Annals of Agricultural Science (2012) 57(2), 117–128

Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

Original Article

In vitro morpho-histological studies of newly

developed embryos from abnormal malformed embryos

of date palm cv. Gundila under desiccation effect

of polyethelyne glycol treatments

Maiad M. El Dawayati a,*, Ola H. Abd EL Bar b, Zeinab E. Zaid a,

Amal F.M. Zein El Dina

a The Central Laboratory of Date Palm Researches and Development, Agriculture Research Center, Cairo, Egyptb Agriculture Botany Department, Faculty of Agriculture, Ain Shams University, Egypt

Received 2 July 2012; accepted 20 July 2012

Available online 26 October 2012

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KEYWORDS

In vitro;

Abnormal embryo;

Date palm;

Desiccation;

Polyethelyne glycol;

Histology

Corresponding author.

-mail addresses: Maiada_dw

hussein@yahoo.com (O.H. A

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niversity.

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Abstract Substantial progress has been made toward micropropagation of date palm species by

somatic embryogenesis. Routine production faced disadvantage due to persisting problems with

the efficiency of embryo maturation and conversion. This was attributed to the formation of

somatic embryos with numerous morphological abnormalities. The present paper was conducted

to study the desiccation effect of poly ethylene glycol (PEG) in relation to embryo maturation,

and germination. Abnormal malformed embryo explants were cultured on germination medium

supplemented with different concentrations of PEG (15, 20 and 25 g l�1) for two periods (15,

30 days) as desiccation treatments. Clear significant changes showed de novo organs as newly direct

somatic embryos (secondary embryos). These embryos could be germinated with shoot formation

from all desiccated abnormal somatic embryo explants with PEG treatments. The addition of PEG

at 20 g l�1 to germination medium for 30 days gave the highest significant number of newly direct

somatic embryos comparing with other treatments. Finally secondary somatic embryos converted

to full plantlets. For the first time histological investigation was made to reveal anatomical devel-

opment of secondary embryos from abnormal malformed embryos under (PEG) treatments. The

first step showed numerous meristematic multicellular centers occurring before treatment with

il.com (M.M. El Dawayati),

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lty of Agriculture, Ain-Shams

g by Elsevier

e, Ain Shams University. Production and hosting by Elsevier B.V.

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Open access under CC BY-NC-ND license.

118 M.M. El Dawayati et al.

(PEG). These centers may serve as an inactive source of embryoids. Some morphological and his-

tological changes were recorded during (PEG) treatments and determined by special developmental

stages (2-celled, 4-celled, globular and bipolar embryos with normal structure) of new somatic

embryos of date palm. The presented data strongly supported that the osmotic stress caused by dif-

ferent concentrations of PEG activates the process of morpho-ontogenetic events to develop new

healthy somatic embryos.

ª 2012 Faculty of Agriculture, Ain Shams University. Production and hosting by Elsevier B.V.Open access under CC BY-NC-ND license.

Introduction

Somatic embryogenesis is the process by which somatic cells,under induction conditions, generate embryogenic cells, under-goes through a series of morphological and biochemical

changes that result in the formation of a somatic embryo.(Francisco et al., 2006) However, somatic embryogenesis isnowadays best known as a pathway to induce regeneration

from in vitro tissue cultures. Some problems have beenreported concerning the multiplication step of embryogeniccalli, among which are the lack of synchrony in embryo devel-opment and the risk of morphological abnormalities. Morpho-

logical alterations occurring in the course of somatic embryodevelopment, such as embryo fusion, lack of suitable apicalmeristem or loss of bipolarity, have been held responsible for

a poor yield of vital embryos in several species (Benelli et al.,2010). Also through the direct induction of somatic embryos(without callus formation) an abnormal somatic embryo for-

mation was observed in grapevine embryos (Bharathy andAgrawal, 2008). Since the earliest successful demonstrationin micropropagation of date palm (Phoenix dactylifera L.)

