Acta agriculturae Slovenica, 121/4, 1–12, Ljubljana 2025
doi:10.14720/aas.2025.121.4.20659 Original research article / izvirni znanstveni članek
Callus-mediatede embryogenesis of leaf celery (Apium graveolens L. var. 
secalinum Alef.) as a response to malt, peptone and yeast extracts appli-
cation
Nancy Danial GIRGIS 1, Sally Farag DESOUKEY 2, Ahmed Mohamed AMER 1 and Nermeen Mohammad 
ARAFA 1, 3
Received November 18, 2024, accepted September 17, 2025
Delo je prispelo 18. november 2024, sprejeto 17. september 2025
1 Department of Plant Biotechnology, Biotechnology Research Institute, National Research Centre, El-Buhouth St., (P. O. Box 12622), Dokki, Cairo, Egypt.
2 Department of Agricultural Botany, Faculty of Agriculture, Cairo University, (P. O. Box 12613), Dokki, Giza, Egypt
3 Corresponding Author: e-mail address: nermeenfouad78@yahoo.com
Callus-mediatede embryogenesis of leaf celery (Apium gra-
veolens L. var. secalinum Alef.) as a response to malt, peptone 
and yeast extracts application
Abstract: Historically, leaf celery (Apium graveolens L. 
var. secalinum Alef. of Apiaceae family is well-known tradition-
al plant that possesses eminent therapeutic characteristics. The 
current work aimed to develop an efficient protocol for celery 
embryogenesis via indirect approach of callus cell cultures us-
ing biotic elicitors (malt, peptone and yeast extract) at different 
concentrations (1.5, 2.5, 3.5 g l-1) of each. The optimal explant 
type to induce calli were the stems and the best medium was 
MS-medium + 0.5 mg  l-1 2,4-D. Fresh mass and growth of 
celery stem calli were proliferated recording the highest values 
after 8 weeks of culture, whereas the highest growth rate was 
achieved after 5 weeks of culture. Malt extract (1.5 g  l-1) per-
formed as the best biotic elicitor for the highest value of callus 
fresh mass. Similarly, growth rate and growth value increased 
to 1.2 ± 0.009 g/day and 12.1 ± 0.12, respectively. Throughout 
the histological investigation, the anatomical structure for all 
elicited treatments of celery stem calli exhibited more potent 
efficiency of malt than peptone for embryogenesis development 
then the yeast extract. Meanwhile, elicitor- free MS-medium 
stopped the callus growth at the stage of globular embryo for-
mation. This work documented the elicitors’ efficiency for cal-
lus growth and somatic embryo development of Apium graveo-
lens. var. dulce (Mill.) Pers.. 
Key words: Apium graveolens L. var. secalinum Alef., cal-
lus, embryogenesis, biotic elicitors, histological investigation
Embriogeneza listne zelene (Apium graveolens L. var. seca-
linum Alef.) iz kalusa z dodatki slada, peptona in izvlečkov 
kvasa
Izvleček: Zgodovinsko gledano je listnata zelena (Apium 
graveolens L. var. secalinum Alef.) iz družine kobulnic (Apia-
ceae) znana tradicionalna rastlina, ki ima izjemne terapevtske 
lastnosti. Cilj dela je bil razviti učinkovit protokola za embrio-
genezo listnate zelene posredno iz kultur kalusnih celic z upo-
rabo biotskih elicitatorjev (slada, peptona in ekstrakta kvasa) 
pri različnih koncentracijah (1,5, 2,5, 3,5 g l-1) vsakega. Opti-
malna vrsta eksplantata za induciranje kalusa so bila stebla, 
najboljše gojišče pa MS-medij + 0,5 mg l-1 2,4-D. Sveža masa in 
rast stebelnega kalusa zelene sta proliferirali z največjimi vred-
nostmi po 8 tednih gojenja, medtem ko je bila najvišja stopnja 
rasti dosežena po 5 tednih gojenja. Izvleček slada (1,5 g l-1) se je 
izkazal kot najboljši biotski elicitor za največjo vrednost sveže 
mase kalusa. Podobno sta se povečali hitrost rasti in vrednost 
rasti na 1,2 ± 0,009 g/dan oziroma 12,1 ± 0,12. Skozi histološko 
preiskavo je anatomska zgradba vseh izzvanih stebelnih kalu-
sov zelene pokazala močnejšo učinkovitost slada kot peptona 
za razvoj embriogeneze , oba pa večjo kot izvleček kvasa. Med-
tem je MS-gojišče brez elicitorja ustavilo rast kalusa na stopnji 
oblikovanja globularnega zarodka. To delo je dokumentiralo 
učinkovitost elicitorjev za rast kalusa in somatski razvoj zarod-
kov listanate zelene.
