108

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

Pelita Perkebunan 38(2) 2022, 108

—

119

Establishment of an Efficient Primary Callus Induction for

Embryogenic Potential of

Coffea arabica

Rina Arimarsetiowati

1,2)

, Budi Setiadi Daryono

, Yohana Theresia Maria Astuti

and Endang Semiarti

2*)

Indonesian Coffee and Cocoa Research Institute, Jl. PB. Sudirman No. 90, Jember 68118, Indonesia

Graduate Study Program, Biotechnology Laboratory, Faculty of Biology, Universitas Gadjah Mada,

Jl. Teknika Selatan Sekip Utara, Sleman 55281, Yogyakarta, Indonesia

Graduate Study Program, Genetic and Breeding Laboratory, Faculty of Biology, Universitas Gadjah Mada,

Jl. Teknika Selatan Sekip Utara, Sleman 55281, Yogyakarta, Indonesia

Faculty of Agriculture, Institut Pertanian Stiper (Instiper), Jl. Nangka II, Maguwoharjo,

Depok, Sleman, Yogyakarta 55282, Indonesia

Corresponding author: endsemi@ugm.ac.id

Received: 8 July 2022 / Accepted: 21 July 2022

Abstract

Coffea arabica L. is a species of coffee that contribute for more than seventy

percent of world coffee production. Various attempts have been made to obtain

large quantities of planting material through propagation in vitro somatic embryo-

genesis technology. Producing embryo somatic depends on the embryogenic

callus formation from the primary callus. The problem is that not all primary calluses

meet the embryogenic criteria for developing into an embryo somatic. The objective

of this experiment was to evaluate the effect of different plant growth regulators

(PGRs) on callus induction (indirect somatic embryogenesis) in AS2K clone of

Arabica coffee. Mother plants of Arabica coffee were established in coffee experi-

mental field of Indonesian Coffee and Cocoa Research Institute at Andungsari,

Bondowoso, East Java, Indonesia. Leaf explants were cultured on a half-strength

Murashige and Skoog (MS) medium supplemented with various concentrations

of 1.0, 2.0, 3.0 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0, 2.0, 3.0 mg L

-1

1-phenyl-3-(1, 2, 3-thidiazol-5-yl)-urea (thidiazuron) in combination with 1.0 mg L

-1

6-benzylaminopurine (6-BAP). All the experiments were organized in completely

randomized design and repeated three times, each experimental unit using minimum

seven replicates (a total of 21 explants per combination mediums). The morphology

of the different types of callus was observed under a stereo-zoom binocular micro-

scope. Histological analysis on callus formation was observed on the surface of explants

by anatomic preparation using the paraffin. The percentage of callus formation was

recorded every two weeks until eight weeks. The highest percentage of callus formation

(almost 60%) was in medium containing 1 mg L

-1

2,4-D dan 1 mg L

-1

BAP. In

medium containing TDZ and BAP, the percentage of callus formation was low

between 2-10% in all periods of culture and the calli was growing slowly. Morpho-

logical and histological studies prove that the callus has a friable and embryogenic

texture and begins to develop various stages of somatic embryo formation.

Keywords: callus, Coffea arabica L., in vitro culture, plant growth regulators, somatic

embryo

ISSN: 0215-0212 / e-ISSN: 2406-9574

DOI: 10.22302/iccri.jur.pelitaperkebunan.v38i2.510

109

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

INTRODUCTION

Plant tissue culture is important in the

production of agricultural and ornamental

plants, as well as in plant manipulation for

enhancing agricultural performance. Plant

cell and tissue in vitro culture have triggered

tremendous interest because it facilitates the

investigation of plant physiological and genetic

processes, as well as the potential to support

the breeding of improved cultivars by increasing

genetic variability.

Somatic embryogenesis (SE) is an asexual

reproduction approach that grows naturally

in many plants species and is widely used

for rapid multiplication, crop transformation,

and regeneration. Somatic embryognesis and

zigotic embryogenesis share some develop-

mental and physiological similarities because

it involves common factors of hormonal,

transcriptional, developmental, and epigenetic

controls (Salaün et al., 2021). SE also offers an

appropriate in vitro regeneration procedure

as a primary step in plant genetic improve-

ment (Loyola-Vargas, 2016). SE is associated

with the formation of haploid or diploid

somatic embryos without gamete fusion. SE

is influenced by various of factors, such as

genotype, explant type, and plant growth

regulators. Suitable conditions for SE induc-

tion are generally conducted experimentally

via trial and error experiments (Loyola-

Vargas, 2016). The morphological and histo-

logical examination could reveal the somatic

embryogenesis mechanism and provide insights

for enhancing procedures that influence this

regeneration method (Silva et al., 2015).

