Egyptian Journal of Biotechnology, 2008 Vol.

30: 47-56
http://esbiotech.org/index.php/egyptian-journal-of-biotechnology

EFFECT OF PLANT GROWTH REGULATORS AND

POLYETHYLENE GLYCOL ON MATURATION OF DATE
PALM SOMATIC EMBRYOS
BY
*Zeinab E. Zaid., **Adel A. Abul-Soad and *Rehab A. Sidky
FROM
*The Central Laboratory for Date Palm Researches and Development, Agriculture
Research Center, Egypt
** Department of Tropical Fruit, Horticulture Institute, Agriculture
Research Center, Egypt

ABSTRACT
This study aim to use plant growth regulators and PEG to enhance embryo
maturation and obtain a vigorous plantlets of Phoenix dactylifera L. cv. Malakaby.
Embryonic nodular callus 0.5 g were treated with1 µM ABA or JA with BAP or NAA
and PEG at 0 or 0.25%. Addition 1µm ABA into maturation medium was more
effective to produce somatic embryo numbers (11.60) compared to 1µm JA (6.11).
Addition PEG into maturation medium did not enhance significantly somatic embryo
number (10.33 and 7.38 respectively). The embryos length increased by addition ABA
into maturation medium compared to JA as well as, addition BA with ABA into
maturation medium encouraged embryos length. Osmolarity affected by PEG seemed
the main component required for protein synthesis. Furthermore, the plantlets which
treated with PEG treatments appeared vigor and healthy compared to the plantlets
which untreated with PEG.

INTRODUCTION
Somatic embryos are bipolar
structures similar to zygotic embryos.
They originate from somatic cells and
develop through the same ontogeny as
zygotic embryo (Senaratna, 1993).
Somatic embryos do not mature
completely and germinate precociously
(Gray Dj and Purolit, 1991). The
controls of the process of embryo
maturation are complex and not fully
understood. A number of factors, both
chemical and physical affect
maturation. These include several
growth polyethelenglycol and nutrients
(Bomnan, 1993). The effects of some
of these factors are not well
understood, but the cue for their
application has been taken from a
desire to mimic the changes that occur
during maturation of zygotic embryo
(Madakadze and Senaratna, 2000).
Tisserat, 1982 cultured embryonic
callus of date palm on media devoid of
hormones and found it to be
heterogenous in composition,
containing numerous asexual embryos
and plantlets in various stages of
development. Typically, only a few (5-
20) plantlets with distinct foliar leaf
and primary root are produced per
culture at the end of the 8 weeks
incubation period. Plantlets and
embryo production from callus was not
a synchronous process. Embryo
development and germination of date
palm form a continuous process with
no dormant phase. The plant growth
regulators was determining factor on
survival. JA reduced survival to the
minimal level, whereas ABA achieved
the highest survival rates,
presumptively by the better quality of
embryos induced by exposure to it
Nadina Nieves et al., 2001.Early
studies showed that exogenous JA or
its methylated form (MeJA) could
promote senescence and act as a
growth regulator. Subsequent research
revealed that JA specifically alters
gene expression and that wounding and