Ključne besede: Apium graveolens L. var. secalinum Alef., 
kalus, embriogeneza, biotski elicitorji, histološka preiskava
2
N. D. GIRGIS et al.
Acta agriculturae Slovenica, 121/4 – 2025
1 INTRODUCTION
Apium graveolens L. var. secalinum Alef. leaf cel-
ery, belongs to family Apiaceae’ and it is one of biennial 
branching herbs that grow in Europe and East Asian 
countries. This aromatic plant was used for medicinal 
purposes and then it had been cultivated as a food ad-
ditive (Hassanen et al., 2015). Also, celery seeds con-
tain an essential oil that can be used in industry or as 
flavoring or spice (Toth & Lacy, 1992). Celery is a rich 
source of some bioactive compounds such as pheno-
lic acids, flavonoids in addition to carotene, vitamins 
and cellulose. So, the different parts of the celery plant 
were reported to possess different therapeutic effect 
including anti-inflammatory, antioxidant, antifungal 
and antibacterial properties as it contains 22 volatile 
compounds (Zahra et al., 2011). Tissue culture propa-
gation through somatic embryogenesis is an efficient 
technique for developing micropropagation and for in-
dustrial production of celery (Nadel et al., 1995). The 
addition of plant growth regulators to in vitro cultures 
have different effects on cell division, cell differentia-
tion and growth developing. Auxins are used for callus 
induction, the most commonly used is 2,4-D. Media 
fortified with 1 mg l-1 2,4-D gave the best callus in-
duction and shoot regeneration of Bunium persicum 
(Boiss.)F. F.Dtsch , a member of the Umbellifera family 
(Valizadeh & Kazemi, 2009). Chen (1976) found that 
2,4-D and kinetin combinations were the most effective 
for celery callus initiation. MS medium with 0.5 mg/l 
2,4-D  and 0.5 mg/l kinetin successfully produced fri-
able calli from petiole and leaf explants as reported by 
Bruznican et al. (2017). While Hassan (2019) reported 
that the optimum plant growth regulator for callus ini-
tiation and regeneration was BAP (1 mg l-) and 2,4-D 
(0.5 mg l-1 ). Also, some reports indicated that transfer-
ring callus to media with 1 mg l-1 2,4-D (Wakhlu et al., 
1990) or 0.5 mg l-1 2,4-D (Bonianpoor, 1995) resulted 
in embryogenesis formation. Somatic embryogenesis 
can be conducted in a direct or indirect method. In 
the process of indirect somatic embryogenesis, it starts 
with embryogenic calli formation.  These embryonic 
cells are characterized by large nuclei and cytoplasm 
with high metabolic activity (Jim’enez & Bangerth, 
2001). Induction of somatic embryogenesis is affected 
by many factors, like plant phenotype, explant type, 
medium composition, plant growth regulators, organic 
additives and incubation conditions (Wojcikowska & 
Gaj, 2017, Rizwan et al., 2020, Gulzar et al., 2020, and 
Araujo et al., 2021). To induce somatic embryogenesis 
significantly, high concentrations of auxins such as 2,4-
D should be added, while embryo development often 
needs lower auxin levels (Edwin et al., 2008). Reinert 
et al. (1966) first reported the induction of adventi-
tious embryos in celery. Al-Abta & Collin (1978) used 
cell suspension cultures of celery for the somatic em-
bryos production. In addition, embryoid formation in 
cell suspension cultures of celery was dependent on 2, 
4-D addition. Somatic embryogenesis was successfully 
induced from nodal segments using 1 and 4 mg l-1 of 
2,4-D alone (Chagas et al., 2023). Embryogenic calli 
were highly proliferated on culture medium with 1.5 
mg l-1 2,4-D (Zhang et al., 2021). The maximum em-
bryogenesis percentage from hypocotyl explants of B. 
persicum was found on 0.5 mg l-1 BAP + 1 mg l-1 2,4-
D, 0.5 mg l-1 BAP + 2 mg l-1 2,4-D, and 1 mg l-1 2,4-D 
treatments (Ouzhand et al., 2023). Mahadi et al. (2016) 
showed that the addition of 2 mg l-1 2,4-D and 2 mg 
l-1 BAP gave the best result for embryogenic callus of 
Calamansi (Citrus x microcarpa Bunge), while, using 
1.0  mg l-1 2,4-D and 2.0 mg l-1 BAP gave the best em-
bryo production from cell cultures of Tecoma stans L. 
(Omar et al., 2024). Somatic embryogenesis was also 
improved by the addition of organic compounds, like 
casein hydrolysate, yeast extract, malt extract, coco-
nut water, peptone and tryptone, where they provide 
cultures with sugar, sugar is typically supplied by su-
crose, amino acids, phosphate, several microelements 
and vitamins to enhance growth and development of 
embryogenic cultures (Al-Khayri, 2011). Malt extract 
has been applied for callus and somatic embryogenesis 
induction in many Citrus species (Amin & Shekafan-
deh, 2015). For example, the best result for embryo-
genic callus formation of sweet orange ‘Washington 
Nave’ was obtained on the medium supplemented 
with 500 mg l-1 malt extract (Mazri & Belkoura, 2021). 
While, addition of 1g l-1 of malt extract improved the 
induction of mulberry secondary somatic embryos 
(Agarwal, 2004). Also, Borpuzari & Borthakur (2016) 
reported that maximum callus formation and matura-
tion of globular embryos resulted by addition of 100.0 
mg l-1 of yeast extract combined with 2,4-D and IAA 
of Plumbago rosea L. inter node explants. Sherif et al. 
(2018) mentioned that biotic elicitors such as peptone 
(50 mg l-1), and coconut water (5 %) which were added 
individually or together are necessary for embryogen-
esis from intermodal explant of Anoectochilus elatus 
Lindl., while, 0.5 g  l-1 or 1 g  l-1  peptone successfully 
promoted the embryogenesis from leaf tip segments of 
Oncidium ‘Gower Ramsey’ (Chen & Chang, 2002). The 
histological analysis of somatic embryogenesis through 
various techniques provides detailed knowledge of var-
ious stages of somatic embryogenesis like initiation, 
proliferation, maturation, and conversion and different 
phases of growth (globular, heart, torpedo, and coty-
ledonary). Besides, it allows to identify and monitor 
3
Callus-mediatede embryogenesis of leaf celery (Apium graveolens L. var. secalinum Alef.) ... to malt, peptone and yeast extracts application
Acta agriculturae Slovenica, 121/4 – 2025
the early events of embryogenetic cells such as denser 
cytoplasm, high nucleus-to-cytoplasm ratio and large 
nucleolus (Gomes et al., 2017, Campos et al., 2017), 
this requires more studies to achieve a promising stu-
Generally, the objective of our work was to perform a 
successful protocol of somatic embryogenesis devel-
opment via screening the efficiency of diverse biotic 
elicitors (malt, peptone and yeast extracts) at different 
concentrations on somatic embryos formation and de-
velopment of Apium graveolens L.
2 MATERIAL AND METHODS
2.1 SEEDS STERILIZATION AND GERMINATION 
OF APIUM GRAVEOLENS 
Seeds of leaf celery (Apium graveolens L. var. seca-
linum Alef.) were obtained from Horticulture Research 
Institute, Agricultural Research Center, Ministry of 
Agriculture and Land Reclamation, Egypt. The inves-
tigation was carried out in National Research Centre. 