Somatic embryogenesis (SE) has been

effectively conquered on an industrial level

for the Coffea arabica L. (Bobadilla Landey

et al., 2013). In coffee, this process has been

achieved via direct somatic embryogenesis

from pro-embryogenic cells of leaf tissue in

the absence of conspicuous callus proliferation

or by indirect somatic embryogenesis (ISE)

via friable embryogenic callus formation

(Molina et al., 2002). ISE in coffee comprises

a sequence of steps including callus induction

and proliferation, and embryo development,

as well as germination and conversion into

plants (van Boxtel & Berthouly, 1996).

Growth regulators are critical in regulating

the formation of somatic embryos in coffee

leaf explants. The auxin/cytokinin combi-

nation is commonly used for IES induction

in C. arabica. The most popular auxin 2,4-

D was utilized to stimulate callogenesis in

C. arabica leaf explants. Because auxin

inhibits embryo emergence, cytokinins with-

out auxin were used for direct SE induction.

The efficiency of the direct SE response,

on the other hand, may vary depending on

the type and concentration of cytokinins used

(Andrea, 2020). A concentration of 5 M of

the synthetic cytokinin 6-benzylaminopurine

(6-BAP) was sufficient for direct SE induction

on C. arabica explants (Rojas-Lorz et al.,

2019). In leaf explants of the Mundo Novo

de C. arabica cultivar, 6-BAP at 30 M resulted

in higher somatic embryo production than at

10 and 20 M 6-BAP (Almeida & Silvarolla

MB, 2009). Despite the high 6-BA concen-

tration, embryo production was reduced, and

the process took a long time. The TDZ cyto-

kinin has also been used to induce direct

somatic embryo regeneration in C. arabica

(Kahia et al., 2016; Yi-Chieh et al., 2018),

with a concentration of 1M TDZ resulting

in a 100% embryogenic percentage. TDZ has

a significant impact on embryogenic callus

formation and embryo multiplication.

The formation of embryogenic callus

from the primary callus is required for the

production of somatic embryos. The issue is

that not all primary calluses satisfy the embryo-

genic criteria for developing into a somatic

embryo. The goal of this research was to figure

out the best medium formulation for induction

and callus growth from leaves Arabica coffee

(Coffea arabica L.) explants by studying the

110

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

influence of plant growth regulator through

indirect somatic embryogenesis system at

various of media compositions of 2,4-D,

thidiazuron and BAP to be subsequently used

for plantlet regeneration.

MATERIALS AND METHODS

Planting Material Resource

Mother plants of Arabica coffee were

established in coffee experimental field of

Indonesian Coffee and Cocoa Research

Institute at Andungsari, Bondowoso, East

Java, Indonesia. GPS coordinate and eleva-

tion of plot were recorded with data showing

S07° 55' 42.7" and E113° 41' 30.2" and 1451

m asl. The second pairs of young leaves

(i.e., 100 explants) from the tip on plagio-

tropic branches were collected from healthy

mother plants growing in greenhouse as plant

material.

Medium

In order to develop an efficient and reli-

able procedure for somatic embryogenesis,

leaf explants were cultured on MS (Murashige

and Skoog) medium supplemented with various

hormones in different concentrations and

combinations for a certain stages of in vitro

micropropagation of somatic embryogenesis

based on Sanglard et al. (2019) modified

protocol (Table 1).

Surface Sterilization of Explants

The leaves were washed under running

tap water and rinsed with sterile distilled

water, followed by surface sterilization in

30% sodium hypochlorite solution (5.25%

active chlorine) for 15 min and rinsed thrice

using sterile distilled water. The leaf explants

were subsequently immersed in 70% (v/v)

ethanol for 20-30 s then rinsed thrice with

sterile distilled water. The margin and the

mid-rib of each leaf were removed and the

remaining leaf tissue was cut into 1.0 cm

pieces to be used as explants. The explants

were incubated on a half-strength MS medium

with the adaxial side down in the culture

vessel. These materials were used as source

leaf explants for indirect somatic embryo-

genesis under in vitro conditions in a laminar

flow chamber (Arimarsetiowati, 2011).

