Biotechnological

Communication

Biosci. Biotech. Res. Comm. 9(4): 702-711 (2016)

In uence of foliar application with amino acids and

citric acid on physiological and phytochemical responses

Artemisia abrotanum

produced by in vitro culture

Mohammad M. EL-Zefzafy

, Ismail M. A. Shahhat

1,2

, Rania S. Yousef

and

Eman R. Elsharkawy

4,5

1,2

National Organization for Drug Control and Research (NODCAR), Medicinal Plants Dept.

(

Tissue Culture &

Plant Physiology), Egypt.

Northern Border University, Biology Dept. Division of Botany (Plant Physiology), Faculty of Science, KSA.

Cairo University, Faculty of Agriculture, Biochemistry Dept., Egypt.

Department of Environment and Plant Pasture, Desert Research Center, Mathef El-Mataria, Egypt.

Northern Border University, Chemistry Department, Faculty of Science, Girl Division, Arar- Kingdom of

Saudi Arabia

ABSTRACT

The physiological and phytochemical responses of Artemisia abrotanum to foliar application of different concentra-

tions of amino acids (tryptophan and phenylalanine) and citric acids acid were studied during two successive seasons

(2014-2015). The results showed that foliar application of either amino acids or citric acid signi cantly promoted the

growth parameters in terms of plant height, a number of branches, fresh, and dry biomass. All treatments led to sig-

ni cant increments in essential oil content and yield. Plant growth and essential oil parameters gradually increased

with amino acids or citric acid concentrations. Gas-liquid chromatographic analysis revealed that the major identi-

 ed components of essential oil were Chamazulen, p-Cymen-8-ol and piperitone. Organic and amino acid treatments

resulted in improving quantity and quality of essential oil components.

KEY WORDS: AMINO CIDS AND CITRIC ACID, ARTEMISIA ABROTANUM L., IN VITRO CULTURE

702

ARTICLE INFORMATION:

*Corresponding Author: elsharqawyeman@hotmail.com

Received 29

Nov, 2016

Accepted after revision 28

Dec, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Mohammad M. EL-Zefzafy et al.

INTRODUCTION

The genus Artemisia belongs to one of the largest and

most widely distributed genera of the family Asteraceae.

It is a diverse and economically important genus and

it has more than 500 species. Artemisia is a wind pol-

linated cosmopolitan genus, mainly distributed in tem-

perate areas of mid to high latitudes of the northern

hemisphere, colonizing in arid and semiarid environ-

mental landscape. Most plants within this genus pos-

sess ethnomedical and biological properties related to

antiviral andantimalarial. antifungal, anticoagulant,

hypoglycemic, and antispasmodic activities, some plants

of the genus are used as foodstuff, ornamentals or soil

stabilizers, some are allergenic or toxic, and some are

weeds growing in the  elds, (Zafar, et al.,1990 Nin,et

al 1997, Valles & McArthur 2001, Bnouham et al., 2002

and Hayat et al., 2009).

Artemisia species invariably found as small fragrant

shrubs or herbs and most yield essential oils. Some of

these oils found used medicine .The plants themselves

are popular among gardeners as cultivated ornamentals.

essential oil of Artemisia arborescens has been used in

the treatment of in ammation, intestinal trouble, and

diarrhea and antioxidant. A. abrotanum from Cuba had

trans-sabinyl acetate and -terpineol as main oil com-

pounds. The same species collected in Serbia displayed

silphiperfol-5-en-3-one A (14.6%), ascaridole (13.1%),

1,8-cineole (10.5%), -bisabolol oxide A acetate (8.7%)

as main oil components . Similarly, an oil from North-

western Italy had 1,8-cineole (34.7%), bisabolol oxide

(18.4%) and ascaridole (16.0%) as main compounds.

The dominant components in the oil from the Crimea

were 1,8-cineole and camphor. A German A. abrota-

num showed 1,8-cineol as main oil compound . Plant

cultivated in Poland were rich in piperitone (17.5%),

davanone (16.8%), 1,8-cineole (12.5%) and silphiperfol-

5-en-3-ol A (6.3%) (Kordali et al 2005 and Jerkovic et

al., 2003, Orav, et al., 2006, Lopes-Lutz et al., 2008 Sha-

ropov et al 2012, and Ghasemi Pirbalouti et al., 2013).

Amino acids as organic nitrogenous compounds are

the building blocks in the synthesis of proteins (Houn-

some et al., 2008). Amino acids are particularly impor-

tant for stimulation cell growth, they act as buffers

which help to maintain favorable PH value within the

plant cell, since they contain both acid and basic groups;

they remove the ammonia from the cell. (Abdel Aziz et

al., 2010). Amino acid formulations, mixtures of nutri-

ents, hydrolyzed proteins, humic acids, seaweed extracts

and brassinolides are proposed as a commonly used

growth promoters (Thomas et al., 2009). The application

of amino acids can stimulate the performance of plant

(Abdel-Mawgoud et al., 2011). The role of Tryptophan is

well known: it has an indirect role in the growth via its

in uence on auxin synthesis, (Talaat, et al, 2002).The

yield-contributing characters and quality of plants could

be improved by foliar application of glutamine (Amin

et al., 2011).

