Indian Journal of Experimental Biology

Vol. 51, September 2013, pp. 739-745

Somatic embryo-like structures of strawberry regenerated in vitro on media

supplemented with 2,4-D and BAP

Genesia F Omar1, Fouad H Mohamed

1, Klaus-Thomas Haensch

2*, Sawsan H Sarg

1 & Mohamed M Morsey

1

1Department of Horticulture, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt 2Department Plant Propagation, Leibniz-Institute of Vegetable and Ornamental Crops,

Kühnhäuser Straße 101, D-99090 Erfurt, Germany

Received 18 January 2013; revised 5 June 2013

Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of

strawberry (Fragaria × ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations

of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these

structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2 - 4 mg L-1 2,4-D in

combination with 0.5 - 1 mg L-1 BAP and 50 g L-1 sucrose. Histological examination of SELS revealed the absence of a root

pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture

conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of

strawberries via somatic embryogenesis is discussed.

Keywords: Auxin, Cytokinin, Fragaria × ananassa, Somatic embryogenesis

Strawberries (Fragaria × ananassa Duch) are one of

the most important fruits. In 2010 the worldwide

production of strawberries has been reported to be

around 4.3 million tonnes1. Such a huge production

is dependent upon efficient nursery production of

certified transplants. This in turn requires enough

in vitro disease-indexed plants from nuclear stocks

for further propagation in the nursery fields.

Micropropagation is a tool for the multiplication

of disease-free strawberry plants2,3

. Potentially

micropropagation of strawberry plants can be

carried out via somatic embryogenesis4-12

. Somatic

embryogenesis is a better alternative because of its

high propagation rates, the presence of both root

and shoot meristems in the same propagation unit and

the easy manageability for scale up as well as

other manipulations since embryos can be grown

freely floating in liquid cultures13

.

Plant growth regulators play a key role in the

induction of somatic embryogenesis14

. Raemakers

et al.15

estimated that in dicotyledonous species

among the different growth regulators applied for

somatic embryo induction, 2,4-dichlorophenoxyacetic

acid (2,4-D) has been the most commonly applied

auxin (49%) and 6-benzylaminopurine (BAP) the

most frequently used cytokinin (57%).

Auxins and cytokinins have also been used

to induce somatic embryogenesis in strawberry.

The auxins 2,4-D4,6,9-12

, 4-amino-3,5,6-trichloro-2-

pyridinecarboxylic acid (picloram)9, indole-3-butyric

acid (IBA)5,9

and α–naphthalene acetic acid

(NAA)4,6,9-11

alone or in combination with the

cytokinins BAP4-6,10,11

or thidiazuron (TDZ)12

have

been reported to induce embryogenesis.

All of the previous research dealing with

strawberry somatic embryogenesis included only

limited histological examination of the obtained

structures which were classified as somatic embryos.

However somatic embryos are defined as bipolar

structures, which do not have a vascular connection

with the mother tissue at any time of their life16

and develop through characteristic embryological

stages17

. None of the above mentioned reports

dealing with somatic embryogenesis in strawberry

presented unambiguous histological proof of somatic

embryogenesis. Pallavi et al.12

presented squash

preparations of callus with structures designated as

pro-embryos but the applied method was unsuitable

to show the undisturbed anatomy of these

early formations. Kordestani and Karami9 published

——————

*Correspondent author

Telephone: +49 (0) 36201-785 224

Fax: +49 (0) 36201-785 250

E-mail: haensch@erfurt.igzev.de

INDIAN J EXP BIOL, SEPTEMBER 2013

740

histological sections of structures which they

classified as globular somatic embryos but this

classification is not convincing because these

structures are broadly connected to the explant and

no information was given concerning the anatomy of

the more developed structures. The only histological

examination of a putative advanced stage of a somatic

embryo in strawberry is presented by Biswas et al.6

which shows anatomy of the structures classified as

early torpedo shaped embryos. Also this examination

represents no proof of somatic embryogenesis in

strawberry because the longitudinal section shows no

cotyledons and a root meristem consisting of small

cells with a dense cytoplasm and large nuclei.