Callus Induction

To evaluate the effect of different plant

growth regulators (PGRs) on callus induction

(indirect somatic embryogenesis), leaf explants

were cultured on a half-strength MS medium

supplemented with various concentrations of

1.0, 2.0, 3.0 mg L

-1

of 2,4-Dichlorophenoxyacetic

acid (2,4-D) or 1.0, 2.0, 3.0 mg L

-1

1-phenyl-3-

(1, 2, 3-thidiazol-5-yl)-urea (TDZ) in combi-

nation with 1.0 mg L

-1

N6-benzyladenine (BAP).

The pH of all media was adjusted to 5.6, which

was consistent throughout all experiments

(Table 1). The culture was incubated in a dark

Table 1. Composition of callus inducing medium (CIM) for indirect somatic embryogenesis from leaf explants of C. arabica L

Medium components

Callus inducing medium (CIM)

CIM 1 CIM 2 CIM 3 CIM 4 CIM 5 CIM 6

MS macro and micro salt ½ ½ ½ ½ ½ ½

Gamborg’s B5 vitamins + + + + + +

BAP, mg L

-1

1 1 1 1 1 1

2,4-D, mg L

-1

1 2 3 - - -

Thidiazuron, mg L

-1

- - - 1 2 3

L-cysteine, g L

-1

0.08 0.08 0.08 0.08 0.08 0.08

Malt extract, g L

-1

0.4 0.4 0.4 0.4 0.4 0.4

Casein hydrolysate, g L

-1

0.1 0.1 0.1 0.1 0.1 0.1

Sucrose, g L

-1

30 30 30 30 30 30

Gellan gum, g L

-1

2.8 2.8 2.8 2.8 2.8 2.8

pH 5.6 5.6 5.6 5.6 5.6 5.6

111

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

growth room at 25±2

C. The percentage of

callus formation was recorded every two

weeks until eight weeks.

Experimental Design and Data Analysis

The treatments is the combination of

medium between 2,4-D and BAP or 2,4-D

and thidiazuron. All the experiments were

organized in a completely randomized design

and repeated three times, each experimental

unit using seven replicates (total of 21 explants

per treatment). All statistical analyses were

performed using RStudio software (Version

1.4.1106). One-way analysis of variance

(ANOVA) was used to test for statistical

significance and followed by Tukey’s test

at 5% probability level (P 0.05).

Morphological and Histological

Analysis

The morphology of the different types

of callus was observed under a stereo-zoom

binocular microscope (SZ 4045 TR Olympus

Tokyo, Japan) coupled to the computer running

image capture software (DP2-BSW). Histo-

logical analysis on callus formation was observed

on the surface of explants by anatomic prepa-

ration using the paraffin method according to

Ruzin (1999). The anatomic samples in the glass

slides were observed under a light microscope

(Olympus, Japan).

RESULTS AND DISCUSSION

Plant Growth Regulators Effects on

Callus Formation

The success of AS2K clone Arabica

coffee propagation through indirect somatic

embryogenesis can be done using leaf explants

through callus formation. The advantage of

indirect somatic embryogenesis is fast, uniform,

and large scales. Moreover, the higher callus

embryogenic production, the greater the

number of embryo will be achieved, and the

plantlet will regenerate excessively. The type

and concentration of auxin and cytokinin and

or their combination differ from each species

in producing the best medium (Méndez-

Hernández et al., 2019). Auxin is a growth

regulator that can trigger cell division, cell

growth development and meristem organi-

zation for callus formation (Kumar et al.,

2016). Auxin is commonly used to stimulate

elongation and cell division, as well as to

induce the formation of callus, culture suspen-

sion, and roots. Cytokinins are compounds

that can promote the leaf’s cell division,

growth, and development. The combination

of 2,4-D (auxin) with BAP (cytokinin) will

stimulate cell growth and division (Mayerni

et al., 2020). Explant type, age, genotype,

nutritional status, and interactions between

endogenous and exogenous hormones can

influence the development of somatic embryo-

genesis (Sujatha and Visarada, 2013; Singh

et al., 2016; Raji et al., 2018).

This study used young leaves because

it was more effective than other somatic tissue

explants (root, hypocotyl, epicotyl). Callus grew

on the explant side of the cut that was in direct

contact with the medium. It is suspected

that in leaf explants, cytokinins and auxins

are not absorbed through the leaf epidermis,

but are only absorbed by tissues that are

in direct contact with the medium and are

not translocated throughout the leaf tissue.