Seeds of celery were disinfected by 70 % ethyl alcohol 
for 1 min then washed 3 times with sterile distilled wa-
ter, then soaked in 45 % sodium hypochlorite (NaOCl) 
for 20 min before rinsing three times with sterile wa-
ter. Seeds were placed on MS medium (Murashige & 
Skoog, 1962) containing 3 % (w/v) sucrose and 0.7 % 
(w/v) agar. The pH of the culture medium was adjusted 
to 5.8 before autoclaving. The cultures were maintained 
in the growth chamber at 25 ± 2 °C with 16 h/8 h pho-
toperiod of fluorescent, 100 μmol photons m−2 s−1 white 
light illumination.
2.2 CALLUS INDUCTION OF APIUM GRAVEO-
LENS
Stem and leaf segments were separated from the 
grown seedlings (4 weeks old) as explants for callus in-
duction. Both stem and leaf segments were cultured on 
solidified MS medium containing various supplemen-
tation: 0.5 mg l-1 dichlorophenoxyacetic acid (2, 4-D), 
0.5 mg l-1 2, 4-D +1.0 mg l-1 benzyl aminopurine (BAP) 
and 0.5 mg l-1 2, 4-D +1.0 mg l-1 kinetin (kin). Cul-
tures were incubated under temperature of 25 ± 1  °C 
and light conditions of 16 h/8 h photoperiod and 100 
μmol  m-2 s 1. Callus fresh mass were recorded every 
week through 8 weeks.
2.3 ELICITATION OF APIUM GRAVEOLENS CAL-
LUS CULTURES BY DIFFERENT BIOTIC ELICI-
TORS.
Several levels (1.5, 2.5 and 3.5 g l-1) of malt extract, 
yeast extract and peptone extract as biotic elicitors were 
supplemented after callus induction to the callus culture 
medium fortified with 0.5 mg l-1 2, 4-D. Cultures were 
kept in the growth chamber under temperature of 25 ± 
1  °C and light conditions of 16 h photoperiod and 100 
μmol m-2 s-1. Callus fresh mass were recorded every 10 
days through 60 days.
2.4 PARAMETERS OF APIUM GRAVEOLENS CAL-
LUS CULTURE GROWTH 
The cultures were implemented in triplicates and 
the growth parameters were calculated using two param-
eters as follows:
(a) Growth rate: GR = Pt–P0/10 (g/day).
(b) Growth value: GV = Pt–P0/P0
Pt = mass (g) of calli at the end of every ten days
P0 = starting mass (g) of the callus.
2.5 HISTOLOGICAL INVESTIGATION OF APIUM 
GRAVEOLENS CALLUS CULTURES.
The anatomical structure of Apium graveolens stem 
calli treated with the different elicitors was studied. Mi-
crotechnique practices were conducted at the Depart-
ment of Agricultural Botany, Faculty of Agriculture, 
Cairo University, within the second season. Samples were 
fixed for at minimum forty-eight hours in (formalde-
hyde, acetic acid, alcohol mixture solution (F.A.A), dehy-
drated, and then embedded in paraffin wax (Sass, 1951). 
Parts that were cut on a rotary microtome at a thickness 
of 15-20 microns were stained with crystal violet/eryth-
rosine before mounting in Canada balsam. Slides were 
investigated microscopically and photomicrographically.
2.6 STATISTICAL ANALYSIS
IBM SPSS statistics subscription software was used. 
The significance level was investigated by the analysis 
of variance analysis (ANOVA). Three replicates of 5 ex-
plants per replicate were used in each treatment. Data are 
shown as means ± standard errors (SE) and compared by 
LSD test at p value less than 0.05.
4
N. D. GIRGIS et al.
Acta agriculturae Slovenica, 121/4 – 2025
3 RESULTS 
3.1 IN VITRO SEEDLINGS GERMINATION OF 
APIUM GRAVEOLENS.
Apium graveolens seeds were cultured on MS medi-
um under sterilization conditions, the application of ster-
ilization procedure in our experiment was efficient for 
further micropropagation; this examination was similar 
to those mentioned by Bruznican et al. (2017) who used 
70 % ethanol following by sinking in 5 % NaOCl contain-
ing 3 drops of Tween 20 for fifteen minutes to decrease the 
contaminants on the seed surface. Beginning of Apium 
graveolens seed germination was obtained on MS basal 
medium after 2 weeks culture (Fig. 1), and continued to 
germinate at 3 and 4 weeks of culture (Figs. 2, 3). Celery 
in vitro seedlings were obtained after 8 weeks of seeds 
cultivation (Fig. 4) without any auxin or cytokinin in cul-
ture medium. This is close to the results of Bruznican et 
al. (2017) who obtained seedlings on hormone-free me-
dium. Data tabulated in Table 1 showed the germination 
percentage of celery seeds and their lengths for 8 weeks 
of culture; maximal percentage of seeds germination 
was between the seventh and the eighth week of culture 
(83.10 ± 0.59, 86.57 ± 0.87, respectively), with seedling 
lengths (4.77 ± 0.15cm and 5.00 ± 0.58cm, respectively). 
A clear difference was observed in either seeds germina-
tion percent or seedlings length (cm) at the eighth week 
of culture comparable to the other periods, whilst there 
was no significance in seedlings length from the fifth to 
the seventh week of culture, and there was slightly sig-
nificance in seeds germination percent among 5, 6 and 
7 weeks of culture. Therefore, it was recommended to let 
seedlings grow for 8 weeks to obtain normal and healthy 
considerable amounts of celery in vitro seedlings.
3.2 CALLUS INDUCTION OF APIUM GRAVEO-
LENS IN VITRO SEEDLINGS.
About 1 cm of healthy leaf and stem of celery seed-
lings were aseptically excised and then sub-cultured on 
MS-medium including 0.5 mg l-1 2, 4-D alone or com-
bined with 1 mg BAP-1 or 1 mg l-1 kin for callus initia-
tion stage. All treatments exhibited different percentag-
es of callus induction with variable significance of both 
of stem and leaf explants as shown in Table 2. It was ob-
served that, maximal percent of calli (83.3 ± 0.58 %) in 
high significant difference clearly appeared using stem 
explant at treatment of 0.5 mg l-1 2,4-D compared to the 
leaf explant (55 ± 5.77) on the same distinct medium. 