The aim of the present study has been therefore to

check the embryonic nature of the somatic embryo-

like structures (SELS) that are regenerated in in vitro

cultures of strawberry on media supplemented with

different combinations of 2,4-D and BAP.

Materials and Methods Regeneration in vitro—Young leaves and petioles

from two strawberry cultivars namely Sweet Charlie

and Chandler (Fragaria × ananassa Duch), were

obtained from greenhouse-grown plants during

November. These were rinsed several times with

normal tap-water, surface sterilized in sodium

hypochlorite (14% active chlorine; VWR

International GmbH Deutschland) for 10 min,

followed by a treatment with 70% ethanol (VWR

International GmbH Deutschland) for 1 min and

rinsed three times in sterilized distilled water.

Leaf disks (5 × 5 mm) and petiole segments

(10 mm) were cultured on the surface of media

solidified with agar under sterile conditions in a

50 mL glass jar containing 10 mL medium. The basal

medium was MS medium18

with additional 500 mg L-1

casein hydrolysate. This medium was supplemented

with different concentrations and combinations of

2,4-D, BAP and sucrose. For each cultivar all possible

combinations of five concentrations (1.0, 2.0, 3.0,

4.0 and 5.0 mg L-1

) of 2,4-D, three concentrations of

BAP (0.5, 1.0 and 2.0 mg L-1

) and two levels of

sucrose (30 and 50 g L-1

) were prepared. Media were

solidified with 7.0 g L-1

agar and the pH was adjusted

to 5.7 before autoclaving (121 °C, 15 min).

Cultures were incubated at 25 °C in dark for two

weeks and then transferred under light (16/8 h

photoperiod at 69 µmol m-2

s-1

white fluorescent light)

at 18 °C for three weeks. After this time cultures were

transferred to MS medium without any plant growth

regulators with 30 g L-1

sucrose and grown for three

weeks (same light conditions as above and 25 °C

temperature). Three replicates were used per

treatment and each replicate comprised 10 glass jars.

Callus formation and regenerating SELS were

observed after the total time of eight weeks using

a stereo microscope.

Histological examination—Histological examination

was carried out following Haensch19

. Altogether eight

representative explants with regenerating SELS were

examined. The material was fixed using FAA, a

solution of 5.4 mL formalin (37%), 65.6 mL ethanol

(96%), 5 mL glacial acetic acid and 24 mL distilled

water20

. Parts with representative structures

were embedded in hydroxyethylmethacrylate (Histo-

technique-set Technovit 7100; Kulzer, Wehrheim,

Germany). The specimens were dehydrated in steps of

2 h through a graded series of ethanol (70, 90, 96 and

100%), then pre-infiltrated overnight with a mixture

of equal parts of 100% ethanol and Technovit

7100 base liquid and subsequently transferred into an

infiltration solution of 100 mL Technovit 7100 base

liquid and 1 g hardener I for 1 day. For proper

infiltration a vacuum was applied for 30 min at the

start of the last two processes. Explants were put in

Teflon molds with a mixture consisting of 15 parts

infiltration solution and one part hardener II and

polymerized for 1 h at room temperature and

afterwards for 6 h at 37 °C. The explants were then

mounted on block holders using Technovit 3040.

Afterwards they were cut into 6 µm slices at room

temperature. For this purpose a Jung CM1800

microtome with type 818 disposable microtome

blades was used (both from Leica Instruments,

Nussloch, Germany). Sections were stretched on the

surface of distilled water, then mounted on slides

and stained with 0.05% toluidine blue O (Serva,

Heidelberg, Germany) which was dissolved in

1% sodium tetraborate decahydrate buffer following

Hutchinson et al21

. The slices were then rinsed with

distilled water, dried and covered with Entellan

(Merck, Darmstadt, Germany) and a cover slip. This

technique stains the cytoplasm and unlignified cell

walls red and the lignified cell walls and DNA-

containing structures blue20

. Microscopic analysis was

performed using an Axio Imager A1 microscope

(Zeiss, Jena, Germany).