The same thing also happened to Robusta

coffee culture, where callus only formed on

the side of the leaf. However, in some leaf

explants, callus can form on the surface or side

of the leaf. The ability of morphogenesis is

related to the place of competent cells. With

the proper stimulation of growth regulators,

these competent cells then regenerate. The

performance of callus growth is different

from each medium composition until 56 days

after culture in CIM (Figure 1).

112

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

In this study, all concentrations and

combinations of growth regulators containing

2,4-D, Thidizuron, and BAP could produce

callus formation (indirect somatic embryo-

genesis) with different percentage responses.

The first changes in leaf explant culture after

being transferred to MS medium containing

2,4-D, Thidizuron, and BAP were swelling

of the leaf explants on the 7

day and then

callus formation was started on the14

day

of culture (Figure 2). The highest callus

formation was achieved in medium containing

1 mg L

-1

2,4-D and 1 mg L

-1

BAP (CIM 1)

after 14 days of callus initiation with a percentage

of 58%. Furthermore, on the 28

and 42

days,

there was a decrease in the percentage of callus

formation on CIM 1 medium because some

callus turned brown and died. In addition,

the percentage of callus formation increased

on the 56

day with a percentage of almost 60%

on CIM 1 medium. Callus formation in medium

containing 2,4-D and BAP resulted in a high

percentage compared to medium containing TDZ

and BAP (Figure 2). In medium containing

TDZ and BAP (CIM 4, CIM 5, and CIM 6),

the percentage of callus formation was low

between 2-10% in all periods of culture and

the calli was growing slowly. The combina-

tion of 2,4-D and BAP was the most effective

composition in the callus formation of AS2K

clone Arabica coffee. Previous studies have

shown that 2,4-D is the best auxin in callus

formation (Irene et al., 2019). Among chemical

compounds, 2,4-D is the dominant one to

induce embryogenic callus in coffee species.

This compound can stimulate an increase in

endo-genous natural auxin levels, can stimulate

cell proliferation and produce mass embryo-

genic callus formation. Callus can form pre-

embryoids, which are small white dots that

appear on leaf explants. Somatic embryo

induction and regeneration is a very sensitive

process to various culture conditions. Embryo-

genic callus will naturally be produced after

60 days of culture on CIM medium with

the characteristics of crumb, friable and yellowish

color. In addition to CIM 1 medium, CIM 2

medium also produced a high percentage of

callus formation. This is presumably due to

the interaction and balance between growth

regulators added to the medium and those

produced by endogenous cells that will

determine the direction of development of

a culture. The addition of auxin to the medium

Figure 1. Callus formation through indirect somatic embryogenesis in Arabica coffee AS2K

clone on 56 days after culture in CIM (callus inducing medium)

CIM 5 CIM 6CIM 4

CIM 2 CIM 3CIM 1

113

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

will change the ratio of endogenous growth

regulators which then becomes a determining

factor for the growth process and morpho-

genesis of explants.

Growth Type, Structure, and Color of

Callus

The effect of different combinations of

PGRs and their interaction on callogenesis

are presented in Figure 3 and 4. The experimental

result clearly showed that the percentages and

the characteristic of callus formation of AS2K

clone Arabica coffee based on place of callus

growth, type of callus growth, structure of

callus, and color of callus, differed, based

on the extent of the interaction between the

combination of PGRs. The interaction between

the callus induction percentage, the place where

callus formed (whole leaf edge and certain

part of the leaf) and the type of callus growth

(low, intermediate, and high), varied widely

on 14, 28, 42, and 56 days after culture in

CIM (Callus Inducing Medium) medium

(Figure 3). The highest percentages of callus

formation (around 50%) was reached in

whole leaf edge on CIM 2 medium on 28,

42, and 56 days after cultures with the callus

growth were low, intermediate, and high,

respectively and not significantly different

between the period of culture. Furthermore,

the interaction between the callus induction

percentage, the structure (morphogenic and non-

morphogenic) and color of callus (brownish,

creamish, and whitish), varied widely on 14,

28, 42, and 56 days after culture in CIM (Callus

Inducing Medium) medium (Figure 4). The

highest percentage of the morphogenic callus

(almost 60%) was reached in CIM 2 medium

on 56 days after cultures with creamish color,

but it was not a significant difference com-

pared to the CIM 1 on 14 days after cultures.

