الفهرس 
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Abstract
S1lJMMlARY awJl OOl’«1JJSION
This investigation was carried out during three successive years of
.1991, 1992 and 1993, at Tissue Culture Laboratory of Horticulture
Department, Faculty of Agriculture, Moshtohor, Zagazig University. Various
date palm explants of Zaghloul cv. were the plant material used in this work to
study the following>
1- Phenolic compounds in four explants as affected by
antioxidant and charcoal inclusion:-
.,
Phenolic compounds were determined in, shoot tip, sub-shoot tip,
axillary bud and leaf primordium before and after soaking in antioxidant
solutions. Moreover, phenols level in both cultured explants and employed
medium itself in response to adding charcoal to nutrient media was
periodically detennincd 2 and 4 weeks from culturing.
2- Some factors affecting producing ability and
characteristics of callus from different explants :-
In this regard four main factors were included and some experiments
were conducted to study their influences as follows:-
2.1. Nutrient media constituents: ~
2.l.a. Auxins (source and rates):
In this connection five factorial experiments were done to evaluate the
interaction effect between three different kinds of Zaghloul date palm explants
i.e., shoot tip, sub-shoot tip and leaf primordium from one side and any of:-
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a)- three auxins i.e., ~ NAA and 2,4-D each at 10 mglliter, b)· two levels
of , IAA (2.0/4.0 mglliter) or c)- NAA level (5.0/10.0 mg/liter) from the other
side in Ill, znd and 3m.experiments, respectively. While the fourth and fifth
experiments were developed for studying the effect of different combinations
between two auxins namely 2,4-D (0.1 or 5.0 mglliter) and IAA (1.0,2.0,4.0
or 6.0 mglliter) on callus formation from shoot tip or sub-shoot tip explants in
former and latter experiments, respectively.
2.t.b. Effect of cytokinen ” Benzylamino purineIBAP”:
For this purpose an experiment was conducted to investigate two levels
of BAP (0.0 & 0.1 mglliter) as a component of nutrient culturing medium,
since both levels of cytokinen were combined with the above mentioned three
explants of Zaghloul cv. (shoot tip, sub-shoot tip and leaf primordium).
2.t.c. Effect of some complex additives:
Addition of coconut milk to nutrient medium [(0.0 and 5°,10 (v:v)] in
combination with three explants (shoot tip, sub-shoot tip and leaf primordium)
was studied. Besides, three other undefined complex matters namely malt
extract, casine hydrolesate and yeast extract at 150, 200 and 150 mg/liter,
respectively were also studied regarding their effect on producing callus from
four explants (shoot tip, sub-shoot tip, leaf primordium and axillary bud).
2.2. Effect of explant type :-
Three explant kinds of Zaghloul date palm cultivars namely shoot tip,
sub-shoot tip and leaf pmnordium were evaluated regarding characteristics and
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producing ability of callus. Beside segments of cotyledonary sheath as clonal
explant was also used for the same purpose.
2.3. Effect of explanting date:
Six excision dates of shoot tip explant at two months interval all the
year around i.e., (January, March, May, July, September and November) in
combination with 2 rates (0.1 or 5 mg/lite~) of 2,4-D were compared through
culturing on base medium containing four IAA levels i.e., 1,2,4, 6 mg/liter in
experiment 1, 2, 3 and 4, respectively.
2.4. Effect of some preculturing treatments:
Shoot tip segments were soaked for two hours just before culturing in
one of the following solutions:
a- Kinetin at (2, 4 or 8 mg/liter).
b- IAA at (2,4 or 8 mg/liter),
c- Vitamin mixture.
d- Coconut milk (100/0, v:v).
e- Kinetin + IAA each at 21.0 mglliter.
f- Vitamin mixture + IAA (2 mg) + kinetin 2 mg.
g- Distilled water (control). ..
3- Availability of establishlng somatic embryogenesis:
Establishment of somatic embryogenesis (embryogenic callus) from the
yellowish aggregated typ~ callus through culturing and alternate sub-culturing
on different nutrient medi~ was studied. Such employed media were charcoal
omission but including alrelative higher adenine sulfate level. However, both
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auxins and cytokinen were presented at lower rate or completely omitted.
Since. five nutrient media were prepared for this purpose i.e., ,. kinetin and
auxins omission base medium. 2- base medium supplemented with (kinetin +
malt extract + IAA at 0.5. 300 and 0.1 mg/liter, respectively). 3- kinetin
omission base mediwn supplemented with [ BAP (0.0, 0.1 and 0.5 mg) + NAA
+ malt extract at 0.1 and 300 mg/liter, respectively], 4- Base medium
supplemented with lAA + IBA each at 1.0 mg/lirer, and 5- medium of M &
S. salts f IAA + IBA each at 1.0 mg + vitamin mixture.
4- Histological examination :-
Developmental stages of callus initiated by shoot tip into somatic
embryogenic callus was histologically studied.