Meanwhile, addition of 1 mg l-1 BA or kin with 0.5 mg l-1 
2, 4-D decreased significantly callus induction in both 
stem and leaf explants (Fig. 2). So, it was recommended 
to use stem as the optimal explant rather than the leaf 
explant (Fig. 3) to be cultured in medium fortified with 
little concentration of 2,4-D (0.5 mg l-1) alone to induce 
callus cultures of celery for two months of cultivation.
On the basis of previous results, stem calli of 
Apium graveolens were re-cultured on the same cer-
Incubation 
period Seeds germination % Seedlings length (cm)
Week 1 1.00 ± 0.29g 0.75 ± 0.14e
Week 2 46.97 ± 1.16f 0.75 ± 0.14e
Week 3 65.10 ± 2.89e 2.73 ± 0.15d
Week 4 70.00 ± 5.77 de 3.77 ± 0.15c
Week 5 73.10 ± 1.16cd 4.27 ± 0.15bc
Week 6 78.00 ± 0.58bc 4.27 ± 0.15bc
Week 7 83.10 ± 0.59ab 4.77 ± 0.15bc
Week 8 86.57 ± 0.8a 5.00 ± 0.58a
LSD 0.05 5.91 0.61
Table 1: Percentage of seeds germination and seedlings length 
during 8 weeks of culture 
Mean values accompanied with different superscript letters were sig-
nificantly different (p < 0.05).
Figure 1: Phases of seedlings formation of celery 
Acta agriculturae Slovenica, 121/4 – 2025 5
Callus-mediatede embryogenesis of leaf celery (Apium graveolens L. var. secalinum Alef.) ... to malt, peptone and yeast extracts application
tain medium (0.5 mg l-1 2, 4-D) for callus induction, 
and maintained for 8 weeks to study these parameters; 
fresh mass, growth rate and growth value (Table 3). The 
highest values of fresh mass and growth value were re-
corded with clear significant differences (7.2  ±  0.06  g 
and 13.4 ± 0.16, respectively) after the eighth week of 
culture. However, the growth rate increased continu-
ously recording the optimal value (2.07 ± 1.46 g/day) at 
the fifth week of culture before decreasing significantly 
from the sixth to the eighth week. Among all tested pe-
riods of culture, a considerable significant difference in 
all determined parameters for callus cultures produc-
tion through the eight weeks of subculture was noticed. 
Subsequently, the fifth week of culture was chosen as the 
preferable period to make subculture of stem calli due 
to the successive proliferation of callus recovery com-
parable to other periods. Contrarily, the mass produc-
tion of callus cultures was achieved after eight weeks of 
culture (Fig.4). 
Treatments Callus induction%
Stem Leaf
0.5 mg l-1 2,4-D 83.30 ± 0.58a 55.00 ± 5.77ab
0.5 mg l-1 2,4-D + 1 mg 
l-1 BA
53.30 ± 0.58ab 25.00 ± 0.58bcd
0.5 mg l-1 2,4-D + 1 mg 
l-1 Kin
16.70 ± 0.58cd 10 ± 0.58d
LSD 0.05 36.42
Table 2: Callus induction of celery stem and leaf explants after 
two months of cultivation 
Mean values accompanied with different superscript letters were sig-
nificantly different (p < 0.05).
Incubation 
period of 
cultures Fresh mass (g)
Growth rate (g/
day) Growth value
Week 1 1.50 ± 0.29h 0.14 ± 0.04g 2.00 ± 0.58g
Week 2 1.80 ± 0.06g 0.19 ± 0.01g 2.60 ± 0.12g
Week 3 3.00 ± 0.06f 0.36 ± 0.01f 5.00 ± 0.12f
Week 4 3.80 ± 0.06e 0.47 ± 0.02e 6.60 ± 0.12e
Week 5 4.50 ± 0.29d 2.07 ± 1.46a 8.00 ± 0.58d
Week 6 4.90 ± 0.06c 0.63 ± 0.01d 8.80 ± 0.12c
Week 7 5.80 ± 0.06b 0.76 ± 0.01c 10.60 ± 0.12b
Week 8 7.20 ± 0.06a 0.96 ± 0.01b 13.40 ± 0.16a
LSD 0.05 0.38 0.05 0.75
Figure 2: Stem explants of celery after one month of culture on 
0.5 mg l-1 2, 4-D + 1 mg l-1 BA (a), 0.5 mg l-1 2,4-D + 1 mg l-1 Kin 
(b) and 0.5 mg l-1 2,4-D (c).
Figure 3: Callus induction from celery leaf (a) and stem (b) ex-
plants on MS-medium + 0.5 mg l-1 2, 4-D after two months of 
culture
Table 3: Fresh mass, growth rate and growth value of celery 
stem calli grown on medium containing 0.5 mg l-1 2.4-D during 
8 weeks of culture 
Mean values accompanied with different superscript letters were sig-
nificantly different (p < 0.05).
Figure 4: The produced calli from celery stem explants on MS-
medium + 0.5 mg l-1 2, 4-D after 8 weeks of culture
Acta agriculturae Slovenica, 121/4 – 20256
N. D. GIRGIS et al.
3.3 EFFECT OF BIOTIC ELICITORS ON GROWTH 
DYNAMIC PARAMETERS OF APIUM GRAVEO-
LENS STEM CALLI
In this part of work, clusters of Apium graveolens 
stem calli were studied for their propagation capacity by 
re-culture onto the same callusing medium (0.5 mg l-1 
2,4-D) supplemented with some biotic elicitors such 
as malt extract, yeast extract and peptone extract at 
concentrations; 1.5, 2.5, 3.5 mg l-1 of each. Fresh mass, 
growth rate and growth value are the determinant pa-
rameters which had been calculated for callus growth 
dynamic parameters of Apium graveolens during 60 
days of culture as illustrated in Tables 4, 5 and 6.