which is considered to be one of the oldest cultivatable cropsand important multipurpose tree significant progress has beenmade to improve plant regeneration through adventitiousorganogenesis and somatic embryogenesis. Several studies

were conducted to optimize the regeneration efficiency. Theproduction on a wide scale of normal somatic embryos isnot always possible for most date palm species, which the

development and germination of normal bipolar embryos fromembryogenic callus did not induce easily. Zaid (2003) deter-mined the percentage of abnormalities of date palm somatic

embryos, and described visually the abnormal structures of so-matic embryos which appeared among differentiated somaticembryo cultures of date palm as (multi-head, shrink-coat,muli-tails-abortion and malformed) and confirmed that these

abnormal structures of somatic embryos failed completely toconvert to plantlets. Also she found the relation betweenabnormality percentage and reculture number, Langhansova

et al. (2004) found in Panax ginseng many anomalous struc-tures especially in the torpedo stage. Also various malformedembryoids were observed by other authors without detailed

histological studies (Chang and Hsing, 1980; Choi, 1988).Desiccation was essential for promoting plantlet regenera-

tion following maturation with high osmoticum (Misra et al.,

1993). Improvement of embryo quality can be achieved underosmotic stress, which is an important factor for directing em-bryo development and maturation both in vivo and in vitro(Capuana and Debergh, 1997; Troch et al., 2009). There are a

number of reports in which desiccation tolerance is achievedto improve the maturation of somatic embryo development insome plant species by Abscisic acid (ABA), Polyethelyne glycol

(PEG), sorbitol and Jasmonic acid (Tissa et al., 1989; Walkerand Parrott, 2001; Nadina et al., 2001 and Langhansova et al.,

2004). PEGmolecules are too large tomove through the cell walland do not cause plasmolysis (Walker andParrott, 2001). Attreeet al. (1995) reported that non-plasomlyzing osmotica are more

effective in promoting somatic embryomaturation in some coni-fer species. The effect of PEG mimics the naturally occurringwater stress on seeds during the late stages ofmaturation.Waterstress caused by both of PEG and high concentrations ofABA is

essential for somatic embryo development to accumulate ofstorage compounds and inhibition of precocious germination(Stasolla and Yeung, 2003). PEG enhanced somatic embryo

maturation in several species, including P. glauca (Attreeet al., 1995), Abies numidica (Vookova and Kormut’ak, 2001)andAbies hybrids (Salaj and Salaj, 2003; Salaj et al., 2004). Fin-

kelstien andCrouch (1986) have shown that embryomaturationis frequently associated with a low osmotic potential in tissue ormedium surrounding the embryo. The importance of water rela-tions was proposed by Fischer et al. (1987), and has been sup-

ported by evidence from both embryo culture experiments (Xuet al., 1990). The anatomical investigation of the sections of so-matic embryos ofTilia cordata showed that some epidermal cells

were densely cytoplasmic with large nuclei a prominent featureof embryogenic cells (Sharp et al., 1980). Mitotic activity led tothe development of secondary embryos from these meristematic

cells. From this concept the aimof the present workwas to studythe relation of desiccation effect of poly ethylene glycol (PEG)on maturation, and germination of malformed embryos and

to reveal changes of these explants from undesirable structuresto regenerative sources by developing secondary embryos thatwere able to convert to complete healthy plantlets. The lattercan be transferred to green house. Furthermore a histological

analysis was made to reveal the morphological changes ofmalformed embryos during (PEG) treatment and to define the dif-ferent developmental stages of new somatic embryos of date palm.

Materials and methods

This study has been carried out at TheCentral Laboratory ofDatePalm Researches and Development, Agriculture Research Center(ARC),Giza, Egypt and themorphological and anatomical exam-

inations were achieved in The Plant Microtechnique Laboratory,Agriculture Botany Department, Faculty of Agriculture, AinShamsUniversity, during 2010–2012 onP. dactylifera cv.Gundila.

Explant material

Abnormal embryos were collected from new proliferated indi-

rect somatic embryos produced from the embryonic callusof date palm (P. dactylifera L.) Gundila cv. on germinationmedium. Embryonic calli were obtained from sterilized

In vitro morpho-histological studies of newly developed embryos 119

meristematic tissues of the shoot tip and its primordial leaves as

described by Tisserat (1981), Matter (1986), Eldawayti (2000),Zaid (2003) and Zein El Din (2010).

