinin is responsible for the cell aggregation that leads to compact structured callus [⁴⁸]. Similar results were also observed by Elaleem and coauthors [⁴⁹] who investigated the medium preparation, concentration and combination of PGR’s affect the callus texture, color and nature. Rajaram and coauthors [⁵⁰] achieved green compact callus of caralloma fimbriata callus in the MS medium supplemented with 2, 4-D and BAP. Furthermore, Sreelatha and coauthors [⁵¹] attained a compact, green callus from an explant of Caraloma stalagmifera cultured on MS medium using 2, 4-D and Kn.

Effect of melatonin on the callus growth and accumulation of phenol, flavonoids and antioxidant activities in callus culture of S. microftiana.

Fresh and dry weight

The fresh and dry weight of the callus was significantly influenced by the different concentrations of melatonin and in combination with the contact level of PGR’s auxins. A significant increase in S. moorcroftiana callus fresh weight was obtained under different concentrations of melatonin and in combination with constant levels of PGR’s (1 mgL^-1 2, 4-D and 1 mgL^-1 BAP). The maximum fresh and dry weight accumulation (2.53 and 1.92 mg L^-1) was recorded on MS medium fortified with 1.5 mgl^-1 melatonin, respectively, followed by fresh (2.11 mg L^-1) and dry (1.28 mg L^-1) weight in tissue culture media fortified with melatonin at 1 mg L^-1. Whereas minimum fresh and dry weight biomass accumulation (1.66 and 0.75 mg L^-1) was observed on MS medium containing 2 mg L^-1 melatonin. This indicated that melatonin is suitable for better fresh and dry weight of callus (Table 4 and Figure 4).

Table 4
Effect of various concentrations of melatonin for callus fresh and dry weight of S. moorcroftiana

Melatonin (mg L-1)	callus fresh weight (mg L-1)	callus dry weight (mg L-1)	DPPH activity	Phenolics (mg g-1 DW)	Flavonoids (mg g-1 DW)
0	1.87±0.02c	0.86±0.01d	80.98±0.6C	0.86 ±0.01D	0.89±0.01D
0.5	1.74±0.02d	1.16±0.01c	80.02±0.5D	1.12 ±0.01C	1.12 ±0.01C
1	2.11±0.02b	1.29±0.01b	82.11±0.8B	1.29±0.01B	1.33±0.01B
1.5	2.53±0.03a	1.92±0.01a	87.20 ±0.7A	1.92±0.02A	2.19±0.02A
2	1.66±0.01e	0.75±0.01e	79.83 ±0.7D	1.05±0.01C	1.12 ±0.01C
ANOVA	***		***	***	***

Figure 4
Callus fresh weight (A), dry weight (B), DPPH (C), total phenols (D) and total flavonoids (E) of S. moorcroftiana as affected by various concentrations of melatonin. W1: Control, W2: 0.5 mg L^-1 melatonin, W3: 1.5 mg L^-1 melatonin, W4: 1.5 mg L^-1 melatonin, W5: 2.0 mg L^-1 melatonin.

Melatonin is considered as a promoter of plant growth, development and adaptation [⁵²] and is known to have a stimulatory effect on callus fresh weight and growth which may be because the growth is triggered due to the melatonin stimulated biosynthesis of IAA [⁵³]. Melatonin in combination with auxin also induces the organogenic pattern of plants [⁵⁴]. Melatonin plays an important role in regulation of photosynthesis, callus growth, formation and root regeneration of explants [⁵⁵-⁶⁵]. The increase in fresh and dry biomass after treatment with melatonin is due to the role of melatonin in cell expansion which increases the cell volume thereby increasing the callus biomass [⁵⁷]. Furthermore, melatonin has also a role in inhibition of ACC oxidase activity, hence showing greater similarity to auxin in some physiological responses [⁵⁸], resulting in increased the fresh and dry biomass. In the present study it was also observed that increase in melatonin content from 1.5 to 2 mg L^-1 decreased the fresh and dry biomass. This might be due to the fact that melatonin in higher concentrations generates ROS which leads to cell apoptosis thereby preventing cellular growth and proliferation [⁵⁹]. The present results are in conformity with Hernández-Ruiz and Arnao [⁵⁸] in Lupin cotyledons who reported that a significant increase in biomass was noted in the presence of melatonin. Similar results were also recorded in Cucumis sativus [⁵⁵], Punica granatum [⁶⁰] and Oryza sativa [⁶¹]. Moreover, a decrease in biomass accumulation of Fagonia indica was observed with a higher concentration of melatonin [⁶²].