Similarly, the morphogenic callus formation

was reached almost 60% in CIM 1 medium

on 56 days after culture but the brownish color.

The application of cytokinins affected

callogenesis by resulting in decrease of cell

wall lignification, facilitating callus initiation

and growth in vitro. It has been observed

that callus proliferation usually started from

the cut surface of the explants and finally

covered the whole explants. At the beginning

of two weeks of incubation, the colors of calli

Figure 2. Percentage of callus formation through indirect somatic embryogenesis in Arabica coffee

AS2K clone on 14, 28, 42, and 56 days after culture in CIM (callus inducing medium)

114

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

were whitish, but some of them are creamish

(Figure 5A). At the end of culture on 56

days, some of the callus were still creamish

and later turned into brownish (Figure 5B).

After 56 days of incubation, the brownish calli

grew yellowish friable calli on the surrounding

callus (Figure 5C). However, some of the

callus are compact with hyperhydric look.

Calli with hyperhydric exudates induced

necrosis soon after. The growth of some

calli showed high lignification, including of

their hard texture, whereas others were

embryogenic and separated easily into small

fragments (Figure 5C). Similarly colored and

structured calli from leaf explants in C. arabica

have been reported by Bartos et al. (2018).

It was confirmed that the embryogenic calli

were yellowish and friable, with the potential

to become embryo somatic.

Figure 3. Characteristics of callus formation of Arabica coffee AS2K clone based on location

(whole leaf edge and certain part of the leaf) and type of callus growth (low, intermediate,

and high) on 14, 28, 42, and 56 days after culture in CIM (callus inducing medium)

Figure 4. Characteristics of callus formation of Arabica coffee AS2K clone based on the structure

(morphogenic and non-morphogenic) and color of callus (brownish, creamish, and

whitish) on 14, 28, 42, and 56 days after culture in CIM (callus inducing medium)

115

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

Callus Type Based on Morphology

and Histology

In our study, explants responded by

forming three types of callus which varied

in color, textures, and friability (Figure 5).

Morphologically, the calli were compact

cream-colored (Type I calli; Figure 5A) after

14 days of cultivation in callus inducing

medium, spongy brown structures (Type II

calli; Figure 5B) after 30 days of cultivation

in callus inducing medium, and friable white-

yellowish (Type III calli; Figures 5C) after

120 days of cultivation. Both Type I and

II calli possessed degenerated contracted

cells without a distinct nucleus or a dense

cytoplasmic region (Figure 5D and 5E).

In contrast, Type III of friable calli were

composed of small isodiametric cells with

a conspicuous nucleus and a dense cytoplasm

(Figure 5F). Similar observations of the cellu-

lar structures of histological analyses of

embryogenic callus in C. arabica have been

reported in Bartos et al. (2018).

Type I primary calli (Figure 5A) is mostly

made up of large parenchyma cells with

developed vacuoles, reduced intercellular

spaces, and relatively thick cell walls. Tores

et al. (2015) revealed the existence of parts

characterized by the appearance of large,

elongated cells and well-developed vacuoles

in histological changes of cell masses with

no embryogenic aspects, such as the primary

calli presented in this report. Morphological

observations also revealed that some of the cells

composed of the primary calli already exhibit

cell dedifferentiation and strong evidence of

mitosis during this stage of embryogenic

development (Figure 5D).

The majority of Type II primary calli

(Figure 5B) are elongated parenchyma cells

with large intercellular spaces, developed

vacuoles, and cell walls (Figure 5E). The

frequent cell multiplication process was also

noticeable in these cultivations, which explains

the extreme and irregular growth of these cell

masses. Similarly, Ardiyani (2015) identified

Figure 5. Three types of callus structures induced from leaves of C. arabica cultured on callus

inducing medium. Morphologically, compact cream-colored, Type I primary calli (A);

spongy brown structures, Type II primary calli (B); and friable white-yellowish, Type

III embryogenic calli (C). Histological section of cream compact structure calli (D); brown

spongy calli (E); and calli with embryogenic structure (F)

116

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

that most of the cells that make up these

propagules are parenchyma cells with lacking

nuclei and protoplasm, destroyed cell walls,

and a large amount of intercellular spacing

while examining the histologic characteris-

tics of non-embryogenic calli of Coffea liberica

with morphological characteristics.