elicitors could cause JA/MeJA
accumulation in plants. These results
implied a role for Jasmonate in plant
defence that has recently been
confirmed (Creelman and Mullet,
1997). (Zaid et al., 2004) reported
that, addition of abscisisc acid to
differentiation medium date palm
improved embryos maturation and
repressed precocious germination. The
aim of this study was to investigate
interactions of abscisic acid (ABA),
jasmonic acid (JA), naphtalenacetic
acid (NAA), and benzylaminopurine
(BAP) with polyethelenglycol (PEG)
on maturation of date palm somatic
embryos cv. Malakaby.
MATERIALS AND METHODS
Plant materials
The experiments were carried out
by using embryonic callus which
obtained from shoot apex (1-2 cm in
length) of date palm genotype
Malakaby. Shoot apex were sliced
longitudinally into 4 pieces and then
cultured on Murashige and Skoog
(MS) basal nutrient medium (1962)
supplemented with 170 mg
NaH2Po4.2H2O; 200 mg glutamine; 40
mg adenine sulfate; 0.4 mg Thiamine
HCL, 3g activated charcool (AC), 30g
sucrose; 6g agar and 100 mg 2,4-
D+3mg 2ip /liter for 6 months as
described by Mater (1986).
Maturation of somatic embryos
To study the effect of plant growth
regulators and PEG on maturation of
somatic embryos of date palm cv.
Malakaby, approximately 0.5 g
embryonic nodular callus were treated
with 1 µM ABA or JA with 1 µM BAP
or NAA and PEG at 0 or 0.25%. Each
treatment included 3 replicates each
replicate included 3 small jars (150 ml)
and each jar contained one explant.
Culture jars were incubated in a
temperature-controlled room at 24±1
°C under16 h daily exposure to low
light intensity of 1000 lux illumination.
Data of a average number of
somatic embryos and average somatic
embryos length (cm) were calculated
after 6 weeks from culturing. Protein
analysis
Somatic embryos were harvested
into 1.5 ml microcentrifuge tubes
freeze-dried and stored at -70°C until
analysis. Each sample of embryos was
approximately 60 mg as dry weight.
Proteins were extracted from somatic
embryos by grinding 350 µl of 25 mM
potassium phosphate buffer containing
1.0 M sodium chloride, pH 7.0 in the
presence of protease inhibitors 1 mM
phenylmethylsulphonyl fluoride
(PMSF) and 10 µM leupeptin. The
samples were centrifuged at 14000 rpm
for 5 min and the supernatant was
collected. Protein quantification was
carried out using the bicinchoninic acid subjected to analysis of variance.
assay (BCA) (Simth et al., 1985) with Separation of means among treatments
bovine serum albumin as the standard. was determined using L.S.D test at 5%
according to Snedecor and Cochran
Conversion of somatic embryos
(1972).
After 6 weeks on embryo
maturation the pro embryo were
transferred into regeneration medium
RESULTS AND DISCUSSION
containing 0.1 mg/l NAA +200 mg/l
Glutamin+30 g/l sucrose (Mater, 1986) Addition of different
in order to determine conversion of combinations of plant growth
somatic embryo into plantlets. The data regulators and PEG to the culture
were calculated after 6 weeks. medium resulted in wide-range
responses of data showed in Table 1.
The randomized factorial
design was used and data were