Citric acid is a six carbon organic acid, having a cen-

tral role in CA cycle in mitochondria that creates cellular

energy by phosphorylative oxidation reactions. It is cre-

ated by the addition of acetyl-CoA to oxaloacetic acid

that is converted to succinate and malate in next steps

(Wills et al., 1981). Despite the proposed bene ts of the

application of amino acids on plant growth, there is not

many studies about the physiological changes induced

by foliar applied amino acids and organic acid together ,

in medicinal plants in which their biochemical constitu-

ents are important. Especially essential oil.

This research was conducted to evaluate the physi-

ological changes induced by foliar applied active formu-

lations of amino acids and citric acid on the Artemisa

plant produced by in vitro culutur. The foliar applica-

tions of three different concentrations of Aminol-Forte

(50,150, 250.) were applied to plant to study their effect

on the growth, presence of phytochemical compounds

and improving of quality and quantity of essential oil.

MATERIAL AND METHODS

A  eld experiment was carried out for two successive

seasons (2014 – 2015) in Applied Research Center of

Medicinal Plants (ARCMP), National Organization for

Drug Control and Research (NODCAR), Egypt in order to

examine the physiological and phytochemical responses

of Artemisa plants to foliar applied of amino acids and

citric. To achieve the study purpose, healthy plantlets

(rooting stage in average tall 6 – 8 cm) were selected

from tissue culture laboratory, (ARCMP), National

Organization for Drug Control and Research (NODCAR),

Egypt. Plant materials were washed from phytagel under

running tap water and soaked in a solution of fungicide

(0.2 % Benlet), then transferred to green house in plas-

tic containers (5 cm) full of peat moss/sand (2/1 –V/V)

for  ve weeks. Then plants were transferred to plastic

containers (10 cm) full of peat moss /sand (2/1- V/V) for

 ve weeks too.

After an acclimatization period  nished (ten weeks),

plants were transferred from a greenhouse to open  eld

conditions in 1st Marsh of 2014/2015 season. The experi-

mental soil was prepared and divided into ten plots, each

plot area was 7 m2 (3.5 × 2), with six rows at 60 cm apart

and 40 cm between hills. The mechanical and chemi-

cal analysis of the soil were carried out before planting,

according to Jackson (1967), the obtained results were

as shown in Table 1. Ammonium nitrate 33.5% N (200

Kg/fed), Calcium superphosphate 16 %P2O5 (150 Kg/

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID 703

Mohammad M. EL-Zefzafy et al.

fed) and potassium sulfate 48% K2O (50 Kg/fed) were

added in three doses to fertilize the Artemesia plants.

The  rst dose was added at 21 days before the  rst cut,

the second and third doses were added after 21 days of

the  rst and second cuttings, respectively.

The plants were sprayed twice after 60 days from

planting and after  rst cut with freshly prepared solu-

tions of amino acids (Tryptophan or phenylalanine) and

citric acid at the concentrations of 50, 150 and 250 mg/l

as well as untreated plants (control; distilled water). The

spraying was done manually using a spraying bottle.

Physiological response parameters:

Two cuts were taken during the growing seasons (the  rst

one on June and the second on September).The physi-

ological response parameters were recorded at each cut

as plant height (cm), a number of branches / plant, herb

fresh weight (g/plant) and herb dry weight (g/plant).

Phytochemical response parameters: Determination of

essential oil percentage, %:

The percentage volatile oil of air dried herb for each

treatment was determined by hydro-distillation of veg-

etative parts of plants (100g) of each treatment for 3h

according to British pharmacopeia (1980). The essential

oil was dried over anhydrous Na

, stored in a dark

glass bottle and kept at 4°C until it was analyzed.

Chemical analysis of the volatile oil:

The analysis of the volatiles was performed using a

Hewlett-Packard 6890 GC linked to a Hewlett- Packard

5973 mass-selective detector. For the analysis a Zebron

ZB-5MS, capillary column (27m × 250μm i. d., 0.25μm

 lm thickness) was used. The carrier gas was helium at

1.3 ml/min in constant  ow mode. The injector tem-

perature was 250°C, the injection volume 1μl, and the

split ratio 1:20. The initial oven temperature of 60°C was

held for 1minute, then increased at a rate of 5°C/min

up to 220°C, and subsequently at a rate of 15°C/min up

to 280°C, and  nally was held isothermal for 1min. The

transfer line to the MSD was set at 280°C and the scan

conditions were: M/Z 40–300, at 1.75 scans/sec. Prior

to analysis 900μl of the volatile fractions were mixed

with 100 μl of biphenyl (2.0 mg/ml in hexane) as an

internal standard. The components of essential oils were

identi ed by comparing their relative retention times

and mass spectra with those of Registry of Mass Spectral

Data and literature citations.