3.3.1 Fresh mass of the enhanced stem calli of Apium 
graveolens by biotic elicitors.
In Table 4, all biotic elicitors (malt extract, yeast 
extract, peptone extract) in all their various concen-
trations (1.5, 2.5, 3.5 g  l-1) increased significantly the 
fresh calli growth from 10 days of culture till recording 
the highest values at the end of experiment (60 days). 
Likewise, medium devoid of elicitors (control) behaved 
the same. Furthermore, it was observed apparent sig-
nificance differences among all augmentation treat-
ments in all determined time periods. Both of malt (1.5 
g l-1) and peptone (1.5 and 2.5 g l-1) extracts augmented 
significantly callus cultures in all different periods of 
time. Maximum values with high significant differ-
ence were achieved in callus fresh mass (13.1 ± 0.15 g, 
12.9 ± 0.12 g) at the treatments of 1.5 g l-1 malt extract 
and 1.5 g l-1 peptone extract, respectively after 60 days 
of culture compared to the non-elicited treatment 
(9.5 ± 0.13 g). Data resulted from Table 4 reveal that all 
used elicitors increase the callus cultures proliferation of 
celery compared to the control; except the treatment of 
yeast extract at 1.5 g l-1 concentration which decreased 
the callus fresh mass values (6.7 ± 0.11 g, 7.4 ± 0.12 g, 
8.5 ± 0.15 g) compared to the control (9.5 ± 0.13 g) at 
the periods of 40, 50 and 60 days of culture, respectively. 
3.3.2 Growth rate of the enhanced stem calli of Api-
um graveolens by biotic elicitors.
Data in Table 5 revealed in the growth rate values of 
Apium graveolens stem calli which had been augmented 
by malt, yeast and peptone extracts in all their different 
concentrations. Callus growth rate was directly propor-
tional to boost in the periods of time from 10 days till 
60 days of culture for either elicitors-containing media 
or the control, except the treatment of yeast extract at 
3.5 g l-1 concentration exhibited high value till 50 days 
of culture. Similar to the achieved results in Table 4, 1.5 
Incubation 
period of 
cultures Control Malt extract Yeast extract Peptone extract
1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1
10 days 5.80 ± 
0.07A-a
7.30 ± 
0.17A-b
7.10 ± 
0.12A-b
6.50 ± 
0.11A-a
6.60 ± 
0.12A-a
6.50 ± 
0.15A-a
5.90 ± 
0.11A-a
7.30 ± 
0.11A-b
7.20 ± 
0.15A-b
7.10 ± 
0.11A-b
20 days 6.10 ± 
0.09A-a
8.50 ± 
0.1B-d
8.20 ± 
0.17B-d
7.50 ± 
0.14B-c
7.40 ± 
0.12B-c
6.70 ± 
0.11A-b
6.10 ± 
0.12A-a
7.40 ± 
0.14A-c
8.20 ± 
0.10B-d
7.40 ± 
0.12A-c
30 days 6.60 ± 
0.12B-a
10.40 ± 
0.09C-d
9.80 ± 
0.12C-c
8.60 ± 
0.17C-b
8.50 ± 
0.15C-b
8.40 ± 
0.17B-b
6.60 ± 
0.09B-a
8.10 ± 
0.15B-b
9.80 ± 
0.11C-c
9.70 ± 
0.15B-c
40 days 7.40 ± 
0.1C-b
11.00 ± 
0.12D-g
10.50 ± 
0.09D-f
8.90 ± 
0.14C-d
9.10 ± 
0.12D-d
9.00 ± 
0.15C-d
6.70 ± 
0.11B-a
8.20 ± 
0.13B-c
10.00 ± 
0.1C-e
9.90 ± 
0.11B-e
50 days 8.50 ± 
0.15D-b
11.60 ± 
0.12E-e
11.40 ± 
0.12E-e
9.50 ± 
0.11D-c
9.60 ± 
0.09E-c
9.60 ± 
0.11D-c
7.40 ± 
0.12C-a
10.90 ± 
0.11C-d
10.70 ± 
0.12D-d
10.80 ± 
0.15C-d
60 days 9.50 ± 
0.13E-b
13.10 ± 
0.15F-e
11.80 ± 
0.09F-d
10.40 ± 
0.12E-c
10.40 ± 
0.15F-c
10.30 ± 
0.12E-c
8.50 ± 
0.15D-a
12.90 ± 
0.12D-e
12.80 ± 
0.15E-e
11.90 ± 
0.12D-d
Table 4: Fresh mass of celery stem calli grown on MS-Medium containing malt, yeast and peptone extracts 
Values are introduced as means ± standard errors of three replicates. Values introduced with different capital letters within a column or with different 
small letters within a row are significantly different at p < 0.05.