Table (1): Effect of plant growth regulators and PEG on the number and
embryos length (mm) of date palm somatic embryos cv. Malakaby
Avr. number embryo Avr. embryo length (mm)
(A) (B) (C) g/l (C) g/l
1µm 1µm Mean Mean
0.0 2.5 0.0 2.5
(AB) (AB)
PEG PEG PEG PEG
Control(0.0) 13.66 7.00
0.0 20.66 12.00 16.33 3.66 8.00 5.83
ABA BA 11.00 8.66 9.83 8.66 8.33 8.49
NAA 9.66 7.66 8.66 5.33 7.33 6.33
Mean(A) 13.77 9.44 11.60 5.88 7.88 6.88
0.0 11.00 5.00 8.00 3.66 3.66 3.66
JA BA 5.00 5.00 5.00 3.66 5.00 4.33
NAA 4.66 6.00 5.33 2.00 3.33 2.66
Mean(A) 6.88 5.33 6.11 3.10 3.99 3.55
Mean(B) 12.16 7.41 6.99 4.74 6.41 4.49
Mean© 10.33 7.38 4.49 5.94

Addition 1µm ABA into stated that ABA plays an important

maturation medium was more effective role in both somatic and zygotic
to produce maximum number of embryos maturation, while Nadina
somatic embryos (11.60) compared to Nieves et al., 2001, reported that the
1µm JA (6.11). Dunstan et al., 1995 treatment with JA, only and in
combination with ABA was no embryo
survival was observed during
maturation stage. Moreover, inclusion
BA or NAA did not significantly
improve somatic embryos formation
during maturation stage. This result is
in line with Madakadze and Senaratna
(2000), they mentioned that BA and
NAA do not enhance ABA effects on
maturation frequency of geranium
somatic embryos. On the other hand,
addition PEG into maturation medium
did not enhance significantly somatic
embryo number (10.33 and 7.38
respectively). But all other treatments
which containing PEG formed globular
structures uncounted (data
unpublished). High molecular weight
osmoticum such as polyethylene glycol
(PEG) was successfully used for the
maturation in Picea glauca (Attree et
al., 1991) and Abies hybrids (Vookova
et al., 1997). Although in mentioned
studies a positive effect of PEG was
demonstrated, in pinus strobes PEG
treatments did not results in the
stimulation of somatic embryo
maturation and plantlet regeneration
(Terezia Salajova et al., 1999).
Regarding, the effect of plant
growth regulators and PEG on
embryos length of date palm
cv.Malakaby, data revealed that,
embryos length increased by addition
ABA into maturation medium
compared to JA and also, addition BA
with ABA into maturation medium
encouraged embryos length This result
agreed with Zaid et al., 2004 they
reported that 1.0 mg/l ABA +0.1 mg/l
BA was more effective to enhance the
embryo length during maturation stage
of date palm somatic embryogenesis.
On the other hand, it was clearly
from data presented in Table1. that the
highest significant value of embryos
length was 5.9mm when was added
PEG into maturation medium. While
the lowest significant value of embryos
length was observed when the explants
(embryogenic callus) was cultured on
free-PEG medium (4.4 mm).
Langhansova, et al., 2004 studied the
effect of PEG and ABA on somatic
embryogenesis and plantlet
regeneration, they stated that the
attempt to increase the quality of
somatic embryos by using the high
molecular mass osmoticum, PEG
4000,and ABA was accomplished by
insertion of a maturation phase of
culture between multiplication
(maintenance) and regeneration phases.
Also they reported that, the cultures
treated with PEG 4000 and ABA
exhibited better development. Similar
results were obtained for both PEG
concentration tested.
Protein analysis
Data presented in Figure1.
indicated that addition of ABA to
media containing PEG significantly
improved protein presence of BA or
NAA treatments compared to the
control and treatment without PEG.
When JA was included in the media all
treatments used significantly improved
protein content compared to the
control. The highest increase was
observed when JA alone was added to
the maturation media (Figure2.).
These results are in line with
Madakadze and Senaratna, 2000 they
decided that, ABA and NAA in
combination with JA improve protein
content of geranium somatic embryos.
Osmoticum effected by PEG seems the
main component required for protein
synthesis. In contrary, Nadina Nieves
et al., 2001, reported that JA treatment
during the induction-maturation period
caused a decrease in protein level.
 140

120

100
Protien cotent

80

60

40

20

0
Control ABA BA NAA
Treatments

control ABA ABA+PEG

Figure (1): Effect of plant growth regulators (ABA, BA and NAA ) and PEG
concentration supplemented in the maturation medium on the
protein content of data palm (cv. Malakaby) embryos.

180

160

140

120
Protien cotent

100

80

60

40

20

0
Control JA BA NAA
Treatments

control JA JA+PEG

Figure (2): Effect of plant growth regulators (JA, BA and NAA ) and PEG
concentration supplemented in the maturation medium on the
protein content of data palm (cv. Malakaby) embryos
Conversion of somatic embryos
The somatic embryos were
harvested from all treatments and were
cultured on regeneration medium
containing 0.1 mg/l NAA +200 mg/l
glutamin+30 g/l sucrose in order to
determine the conversion of somatic
embryos into distinct plantlets after 6
weeks.
All treatments used which
containing ABA significantly
improved conversion process
compared to the control whereas, all
treatments which containing JA did not
significantly improve conversion
compared to control (Table2.). Hence,
the average number of shoot was 49.77
in ABA treatments, 16.60 in JA
treatments and 44.33 in control
treatment. Gutmann et al., (1996),
indicated that somatic embryos
matured in the presence of ABA
exhibited normal histogensis, they
consisted of small cells with high
division activity leading to the
formation of normal mature embryos
capable of germination. Somatic
embryos developed without ABA
exhibited abnormal cell expansion and
germinated precociously. To avoid the
formation of abnormal shapes of date
palm somatic embryos (off type), was
added abscisic acid (ABA) onto
differentiation medium to produce
mature somatic embryos that were
visually normal and did not germinate
precociously (Zaid et al., 2004).