DPPH free radical scavenging activity

Different concentration of melatonin with constant level of plant growth regulators (1 mg L^-1 2, 4-D and 1mg L^-1 BAP), the antioxidant activity (DPPH) in terms of free radical scavenging activity was determined in S. moorcroftiana callus (Table 4). The maximum antioxidant activity (87.197 %) was observed in callus fortified with 1.5 mg L^-1 melatonin in combination with 1mgL^-1 2,4-D and 1mg L^-1 BAP which was followed by 1 mgL^-1 melatonin concentration (82%) in combination with 2,4-d and BAP (1mg L^-1), while that of minimum DPPH radical scavenging activity (80%) was recorded in 2 mg L^-1 melatonin in combination with PGR’s which is statistically at par with 0.5 mgL^-1 melatonin (80.81%).

The DPPH assay reflects the capability to scavenge free radicals, which incorporates hydrogen radicals from potential antioxidants [¹⁰]. Melatonin has been reported to enhanced phytochemical constituents and improved bio-reductive capacity [¹⁷]. Callus produced in the presence of melatonin proved to have the highest reducing power (RP) because melatonin enhanced the phytochemical constituents (flavonoids and phenolics) in callus cultures by acting as a good reductone (reductones are terminators of free radical chain reactions). Melatonin has been reported to have higher antioxidant activity in different plants than other PGRs [⁶³]. Melatonin also activities antioxidant enzymes [⁶⁴] which protect plants from oxidative damage and increases the efficiency of the mitochondrial electron transport chain [⁶⁵] which results in improved antioxidant activity. Bioactive compounds like flavonoids and phenolics acted as an electron donor and scavenge free radicals by converting them into more stable products and electron donation capacity of the plant depends on the amount of bioactive compounds present in it. Similar results were also found by Arnao and Hernandez-Ruiz [¹⁶] in Lupinus albus. Moreover, Liang and coauthors [⁶⁶] also confirmed the present results, who reported improved phenolic and antioxidant compounds in the leaves of Actinidia spp. (kiwifruit) by exogenous application of melatonin.

Total phenolic content

Under various concentrations of melatonin with a combination of constant levels of plant growth regulators (1 mg L^-1 2,4-D and 1mg L^-1 BAP) (Table 4). The total phenolic content of S. moorcroftiana callus was recorded. The maximum total phenolic content was accumulated in callus proliferated on MS medium augmented with 1.5 mg L^-1 melatonin (1.92 mg g^-1 DW) in combination with PGR’s, which was followed by 1 mg L^-1 melatonin (1.29 mg g^-1 DW). However, the minimum total phenolic content yield (0.86 mg g^-1 DW) was recorded on controlled treatment. While that of maximum melatonin concentration (2 mgL^-1) gives the yield of (1.05 mg g^-1 DW) which is statistically similar to 5 mg L^-1 melatonin concentration (1.12 mg g^-1 DW).