Statistical analysis—Quantitative data were analyzed

using analysis of variance (software STATISTICA 8,

OMAR et al.: SOMATIC EMBRYO LIKE STRUCTURES IN STRAWBERRY

741

StatSoft 2009), and the means were compared by

Least Significant Difference Test (LSD) at 5% level

of probability. Ordinal data were analyzed in the

same way following Thöni22

.

Results Regeneration in vitro—The culture of the leaf and

petiole segments of both the cultivars in all the

treatments resulted in formation and proliferation of

callus (Fig. 1 A-F). However, the callus was creamy,

yellowish or brownish and granular depending

on the hormone combination. The degree of callus

formation varied between slight and massive

(Table 1). There were many treatments reaching the

highest possible rating for callus production without

showing significant differences to each other. A

center within this experimental scheme with many of

such very suitable treatments was observed in both

explant types of cv. Sweet Charlie in the range of 3 –

4 mg L-1

2,4-D in combination with 0.5 – 1 mg L-1

BAP and 50 g L-1

sucrose. In cv. Chandler most

treatments with this highest possible rating were

found in leaf disk cultures in a range of treatments

with 1 - 2 mg L-1

2,4-D and 2 mg L-1

BAP or with 2 -

Fig. 1—Representative examples of callus formation and regeneration of somatic embryo-like structures (SELS) showing a high

variability in shape. Bar = 1 mm (A-F)

INDIAN J EXP BIOL, SEPTEMBER 2013

742

3 mg L-1

2,4-D and 0.5 mg L-1

BAP each combined

with 50 g L-1

sucrose.

Regenerating structures were observed on both the

explant types of both the cultivars. These structures

resembled somatic embryos (Fig. 1 A-F). They

were globular- to leaf-shaped or similar to shoots,

yellowish or greenish and sometimes covered with

hairs. However, a root pole was never visible. A clear

classification of these structures was not possible

without histological examination. These structures

were designated as “somatic embryo-like structures”

(SELS). This regeneration response was obtained with

a broad spectrum of different combinations of the

applied growth regulators and sucrose but, in contrast

to callus formation, regeneration of SELS did not

occur in all the treatments (Table 2). The SELS

induced on different combinations were similar in

external characters. The best response in terms of

regeneration was observed with petioles of cv. Sweet

Charlie and leaf disks of cv. Chandler on media with

2 - 4 mg L-1

2,4-D in combination with 0.5 - 1 mg L-1

BAP and 50 g L-1

sucrose and with leaf disks of cv.

Sweet Charlie on a medium with 3 mg L-1

2,4-D in

combination with 1 mg L-1

BAP and 50 g L-1

sucrose.

Histological examination—The light microscopic

examination of the histological sections revealed that

the regeneration of SELS occurred both when the

callus was slight as well as when callus formation was

much more extensive (Fig. 2 A, B). In case of slight

callus formation the regenerating structures originated

near the vasculature of the original explant (Fig. 2 A).

In case of extensive callus formation the origin of the

regenerating structures was difficult to determine

because some of the observed structures represent

transitions between callus and shoot like structures.

For example Fig. 2 B shows a remaining leaf disc

explant and on its right margin three structures. The

upper one is a callus clump showing irregular tissues.