Table (2): Effect of plant growth regulators and PEG on conversion of date palm
somatic embryos cv. Malakaby.
Avr. shoot number Avr. shoot length (cm)
(A) (B) (C) g/l (C) g/l
1µm 1µm Mean Mean
0.0 2.5 0.0 2.5
(AB) (AB)
PEG PEG PEG PEG
Control(0.0) 44.33b 13.00b
0.0 51.33 46.66 48.99 8.33 15.00 11.66
ABA BA 53.66 49.00 51.33 15.33 17.66 16.49
NAA 46.66 51.33 48.99 17.00 20.33 18.66
Mean (A) 50.55 48.99 49.77a 13.55 17.66 15.60a
0.0 22.33 15.33 18.83 7.33 8.66 7.99
JA BA 15.33 15.33 15.33 7.00 10.33 8.66
NAA 12.66 18.66 15.66 8.66 15.66 12.16
Mean (A) 16.77 16.44 16.60c 7.66 11.55 9.60c
Mean (B) 33.91a 33.33a 32.32b 9.82c 12.57b 15.41a
Mean (C) 33.66a 32.71a 10.60b 14.60a
 1 2
Figure (3): Effect of ABA, BA and NAA combination with PEG on maturation
embryo. 1 = (ABA+NAA+PEG), 2 = (ABA+PEG), 3 =
(ABA+BA+PEG)
Moreover, the PEG treatments
were ineffective on average number of
shoot, whereas the data was recorded
no significantly differences between
PEG treatments (32.71) and free-PEG
treatments (33.66).
On the other hand, ABA
treatments enhanced the average shoot
length (15.6mm) compared to JA
treatments (9.6mm) or control
(13.00mm). As well as, addition NAA
with ABA encouraged shoot
elongation (15.41mm) compared to BA
(12.57mm) or ABA alone (9.82mm)
Figure3. These results are agreement
with Abul-Soad et al., 2006 they
reported that, the small embryos of
date palm after differentiation can
elongate considerably to reach 20 cm
3
after 18 weeks in culture onto the
elongation medium which composed of
MS basal salts supplemented with 0.1
mg l-1 NAA and 3.0 g l-1 AC. Finally, it
could be the best result for shoot
number was addition BA and ABA
without PEG however, the best results
for shoot length was addition NAA and
ABA with PEG. Furthermore, it found
that, the plantlets which treated with
PEG treatments appeared vigor and
healthy compared to the plantlets non
treated with PEG.
In conclusion, ABA is very
important to maturation stage of date
palm cv. Malakaby. While, JA is not
effectiv to maturation stage. BA and
NAA addition enhanced ABA effects
during convert the embryos to intact
plantlets. Osmoticum effects by PEG Creelman, R.A. and Nullent, J.E.
addition seemed the main component (1997): Biosynthesis and
required for protein synthesis action of jasmonates in plants
Annu. Rev. Plant physiol.
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Gutmann, M.; Von Aderkas, P.;
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Label, P. and Lelu, M.A.
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Attree, S.M.; Moore, D.; Sawhney, maturation of hybrid Iarch. J.
V.K. and Fowke, L.C. Exper. Bot. 47 (305): 1905-
(1991): Enhanced maturation 1917.
and desiccation tolerance of
Lai, F.M. (1994): Maturation and
white spruce [Picea glauca
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Depatment Crop Science,
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519-525.
Langhansova, L.; Konradova, H. and
Bomman, CH. (1993): Maturation of
Vanek, T. (2004):
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Polyethylene glycol and
Redenbaugh K (ed) Synseeds
abscisic acid imbrove
– Applications of Synthetic
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Ann Arbor: CRC-Press Boca
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F. and Coudret, A. (1997):
Effect of abscisic acid and
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Sci 124: 183-191.
Madakadze, R.M. and Senaratna, T.
(2000): Effect of growth
regulators on maturation of
geranium (Pelaragonium x
Hortorum) somatic embryos.
Plant Growth regulation 30:
55-60.
Mater, A.A. (1986): In vitro
propagation of (Phoenix
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4(2): 137-152.
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(1962): A revised medium
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tissue cultures. Physiol.
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N.M.; Olson, B.J. and
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 ‫‪ARABIC SUMMARY‬‬