Phenolics are low molecular weight anti-oxidative compounds found in various plant species, which are useful against several disorders [⁶⁷-⁶⁹]. Physiological responses are positively affected by exogenously applied melatonin [⁶⁶]. Melatonin also has a beneficial function in signaling by inducing various metabolic pathways and stimulating the production of different secondary metabolite plant substances [⁷⁰]. Biomass accumulation and phenolic production are directly proportional and strongly dependent on the activation of key enzymes (tyrosine ammonia lyase) responsible for phytochemical production [⁷¹] which is greatly activated by melatonin thereby increasing phenolic and antioxidant activity. Melatonin is considered as an antioxidant which directly detoxifies ROS and reactive nitrogen species and also stimulates many antioxidant enzymes indirectly which suppresses pro-oxidant enzyme activity thereby increasing antioxidant activity such as phenolic [⁷²] etc. The up regulation of phenylpropanoid genes by melatonin also results in polyphenol metabolism thereby increasing the phenolic content [⁷³]. The results are in agreement with Riaz and coauthors [⁷⁴] who observed that Melatonin significantly increased TPC values. Similar results were also found by Sumaira and coauthors [⁷⁵] and Sheshadri and coauthors [⁷⁶] also reported that Melatonin greatly enhances the biosynthesis of phytochemicals.

Total flavonoid content (TFC)

In culture media addition of melatonin in combination with constant level of plant growth regulators (1 mg L^-1 2,4-D and 1 mg L^-1 BAP) resulted in significant variation of total flavonoids content accumulation in S. moorcroftiana callus culture. Maximum total flavonoid production (2.19 mg g^-1 DW) was obtained in callus cultured on MS medium supplemented with 1.5 mg L^-1 melatonin, which was closely followed by 1mg L^-1 melatonin (1.33 mg g^-1 DW). However, the maximum level of melatonin is statistically at par with 0.5 mg L¹ melatonin concentration (1.12 mg g^-1 DW) and (1.12 mg g^-1 DW) respectively. However, the minimum total flavonoid content yield is recorded on control treatment with 0 level of melatonin (0.89 mg g^-1 DW) (Table 4). Flavonoids are plant secondary metabolites that show in vitro and in vivo antioxidant activity due to the availability of free hydroxyl (OH^-) groups, particularly 3-OH [⁷⁷-⁷⁸]. Flavonoids are a class of phenolic compounds having antimicrobial, antioxidant, anti-inflammatory and allergenic activities [⁷⁹-⁸³]. Callus having different TFC strongly depends upon PGR type and concentration [⁸²]. The results are in line with those of Riaz and coauthors [⁷⁴] who reported that application of Melatonin in combination with NAA significantly increased the total flavonoids content in the callus. Similarly, Sheshadri and coauthors [⁷⁶] also reported that melatonin greatly enhances the biosynthesis of phytochemicals.

CONCLUSION

The current results suggest that using various PGRs and melatonin concentrations significantly enhanced the callus induction and secondary metabolites production in the callus culture of S. moorcroftiana (Figure 5). The application of 2,4-D+BAP at rate of 1+1 mg L^-1 significantly increased the callus induction and callus growth. Callus culture supplemented with 1.5 mg melatonin L^-1 significantly increased the phenols, flavonoids and DPPH scavenging activity of the callus culture.

Figure 5
Graphical abstract that shows the effect of different concentration of melatonin on callus shape and color.

• Funding: This research received no external funding.