Acta agriculturae Slovenica, 121/4 – 2025 7
Callus-mediatede embryogenesis of leaf celery (Apium graveolens L. var. secalinum Alef.) ... to malt, peptone and yeast extracts application
Incubation 
period of 
cultures Control Malt extract Yeast extract Peptone extract
1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1
10 days 0.40 ± 
0.01A-a
0.60 ± 
0.01A-c
0.60 ± 
0.02A-c
0.56 ± 
0.01A-b
0.56 ± 
0.02A-b
0.55 ± 
0.01A-b
0.50 ± 
0.01A-a
0.63 ± 
0.29A-c
0.64 ± 
0.01A-c
0.63 ± 
0.01A-c
20 days 0.50 ± 
0.01B-a
0.70 ± 
0.01B-d
0.70 ± 
0.02B-d
0.65 ± 
0.01B-c
0.58 ± 
0.02A-b
0.57 ± 
0.01A-b
0.52 ± 
0.01A-a
0.72 ± 
0.02B-d
0.65 ± 
0.11A-c
0.66 ± 
0.01A-c
30 days 0.50 ± 
0.01C-a
0.90 ± 
0.02C-e
0.80 ± 
0.02C-d
0.74 ± 
0.02C-c
0.62 ± 
0.02A-b
0.73 ± 
0.02B-c
0.56 ± 
0.01B-a
0.87 ± 
0.02C-d
0.88 ± 
0.01B-d
0.86 ± 
0.01B-d
40 days 0.60 ± 
0.01D-b
1.00 ± 
0.02C-e
0.90 ± 
0.02D-d
0.79 ± 
0.03C-c
0.80 ± 
0.01B-c
0.80 ± 
0.01C-c
0.57 ± 
0.01B-a
0.94 ± 
0.01D-d
0.92 ± 
0.02B-d
0.91 ± 
0.02B-d
50 days 0.70 
0.01E-b
1.00 ± 
0.01D-e
0.90 ± 
0.01E-d
0.86 ± 
0.0D-c
0.87 ± 
0.01C-c
0.86 ± 
0.01D-c
0.63 ± 
0.02C-a
0.97 ± 
0.02D-d
0.99 ± 
0.01C-d
0.98 ± 
0.01C-d
60 days 0.80 ± 
0.01F-b
1.20 ± 
0.01E-f
1.00 ± 
0.02F-d
0.93 ± 
0.02E-c
0.94 ± 
0.01D-c
0.93 ± 
0.01E-c
0.65 ± 
0.03C-a
1.18 ± 
0.01E-e
1.17 ± 
0.02D-e
1.09 ± 
0.02D-d
Incubation 
period of 
cultures Control Malt extract Yeast extract Peptone extract
1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1 1.5 g l-1 2.5 g l-1 3.5 g l-1
10
days
4.90 ± 
0.07A-a
6.30 ± 
0.07A-c
6.20 ± 
0.12A-c
5.60 ± 
0.09A-b
5.50 ± 
0.17A-b
5.60 ± 
0.15A-b
5.00 ± 
0.09A-a
6.40 ± 
0.17A-c
6.30 ± 
0.15A-c
6.30 ± 
0.09 A-c
20
days
5.10 ± 
0.06A-a
7.50 ± 
0.06B-c
7.30 ± 
0.09B-c
6.50 ± 
0.09B-b
6.30 ± 
0.22B-b
6.40 ± 
0.12B-b
5.20 ± 
0.15A-a
7.40 ± 
0.2B-c
7.30 ± 
0.18B-c
6.40 ± 
0.1A-b
30
days
5.50 ± 
0.07B-a
9.40 ± 
0.10C-e
8.80 ± 
0.13C-d
7.50 ± 
0.15C-c
7.40 ± 
0.23C-c
7.40 ± 
0.17C-c
5.30 ± 
0.17A-a
8.70 ± 
0.12C-d
8.80 ± 
0.12C-d
6.70 ± 
0.12B-b
40
days
6.50 ± 
0.07C-b
10.10 ± 
0.12D-e
9.40 ± 
0.15D-d
8.20 ± 
0.12D-c
8.30 ± 
0.18D-c
8.20 ± 
0.12D-c
6.00 ± 
0.17B-a
9.30 ± 
0.10D-d
9.50 ± 
0.12D-d
9.40 ± 
0.17C-d
50
days
7.50 ± 
0.06D-b
10.60 ± 
0.1E-e
9.90 ± 
0.0E-d
8.40 ± 
0.10D-c
8.50 ± 
0.15D-c
8.40 ± 
0.10D-c
6.10 ± 
0.1B-a
9.90 ± 
0.10E-d
9.70 ± 
0.20D-d
9.70 ± 
0.19C-d
60
days
8.60 ± 
0.09E-b
12.10 ± 
0.12F-e
10.80 ± 
0.12F-d
9.20 ± 
0.09E-c
9.30 ± 
0.17E-c
9.40 ± 
0.12E-c
7.40 ± 
0.23C-a
11.00 ± 
0.12F-d
10.90 ± 
0.23E-d
10.90 ± 
0.10D-d
Table 5: Growth rate of celery stem calli grown on MS-Medium containing malt, yeast and peptone extracts 
Values are introduced as means ± standard errors of three replicates. Values introduced with different capital letters within a column or with different 
small letters within a row are significantly different at p < 0.05.
Values are introduced as means ± standard errors of three replicates. Values introduced with different capital letters within a column or with different 
small letters within a row are significantly different at p < 0.05.
Table 6: Growth value of celery stem calli grown on MS-Medium containing malt, yeast and peptone extracts 
Acta agriculturae Slovenica, 121/4 – 20258
N. D. GIRGIS et al.
g  l-1 of both of malt and peptone extracts gave higher 
values of callus growth rate (1.2 ± 0.01 and 1.18 ± 0.01, 
respectively) than 1.5 g l-1 of yeast extract (0.94 ± 0.01), 
likewise 3.5 g l-1 yeast extract decreased significantly the 
callus growth rate (0.65 ± 0.03) than elicitors free me-
dium (0.8 ± 0.01).
3.3.3 Growth value of the enhanced stem calli of 
Apium graveolens by biotic elicitors.
Data tabulated in Table 6 revealed significantly 
an ascending growth value for Apium graveolens stem 
calli grown on elicitors containing media, along with 
the medium devoid of elicitors during 60 days of cul-
ture. There were non-significant differences between 
40 and 50 days of culture in all concentrations of yeast 
extract, 3.5 g l-1 malt extract, 2.5 and 3.5 g l-1 peptone 
extract. Likewise, there were non-significant differ-
ences between 10 and 20 days of culture in treatments 
of yeast and peptone extracts at 3.5 g l-1 concentration 
of each, beside elicitors free medium. On the other 
hand, the highest growth value of celery callus cultures 
was verified significantly using 1.5 g  l-1 malt extract 
(12.1 ± 0.12), followed by using 1.5 g  l-1 peptone ex-
tract (11 ± 0.12) after 60 days of culture. Surprisingly, 
among all these augmentation treatments to increase 
callus growth value, it was observed that the highest 
concentration of yeast extract (3.5 g  l-1) reduced cal-
lus growth value significantly at 40 (6.0  ±  0.17), 50 
(6.1 ± 0.12), 60 (7.4 ± 0.23) days of culture compared 
to the control (6.5 ± 0.07, 7.5 ± 0.06, 8.6 ± 0.09, re-
spectively).