Experiamental design includes morphological and anatomicalstudy

Morphological studyAbnormal somatic embryo explants with malform shape asdescribed by (Zaid, 2003) were cultured on germination medium

supplemented with three concentrations of Poly Ethylene Glycol8000 (PEG) treatments (15, 20 and 25 g l�1) as desiccation agentfor a time period of 15 and 30 days. All explants from all different

PEG concentrations and the two studied periods were sub-cultured on germinationmedium to followup their recovery afterdifferent PEG desiccation treatments. Germination medium wascomposed ofMS basal nutrient medium and vitaminsMurashige

and Skoog (1962), 170 mg l�1 NaH2PO4, 200 mg l�1 glutamine,40 g l�1sucrose, 0.1 mg l�1 NAA and 0.05 mg l�1 BA medium

was solidified by adding 5.5 g l�1 agar. The pH of the medium

was adjusted to 5.7 ± 0.1 prior to agar addition. The mediumwas distributed into small culture jars 150 ml (40 ml/jar) beforeautoclavingat 120 �Cand15 Ibs/in2 for 20 min.Culture jarsweredivided into two groups for the time period of desiccation by dif-

ferent PEG tested concentrations (15 and 30 days) each group di-vided into three treatments of PEGdifferent concentrations, eachtreatment consists of three replicates and each replicate consists

of three cultures jars. Each culture jar contained three abnormalmalformed somatic embryo explants.Culture jarswere incubatedat 27 ± 2 �C with a 16/8 h photoperiod with 80 lmol m2 s�1

fluorescent lighthouse.Results were recorded according to the morphological

changes of abnormal malformed embryos as affected by the

desiccation period for (15 and 30 days) with PEG and differentconcentrations of PEG. The number of newly emerged directsomatic embryos (secondary embryos) and their germinationby emerging inducing shoots were also recorded. See diagram

of experimental design.

Fig. 1 Morphology of the malformed embryo showing disorga-

nized cellular mass (bar = 0.5 cm). 2–7 Anatomy of the mal-

formed embryo.

Fig. 2 Cross section of malformed embryo showing the undu-

lated margin, exodermal layers, parenchymatous cells and the

embedded vascular bundles (v.b) (bar = 100 lm).

Fig. 3 Cross section of the malformed embryo showing meri-

stematic nature of subepidermal layers (small cells with dense

contents). Larger and more vacuolated cells in the inner zone

(bar = 100 lm).

Fig. 4 Enlarged area from Fig. 3 showing proliferation of the

subepidermal cell layers of the malformed embryo (bar = 50 lm).

120 M.M. El Dawayati et al.

Histological study

Samples of the malformed embryo (the explant) were takenbefore and after the treatment of PEG at 15 and 30 days ofduration with the three different concentrations. All previoussamples were killed and fixed in FAA solution (Formaline,

acetic acid and 50% ethyl alcohol, 5:5:90 by volum) for 24 h.The schedule of the paraffin method as described by Johansen(1940) was followed. Serial transverse and longitudinal sec-

tions (6–8 lm) in thickness were made by LEICA rotarymicrotome model RM 2125 RTS and fixed on slides by meansof Haupts adhesive (Sass, 1951). The sections were stained

with a Safranin–Fastgreen combination, and then mountedin Canada balsam (Sass, 1951).

Anatomical examination and photomicrograph wereachieved using OPTICA binocular model SZM-2 and a LEI-CA light microscope research (microscope model DM 2500supplied with a digital camera).

Recovery of desiccated malformed embryo explants

All malformed embryo explants desiccated with different PEGtested concentrations for two time periods of treatment werereturned to culture on normal germination medium for two

Fig. 5 Cross section of themalformed embryo showing embedded

cellular protrusions varied in width, length and shape. Note the

procambium strand in these protrusions (arrows) bar = 100 lm.

Fig. 6 Enlarged area from Fig. 5 showing intense periclinal cell

divisions of the peripheral cell layers (bar = 50 lm).

Fig. 7 Enlarged from Fig. 5 (the oval area) showing the

meristematic centers occurred within the malformed embryo.