REFERENCES

• ¹ Mamadalieva NZ, Akramov DK, Ovidi E, Tiezzi A, Nahar L, Azimova SS, et al. Aromatic Medicinal Plants of the Lamiaceae Family from Uzbekistan: Ethno-pharmacology, Essential Oils Composition, and Biological Activities. Medicines. 2017;4(1):8.
• ² Bolta Z, Baricevic D, Bohanec B, Andrensek S. A preliminary investigation of ursolic acid in cell suspension culture of Salvia officinalis Plant Cell, Tissue Organ Cult. 2000; 62: 57-63.
• ³ Slusarczyk S, Zimmermann S, Kaiser M, Matkowski A, Hamburger M, Adams M. Antiplasmodial and antitrypanosomal activity of tanshinone-type diterpenoids from Salvia miltiorrhiza Planta Med. 2011; 77: 1594-6.
• ⁴ Kontogianni VG, Tomic G, Nikolic I, Nerantzaki AA, Sayyad N, Stosic-Grujicic S, et al. Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity. Food Chem. 2013;136(1):120-9.
• ⁵ Dhifi W, Bellili S, Jazi S, Bahloul N, Mnif W. Essential oils’ chemical characterization and investigation of some biological activities: a critical review. Medicines. 2016; 3:25.
• ⁶ Avato P, Fortunato IM, Ruta C, D'Elia R. Glandular hairs and essential oils in micropropagated plants of Salvia officinalis L. Plant Sci. 2005;169:29-36.
• ⁷ Skala E, Wysokinska H. In vitro regeneration of Salvia nemorosa L. from shoots tips and leaf explants. In vitro Cell Dev Biol Plant. 2004;40(6):596-602.
• ⁸ Ashry NA, Ghonaim MM, Mohamed HI, Mogazy AM. Physiological and molecular genetic studies on two elicitors for improving the tolerance of six Egyptian soybean cultivars to cotton leaf worm. Plant Physiol Biochem. 2018; 130:224-34.
• ⁹ Alam M, Hayat K, Ullah I, Sajid M, Ahmad M, Basit A, et al. Improving okra (abelmoschus esculentus l.) growth and yield by mitigating drought through exogenous application of salicylic acid. Fresenius Environ Bull. 2020; 29:529-35.
• ¹⁰ Abu-Shahba MS, Mansour MM, Mohamed HI, Sofy MR. Comparative cultivation and biochemical analysis of iceberg lettuce grown in sand soil and hydroponics with or without microbubble and microbubble. J Soil Sci Plant Nutri. 2021; 21:389-403.
• ¹¹ Moustafa-Farag M, Mohamed HI, Mahmoud A, Elkelish A, Misra AN, Guy KM, et al. Salicylic acid stimulates antioxidant defense and osmolyte metabolism to alleviate oxidative stress in watermelons under excess boron. Plants. 2020; 9 (724); doi:10.3390/plants9060724.
  » https://doi.org/10.3390/plants9060724
• ¹² Naeem M, Basit A, Ahmad I, Mohamed HI, Wasila H. Effect of salicylic acid and salinity stress on the performance of tomato. Gesunde Pflanz.2020; 72:393-402
• ¹³ Mohamed HI, Mohammed AHMA, Mohamed NM, Ashry NA, Zaky LM, Mogazy AM. Comparative effectiveness of potential elicitors of soybean plant resistance against Spodoptera Littoralis and their effects on secondary metabolites and antioxidant defense system Gesunde Pflanz. 2021; 73:273-85
• ¹⁴ Cuenca S, Amo-Marco JB. In vitro propagation of two Spanish endemic species of Salvia through bud proliferation. In vitro Cell Dev Biol Plant. 2000; 36: 225-9.
• ¹⁵ Misic D, Grubisic D, Konjevic R. Micro-propagation of Salvia brachyodon through nodal explants. Biology of Plants. 2006; 50:473-6.
• ¹⁶ Arnao MB, Herna´ndez-Ruiz J. Melatonin: possible role as light-protector in plants. In UV Radiation: Properties, Effects, and Applications. Physics Research & Technology Series (In: Radosevich, J.A., ed.), 2014; pp. 79-92. Nova Science Publishing.