3.4 SCREENING OF SOMATIC EMBRYOS DE-
VELOPMENT AND DIFFERENTIATION IN 
APIUM GRAVEOLENS STEM CALLI BY HIS-
TOLOGICAL INVESTIGATION
In this part of the study, the augmented biomass 
of celery stem callus cultures which had been deliv-
ered from the previous work were examined for their 
ability for differentiation and embryogenesis to form 
somatic embryos. Clusters of stem calli which were 
grown for 4 weeks on MS-Medium elicited by malt, 
yeast and peptone extracts (1.5, 2.5, 3.5 g l-1 of each) 
were screened for their possibility for embryogenesis 
and differentiation by histological assay. Data result-
ed from the histological examination for all elicited 
treatments revealed these findings: calli on elicitors 
free MS-medium (0.5 mg l-1 of 2, 4-D, control) led to 
globular embryo formation (Fig.5). Meanwhile, MS-
medium supplemented with 3.5 g l-1 of malt extract as 
biotic elicitor was the optimal structure to form a tor-
pedo shaped embryo (Fig. 6) as an advanced stage of 
indirect somatic embryogenesis. So, we recommend 
malt extract at 3.5 g l-1 concentration as the best biotic 
elicitor for embryogenesis development. However, 
peptone extract containing medium at 1.5 g  l-1 con-
centration led to formation of an early heart (Fig. 7, 
a) and late heart shaped embryo (Fig. 7, b). So, 1.5 g l-1 
of peptone could be considered as good biotic elicitor 
for synchronization in the indirect somatic embryo-
genesis. On the other hand, MS-medium fortified 
Figure 5: Celery late globular embryo derived from stem calli 
under the effect of plant growth regulator (0.5 2,4-D) as a con-
trol without elicitor (a). Magnified portion showing the forma-
tion of xylem vessels with annular thickening (b) after 4 weeks 
of celery stem calli culture.
Figure 6: Torpedo shaped embryo formed as an advanced stage 
of indirect somatic embryogenesis using malt as an elicitor after 
4 weeks of celery stem calli culture 
Acta agriculturae Slovenica, 121/4 – 2025 9
Callus-mediatede embryogenesis of leaf celery (Apium graveolens L. var. secalinum Alef.) ... to malt, peptone and yeast extracts application
with 2.5 g l-1 of yeast extract helped in heart shape for-
mation (Fig. 8), therefore yeast extract (2.5 g l-1) acted 
as valuable biotic elicitor for embryogenesis progres-
sion. All other treatments of biotic elicitors exhibited 
normal parenchyma cells without any differentiation 
structure.
4 DISCUSSION 
The obtained results in Tables 2 and 3 considered 
2,4-D addition individually in callus culture medium as 
the optimal auxin in celery calli propagation. This find-
ing is in accordance with Soorni et al. (2012) who con-
firmed by statistical analysis the substantial role of 2,4-D 
at 1 mg  l-1 concentration on callus induction efficiency 
of Cuminum cyminum L. leaf explants. Also, Babiker et 
al. (2021) who gained callus cultures of Chrysanthemum 
from leaf or internode explants using 2,4-D at 0.5 mg l-1 
or 2 mg l-1 concentrations. As well Mahood et al. (2022) 
who obtained the highest percentage of callus induction 
(90  %) using stem explants following by leaf explants 
(80 %) using 1 mg l-1 2,4-D in Gazania rigens (L.) Gaertn. 
callus culture medium after four weeks of cultivation. 
From the previous findings, it could be recommended 
to use 2,4-D as stimulator for callus growth, 2,4-D had 
distinctive characteristics; it was hardly decomposed by 
heating during the sterilization process or by the en-
zymes resulted from the used explants (George et al., 
2008, Al-Khayri, 2011). 
On basis the obtained results in Tables 4, 5, and 6, 
a positive relationship between the propagation incre-
ment of celery callus cultures and the time periods incre-
ment for these cell cultures incubation could be observed 
throughout the experiment. Elicitor containing media 
with their different concentrations raised significantly 
the calculated growth measurements (fresh mass, growth 
rate and growth value) for celery callus cultures more 
than medium devoid of elicitors (control). In this re-
gard, supply of biotic elicitors (malt, yeast, and peptone) 
in celery calli medium increased the callus proliferation 
dependent on lower concentrations of those elicitors. 
This suggestion matches the findings in Tables 4, 5 and 
6 which indicated the highest elicitors’ effectiveness on 
mass production of celery callus cultures which had been 
investigated for fresh mass and growth rate at the low-
est concentration of each elicitor. Among the achieved 
results previously, malt extract followed by peptone ex-
tract at 1.5 g l-1 concentration of each contributed to at-
taining the biggest amount of cellular biomass of celery 
compared to the others. This was attributing to consider 
that malt and peptone extracts act as efficient elicitors for 
callus cultures propagation of Apium graveolens when 
used in low concentrations. The significant importance 
of biotic elicitors (malt, yeast and peptone) in cell cul-
tures propagation could be due to considering malt ex-
tract as enriched carbohydrate source; yeast extract as 
good source of minerals, vitamins and amino acids, and 
peptone extract as high organic nitrogen source (Badr-
Elden, 2017, Vasil & Hildebrandt, 1966, Parc et al., 2007). 
Based on the obtained results, we recommend adding bi-
otic elicitors in celery callus culture medium, particular 
malt and peptone extracts which were more preferable 
than yeast extract for celery biomass production. This 
recommendation is close to what have been achieved by 
Sawy et al. (2005), Hussain et al. (2016) and Badr-Elden, 
(2017) who attained the highest callogenesis in ovules of 
Citrus and kinnow mandarin using high concentration 
(500 mg l-1) of malt extract in callus induction medium. 