The Type III embryogenic calli were shown

to be entirely composed of meristematic areas

surrounded by small cells with isodiametric

diameter, dense cytoplasm, visible nucleus,

and narrower cell walls (Figure 5C). We even

found cells with two nuclei in these propagules,

indicating incomplete cytokinesis in the cell

division process, along with cells at the onset

of linearization, indicating the beginning of

the organization involved in the somatic

embryo formation process (Figure. 5F). The

development of embryogenic calli clusters

in this species is composed of small isodia-

metric cells with dense cytoplasm and promi-

nent nuclei, and that somatic embryos are

produced through a series of organized cell

divisions. Ribas et al. (2011) observed, in

histological sections of yellowish embryo-

genic calli from somatic embryogenesis of

C. arabica variety Caturra, the proliferation

of a homogeneous meristematic region

mainly consisting of aggregates of small cells

with a high nuclear-cytoplasmic proportion,

dense central nucleus, and thicker cytoplasm,

as well as the development of small somatic

proembryos.

These findings support those of Torres

et al. (2015), who showed in the somatic

embryogenesis of young C. arabica leaves

of variety Catiguá that meristematic cells that

form the embryogenic calli, when associated

with optimal growth conditions, implement

a frequent cell division process, which over

period results in the production of embryo-

genic cell clusters that compose the somatic

embryos of the species.

C. arabica leaf segments of approxi-

mately 1 cm

were cultures in primary calli

induction medium for 30 days to induce

somatic embryogenesis. It was confirmed

at this stage of cultivation that calli forma-

tion began on the seventh day of culture

(Figure 6). Histological examinations of leaf

segments responsive to callus formation

revealed that the development of these calli

masses was strongly linked with the vascular

bundle in 100% of the evaluated propagules

(Figure 6D). It was identified that calli formation

primarily happened in the first place near

the edge of the excised leaves in these cultures

because these areas have stronger contact

of the vascular explant tissue with the ingre-

dients of the nutrient medium.

During this stages of C. arabica somatic

embryogenesis, we confirmed that the cell

divisions that resulted in the primary calli

began in regions of the vascular bundle where

small size cells with dense cytoplasm and

visible nuclei were observed (Figure 6A),

Figure 6. Histology of primary callus formation in C. arabica L. leaf segments; A.Explants at 7

days of culture, B. Explants at 14 days of culture C. Explants at 21 days of culture, D.

Explants at 30 days of culture; Abbreviations: ab = abaxial face, ad = adaxial face, c =

callus, pp = palisade parenchyma, sp = spongy parenchyma, ve = vessel element

117

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

i.e., from procambium cell division (Figure

6B-C-D). This finding suggests that using

leaves at very advanced stages of development

as an explants donor source for the induction

of indirect somatic embryogenesis may not

be worthwhile.

Cell proliferation resulted in a greater

leaf area during cultivation, which was directly

caused by the majority of meristematic divi-

sions, which enabled the development during

of differentiated cells. Moreover, as callogenesis

progressed, we noticed that the effect to which

the primary cells were separated from these

promeristematic regions started to increase

in size (Figure 6C-D).

As a result of their high pressure against

the epidermal cells surrounding the leaf, the

cells of the palisade parenchyma attached

to the primary calli started showing a signifi-

cant rise in width and a significant decrease

in height, demonstrating straightening mecha-

nisms. Nevertheless, it was not recognized

in the spongy parenchyma cells adjoining

the calli that persisted isodiametric even after

cultivation development (Figure B-C). In

contrast, we found no evidence of cell divi-

sion or cellular dedifferentiation in the paren-

chymal tissue at the start of the development

of primary calli. These findings support the

theory that procambium cells initiated

callogenesis in C. arabica leaf explants. The

first stages of somatic embryogenesis were

studied using leaf explants from eight different

varieties of C. arabica. According to Futura

et al. (2014), these findings are primarily due

to the procambium possibly being meristematic

tissue, which implies that its cells have a

higher mitotic potential, enhancing the forma-

tion of primary calli. Furthermore, it is prob-

ably that these findings are related to the

fact that the procambium is located close

to phloem, which consists plant growth

regulators and thus advantages callogenesis

induction. The cell divisions that stimulated

callus formation began in the perivascular

tissue of the rib, even if they could have

proceeded in the spongy parenchyma as well.

With this comprehensive knowledge, morpho-

logical markers for each stage of C. arabica

somatic embryogenesis can be developed.

As a consequence, the optimal duration for

culture transfer to the next step can be deter-

mined more easily and effectively, shortening

the time.