• ¹⁷ Arnao MB, Hernández-Ruiz J. Functions of melatonin in plants: a review. J Pineal Res. 2015; 59:133-50.
• ¹⁸ Sofy AR, Sofy MR, Hmed AA, Dawoud RA, Refaey EE, Mohamed HI, et al. Molecular characterization of the Alfalfa mosaic virus infecting Solanum melongena in Egypt and control of its deleterious effects with melatonin and salicylic acid. Plants. 2021;28;10(3):459. doi: 10.3390/plants10030459.
  » https://doi.org/10.3390/plants10030459
• ¹⁹ Shi H, Chen K, Wei Y, He C. Fundamental issues of melatonin-mediated stress signaling in plants. Front Plant Sci. 2016; 7:1124.
• ²⁰ Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am J Enol Vitic. 1965; 16: 144-58.
• ²¹ Park HH, Lee S, Son HY, Park SB, Kim MS, Choi EJ, et al. Flavonoids inhibit histamine release and expression of proinflammatory cytokines in mast cells. Arch Pharm Res. 2008; 31(10):1303-11
• ²² Ahmad M, Khattak MR, Jadoon SA, Rab A, Basit A, Ullah I, Khalid MA, Ullah I, Shair M. Influence of zinc sulphate on flowering and seed production of flax (Linum usitatissimum L.): A medicinal flowering plant. Inter J Biosci. 2019; 14: 464-76.
• ²³ Steel RGD, Torrie JH, Dickey DA. Principles and Procedures of Statistics, A Biometrical Approach, 3^rd Edn. New York, NY: McGraw Hill Book Int. Co, 1997; 172-7.
• ²⁴ Anjusha S, Gangaprasad A. Callus culture and in vitro production of anthraquinone in Gynochthodes umbellata L. Razafim and B. Bremer (Rubiaceae). Ind. Crops Prod. 2017; 95: 608-14.
• ²⁵ Pasternak T, Miskolczi P, Ayaydin F, Mészáros T, Dudits D, Fehér A. Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa. Plant Growth Regul. 2000; 32: 129-41.
• ²⁶ Rahman NNA, Rosli R, Kadzimin S, Hakiman M. Effects of auxin and cytokinin on callus induction in Catharanthus roseus (L.) G. Don. Fundamental App Agri. 20019; 4(3): 928-32.
• ²⁷ Hesami M, Daneshvar MH. Indirect organogenesis through seedling-derived leaf segments of Ficus religiosa- a multipurpose woody medicinal plant. J Crop Sci Biotechnol. 2018; 21:129-36.
• ²⁸ Huang, Staden JV. Salvia chamelaeagnea can be micropropagated and its callus induced to produce rosmarinic. South African J Bot. 2002; 68: 177-80.
• ²⁹ George E, Hall MA, Klerk J. The components of plant tissue culture media I: macro- and micro-nutrients. E. George, M.A. Hall, G.-J. Klerk (Eds.), Plant Propagation by Tissue Culture, Springer, Netherlands. 2008; pp. 65-113
• ³⁰ Sugimoto K, Jiao Y, Meyerowitz EM. Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell. 2010; 18:463-71.
• ³¹ Shah MI, Musarrat J, Ihsan I. In-vitro callus induction, its proliferation and regeneration in seed explants of wheat (Triticum aesitivum) var. LU-26S. Pak J Bot. 2003;35(2):209-17.
• ³² Mastuti R, Munawarti A, Firdiana E. The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. - A medicinal plant. AIP Conference Proceedings 1908, 040007 (2017); https://doi.org/10.1063/1.5012721
  » https://doi.org/10.1063/1.5012721
• ³³ Kintzios S, AN, Skoula M. Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures. Plant Cell Rep. 1999;18(6):462-6.
• ³⁴ Raj D, Kokotkiewicz A, Drys A, Luczkiewicz M. Effect of plant growth regulators on the accumulation of indolizidine alkaloids in Securinega suffruticosa callus cultures. Plant Cell Tissue Organ Cult. 2015;123(1):39-45. doi:10.1007/s11240-015-0811-6