‫تأثير منظمات ا نمو ا نباتية وا بو ى إيثيلين جلي ول على نضج اأجنة ا جسمية نخيل ا بلح‬

‫لسادة ا د اترة‬
‫‪1‬‬
‫زي ب ا سيد زايد‪ - 1‬عادل أحمد أبو ا سعود‪ - 2‬رحاب أحمد صدقي‬

‫مــــــــن‬
‫ا معمل ا مر زي إبحاث و تطوير خيل ا بلح – مر ز ا بحوث ا زراعية – مصر‪.‬‬ ‫‪1‬‬

‫قسم بحوث ا فا هة ااستوائية – معهد بحوث ا بساتين‪ -‬مر ز ا بحوث ا زراعية‪ -‬مصر‪.‬‬ ‫‪2‬‬

‫‪ -‬فثةا ين‬ ‫ا جسةمو‬ ‫اأبسيسةي ‪ -‬حمة‬ ‫ا لمات ا دا ة‪ :‬خيل ا بلح‪ -‬ا ضج‪ -‬شؤ اأج ةة ا جسةمية‪ -‬حمة‬
‫اسيت اسيد‪ -‬ب زيل ادي ين‪ -‬ا بو ى ايثيلين جلي ول‪.‬‬

‫تسعى هذ ا دراسة إ ى تحسين مرحلة ضج اأج ة ا جسمية خيل ا بلح ص ف ا ما ةابى وا حصةول علةى‬
‫بيتةةات قويةةة عةةن طريةةم اسةةتخدام م مةةات ا مةةو ا باتيةةة وا بةةو ى ايثيلةةين جلي ةةول‪ .‬عوملةةت ‪5.0‬جةةم مةةن ا ةةا‬
‫با تفاعةةل م ةع ب زيةةل ادي ةةين و فثةةا ين اسةةيت اسةةيد بتر يةةز ‪1‬‬ ‫ا جسةةمو‬ ‫اأبسيسةةي أو حم ة‬ ‫ا ج ي ةةى بحم ة‬
‫ا تفاعل مع ا بو ى ايثيلين جلي ول بتر يةز ‪ .%5.20‬وجةد أن إضةا ة ‪ 1‬مي رومةول مةن حمة‬ ‫مي رومول و ذ‬
‫اأبسيسةةي إ ةةى بيئةةة ا ضةةج ا ةةت مةةؤثرت جةةدا ةةى ا تةةاة عةةدد بيةةر مةةن اأج ةةة ا جسةةمية (‪ )11.15‬مقار ةةة مةةع‬
‫(‪ .)1.11‬إض ةةا ة ا ب ةةو ى ايثيل ةةين جلي ةةول إ ةةى بيئ ةةة ا ض ةةج ةةم يحس ةةن مع وي ةةا ع ةةدد اأج ةةة‬ ‫ا جس ةةمو‬ ‫حمة ة‬
‫اأبسيسةةي إ ةةى بيئةةة ا ضةةج‬ ‫(‪ 8.17 ,15.11‬علةةى ا ت ةوا ى)‪ .‬زاد طةةول اأج ةةة ا جسةةمية بواسةةطة إضةةا ة حم ة‬
‫ا جسمو ‪ .‬أ هرت ا د ارسةة أن تةيثير اأجهةاد بواسةطة ا بةو ى ايثيلةين جلي ةول يعتبةر ا مر ةب‬ ‫مقارة مع حم‬
‫وجد أن ا بيتات ا تى عوملت بواسطة معامات ا بةو ى‬ ‫ا رئيسى واأ ثر احتياجا ب اء ا بروتين‪ .‬عاوت على ذ‬
‫ايثيلين جلي ول أ هرت قوت وصحة مقارة با بيتات ا تى م تعامل‪.‬‬