CONCLUSIONS

This research concluded that propaga-

tion of AS2K clone Arabica coffee through

indirect somatic embryogenesis technique was

successfully developed using leaf explants

inducing the formation of Type I primary

callus from procambium cell divisions. The

propagation step of coffee through indirect

somatic embryogenesis begins with callus

induction. The best combination medium for

inducing callus formation was 1 mg L

-1

2,4-D

and 1 mg L

-1

BAP (CIM 1). These propagules

differentiate into embryogenic calli, cell

masses made entirely of meristematic cells

after about 120 days of cultivation. The

combination of 2,4-D and BAP was more

effective composition in the callus forma-

tion of AS2K clone Arabica coffee than the

combination of thidiazuron and BAP.

ACKNOWLEDGEMENTS

We would like to thank the Ministry of

Research, Technology and Higher Education

of the Republic of Indonesia through PPD

UGM research (grant: 2232/UN1/DITLIT/

DIT-LIT/PT/2021) for financial support.

118

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Arimarsetiowati et al.

REFERENCES

Almeida, .A.S. & M.B. Silvarolla (2009). Induction

of somatic embryos of Coffea arabica

genotypes by 6-benzyladenine. Inter-

national Journal of Plant Develop-

mental Biology, 53, 5-8.

Ardiyani, F. (2015). Morphological characteriza-

tion and identification of Coffea liberica

callus of somatic embryogenesis propa-

gation. Pelita Perkebunan, 31(2), 81–89.

Arimarsetiowati, R. (2011). Pengaruh auksin 2,4-D

dan sitokinin 2-ip terhadap pembentukan

embriogenesis somatik langsung pada

eksplan daun Coffea arabica L. Pelita

Perkebunan, 27(2), 68–76.

Bartos, P.M.C.; H.T. Gomes; S.M. Gomes;

S.C.V. Filho; J.B. Teixeira & J.E.S Pereira

(2018). Histology of somatic embryo-

genesis in Coffea arabica L. Biologia,

73, 1255–1265.

de Almeida, J.A.S. (2020). Observations on

Somatic Embryogenesis in Coffea

arabica L. Coffee - Production and

Research. Dalyse Toledo Castanheira,

Intech Open.

Furuta, K.M.; E. Hellmann & Y. Helariutta

(2014). Molecular control of cell speci-

fication and cell differentiation during

procambial development. Annual

Review of Plant Biology, 65, 607–638.

Irene, W.M.; H.L. Alumiro, K.K. Asava;

C.O. Agwanda & S.E. Anami (2019).

Effects of Genotype and Plant Growth

Regulators on Callus Induction in Leaf

Cultures of Coffea arabica L. F1 Hybrid.

Journal of Plant Biochemistry &

Physiology, 7(2), 236.

Kahia, J.; M. Kirika; H. Lubabali & S. Mantel

(2016). High-frequency direct somatic

embryogenesis and plantlet regenera-

tion from leaves derived from in vitro-

germinated seedlings of a Coffea

arabica hybrid cultivar. Hortscience,

51, 1148–1152.

Kumar, S.; R. Singh; S. Kalia; S.K. Sharma &

R. Kalia (2016). Recent advances in

understanding the role of growth regu-

lators in plant growth and development

in vitro-I conventional growth regu-

lators. Indian Forester, 142, 459–470.

Landey, R.B.; A. Cenci; F. Georget; B. Bertrand;

G. Camayo; E. Dechamp; J. Simpson;

J.C. Herrera; S. Santoni; P. Lasherme

& H. Etienne (2013). Highgenetic and

epigenetic stability in Coffea arabica

plants derived fromembryogenic suspen-

sions and secondary embryogenesis as

eevealed by AFLP,MSAP and the phe-

notypic variation rate. PLoS One, 8(2)

e56372.

Loyola-Vargas, V.M. (2016). The history of

somatic embryogenesis. In: Somatic

Embryogenesis: Fundamental Aspects

and Applications. (V. M. Loyola-Vargas

& N. Ochoa-Alejo, Eds.), Springer Inter-

national Publishing AG. Switzerland.

Mayerni, R.; B. Satria; D.K. Wardhani &

S.R.O.S. Chan (2020). Effect of auxin

(2,4-D) and cytokinin (BAP) in callus

induction of local patchouli plants

(Pogostemon cablin Benth.). IOP Con-

ference Series: Earth Environmental

Science, 583 012003.