The structure beneath looks on the whole like a very

malformed shoot, which is connected to the maternal

explant through vasculature (see this detail in Fig. 2 C)

and has meristem-like tissues and very callus-like

leafy substructures. The structure to the right hand

side with its cauliflower-like appearance and small

round formations on its surface represents a transition

between the two structures described above. Sections

of such malformed structures at different positions of

the explant show the regeneration of further structures

Table 1—Effects of plant growth regulators and sucrose on callus formation in leaf disk and petiole cultures of two cultivars of

Fragaria × ananassa

[Values are means]

Degree of callus formation

cv. Sweet Charlie cv. Chandler

Leaf disk Petiole Leaf disk Petiole

2,4-D

(mg L-1)

BAP

(mg L-1)

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

1.0 0.5 2.3ef 3.0cd 3.0cd 2.7de 3.0cd 1.3gh 1.0h 2.0efg

1.0 1.0 3.3bc 3.0cd 2.3ef 2.0f 2.7cde 3.0cd 2.0efg 2.0efg

1.0 2.0 3.0cd 2.7de 3.7ab 4.0a 3.3abc 4.0a 3.3abc 1.0h

2.0 0.5 3.0cd 3.0cd 3.0cd 3.7ab 3.0cd 4.0a 2.3def 3.0cd

2.0 1.0 3.0cd 2.0f 2.0f 3.3bc 3.3abc 3.0cd 3.0cd 3.3abc

2.0 2.0 3.0cd 2.0f 3.3bc 3.3bc 2.0efg 4.0a 2.3def 3.0cd

3.0 0.5 3.0cd 4.0a 3.0cd 4.0a 3.3abc 4.0a 3.0cd 2.3def

3.0 1.0 3.3bc 4.0a 2.0f 4.0a 3.7ab 3.0cd 1.0h 4.0a

3.0 2.0 2.0f 3.0cd 2.0f 3.7ab 4.0a 2.3def 2.0efg 3.0cd

4.0 0.5 4.0a 4.0a 3.7ab 4.0a 3.3abc 3.7ab 1.3gh 3.0cd

4.0 1.0 3.7ab 4.0a 4.0a 4.0a 3.0cd 3.0cd 2.0efg 3.0bcd

4.0 2.0 3.0cd 3.0cd 3.0cd 2.0f 3.3abc 3.0cd 2.0efg 3.0bcd

5.0 0.5 3.3bc 2.0f 4.0a 4.0a 2.0efg 4.0a 3.0bcd 1.0h

5.0 1.0 2.3ef 3.0cd 3.0cd 3.0cd 1.0h 4.0a 3.0bcd 1.0h

5.0 2.0 2.0f 3.0cd 2.0f 4.0a 1.0h 1.0h 2.0efg 1.7fgh

Means followed by the same letter are not significantly different within each cultivar based on LSD test at P=0.05. Degree of callus

formation: 1 = slight, 2 = minor moderate, 3 = moderate, 4 = massive]

OMAR et al.: SOMATIC EMBRYO LIKE STRUCTURES IN STRAWBERRY

743

on their surface with a globular shape (Fig. 2 D, H),

heart shape (Fig. 2 G) or longish and leafy shape (Fig.

2 G, H). All these structures were broadly connected

to the maternal explant. Fig. 2 E and F show that such

structures were linked to their tissue of origin by

vascular strands. None of the more advanced stages of

such structures showed a root pole. Thus the anatomical

studies show that the regenerating structures can best

described as SELS as arguably thought.

Discussion

Many reports have suggested that micropropagation

of strawberry can be carried out potentially via

somatic embryogenesis4-12

but none of these reports

have presented an unambiguous histological proof

of somatic embryogenesis. The aim of the present

examination has been therefore to check the embryonic

nature of somatic embryo-like structures (SELS)

formed on media supplemented with 2,4-D and BAP

because combinations of these growth regulators

have been used in different protocols for somatic

embryogenesis in strawberry4,6,10,11

. The applied

experimental scheme of different combinations of 2,4-D

and BAP was combined with two levels of sucrose to

consider the influence of sucrose concentration as

shown by Gerdakaneh et al.10

and two explant types.

Furthermore 500 mg L-1

casein hydrolysate was added

to the medium to meet the needs of the cultures for

different amino acids as reported by Gerdakaneh

et al11

. At the beginning the explants have been

cultivated in darkness like in the procedure of

Donnoli et al.5 and of Biswas et al.