Note the thick walled fragmentation lines demarcating each center

(bar = 50 lm).

Fig. 8 Somatic embryogenesis of date palm section through

malformed embryo after treatment of PEG showing transverse

division in the cells of the meristematic centers to yield a two celled

embryo (TE). Further division yields a four celled embryo (FE)

(bar = 50 lm).

In vitro morpho-histological studies of newly developed embryos 121

subcultures after different PEG tested treatments to study fur-ther effects of PEG desiccation to indicate the ability of theseabnormal structures to regenerate after treatments by record-

ing the number of newly appeared secondary somatic embryoswith developing shoots.

Statistical analysis

The randomized factorial design was used and data were sub-jected to analysis of variance. Separation of means among

treatments was determined using L.S.D test at 5% accordingto Snedecor and Cochran (1972).

Results

Morphological study

Malformed embryo explants were cultured on germinationmedium supplemented with different concentrations of PEG

Fig. 9 Somatic embryogenesis of date palm Transverse section

of multicellular ovate to globular proembryo (bar = 50 lm).

Fig. 10 Somatic embryogenesis of date palm Early stage of the

bipolar proembryo which is characterized with high dense

cytoplasmic meristematic region and a less meristematic vacuo-

lated region (bar = 100 lm).

Fig. 11 Somatic embryogenesis of date palm Transverse section

of proembryo reveals its cylindrical shape. The arrow indicates a

procambial strand embedded in the center, most of this structure

represents the cotyledon while the embryo axis is not obvious

(bar = 100 lm).

Fig. 12 Somatic embryogenesis of date palm The embryo

continued to elongate particularly the cotyledone primordia (c)

(bar = 300 lm).

Fig. 13 An enlarged view from Fig. 12 (the rectangle shape)

showing the differentiation of both shoot (s.m) and root apices

(r.m) meristems as well as continuous procambium strands (p.s)

(bar = 100 lm).

122 M.M. El Dawayati et al.

8000 (15, 20 and 25 g l�1) for two time periods (15 and 30 days)as desiccation treatments. Clear significant changes showed denovo organs as new direct somatic embryos and germinated

embryos with shoot formation were emerged on the outersheath of all desiccated abnormal somatic embryo explantswith PEG treatments. We can summarize these significantchanges by our data.

The desiccation effect of PEG concentrations and time per-iod on newly direct somatic embryo number and germinationembryo number from malformed embryo explants of date

palm cv. Gundila are shown in Table 1.Table 1 indicates that the addition of PEG at 20 g l�1 to

germination medium gave the highest significant result by

inducing newly direct somatic embryo number (6.38), whereasthe lowest significant embryo number (2.49) was achieved by15 g l�1 PEG. On the other hand PEG with different concen-

trations added to germination medium did not exhibit signifi-cant differences among treatments on the new germinatedembryos. However, the effect of PEG concentrations at 15,20 and 25 g l�1 on germinated embryo number was (5.49,

Fig. 15 Germinated somatic embryos. Note the malformed

embryo transformed into brownish color mass (arrow).Fig. 14 Several developing somatic embryos (arrows) raised

from the malformed embryo.

In vitro morpho-histological studies of newly developed embryos 123

6.83 and 4.78) respectively. According to the effect of the pro-longed period of PEG desiccation treatments, data obviouslyshowed that malformed explants desiccated for 30 days with

Table 1 Effect of PEG concentrations and time periods on direct

malformed embryo explants of date palm cv. Gundila.

(A) PEG (g l�1) (B) Time period

Direct somatic embryos number

15 Day 30 Day Mean

15 1.88 3.10 2.49

20 5.88 6.88 6.38

25 1.88 4.33 3.10

Mean (B) 3.21 4.36

LSD0.05 A 1.22

B 0.99

AB NS

Table 2 Effect of PEG treatments on the secondary somatic embryo

two subcultures of date palm cv. Gundila (45 days for each one).