Méndez-Hernández, H.A.; M. Ledezma-Rodríguez;

R.N. Avilez-Montalvo; Y.L. Juarez-Gomez;

A. Skeete; J. Avilez-Montalvo; C. De-la-Peña

& V.M. Loyola-Vargas (2019). Signaling

overview of plant somatic embryogenesis.

Front Plant Science, 10, 77.

Molina, D.M.; M.E. Aponte; H. Cortina & G. Moreno

(2002). The effect of genotype and explant

age on somatic embryogenesis of coffee.

Plant Cell, Tissue and Organ Culture,

71, 117–123.

Raji, M.R.; M. Lotfi; B. Tohidfar Zahedi; A Carra;

L. Abbate & F. Carimi (2018). Somatic

embryogenesis of muskmelon (Cucumis

melo L.) and genetic stability assessment

of regenerants using flow cytometry and

ISSR markers. Protoplasma, 255, 873–883.

Ribas, A.F.; E. Dechamp; A. Champion; B. Bertrand;

M.C. Combes; J.L. Verdeil; F. Lapeyre;

P. Lashermes & H. Etienne (2011).

119

PELITA PERKEBUNAN, Volume 38, Number 2, August 2022 Edition

Establishment of an efficient primary callus induction for embryogenic potential of Coffea arabica L.

Agrobacterium mediated genetic trans-

formation of Coffea arabica (L.) is

greatly enhanced by using established

embryogenic callus cultures. BMC Plant

Biology, 11, 92.

Rojas-Lorz, L; G. Arrieta-Espinoza; M. Valdez-

Melara; L.F.P. Pereira & A. Gatica-Arias

(2019). Influence of silver nitrate on

somatic embryogenesis induction in

Arabica coffee (Coffea arabica L.).

Brazilian Archives of Biology and

Technology, 62: e19180228.

Ruzin, S.E. (1999). Plant Microtechnique and

Microscopy. Oxford University Press,

New York Oxford. pp. 322.

Salaün, C.; L. Lepiniec & B. Dubreucq (2021).

Genetic and molecular control of

somatic embryogenesis. Plants (Basel).

10 (7), 1467.

Sanglard, N.A.; P.M Amaral-Silva; M.C. Sattler;

S.C. de Oliveira; L.M. Cesário; A. Ferreira;

C.R. Carvalho & W.R. Clarindo (2019).

Indirect somatic embryogenesis in

Coffea with different ploidy levels: a

revisiting and updating study. Plant Cell,

Tissue and Organ Culture (PCTOC).

Silva, G.M.; A.C.F. Cruz; W.C. Otoni; T.N.S Pereira;

D.I. Rocha & M.L Silva (2015). His-

tochemical evaluation of induction of

somatic embryogenesis in Passiflora

edulis Sims (Passifloraceae). In Vitro

Cellular & Developmental Biology–

Plant, 51, 539–545.

Singh, R.; S.P. Kashyap; N. Kumari & M. Singh

(2016). Regeneration of soapnut tree

through somatic embryogenesis and

assessment of genetic fidelity through

ISSR and RAPD markers. Physiology

and Molecular. Biology of Plants, 22,

381–389.

Sujatha, M. & K. Visarada (2013). Biolistic DNA

delivery. pp. 27–44. In: Transformation of

Nuclear DNA in Meristematic and Embryo-

genic Tissues. (S. Sudowe & A.B. Reske-

Kunz, Eds.) Springer, New York.

Torres, L.F.; L.E.C. Diniz, K.G. do Livramento;

L.L. Freire & L.V. Paiva (2015). Gene

expression and morphological charac-

terization of cell suspensions of Coffea

arabica L. cv. Catiguá MG2 in different

cultivation stages. Acta Physiologiae

Plantarum, 37, 175.

van Boxtel, J. & M. Berthouly (1996). High

frequency somatic embryogenesis

from coffee leaves. Factors influencing

embryogenesis, and subsequent pro-

liferation and regeneration in liquid

medium. Plant Cell, Tissue and Organ

Culture, 44, 7–17.

Yi-Chieh, W.; L. Meng-Ze; H. Bin; C. Hsiao-Hang &

C. Jen-Tsung (2018). Thidiazuron enhanced

somatic embryogenesis from callus lines

of Arabica coffee and subsequent plant

regeneration. Acta Biologica Cracoviensia

Series Botanica, 60, 35–44.

**0**