Identi cation of essential oil components:

The components of the were identi ed by comparison

of their mass spectra with those of a computer library or

with the authentic compounds and con rmed by com-

parison of their retention indices with those of authentic

compounds. Kovats, indices (Kova´ ts, 1958) were deter-

mined by co-injection of the sample with a solution

containing a homologous series of hydrocarbons, at a

temperature run identical to that described above.

Statistical analysis:

The experimental treatments were laid out in a rand-

omized complete block design (RCBD) with 3 replicates.

Data on plant height (cm), number of branches per plant,

herb fresh weight (g/plant), herb dry weight (g/plant),

essential oil (%) and yield of essential oil (ml/plant)

were statically analyzed using the COSTAT 6311Win and

means were compared using LSD at a probability level

of 5%.

RESULTS AND DISCUSSION

Artemisia abrotanum plant was sprayed with phenylala-

nine, tryptophan, and citric acid at different concentra-

tions and the results were presented in (Table 2). Results

showed that all treatments signi cantly increased plant

height, the number of branches, fresh and dry weights

of aerial parts per plant. The most pronounced effect

on these growth criteria was obtained at treatment 250

mgl-1 phenylalanine and 250 mgl-1 of citric acid. Foliar

application of phenylalanine resulted in the greatest

effect as compared with other treatments (tryptophan

and citric acid). Data in Table 2 also clearly indicated

1.31

HCO

0.71 Total N 9.38 Fe 1.34

0.73

1.73

4.73 Cu 0.33

1.8

3.38

4.28 Zn 0.89

1.52 Mn 4.52

CaCO

8.19

0.47

2.59

Soluble anions

(m.equ/l)

Macro elements

(ppm)

Micro elements

(ppm)

E.C,

mmhos/cm

Chemical analysis

3.8

Soil texture

Sandy loam

Coarse sand, %

Fine sand %

Silt, %

Clay, %

Mechanical analysis

Soluble cations

(m.equ/l)

15.8

61.4

Table 1: Mechanical and chemical analysis of experimental soil

704 INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mohammad M. EL-Zefzafy et al.

that the lowest records of A. abrotanum growth charac-

teristics were observed in plants cultivated in soil with-

out any foliar application control).

Data in Table 2 show that all applications of amino

acids (phenylalanine or tryptophan) and citric acid sig-

ni cantly enhanced the height of A. abrotanum plants

as compared with control in both seasons, (Fig. 1). The

highest plant height (71.50 and 69.56 cm) was recorded

at 250 mgl-1 of phenylalanine and tryptophan, respec-

tively, while the control achieved a minimum plant

height (50.58 cm). It was also observed that there was

no signi cant difference between control and the results

obtained at 50 mgl-1 of all amino acids and citric acid.

It is obvious from the data in Table 2 that, all amino

acids and citric acid treatments (50, 150 and 250 mgl-1)

gradually increased the number of branches/plant (Fig.

2). Moreover, this increasing was signi cant, except

that at 50 mgl-1 with tryptophan and citric acid, where

there was no signi cant effect of these treatments on the

number of branches. The average minimum of branches

number (31.71±3.55) was recorded with control, while

the high concentration of tryptophan (50 mgl-1) gave

the maximum result of this parameter (52.23±6.37) com-

pared with control. Nevertheless, it was shown that there

was no signi cant difference between tryptophan and

phenylalanine at high concentration (250 mgl-1). Appli-

cation of all treatment concentrations of amino acids

and citric acid observed a higher signi cantly increased

the number of branches in the second season than the

 rst season.

Herb fresh weight signi cantly responded to foliar

application of tryptophan, phenylalanine and citric acid

at different concentrations compared with untreated

plants (Table 2). This response was gradual with acid

concentrations (Fig. 3). Phenylalanine was superior to

the other acids (tryptophan and citric acid) at all con-

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut

22.24 25.57 25.65 27.69 50.58±3.91 13.63 15.57 15.68 18.54 31.71±3.55 32.53 40.35 41.43 46.39 80.35±10.56 12.35 14.57 14.35 18.57 29.92±4.24

50 mg/l 25.45 25.57 28.51 30.42 54.98±5.59 16.35 18.60 19.40 21.80 38.08±4.42 46.27 52.57 51.29 60.47 105.30±9.14 16.24 19.85 19.20 22.64 38.97±4.07

150 mg/l 29.46 31.35 32.49 34.47 63.89±4.35 18.89 20.35 22.59 25.37 43.60±6.17 50.54 57.89 60.75 70.29 119.74±15.99 19.90 22.46 25.30 28.46 48.06±8.06

250 mg/l 31.59 35.23 36.45 39.73 71.5±6.62 22.68 24.47 24.64 30.43 51.11±5.60 67.35 74.35 79.34 82.25 151.70±14.14 22.57 26.35 28.58 31.35 54.43±7.79

50 mg/l 24.46 26.46 26.49 28.63 53.02±2.97 14.13 16.57 16.17 20.54 33.71±4.25 39.57 48.90 44.53 51.88 92.44±5.61 17.35 18.36 19.35 20.36 37.71±2.83