  » https://doi.org/10.1007/s11240-015-0811-6
• ³⁵ Fatima Z, Mujib A, Fatima S, Arshi A, Umar S. Callus induction, biomass growth and plant regeneration in Digitalis lanata Ehrh: influence of plant growth regulators and carbohydrates. Turkish J Bot. 2009; 33(6): 393-405.
• ³⁶ Aloni R, Aloni E, Langhans M, Ullrich CI. Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. Ann Bot. 2006; 97:883-93
• ³⁷ Park WT, Kim YK, Udin MR, Park NI, Kim SG, Young L, et al. Somatic embryogenesis and plant regeneration of lovage (Levisticum officinale Koch). Plant Omics J. 2010; 3:159-61.
• ³⁸ Rehman R, Chaudary MF, Khawar KM, Lu G, Mannan A, Zia M. In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential. Biologia. 2014; 69(3): 341-9.
• ³⁹ Blinstrubiene A, Burbulis N, Juškevičiūtė N, Vaitkevičienė N, Žūkienė R. Effect of Growth Regulators on Stevia rebaudiana Bertoni Callus genesis and influence of auxin and proline to steviol glycosides, phenols, flavonoids accumulation, and antioxidant activity In vitro Molecules 2020; 25:2759; doi:10.3390/molecules25122759
  » https://doi.org/10.3390/molecules25122759
• ⁴⁰ Aghaei P, Bahramnejad B, Mozafari AA. Effect of different plant growth regulator on callus induction of stem explants in Pisticia altanica subsp. Kurdica. Plant knowl J. 2013; 2(3):108-12.
• ⁴¹ Wang J, Bao M. Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus. Sci Horti. 2007;111(3):266-70
• ⁴² Manisha BP, Rajesh SP. Impact of Plant Growth regulators (PGRs) on callus induction from internodal explants of Tecomella undulata (Sm.) Seem- A Multipurpose Medicinal plants. Inter J Sci Res Publications 2013;3(11).
• ⁴³ Andre ST, Mongomake KK, Modeste KK, Edmond KK, Tchoa K, Hilaire KT, et al. Effect of plant growth regulators and carbohydrates on callus induction and proliferation from leaf explant of Lippia multiflora Moldenke (verbeneacea). Int J Agri Crop Sci. 2015;8(2):118-27.
• ⁴⁴ Rahayu S, Ika Roostika I, Bermawie N. The effect of types and concentrations of auxins on callus induction of Centella asiatica Nus Biosci. 2016; 8:283-7.
• ⁴⁵ Baker Siddique A, Ara I, Islam SM, Tuteja N. Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.) Plant Signal Behav. 2014; 9:1-10
• ⁴⁶ Gaspar MT, Kevers C, Rampart F, Pencil C, Greppin H, Dommes J, Creveco M. Changing concept in plant hormones action. J in-vitro Dev Biol Plant. 2003; 39:85-105.
• ⁴⁷ Ren JP, Wang XG, Yin J. Dicamba and sugar effects on callus induction and plant regeneration from mature embryo culture of wheat. Agric Sci China. 2010;9(10):31-7
• ⁴⁸ Machakova I, Zazimolova E, George IEF. Plant growth regulators: introductions of auxins, their analogues, inhibitor. J Plant Pro Tissue Cult. 2008; 1(3):175-20
• ⁴⁹ Elaleem kGA, Modawi RS, Khalafalla MM. Effect of plant growth regulators on callus induction in tuber segment culture of potato (Solanum tubrosum L.) cultivar diament. Afr J Biotechnol. 2009; 8(11): 2529-34.
• ⁵⁰ Rajaram R, Priya D, Sudarshana DVI, Suresh KP. In vitro regeneration of Caralluma fimbriata wall. By organogenesis: A potent medicinal plant. A.J.C.S. 2012; 6(1): 41-5.
• ⁵¹ Sreelatha VR, Thippeswamy M, Pullaiah T. In vitro callus induction and plant regeneration from intermodal explants of Caralluma stalagmifera F. Intl J Adv Res. 2015;3(2): 472-80.