6 to increase callus

formation and regeneration. This scheme was suitable to

allow the assessment of the regeneration response in a

broad range of treatments. A large number of SELS

were observed under different culture conditions (Table 2,

Fig. 1 A-F). The high level of the morphogenetic

response in both cultivars on media with 2 - 4 mg L-1

2,4-D in combination with 0.5 - 1 mg L-1

BAP and

50 g L-1

sucrose was similar to the results of Wang et

al.4 who determined a medium containing 5 mg L

-1

2,4-D combined with 0.5 mg L-1

BAP and 50 g L-1

sucrose as the most effective one. The formed

structures were similar to each other and resembled

those which were classified as somatic embryos by

Wang et al.4 and Biswas et al

6. In contrast to such a

classification the present examination of the regenerated

SELS confirmed histologically the macroscopic observation

that none of their more advanced stages showed a

root pole. This means that the bipolarity as the most

Table 2—Effects of plant growth regulators and sucrose on the regeneration of somatic embryo-like structures (SELS) from leaf disk

and petiole cultures of two cultivars of Fragaria × ananassa

[Values are means ± SD]

Number of SELS

cv. Sweet Charlie cv. Chandler

Leaf disk Petiole Leaf disk Petiole

2,4-D

(mg L1)

BAP

(mg L1)

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

30 g l-1

Sucrose

50 g l-1

Sucrose

1.0 0.5 16.3 ± 4.0f-k

10.7 ± 4.6i-n

6.3 ± 2.1m-p

0.0p 11.0 ± 4.6

fg 2.0 ± 2.0

j 0.0

j 0.0

j

1.0 1.0 0.0p 0.0

p 0.0

p 0.0

p 8.7 ± 4.9

ghi 0.0

j 0.0

j 0.0

j

1.0 2.0 1.3 ± 2.3p 5.7 ± 2.1

m-p 13.7 ± 2.5

g-l 22.3 ± 3.5

def 16.0 ± 5.3

ef 11.3 ± 1.5

fg 0.