150 mg/l 26.90 30.35 29.41 33.41 60.04±3.94 17.35 20.77 21.39 26.72 43.12±7.06 45.23 60.42 55.23 68.56 114.72±12.83 21.57 23.35 23.35 25.35 46.81±2.67

250 mg/l 30.45 33.54 36.39 38.74 69.56±7.88 21.47 26.25 27.43 29.30 52.23±6.37 61.37 71.47 62.37 71.47 133.34±0.71 23.37 29.45 26.39 29.45 54.33±2.14

50 mg/l 23.65 27.10 26.90 29.20 53.43±

3.78

14.13 18.57 16.40 20.35 34.73±2.86 44.78 50.36 46.75 51.34 96.62±2.09 17.35 19.36 18.55 19.36 37.31±0.85

150 mg/l 26.38 30.16 29.47 32.13 59.07±3.58 17.35 22.50 18.79 24.45 41.55±2.40 46.23 55.42 47.40 61.74 105.40±5.30 22.57 25.35 24.59 26.39 49.45±2.16

250 mg/l 30.28 33.35 33.28 36.28 66.60±4.19 19.47 25.87 21.39 27.84 47.29±2.75 63.37 67.47 68.45 71.51 135.40±6.45 25.37 27.45 27.35 30.44 55.31±3.51

1.76 1.73 1.75 1.52 3.46 1.7 1.79 1.69 1.51 3.39 1.75 1.56 1.73 1.78 6.77 1.75 1.47 1.76 1.79 3.13

Second season

First season

Mean

Plant height, cm

Mean

Control

Treatments

Phenylalanine

Tryptophan

Citric acid

Mean

No. of branches per plant

Herb fresh weight, g/ plant

Herb dry weight, g/ plant

Mean

First season

Second season

First season

Second season

First season

Second season

LSD at 0.05

Table 2: Effect of amino acids and citric acid on physiological responses of A. abrotanum

FIGURE 1. Effect of amino acids and citric acid (type and concentration) on plant

height of A. abrotanum.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID 705

Mohammad M. EL-Zefzafy et al.

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut 1

Cut 2

Cut

1.23 1.47 1.55 1.77 3.01 ± 0.44 0.13 0.18 0.22 0.30 0.41 ± 0.15

50 mg/l 1.60 1.68 1.76 1.85 3.45 ±0.23 0.31 0.31 0.31 0.39 0.65 ±

0.06

150 mg/l 1.89 2.01 1.92 2.06 3.94 ±0.06 0.40 0.40 0.45 0.54 0.89 ±0.14

250 mg/l 2.05 2.15 2.24 2.45 4.45 ± 0.35 0.50 0.50 0.60 0.72 1.16 ±0.12

50 mg/l 1.47 1.68 1.69 1.86 3.35 ± 0.28 0.22 0.26 0.29 0.45 0.61 ±0.18

150 mg/l 1.66 2.00 1.81 2.06 3.77 ± 0.15 0.41 0.40 0.39 0.48 0.84 ±0.04

250 mg/l 1.96 2.10 2.08 2.34 4.24 ± 0.25 0.53 0.53 0.51 0.65 1.11 ±0.06

50 mg/l 1.47 1.68 1.64 1.85 3.32 ± 0.24 0.30 0.26 0.27 0.33 0.58 ±0.03

150 mg/l 1.66 1.91 2.07 1.96 3.71 ±

0.19 0.41 0.41 0.43 0.51 0.88 ±

0.08

250 mg/l 1.71 2.16 2.44 2.07 4.19 ± 0.45 0.52 0.52 0.52 0.69 1.13 ±0.12

0.02 0.05 0.01 0.08 0.20 NS NS 0.11 0.12 0.09

Control

Phenylalanine

Tryptophan

Citric acid

LSD at 0.05

Treatments

Essential oil, %

Yeild of essential oil, ml/plant

First season

Second season

Mean

First season

Second season

Mean

Table 3: Effect of amino acids and citric acid on essential oil % and yield of A. abrotanum

FIGURE 2. Effect of amino acids and citric acid

(type and concentration) on No. of branches of

A. abrotanum.

FIGURE 3. Effect of amino acids and citric acid

(type and concentration) on herb fresh weight of

A. abrotanum.

centrations in fresh biomass production. This acid

achieved the highest fresh weight of herb (105.30±9.14

g/plant) with increment about 88.8% at 250 mgl-1 com-

pared with the control which, gave the lowest herb fresh

weight (80.35±10.56 g/plant). According to the statisti-

cal analysis conducted,it was cleared that there was no

signi cant difference between the best treatment, 250

mgl-1, of tryptophan (33.34±0.71 g/plant) and citric acid

(135.40±6.45 g/plant).

FIGURE 4. Effect of amino acids and citric acid (type

and concentration) on herb dry weight of A. abro-

tanum.