• ⁵² Erland LA, Murch SJ, Reiter RJ, Saxena PK. A new balancing act: the many roles of melatonin and serotonin in plant growth and development. Plant Signal Behav. 2015;10: e1096469. doi: 10.1080/15592324.2015.1096469
  » https://doi.org/10.1080/15592324.2015.1096469
• ⁵³ Chen Q, QI W, Reiter RJ, Wei W, Wang B. Exogenously applied melatonin stimulates root growth and raises endogenous IAA in roots of etiolated seedling of Brassica juncea. J Plant Physiol. 2009; 166:324-8.
• ⁵⁴ Arnao MB, Hernández-Ruiz J. Melatonin promotes adventitious and lateral root regeneration in etiolated hypocotyls of Lupinus albus L. J Pineal Res. 2007;42:147-52.
• ⁵⁵ Zhang N, Zhao B, Zhang HJ, Weeda S, Yang C, Yang ZC, et al. Melatonin promotes water-stress tolerance, lateral root formation, and seed germination in cucumber (Cucumus sativus L.). J. Pineal Res. 2013; 54:15-23.
• ⁵⁶ Zhang N, Sun Q, Zhang H, Cao Y, Weeda S, Ren S, et al. Roles of melatonin in abiotic stress resistance in plants. J Exp Bot. 2015; 66:647-56.
• ⁵⁷ Thomas JC, Katterman F. 5-Bromodeoxyuridine inhibition of cytokinin induced radish cotyledon expansion. Plant Sci.1992; 83:143-8.
• ⁵⁸ Hernández-Ruiz, J, Arnao МВ. Melatonin stimulates the expansion of etiolated lupin cotyledons. Plant Growth Regul. 2008; 55:29-34.
• ⁵⁹ Fazal H, Abbasi BH, Ahmad N, Ali M. Exogenous melatonin trigger biomass accumulation and production of stress enzymes during callogenesis in medicinally important Prunella vulgaris L. (Selfheal). Physiol Mol Biol Plants. 2018; 24:1307-15.
• ⁶⁰ Sarrou E. Melatonin and other factors that promote rooting and sprouting of shoot cuttings in Punica granatum var Wonderful. Turk J Bot. 2014; 38:293-301
• ⁶¹ Park S, Back K. Melatonin promotes seminal root elongation and root growth in transgenic rice after germination. J Pineal Res. 2012; 53:385-9.
• ⁶² Khan T, Ullah MA, Garros L, Hano C, Abbasi BH. Synergistic effects of melatonin and distinct spectral lights for enhanced production of anti‐cancerous compounds in callus cultures of Fagonia indica J. Photochem. Photobiol. B: Biol. 2018;190,163‐71.
• ⁶³ Posmyk MM, Janas KM. Melatonin in plants. Acta Physiol Plant. 2009; 31:1.
• ⁶⁴ Li C, Wang P, Wei Z, Liang D, Liu C, Yin L, et al. The mitigation effects of exogenous melatonin on salinity-induced stress in Malus hupehensis J. Pineal Res. 2012; 53:298-306.
• ⁶⁵ Akladious SA, Mohamed HI. Physiological role of exogenous nitric oxide in improving performance, yield and some biochemical aspects of sunflower plant under zinc stress. Acta Biol Hung. 2017; 68(1), 101-114.
• ⁶⁶ Liang D, Shen Y, Ni Z, Wang Q, Lei Z, Xu N, et al. Exogenous melatonin application delays senescence of kiwifruit leaves by regulating the antioxidant capacity and biosynthesis of flavonoids. Front Plant Sci. 2018; 9:426.
• ⁶⁷ El-Beltagi HS, Mohamed HI, Elmelegy AA, Eldesoky SE, Safwat G. Phytochemical screening, antimicrobial, antioxidant, anticancer activities and nutritional values of cactus (Opuntia Ficus Indicia) pulp and peel. Fresenius Environ Bull. 2019; 28(2A):1534-51.
• ⁶⁸ Mohamed HI, Abd-El Hameed AG. Molecular and biochemical markers of some Vicia faba L. genotype in response to storage insect pests infestation. J Plant Inter. 2014;9(1):618-26.