0j 0.0

j

2.0 0.5 18.3 ± 5.5e-h

25.7 ± 4.0c-d

22.3 ± 1.5def

35.7 ± 3.1ab

10.3 ± 6.1fgh

35.7 ± 3.8a 0.0

j 0.0

j

2.0 1.0 14.7 ± 3.2g-k

9.3 ± 1.5k-o

14.0 ± 9.2g-l

11.7 ± 0.6h-m

13.7 ± 5.5efg

13.7 ± 2.1efg

11.0 ± 8.0fg

0.0j

2.0 2.0 3.3 ± 2.9nop

0.0p 5.3 ± 1.2

m-p 0.0

p 0.0

j 4.3 ± 2.5

hij 11.7 ± 1.2

fg 0.0

j

3.0 0.5 10.3 ± 4.0i-n

30.3 ± 2.5bc

15.0 ± 3.6g-k

36.0 ± 2.0ab

27.7 ± 2.5bc

26.7 ± 4.0bc

25.3 ± 2.5bcd

0.0j

3.0 1.0 14.7 ± 2.5g-k

34.7 ± 2.1ab

10.7 ± 2.5i-n

22.3 ± 4.0def

19.0 ± 6.1de

31.0 ± 2.0ab

3.7 ± 2.1ij 13.7 ± 1.5

efg

3.0 2.0 1.3 ± 2.3p 3.0 ± 2.6

op 10.0 ± 1.7

j-o 20.0 ± 7.2

d-g 0.0

j 11.3 ± 2.5

fg 0.0

j 8.3 ± 3.5

ghi

4.0 0.5 31.3 ± 4.7b-c

34.0 ± 1.0b 12.0 ± 1.7

h-m 31.0 ± 6.2

bc 23.3 ± 3.2

cd 35.7 ± 3.1

a 0.0

j 15.0 ± 3.0

efg

4.0 1.0 24.7 ± 2.5cde

16.0 ± 2.7f-k

17.7 ± 4.0e-i

41.0 ± 2.7a 19.3 ± 6.4

de 22.3 ± 6.7

cd 0.0

j 0.0

j

4.0 2.0 9.3 ± 2.9k-o

20.3 ± 3.2d-g

16.7 ± 4.9f-k

10.3 ± 2.5i-n

0.0j 15.7 ± 3.5

ef 0.0

j 0.0

j

5.0 0.5 19.3 ± 6.4d-g

0.0p 19.7 ± 6.8

d-g 7.0 ± 4.4

l-p 9.3 ± 4.0

f-i 28.7 ± 14.0

bc 0.0

j 0.0

j

5.0 1.0 1.3 ± 2.3p 6.0 ± 3.5

m-p 11.3 ± 5.7

h-m 1.3 ± 2.3

p 0.0

j 0.7 ± 1.1

j 0.0

j 0.0

j

5.0 2.0 6.0 ± 10.4m-p

10.7 ± 3.8i-n

17.0 ± 4.4f-j

0.0p 0.0

j 0.0

j 0.0

j 19.0 ± 6.1

de

Means followed by the same letter are not significantly different based on LSD test within each cultivar at P = 0.05]

INDIAN J EXP BIOL, SEPTEMBER 2013

744

Fig. 2—Histology of somatic embryo-like structures (SELS).[(A) Structure regenerating near the vasculature of the original explant.

(B) Structures representing transitions between callus and a shoot-like appearance. (C) Detail from Fig. 2B showing the vascular

connection to the maternal explant. (D-H) Sections of regenerating structures showing their globular shape (D, H), heart shape

(G) or longish and leafy shape (G, H), their broad connection to the maternal explant (G, H) and vascular strands from some of them into

the tissue of origin (E, F). Bar: 0.5 mm (A, B, E, F); 0.1 mm (C, D, G, H)]

OMAR et al.: SOMATIC EMBRYO LIKE STRUCTURES IN STRAWBERRY

745

important characteristic of an embryo could not be

proved and that therefore these SELS cannot be

classified as somatic embryos. These SELS have to be

regarded partially as malformed shoots because the

histology revealed a shoot-like structure (Fig. 2 A, B,

E, F) with meristematic areas (Fig. 2 G, H) and

partially very thick callus-like leaves (Fig. 2 B, E, F).

These shoot-like structures originate near the

vasculature of the original explant (Fig. 2 A) or

develop their own vasculature connection with the

tissue from which they originate (Fig. 2 C). Other

SELS have to be considered as malformed leaf-like

structures having a broad connection to the tissue of

origin but showing only restricted vascular strands

(Fig. 2 H). All these structures resemble somatic

embryos only externally. They are the result of the

two overlapping processes of callus formation and

regeneration as it can be seen from Fig. 2 B. This is

the reason for the malformations and the great

variability of structure shapes which can be observed.

Considering the results from this study as

well as those of previously published examinations

concerning somatic embryogenesis in strawberry

leads to the conclusion that until now there is no proof

for the regeneration of somatic embryos from media

which have been supplemented with combinations of

2,4-D and BAP in this species.

Acknowledgment This investigation was undertaken with the support

of the Channel System of the Arab Republic of Egypt,

the State of Brandenburg, the Free State of Thuringia

and the Federal Republic of Germany. Thanks are due

to Mrs. Barbara Weinlich for technical assistance.

References 1 FAOSTAT 2012, Worldwide Strawberry production 2010,

State on 22nd November 2012, http://faostat.fao.org/site/567/

DesktopDefault.aspx?PageID=567#ancor.

2 Boxus P, The production of strawberry plants by in vitro

micropropagation, J Hortic Sci Biotechnol, 49 (1974) 209.

3 Swartz H J, Galletta G J & Zimmermann R H, Field

performance and phenotypic stability of tissue culture-

propagated strawberries, J Am Soc Hortic Sci, 106 (1981) 667.