The results of herb dry weight presented in Table

2, it was shown clearly that the amino acid and cit-

ric acid had a highly positive signi cant effect on the

average herb fresh yield of A. abrotanum. This effect

was observed in the two growing seasons, as well as the

second season was superior to the  rst. Like all previ-

ous growth parameters, dry weight of Artemesia herb

was also gradually increased by increasing the concen-

trations of all amino acids and citric acid (Fig 4). The

increments of herb dry weight of Artemesia were esti-

mated by range from 24.7% to 84.86% at 50 mgl-1 and

250 mgl-1 respectively, with citric acid compared with

the control. Moreover, untreated plants gave the low-

est herb dry weight (29.92±4.24 g/plant). On the other

hand, it was shown that there were no signi cant differ-

encesbetweenall treatments at the same concentration.

The results of essential oil content (%) and its yield

(ml/plant) were presented in (Table 4, Fig. 5 and Fig. 6).

Data in this table shown that the biosynthesis of essen-

tial oil and its average yield were signi cantly promoted

as a result of foliar spraying with amino acids and citric

acid. The increments in essential oil content are ranged

between 10.3% and 47.84%, while in essential oil yield

706 INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mohammad M. EL-Zefzafy et al.

Table 4: Effect of amino acids and citric acid on chemical comparison volatile oil of

A. abrotanum

L plant the  rst season.

Compounds Control Phenylalanine

50 m g / L

Phenylalanine

150 mg / L

Phenylalanine

250 mg / L

Tryptophan

50 mg / L

Tryptophan

150 mg / L

Tryptophan

250 mg / L

Citric acid

50 mg / L

Citric acid

150 mg / L

Citric acid 250

mg / L

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1 1,8-cineol 8.85 8.05 8.96 8.87 8.97 9.16 9.23 9.26 9.78 9.88 9.98 9.92 10.22 10.25 8.84 8.96 9.13 9.36 9.46 9.41

2 -terpineol 0.86 3.06 2.06 3.18 2.47 2.68 3.02 3.40 2.97 3.07 2.57 2.80 2.03 3.19 2.54 2.65 2.56 2.97 3.15 3.06

3 Nuciferol butanoate 10.96 11.08 10.90 12.00 10.77 11.68 12.20 12.55 10.56 10.92 10.48 11.11 11.53 11.87 11.03 11.31 11.00 11.99 10.60 12.05

4 Geranyl isobutyrate 1.92 2.15 1.97 2.16 1.92 2.06 2.25 2.59 2.03 2.08 2.63 2.33 2.01 2.37 2.03 2.20 2.03 2.36 2.13 2.02

5 Camphene 0.79 0.96 0.78 1.04 0.48 0.66 0.87 1.08 0.83 1.04 1.27 1.61 0.78 0.98 0.83 1.00 1.15 0.88 0.94 1.09

6 Boroneol 2.40 2.50 2.48 2.92 2.59 2.82 3.02 1.06 2.62 2.80 2.96 3.15 2.82 2.86 2.94 2.99 2.48 3.17 2.64 2.90

7 Terpineol 1.57 1.89 1.66 1.73 1.50 1.58 1.83 1.90 1.36 1.70 1.98 2.25 1.70 1.78 1.64 1.61 1.49 1.06 1.62 1.29

8 Chamazulene 19.03 19.20 19.92 20.00 20.88 21.19 22.37 23.56 22.26 22.93 22.53 22.79 22.87 23.40 19.03 19.58 20.78 21.49 20.39 21.36

9 Sesquisabinenehydrate 3.27 3.48 2.91 3.16 2.97 1.33 3.59 3.87 2.86 3.17 3.48 3.03 3.33 3.67 3.03 3.20 3.41 3.63 3.16 3.17

10 Caryophyllene oxide 3.91 3.97 3.79 3.98 3.30 3.70 4.07 4.36 3.46 3.69 3.92 4.17 3.93 4.16 3.44 3.48 3.17 4.11 3.43 3.37

11 binene 1.95 2.05 2.76 2.87 2.97 3.56 3.73 3.86 3.78 3.88 3.78 4.02 3.22 4.25 3.14 3.26 313 3.36 3.16 3.41

12 Geranyl isobutyrate 1.96 3.06 2.06 3.18 2.47 2.68 3.02 3.40 2.97 3.07 2.57 2.80 2.03 3.19 2.54 2.65 2.56 2.97 3.15 3.06

13 Piperitone 11.05 11.25 11.76 11.87 11.97 12.16 12.23 12.26 11.78 12.88 12.78 12.72 12.82 12.95 11.14 11.26 11.13 11.36 11.16 11.41

14 B- Eudesmol

1.65 1.57 1.89 1.46 1.73 1.50 1.58 1.83 1.90 1.36 1.70 1.98 2.25 1.70 1.78 1.64 1.61 1.49 1.06 1.62

15 Borneol acetate 1.79 1.96 0.78 1.04 0.48 0.66 0.87 1.08 0.83 1.04 1.27 1.61 0.78 0.98 0.83 1.00 1.15 0.88 0.94 1.09

16 Nuciferol propionate 5.30 5.45 4.74 4.93 4.77 4.98 5.28 5.59 4.83 4.97 4.54 4.94 5.10 5.55 4.83 4.18 4.38 5.18 4.39 5.00