(AB) Mean PEG (g l�1) (C) Duration period

30 Day 15 Day

11.43 13.10 9.77

17.83 22.44 13.22

13.71 16.77 10.66

14.32a 17.43 11.21

12.83 12.33 13.33

16.16 16.00 16.33

12.49 13.33 11.66

13.82b 13.88 13.77

13.10b 16.99a 12.13c

15.65a 12.49b

ª Time period

30 Day 15 Day

12.71 11.55

19.22 14.77

15.05 11.16

LSD0.05, AB 1.33, AC 1.08, BC 1.33, ABC 1.88.

different PEG concentrations gave higher significant newly

direct somatic embryos and germinated embryos than mal-formed explants desiccated for 15 days with different PEGtested concentrations.

somatic embryo number and germinated embryo number from

Germinated embryos number

(A) 15 Day 30 Day Mean (A)

2.88 8.11 5.49

3.44 10.22 6.83

3.77 5.8 4.78

3.36 8.04

NS

2.22

NS

number from malformed embryo explants after the recovery for

(B) PEG (g l�1) (A) Subculture

15 S120

25

Mean (A)

15 S220

25

Mean (A)

Mean (B)

Mean (C)

(A) PEG (g l�1)

15

20

25

Table 3 Effect of PEG treatments on the number of developing shoots from malformed embryo explants after the recovery for two

subcultures of date palm cv. Gundila.

(AB) Mean (C) Duration period (B) PEG (g l�1) (A) Subculture

30 Day 15 Day

8.66 9.77 7.55 15 S18.66 9.77 7.55 15 S114.49 19.44 9.55 20

9.88 11.33 8.44 25

11.01b 13.51 8.51 15 Mean (A)

11.17 13.77 9.66 20 S216.90 21.10 12.77 25

13.32 16.66 9.99

13.99a 17.17 10.80 Mean (A)

11.60b 15.69a 10.18c Mean (B)

15.34a 9.65b Mean (C)

ª Time period PEG (g l�1)

30 Day 15 Day

11.77 8.60 15

20.27 11.16 20

13.99 9.21 25

LSD0.05, AB NS, AC 1.18, BC 1.44, ABC 2.04.

124 M.M. El Dawayati et al.

Histological study

Histological characterization of the malformed embryos before

being treated with PEG (Figs. 1–7).The malformed embryo is an abnormal somatic embryo

consisting of a disorganized cellular mass. Its outline has ribsand furrows with wavy and undulated margins. The mass sur-

face being rough and undulated consisted of 2–5 rows of elon-gated to isodiametric cells arranged in compact layers withoutintercellular spaces. These layers have a protective characteris-

tic to the presence of suberin in the walls (=exodermis) similarto the exodermal layers of the roots (Figs. 1 and 2).

Sometimes the surface layers were smooth and wavy, con-

sisted of two to many layers. Their cells are small, relativelyisodiametric, rich in cytoplasm, thin walled and minimal vacu-olated, a prominent feature of meristematic cells. These cell

layers frequently able to divide with periclinal and occasionallyanticlinal or oblique planes (Figs. 3 and 4).

Cell proliferation continued to increase the thickness andnumber of cell layers of the malformed embryo. Generally,

the overall cellular mass of the malformed embryo primarilyconsisted of parenchyma cells embedding the vascular strands.The parenchymatous cells have intercellular spaces (Fig. 2).

Successive cross sections in the malformed embryo revealedthe development of folded meristematic cellular masses ran-domly embedded in the external and internal areas. A general

mitotic activity was observed in all these folded masses. The in-tense periclinal cell divisions of the peripheral cell layers gaverise to bulging protrusions varied in width, length and shape

(Figs. 5–7). These highly mitotic areas were characterized byhaving dense staining. In addition to the above-mentionedprotrusions, there are some procambium strands that are dif-ferentiated in Fig. 5 (the arrows).

In some peripheral regions cell proliferation gave rise toseveral meristematic multicellular centers which measuredfrom 10 to 25 lm in diameter (Fig. 7). These centers have def-

inite fragmentation lines produced by thickened cell wall, each

cell had prominent centrally positioned nuclei and divided toproduce the bipolar embryos.

A sequence of embryogenesis could be traced from twocelled to the bipolar embryo after treatment with PEG (Figs.

7–13).No differences could be observed among the developmental

stages of the somatic embryogenesis in all treatments of PEG.