4 Wang D, Wergin W P & Zimmerman R Z, Somatic

embryogenesis and plant regeneration from immature

embryos of strawberry, Hort Science, 19 (1984) 71.

5 Donnoli R, Sunseri F, Martelli G & Greco I, Somatic

embryogenesis, plant regeneration and genetic

transformation in Fragaria spp. Acta Hortic, 560 (2001) 235.

6 Biswas M K, Islam R & Hossain M, Somatic embryogenesis

in strawberry (Fragaria sp.) through callus culture, Plant

Cell Tissue Organ Cult, 90 (2007) 49.

7 Husaini A M & Abdin M Z, Interactive effect of light,

temperature and TDZ on the regeneration potential of leaf

discs of Fragaria × ananassa Duch. In Vitro Cell Dev Biol:

Plant, 43 (2007) 576.

8 Husaini A M, Aquil S, Bhat M, Qadri T, Kamaluddin &

Abdin M Z, A high-efficiency direct somatic embryogenesis

system for strawberry (Fragaria × ananassa Duch.) cultivar

Chandler, J Crop Sci Biotechnol, 11 (2008) 107.

9 Kordestani G K & Karami O, Picloram-induced somatic

embryogenesis in leaves of strawberry (Fragaria ananassa

L.), Acta Biol Crac Ser Bot, 50 (2008) 69.

10 Gerdakaneh M, Mozafari A A, Khalighi A & Sioseh-mardah

A, The effects of carbohydrate source and concentration on

somatic embryogenesis of strawberry (Fragaria × ananassa

Duch.), Am-Eurasian J Agric Environ Sci, 6 (2009) 76.

11 Gerdakaneh M, Mozafari A A, Sioseh-mardah A & Sarabi B,

Effects of different amino acids on somatic embryogenesis of

strawberry (Fragaria × ananassa Duch.). Acta Physiol Plant,

33 (2011) 1847.

12 Pallavi C M; Rekha R & Neelambika T M, Indirect somatic

embryogenesis from petiole segment in strawberry cv. Sweet

Charlie, Indian J. Hortic, 68 (2011) 24.

13 Ammirato P V, Organizational events during somatic

embryogenesis, in Plant Tissue and Cell Culture, edited by

C E Green, D A Sommers, W P Hacket, & D D Biersboer

(Alan R Liss Inc, New York) 1987, 57.

14 Jimenez V M & Bangerth F, Endogenous hormone levels in

explants and in embryogenic and non-embryogenic cultures

of carrot, Physiol Plant, 111 (2001) 389.

15 Raemakers C J J M, Jacobsen E & Visser R G F, Secondary

somatic embryogenesis and applications in plant breeding,

Euphytica, 81 (1995) 93.

16 Haccius B, Question of unicellular origin of non-zygotic

embryos in callus cultures, Phytomorphology, 28 (1978) 74.

17 Williams EG & Maheswaran G, Somatic embryogenesis:

factors influencing coordinated behavior of cells as an

embryogenic group, Ann Bot, 57 (1986) 443.

18 Murashige T & Skoog F, A revised medium for rapid growth

and bio assays with tobacco tissue cultures, Physiol Plant,

15 (1962) 473.

19 Haensch K T, Morpho-histological study of somatic embryo-

like structures in hypocotyl cultures of Pelargonium x

hortorum Bailey, Plant Cell Rep, 22 (2004) 376.

20 Gerlach D, Botanische mikrotechnik (Georg Thieme,

Stuttgart New York) 1984, 32.

21 Hutchinson M J, KrishnaRaj S & Saxena P K, Morphological

and physiological changes during thidiazuron-induced

somatic embryogenesis in geranium (Pelargonium ×

hortorum Bailey) hypocotyl cultures, Int J Plant Sci, 157

(1996) 440.

22 Thöni H, Auswertung von Bonituren: ein empirischer

Methodenvergleich, EDV in Medizin und Biologie, 16

(1985) 108.