17 p-Cymen-8-ol 10.96 11.08 10.90 12.00 10.77 11.68 12.20 12.55 10.56 10.92 10.48 11.11 11.53 11.87 10.96 11.08 10.90 12.00 10.77 11.68

18 Davanone 3.30 3.45 4.74 4.93 4.77 4.98 5.28 5.59 4.83 4.97 4.54 4.94 5.10 5.55 4.83 4.18 4.38 5.18 4.39 5.00

Grouped compounds

Monoterpene hydrocarbons % 0.55 0.75 0.91 0.93 1.07 1.11 1.28 1.58 0.94 0.95 1.17 1.22 1.35 1.61 0.87 0.91 1.10 1.15 1.25 1.52

Oxyenated Monoterpenes % 21.44 21.66 21.93 21.92 21.98 21.99 22.39 22.79 21.95 21.95 21.98 21.99 22.35 22.80 20.90 21.93 20.99 21.97 22.34 22.72

Sesquiterpene hydrocarbons % 5.22 5.82 6.18 6.15 6.21 6.29 6.49 6.75 6.23 6.20 6.21 6.35 6.59 6.77 6.16 6.18 6.25 6.32 6.45 6.71

Oxyenated Sesquiterpene % 17.81 17.93 18.45 18.41 18.47 18.52 18.71 18.94 18.48 18.42 18.47 18.52 18.78 18.95 17.44 18.45 18.49 18.55 18.70 18.91

Di terpenes % 1.11 1.31 1.77 1.79 1.89 1.93 1.98 1.99 1.79 1.80 1.89 1.98 1.98 1.99 1.75 1.77 1.89 1.95 1.95 1.92

Aromatics % 37.55 37.85 37.71 38.67 38.81 38.88 38.92 39.90 37.75 38.70 38.81 38.88 38.95 39.92 36.70 37.71 38.85 38.91 38.90 39.94

Others % 2.77 2.97 3.22 3.33 3.43 3.62 3.72 3.85 3.29 3.37 3.43 3.63 3.75 3.87 3.20 3.22 3.45 3.65 3.70 3.83

Total compounds 85.45 86.99 87.17 91.21 91.86 92.34 93.49 95.80 90.35 91.39 91.96 92.57 93.75 95.92 87.03 90.34 90.83 92.50 93.29 95.55

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID 707

Mohammad M. EL-Zefzafy et al.

was between 41.46% and 182.93% compared with the

control. It is also clear from the obtained data that

essential oil content and its yield gradually increased

with amino acid and citric acid level increases (Fig. 5

and 6). The highest recorded value of the average essen-

tial oil content (4.45±0.35 %) and yield of essential oil

(1.16±0.12 ml/plant) were obtained in the herb of plants

treated with phenylalanine at 250 mg/l. While the con-

trol gave minimum essential oil content (3.01±0.44 %)

and oil yield (0.41±0.15 ml/plant). On the other hand,

there are no signi cant differences between all the three

acids (phenylalanine, tryptophan and citric) at the same

concentration in terms of essential oil contents and

essential oil yield.

In this respect, several investigators have studied the

effect of different amino acids on the total oil percent-

age and oil yield (ml/plant) and found the same results;

Gamal El.Din et al., (1997) on Cymbopogon citratus Hort,

Talaat and Youssef (2002) on basil plant, Karima et al.,

(2005) on Matricaria chamomilla L. Amino acids accu-

mulation in plants plays different roles, an osmolyte,

regulation of iron transport, modulating stomata opin-

ing and detoxi cation of heavy metals. Moreover, amino

acids affect synthesis and activity of some enzymes,

gene expression and redox-homeostasis (Rai, 2002).

Many investigators, Talaat et. al. (2002) and Orner

et.al. (2013), mentioned that foliar application of amino

acids signi cantly increased essential oil percentage and

yield. Also, Taraf et.al.(1999) on lemongrass, Eid et.al.

(2010) on Jasminum grandi orm and Safaa et.al. (2011)

on geranium reported that foliar application of citric

acid caused a pronounced increment in the percentage

and yield of essential oil. It has been documented that

the exudation of citrate and malate from roots of cal-

cicole plants (plants growing in alkaline soils) enables

them to extract P and Fe from such soils (Lopez-Bucio

and Nieto-Jacobo, 2000). Use of citric acid alone or in

combinations with malic acid increased the essential oil

production of sweet basil (Jaafari and Hadavi, 2012).

Foliar sprays of citric acid alone or in combination

with Fe sources have been used to recover many plants

from the iron chlorosis (Eidyan et al., 2014). Later stud-

ies revealed that the effect of citric acid is not just due

to pH change and there are a variety of physiological

responses to applied citric acid.

The results of the GC/MS analysis of the essential oil

of A. abrotanum. during the  rst and second season as

are shown in Table (4,5). There are 18 identi ed com-

pounds. Chamazulen was identi ed as the major com-

pound in the different treatments ranged from 19.3 %

to .23.5 %, the second main component, p-Cymen-8-ol

ranged (12.39% and 12.55. %) in the essential oil fol-

lowed with piperiton which was identi ed as the third

main constituent in the essential oil and its relative

percentage accounted for (12.16% and 12.23. %), while

some compound found in traces amount as Camphen

(0.79% and 1.61%).