The earliest stages of embryogenesis could be identified bytransverse to oblique divisions in the cells of the meristematiccenters to give rise to two then four celled proembryo (Fig. 8).

Further divisions often lead to the formation of a multicellularovate or globular proembryo (Fig. 9). Successive cell divisionsleading to the development of a multicellualr bipolar structure(Fig. 10) which consisted of two regions, one is dense cytoplas-

mic meristematic region while the other is a more vacuolatedone. The following divisions of the multicellualr bipolarstructure took place in different directions and gave rise to a

cylindrical proembryo (Fig. 11). The embryo showed proceed-ing elongation and a distinct shoot apex was visible sur-rounded by developing cotyledon which formed before

elongation of embryo axis. Procambial differentiation was ob-served at this stage and no vascular connection existed betweenthe embryo and the explant tissue (Figs. 12–14).

The embryogenesis stages were characterized by asynchro-nous development in each treatment and between thetreatments.

The parenchyma cells in the middle and peripheral parts of

the malformed embryo were more loosely packed and hadmore intercellular spaces then transformed into shrinkagemass with brownish color (Fig. 15).

The recovery of desiccated malformed embryo explants

All malformed embryo explants desiccated with different PEGtested concentrations for two time periods of treatment werereturned to culture on normal germination medium for twosubcultures after different PEG tested treatments. The ability

Fig. 16 Recovery of desiccated malformed embryo after 30 days

duration of desiccation.

Fig. 17 Plantlets of date palm cv. Gundila after three subcul-

tures from culturing on 1.0 mg/l NAA.

In vitro morpho-histological studies of newly developed embryos 125

of these structures to regenerate by giving secondary somatic

embryos and developing shoots on the malformed embryo ex-plants is recorded in Tables 2 and 3 as follows

The effect of PEG desiccation treatments on the number ofsecondary somatic embryos from malformed embryo explants

of date palm cv. Gundila After recovery for two subcultures(45 days for each one).

Data in Table 2 reveal that all PEG different concentrations

had significant effect on the number of new secondary embryoformation arising from the malformed embryo explants whichcultured on normal germination medium for two subcultures.

Abnormal malformed embryos desiccated with PEG at20 g l�1 gave the highest secondary embryo formation (16.99)when cultured on normal germination medium for two subcul-tures after treatment. As revealed in data abnormal malformed

embryos desiccated with PEG at different tested concentra-tions, long period of PEG treatment (30 days) gave a highernumber of secondary embryos (15.65) than short period

(12.49) of PEG treatment (15 days). This effect was true undertwo sub-cultures (Table 3). Subculture number of the recoverymedium did not affect significantly the number of secondary

embryos produced from desiccated malformed embryoexplants with different PEG tested treatments when returnedto culture on normal germination medium after desiccation

treatments. All studied interactions among the effect of subcul-ture number, the PEG different concentrations and the timeperiod for desiccation gave significant effects on the numberof new secondary embryo formation on the malformed embryo

explants when cultured on normal germination medium forrecovery.

The effect of PEG desiccation treatments on the number of

developing shoots from malformed embryo explants of datepalm cv. Gundila after recovery for two subcultures.

Data presented in Table 3 and Fig. 16 reveal that different

PEG concentrations used for desiccating the abnormal mal-formed embryo explants had significant effect on the numberof developing shoots (produced from newly direct somatic

embryos), and these newly direct somatic embryos whencultured on normal germination medium for two subculturesafter desiccation treatments. It is clear from data that, thenumber of developing shoots on germination medium for

two subcultures recorded the lowest significant result as

(10.18) when PEG was used to desiccate abnormal malformed

embryos. Whereas, the highest significant result as (15.69) forthe number of developing shoots on germination medium wasobtained when malformed embryo explants desiccated withPEG at 20 g l�1. Obviously subculture number of the recovery

medium had a significant effect on the number of developingshoots produced from desiccated malformed embryo explantswith different PEG tested treatments when returning to culture

on normal germination medium as the second subculture washigher significantly as result (13.99) than the first subculture asresult (11.01) in producing a number of developing shoots