• ⁶⁹ Helmi A, Mohamed HI. Biochemical and ulturasturctural changes of some tomato cultivars to infestation with Aphis gossypii Glover (Hemiptera: Aphididae) at Qalyubiya, Egypt. Gesunde Pflanz. 2016; 68:41-50.
• ⁷⁰ Tan DX, Hardeland R, Manchester LC, Korkmaz A, Ma S, Rosales- Corral S, Reiter RJ. Functional roles of melatonin in plants, and perspectives in nutritional and agricultural science. J Exp Bot. 2012; 63: 577-97.
• ⁷¹ Ali M, Abbasi BH. Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L. Ind Crops Prod. 2013; 49:400-6.
• ⁷² Reiter RJ, Mayo JC, Tan DX, Sainz RM, Alatorre-Jimenez M, Qin, LL. Melatonin as an antioxidant: under promises but over delivers. J. Pineal Res. 2016; 61: 253-78.
• ⁷³ Xu L, Yue Q, Bian F, Sun H, Zhai H and Yao Y. Melatonin Enhances Phenolics Accumulation Partially via Ethylene Signaling and Resulted in High Antioxidant Capacity in Grape Berries. Front Plant Sci. 2017; 8:1426. doi:10.3389/fpls.2017.01426
  » https://doi.org/10.3389/fpls.2017.01426
• ⁷⁴ Riaz HR, Hashmi SS, Khan T, Hano C, Guivarc’h NG, Abbasi BH. Melatonin-stimulated biosynthesis of anti-microbial ZnONPs by enhancing bio-reductive prospective in callus cultures of Catharanthus roseus var. Alba, Artificial Cells. J Nanomed Biotechnol. 2018; l 46(2):936-50.
• ⁷⁵ Sumaira, TK, Abbasi BH, Afridi MS, Tanveer F, Ikram Ullah, Bashira S, et al. Melatonin-enhanced biosynthesis of antimicrobial AgNPs by improving the phytochemical reducing potential of a callus culture of Ocimum basilicum L. var. thyrsiflora. This journal is The Royal Society of Chemistry, 2017. RSC Adv. 2017; 7:38699-713.
• ⁷⁶ Sheshadri SA, Sriram S, Balamurugan P et al. Melatonin improves bioreductant capacity and silver nanoparticles synthesis using Catharanthus roseus leaves, RSC Adv. 2015; 5(59): 47548-54.
• ⁷⁷ Abd El- Rahman SS, Mohamed HI. Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits. Acta Physiol Plant. 2014;36(2):343-54.
• ⁷⁸ Mohamed HI, Elsherbiny EA, Abdelhamid MT. Physiological and biochemical responses of Vicia faba plants to foliar application with zinc and iron. Gesunde Pflanz. 2016; 68: 201-12.
• ⁷⁹ Aly AA, Mansour MTM, Mohamed HI, Abd-Elsalam KA. Examination of correlations between several biochemical components and powdery mildew resistance of flax cultivars. Plant Pathol J. 2012;28(2):149-55.
• ⁸⁰ Aly AA, Mohamed HI, Mansour MTM, Omar MR. Suppression of powdery mildew on flax by foliar application of essential oils. J Phytopathol. 2013; 161:376-81.
• ⁸¹ Ghonaim MM, Mohamed HI, Omran AAA. Evaluation of wheat salt stress tolerance using physiological parameters and retrotransposon-based markers. Genet Resour Crop Evol. 2021; 68:227-42.
• ⁸² Arif M, Rauf S, Din AU, Rauf M, Afrasiab H. High frequency plant regeneration from leaf derived callus of Dianthus caryophyllus L. Ajps. 2014; 5: 24-54.
• ⁸³ Abd El- Rahman SS, Mazen MM, Mohamed HI, Mahmoud NM. Induction of defense related enzymes and phenolic compounds in lupine (Lupinus albus L.) and their effects on host resistance against Fusarium wilt. European J Plant Pathol. 2012; 134:105-16

Edited by

• Editor-in-Chief: Alexandre Rasi Aoki

• Associate Editor: Adriel Ferreira da Fonseca

Publication Dates

• Publication in this collection
  28 Mar 2022
• Date of issue
  2022

History

• Received
  01 Apr 2021
• Accepted
  14 Oct 2021