Foliar application of amino acid indicate that pheny-

lalanine at 250 mg/1 revealed that, maximum relative

percentage of Chamazulen was (22.37% and 23.5 % ) for

1st and 2nd cuts, respectively during 1st season). In the

2nd season, the foliar application of phenylalanine at

250 mg/1 resulted in the highest relative percentage of

Chamazulen 2nd cut, (Table, 4, 5) in both seasons. The

data indicated that Cymen-8-ol reached to its maximum

values 11.3% and 12.5. % for 1st and 2nd cuts, respec-

tively) as a result of application of phenylalanine at 250

mg/ L during the 1st season. On the other hand, trypto-

phan at 250 mg /l gave the highest relative percentage

1,8-cineol which ranged between (10.22 to 10.95%) in

the 1st and 2nd cuts during two seasons, as compared

with control. while citric acid did show any signi cant

change in some essential oil concentration than amino

acid, like Eudesmol, pipeitone, Terpenol, camphene,

Gerenyl isobutrate, terpenol and 1.8-cineol in First sea-

son, while second season citric acid showed signi cant

decrease in the concentration of some oil component

especially at low concentration 50 mg of citric acid.

The essential oil composition varies according to cuts

or different treatments and was characterized by a high

percentage of oxygenated compounds ranged from 21.9

to 22.8 %. The components of the essential oil in herb

FIGURE 5. Effect of amino acids and citric

acid concentrations on essential oil content.

FIGURE 6. Effect of amino acids and citric

acid concentrations on essential oil yield.

708 INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mohammad M. EL-Zefzafy et al.

Table 5: Effect of amino acids and citric acid on chemical comparison volatile oil of

A. abrotanum

L plant the second season

Compounds Control Phenylalanine

50 mg / L

Phenylalanine

150 mg / L

Phenylalanine

250 mg / L

Tryptophan

50 mg / L

Tryptophan

150 mg / L

Tryptophan

250 mg / L

Citric acid

50 mg / L

Citric acid

150 mg / L

Citric acid 250

mg / L

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1st

Cut

2nd

Cut

1 1,8-cineol 9.78 9.88 9.87 9.98 9.99 10.16 10.23 10.26 10.78 10.89 10.98 10.92 10.92 10.95 9.89 10.26 10.53 10.47 10.66 10.71

2 -terpineol 1.55 2.06 3.46 3.38 3.77 3.68 3.72 3.80 3.97 3.99 3.57 3.80 3.03 3.19 2.54 2.65 2.56 2.97 3.15 3.06

3 Nuciferol butanoate 10.76 11.08 10.90 12.00 10.77 11.68 12.20 12.55 10.56 10.92 10.48 11.11 11.53 11.87 11.03 11.31 11.00 11.99 10.60 12.05

4 Geranyl isobutyrate 2.92 2.15 1.97 2.16 1.92 2.06 2.25 2.59 2.03 2.08 2.63 2.33 2.01 2.37 2.03 2.20 2.03 2.36 2.13 2.02

5 Camphene 1.79 0.96 0.78 1.04 0.48 0.66 0.87 1.08 0.83 1.04 1.27 1.61 0.78 0.98 0.83 1.00 1.15 0.88 0.94 1.09

6 Boroneol 3.45 2.50 2.48 2.92 2.59 2.82 3.02 1.06 2.62 2.80 2.96 3.15 2.82 2.86 2.94 2.99 2.48 3.17 2.64 2.90

7 Terpineol 2.57 1.89 1.66 1.73 1.50 1.58 1.83 1.90 1.36 1.70 1.98 2.25 1.70 1.78 1.64 1.61 1.49 1.06 1.62 1.29

8 Chamazulene 19.23 19.20 19.92 20.00 20.88 21.19 22.37 23.56 22.26 22.93 22.53 22.79 22.87 23.40 19.03 19.58 20.78 21.49 20.39 21.36

9 (Z) Sesquisabinenehydrate 3.27 3.48 2.91 3.16 2.97 1.33 3.59 3.87 2.86 3.17 3.48 3.03 3.33 3.67 3.03 3.20 3.41 3.63 3.16 3.17

10 Caryophyllene oxide 3.91 3.97 3.79 3.98 3.30 3.70 4.07 4.36 3.46 3.69 3.92 4.17 3.93 4.16 3.44 3.48 3.17 4.11 3.43 3.37

11 binene 2.95 2.05 2.76 2.87 2.97 3.56 3.73 3.86 3.78 3.88 3.78 4.02 3.22 4.25 3.14 3.26 313 3.36 3.16 3.41

12 Geranyl isobutyrate 2.96 3.06 2.06 3.18 2.47 2.68 3.02 3.40 2.97 3.07 2.57 2.80 2.03 3.19 2.54 2.65 2.56 2.97 3.15 3.06