from desiccated malformed embryo explants with differentPEG tested treatments. Present data indicate that, the durationperiod (15 and 30 days) for desiccation with different PEG

tested concentrations effected significantly the number ofdeveloping shoots produced on desiccated malformed embryoswhen they return to culture on normal germination mediumfor two subcultures after treatments. Best results for a number

of developing shoots produced in the recovery medium wereobtained from those malformed embryo explants which desic-cated with different PEG concentrations for (30 days) than for

a time period of (15 days) of desiccation (15.34 and 9.65)respectively. All studied interactions among the effect of sub-culture number, the PEG different concentrations and the time

period for desiccation gave significant effects on the number ofdeveloping shoots produced on the malformed embryo ex-plants when cultured on normal germination medium forrecovery.

Discussion

The histological and morphological results presented in thisstudy showed that the desiccation of abnormal malformed so-matic embryos of date palm cv. Gundila by using different

PEG concentrations within two different time periods of treat-ment could modify the development of these undesirable struc-tures to be a useful source of morphotype multiplication in

somatic embryogenesis cultures. Morphological abnormalitiesare common in plant somatic embryogenesis and distinguishedfrom the others on the basis of their morphology and the inabil-ity to develop into plants Bharathy and Agrawal (2008), Kong

et al. (2012). The anatomical study revealed that the malformedembryo composed of disorganized cellular mass consistedprimarily parenchyma cells with embedding the vascular strands

126 M.M. El Dawayati et al.

(Figs. 1 and 2) and a complete absence of root or shoot meris-

tems. These observations were found to be in accordance withHaensch (2004) who confirmed by histological examination thatabnormal embryo structures lacked bipolarity. Moreover, thecomplete absence of root or shoot meristem in embryos may

be due to delayed establishment to polarity (Halperin, 1964).Zein El Din (2010) reported that the abnormal malformed so-matic embryo (structure) of date palm cv. Sakkoty contained

high levels of total reducing and non-reducing sugars, whichapproximately are similar to those found in the embryogeniccallus. Also, the same author found that the abnormal

malformed somatic embryo contains moderate levels of totalprotein and free amino acids when compared with the embryo-genic callus. Storage compounds are important markers of

physiological quality of somatic embryos and a failure to pro-duce these may affect their final developmental stages and theirconversion to plants (Feirer et al., 1989; Cailloux et al., 1996).Whereas, the level of the total phenols was recorded to be of

low level in this form of abnormal somatic embryo the functionof phenolic acids is incorporated into cell wall help-restrict cellexpansion, which is essential for cell division (Fry, 1986). This

pattern of analysis strongly supported the presented data whichshowed that there are two types of meristematic cells in the mal-formed embryo tissue. Type one: in some surface layers, the cells

exhibited common features characteristic of the meristematiccells (Figs. 3 and 4) and themitotic activity was generally intensein these surface layers (Fig. 6). Type two appears as several mer-istematic multicellualr centers, occur in the peripheral or/and

the entire malform tissues, and have definite fragmentation linesproduced by thickened cell wall (Figs. 5 and 7). Some studieshave demonstrated that malformation does not always inhibit

normal regeneration Bharathy and Agrawal (2008). The analy-sis of the sections of somatic embryos of T. cordata showed thatsome epidermal cells were densely cytoplasmic with large nuclei

a prominent feature of embryogenic cells (Sharp et al., 1980)Mitotic activity led to the development of secondary embryosfrom these meristematic cells. Bharathy and Agrawal (2008)

found that adventitious embryos developed on the hypocotylsat the root–shoot zone, cotyledon and sometimes from the entireembryo in grapevine somatic embryogenesis. Somatic embryosinduced mostly on abnormal embryos which were not capable

to further develop into plants. These embryos formed de novo,mostly without callus formation and could be separated easily.

The developmental structures of somatic embryogenesis

starting with the meristematic multicellular centers, two celled,four celled embryos, globular shaped and bipolar which arerecognized in Figs. 7–12 agree with the same structures

observed by Tisserat and Mason (1980). These finding bipolarembryos are more typically normal and similar to the zygoticembryo and defined shoot and root apical meristems and

produced single plants (Fig. 17).It could be possible that these meristematic multicellular

centers act as a source of embryoids to the initiation of second-ary embryogenesis in the malformed embryo tissues.