Likewise, both Parc et al. (2007) and Nhut et al. (2008) 
referred to the considerable effect of peptone extract in 
cell proliferation medium as the most sufficient elicitor 
for strong biomass increment tobacco and avocado, re-
spectively. Otherwise, Eshaghi et al. (2021) recorded low-
er callus mass (0.152 g) in wheat using yeast extract than 
when using casein hydrolysate (0.230 g). Besides, Sidhar 
& Aswath (2014) mentioned the positive effect of moder-
ate yeast extract concentrations on shoot multiplication 
rate and plant regeneration of Stevia rebaudiana (Berto-
ni) Bartoni. However, Kikowska et al. (2015) added yeast 
extract at 200 mg  l-1 to Eryngium planum L. shoot cul-
tures medium for biomass accumulation enhancement, 
and Nadeem et al. (2018) increased by two-fold the fresh 
and dry weights of Linum usitatissimum L.. Cultures over 
the control using MS-medium supplemented with 200 
mg l-1 of yeast extract. 
In line with the achieved findings as shown in Fig-
ures 5, 6, 7 and 8, we selected specific concentration of 
each biotic elicitor for callus culture medium of Apium 
graveolens for successful somatic embryogenesis and dif-
ferentiation. Our results were in harmony with the oth-
ers who supported this idea; plant growth regulators and 
elicitors played a substantial role in embryogenesis and 
the development stages of somatic embryos dependent 
Figure 7: Early heart shaped embryo (1) and late heart shaped 
embryo (2) showing a synchronization in indirect somatic em-
bryogenesis using peptone as an elicitor after 4 weeks of celery 
stem calli culture.
Acta agriculturae Slovenica, 121/4 – 202510
N. D. GIRGIS et al.
on concentration and type. Herein some of these reports; 
Hwankim & Janick (1989) who observed the critical role 
of 2,4-D in embryogenesis of Apium graveolens accord-
ing to its concentration, where low concentration had 
poor effect on embryos formation while high concen-
tration led to embryos suppression. Besides, Mazri et 
al. (2013) used PGRs for olive somatic embryogenesis, 
Chen & Chang (2002) obtained the best response for 
direct embryos formation in Oncidium ‘Gower Ramsey 
using 0.5-1 g l-1 of peptone and Al-Khayri (2011) gained 
embryos higher than the control using yeast extract at 
1 g  l-1 concentration. As well Mazri & Belkoura (2021) 
affirmed the effectiveness of malt extract concentration 
(500 mg  l-1) on somatic embryos formation and differ-
entiation of citrus calli. Likewise, the development of 
somatic embryogenesis for plant regeneration in vari-
ous citrus species was achieved by Amin & Shekafandeh 
(2015), Sawy et al. (2005) and Gholami et al. (2013).
To sum up, authors indicated the significant impor-
tance of 3.5 g  l-1 malt extract as the best biotic elicitor 
for embryos development, followed by 1.5 g  l-1 peptone 
extract to form late stage of heart shaped embryo, then 
2.5 g  l-1 yeast extract for somatic embryogenesis, to be 
added in MS-medium + 0.5 mg  l-1 2, 4-D to efficiently 
induce somatic embryos with their development for 
differentiation. This recommendation is due to the im-
portance of amino acids content in yeast extract, carbo-
hydrates content in malt extract and organic nitrogen 
content in peptone extract which stimulated the em-
bryogenesis and plant growth. These investigations were 
in agreement with Badr-Elden (2017) and Carimi et al. 
(1998) who reported the promotive impact of malt ex-
tract as carbohydrate source for citrus somatic embryo-
genesis and stimulation of the early cotyledonary stage 
of embryos germination in the in vitro rescue of Citrus 
x aurantium L.. Additionally, proembryo morphology 
of calli and proliferation depended on malt extract con-
centration (Mazri & Belkoura, 2021). Furthermore, the 
biosynthesis of endogenous amino acid at early stage of 
protocorm development could not be adequate for faster 
and healthy growth of orchid protocorm, therefore ad-
dition of amino acids from peptone could enhance the 
growth of orchid protocorm, and the growth of embryo 
which was related to easily absorption of water (Setiari 
et al., 2016). Typically, adding more yeast extract to the 
MS medium at higher concentrations limited growth, but 
adding less yeast extract had shown to have positive ef-
fects (Safwat et al., 2014). Ultimately, in this work we af-
firmed the extreme necessity to add PGRs (0.5 mg l-1 2.4-
D) with biotic elicitors (malt (3.5 g l-1), peptone (1.5 g l-1) 
and yeast (2.5 g l-1) extracts) in callus culture medium of 
Apium graveolens L. for investigation of somatic embryo-
genesis development.  Therefore, addition of exogenous 
PGRs and elicitors interacted with the plant hormones 
and modified their levels, which resulted in cell division, 
differentiation, growth and morphogenesis.
5 CONCLUSION
An efficient and reliable protocol of somatic em-
bryogenesis was performed as permanent natural appli-
cation for obtaining progeny homogeneous plants. We 
employed several biotic elicitors (malt, peptone and yeast 
extracts) with different concentrations for callus induc-
tion, somatic embryogenesis development and embryo 
differentiation. In the present research, a comprehensible 
correlation between the elicitors and somatic embryos 
formation was shown that reflects their importance in 
the somatic embryogenesis development which was de-
pendent on the elicitor type and concentration. So far to 
the authors’ knowledge, the effectiveness of biotic elici-
tors on somatic embryos enhancement of celery has not 
been reported before. Indeed, our research achievements 
provided new insightsthrough the elicitors for boosting 
the embryogenesis development and differentiation. All 
biotic elicitors possessed an impressive performance, in 
particular malt extract at 3.5 g  l-1 concentration. Malt 
extract was a perfect starting point for the study of elici-
tors-based embryogenesis for differentiation and indirect 
plant regeneration promotion to be a boon in regenera-
tion mass production of the identical and homogeneous 
plants of Apium graveolens.
6 STATEMENTS & DECLARATIONS
Availability of original data: The authors declare 
that all original data are included in the manuscript as 
well they read and approved the final draft of the manu-
script
The ethical standards: The authors performed this 
article without any human studies to compliance the eth-
ics procedures 
Conflict of interest: The authors declare that they 
have no conflicts of interest
Funding: Not applicable
Authors’ contributions: All authors designed and 
performed the experiments, analyzed and wrote the orig-
inal draft, and edited the manuscript.  All authors agree 
for submission of manuscript to the journal
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