13 piperitone 11.35 11.25 11.76 11.87 11.97 12.16 12.23 12.26 11.78 12.88 12.78 12.72 12.82 12.95 11.14 11.26 11.13 11.36 11.16 11.41

14 B- Eudesmol 2.65 1.57 1.89 1.46 1.73 1.50 1.58 1.83 1.90 1.36 1.70 1.98 2.25 1.70 1.78 1.64 1.61 1.49 1.06 1.62

15 Borneol acetate 2.79 1.96 0.78 1.04 0.48 0.66 0.87 1.08 0.83 1.04 1.27 1.61 0.78 0.98 0.83 1.00 1.15 0.88 0.94 1.09

16 Nuciferol propionate 6.30 5.45 4.74 4.93 4.77 4.98 5.28 5.59 4.83 4.97 4.54 4.94 5.10 5.55 4.83 4.18 4.38 5.18 4.39 5.00

17 p-Cymen-8-ol 11.96 11.08 10.90 12.00 10.77 11.68 12.20 12.55 10.56 10.92 10.48 11.11 11.53 11.87 10.96 11.08 10.90 12.00 10.77 11.68

18 Davanone 4.30 3.45 4.74 4.93 4.77 4.98 5.28 5.59 4.83 4.97 4.54 4.94 5.10 5.55 4.83 4.18 4.38 5.18 4.39 5.00

Grouped compounds

Monoterpene hydrocarbons % 1.05 1.21 1.33 1.45 1.51 1.35 1.58 1.75 0.91 1.22 1.95 1.35 1.52 1.99 1.10 1.17 1.15 1.25 1.61 1.96

Oxyenated Monoterpenes % 21.45 21.61 21.99 21.95 21.99 22.35 22.79 22.89 21.93 21.99 22.90 22.35 22.72 22.89 20.99 21.98 21.97 22.34 22.80 22.93

Sesquiterpene hydrocarbons % 5.33 5.82 6.38 6.25 6.69 6.59 6.75 6.79 6.18 6.35 6.86 6.59 6.71 6.86 6.25 6.21 6.32 6.45 6.77 6.98

Oxyenated Sesquiterpene % 17.84 16.99 18.49 18.44 18.57 18.78 18.94 18.99 18.45 18.52 18.74 18.78 18.91 18.85 18.49 18.47 18.55 18.70 18.95 18.96

Di terpenes % 1.16 1.31 1.78 1.82 1.95 1.98 1.99 2.91 1.77 1.98 1.95 1.98 1.92 2.79 1.89 1.89 1.95 1.95 1.99 2.58

Aromatics % 37.66 37.81 37.77 38.73 38.88 38.95 39.90 39.93 37.71 38.88 36.78 38.95 39.94 38.98 38.85 38.81 38.91 38.90 39.92 38.96

Others % 2.81 3.92 3.26 3.39 3.69 3.75 3.85 3.88 3.22 3.63 3.79 3.75 3.83 3.88 3.45 3.43 3.65 3.70 3.87 3.85

Total compounds 87.30 88.67 91.10 92.03 92.34 93.75 95.80 96.27 90.34 92.57 92.97 93.75 95.55 96.24 90.83 91.96 92.50 93.29 95.92 96.22

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS INFLUENCE OF FOLIAR APPLICATION WITH AMINO ACIDS AND CITRIC ACID 709

Mohammad M. EL-Zefzafy et al.

for different treatments during 2 cuts during both sea-

sons were shown in Tables (4 and 5). The increment in

oil% might be due to the increase in vegetative growth

and nutrient uptake. Similar results were reported by

Gharib (2006). The chemical composition of A. abro-

tanum oils is very variable, the chemovarieties and the

environmental conditions caused to this fact. The major

components from different origins were found to be lin-

alool, 1,8-cineol, piperitone, davanone and silphiperfol-

5-en-3-ol, several workers reported that essential oil

components of are varied according to growth condi-

tions., foliar application of salicylic of (Ocimum basili-

cum L.) and (Majorana hortensis) increased the produc-

tion of quantity and quality of oil ( Gharib et al 2006).

These results are similar to those obtained by (Orav,

et al., 2006).

CONCLUSIONS

We conclude that there is a pure effect by sprayed of all

treatments enhanced growth parameters, plant height ,the

number of branches, the fresh and dry weight of plant,

oil percentage and yield per plant for two cuttings rela-

tive to untreated controls. from the above explanation the

important role of amino acids in enchaning the growth

and production of different plant constituents which is

in accordance with our results, also our results are in

accordance with many investigators who reported that

the application of amino acids increases the concentra-

tion of essential oil and growth rate. Foliar application

of phenylalanine, gave the best effect on growth param-

eters at 250 mg,and increase the quantity and quality of

essential oil, by incrasing the yield percentage of oil and

increasing the concentration of major compounds in the

oil, while the application of citric acid shows variable

effect according to concentration and seasons.

DISCLOSURE STATEMENT

No potential con ict of interest was reported by the

authors.

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