PEG may enhance the rate of embryo formation becausemeristematic cells may be more responsive to it (Xu et al.,1990).

Dehydration process in the culture medium is essential to in-duce and develop newmeristematic cells onmalformed embryos(Lecouteux et al., 1993) The desiccation tolerance is a feature of

somatic embryos that must be induced and therefore requires apretreatment with abscisic acid or other factors to achieve the

desired response The type of pretreatment, the duration of its

implementation and the status of embryo development are crit-ical factors. Also (Finkelstien and Crouch, 1986) have shownthat embryo maturation is frequently associated with a low os-motic potential in the tissue ormedium surrounding the embryo.

The importance of water relations was proposed by (Fischeret al., 1987) and has been supported by evidence from both em-bryo culture experiments. Stasolla et al. (2003) found that the

transcript levels of several antioxidant enzymes are higher insome stages of PEG-treated white spruce somatic embryosand also they found that the applications of PEG in the matura-

tion medium of embryonic callus of white spruce induced twomajor changes in the gene expression the first in immatureembryos and the second in fully developed embryos. Besides

elucidating the mechanism of action of PEG during somaticembryogenesis these findings may have important implicationsin the identification of target genes or metabolic products forimproving somatic embryo quality by PEG application in date.

Zein El Din (2010) found that increasing PEG concentration to15 g l�1 enhanced the percentage of normal individual and re-peated somatic embryo formation, embryo numbers and at

the same time decreased the percentage of abnormality in datepalm cv. Sakkoty somatic embryogenesis. From our obtaineddata, it could be observed that, inducing newly more developing

direct somatic embryos was the best by adding PEG at 20 g l�1

on abnormal malformed embryo structures. In this concern,(Stasolla andYeung, 2003) reported that the effect of PEGmim-ics the naturally occurring water stress on seeds during the late

stages of maturation. Therefore, water stress caused by PEGand increased concentrations of ABA are essential for somaticembryo development to the accumulation of storage com-

pounds and the inhibition of precocious germination (Dodemanet al., 1997). Furthermore they reported that a remarkablechange occurring during thematuration period is that the devel-

opment programme switches from pattern formation to storageproduct accumulation in order to prepare the young saprophytefor dormancy andpost embryonic development. The rate of syn-

thesis and deposition of storage proteins, lipids and starch in-creases and results in cell expansion in both cotyledons andaxis. An essential regulator of the process is ABA (Georgeet al., 2008).

PEG enhanced somatic embryo maturation in several spe-cies, including P. glauca (Attree et al., 1995),A. numidica (Voo-kova and Kormut’ak, 2001) and Abies hybrids (Salaj and

Salaj, 2003; Salaj et al., 2004). In this study the treatments ofPEG to desiccate the abnormal malformed embryos increasedtheir ability to multiplication as revealed in our data and par-

allel with other opinions as a nonplasmolyzing osmoticum,have become a routine method for stimulating embryo matura-tion (Attree and Fawke, 1993). The rate and degree of desicca-

tion of mature embryos are crucial to their subsequent abilityto germinate (Obendorf et al., 1998; Vıctor, 2001) Secondaryor recurrent embryogenesis, which is reported in at least 80species offers a great potential for in vitro production. These

secondary embryos in our study were converted into normalhealthy plantlets and it can be transferred to the green houseas confirmed with (Shi et al., 2009) who reported that frequent

abnormal somatic embryos such as fused cotyledons and/or analtered number of cotyledons were induced from young zygoticembryos in Cinnamomum camphora, while the plantlets derived

from these abnormal somatic embryos had normal appearancesimilar to zygotic embryos. In Quercus suber, both morpholog-

In vitro morpho-histological studies of newly developed embryos 127

ically normal and abnormal somatic embryos conversed into

plantlets with normal appearance.The results of the present study showed that the malformed

embryo explants which failed to form complete plantlet couldprovide a good material for the regeneration of somatic

embryogenesis by using PEG which increased the process ofmorpho-ontogenetic events of the developing somaticembryos.

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