5- Maturation and development of embryogenic callus into
new plantlets:
In this connection, embryogenic callus (white creamy developed
colonies) were segmented and cultured onto two charcoal omitted media
containing relative higher level of both NaH2P04 and adenine sulfate but an
intermediate rate of kinetin! and auxins.
6- Direct Proliferatien :-
The possibility of both shoot tip and axillary bud as clonal explants,
beside pollen grains (androgenesis) to pro.liferate directly into new plantlets
through culturing each asj intact organ onto 5 various nutrient media was
studied. Variation in such employed media were mainly depended on either
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presence or rates of kinetin, BAP and auxins, beside other additives such as
NaH2P04 and malt extract.
Data obtained could be summarized as follows :-
1- Phenolic compounds :-
Different explants were greatly varied in their phenols content, where
both precultured shoot tip and leaf primordia showed the maximum level
especially free from. On the other hand, phenols level in all antioxidant tested
explants, particularly shoot tip were acutely decreased as compared to
untreated ones, however the decrease was more pronounced in free form of
phenols.
As for periodical changes of phenols in cultured shoot tip and axillary
bud (after 2 and 4 weeks) it was clear, that axillary bud contained higher level
~
than shoot tip, especially after 2 weeks, while the difference after 4 weeks was
obviously minimized. Charcoal was generally ineffective, except with free
form in shoot tip. Meanwhile, phenols content was increased in nutrient
media by time and charcoal increased it particularly when it was cultured by
axillary bud.
2- Factors affecting production, and characteristics of callus
initiated from clonal date palm explants of Zaghloul cv,
2.1. Nutrient media constituents :
2.1.a. Auxins (source and rate):
from the life factorial experiments devoted for this purpose, data
obtained could be concluded into the following topics:
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Specific effect on callusing measurements:
Production and type of callus, both were in closed relationship to
source and rate of auxin, where lAA and NAA were more effective than 2,4-D
as each was added at 10 mg/liter. The higher rate oflAA (4 mg) and NAA
(10 mg) was more desirable than their lower rates i.e., 2 and 5 mg, respectively
(experiment, 2 and 3). On the contrary the lower rate of2,4-D (0.1 mg/liter)
exhibited an abundance of callus with more aggregated type than higher rate (5
mg), however the differences were significant and more pronounced with
shoot tip than sub-shoot tip explants (experiment, 4 and 5).
With regard to both necrosis and browning, the response to auxin
treatments was of minor importance, since no considerable effect could be
noticed in relation to auxin source i.e., IAA,NAA and 2,4-D (experiment, I).
Beside, rates of investigated auxins, i.e. lAA (experiment, 2) NAA
(experiment, 3) and 2,4-D with shoot tip (experiment, 4) or sub-shoot tip
(experiment, 5) all were nearly the same in this concern.
Regarding specific effect of exp.lant kind, results revealed the
superiority of shoot tip as compared to either sub-shoot or leaf primordium
(experiment, 1,2, 3).
Interaction Effect :-
Data obtained revealed that the most preferable combinations for
enhancing callus production and improving its type (nodosity) were as
follows: a- IAA at 10 mg/lirer x shoot tip (experiment, I), b- Combinations
between shoot tip and IAA at 2 or 4 mglIiter (experiment, 2), c- Combinations
of (shoot tip x NAA at 110mgll) followed by (shoot tip x NAA at 5 mg/l),
(experiment, 3), d- Combination of2,4-D at 0.1 mg and lAA either at 1.0 or
~---~-- ~-- -- -.- -- ---
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6.0 mg/l for shoot tip explants (experiment, 4) and e· The combination of 2,4-
D at 5 mg x IAA at 4 mgfl for sub-shoot tip explant (experiment, 5).
2.l.b. Effect of cytokinen (BAP) In combination with 3 date palm
explants:
Specific effect of (BAP):
Obtained data revealed that inclusion of BAP at 0.1 mg/one liter of
nutrient media was of minor importance for both callus production and
necrosis occurrence, but it improved type of callus produced (more aggregated
structure).
Interaction effect (BAP x explant):
It could be concluded that the most desirable combination was that of
(BAP at 0.1 mgll x shoot tip ) where more abundant callus of the best type
(nodular) was related to such treatment.
~
2.l.c. Effect of some organic additives combined with different
explants:
This topic included two factorial experiments, the first dealing with
(coconut milk x 3 explants” shoot tip, sub-shoot tip and leaf primordium”)
while the second [other additives ”malt extract, casine hydrolsate and yeast
extract n x four explants II shoot tip, sub-shoot tip, axillary bud and leaf
primordium”].
---,------ ._- ._-- --------- ---_._~-- ----
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Specific effect of organic additives :-
from both experiments, data obtained revealed that :-
1- Coconut milk not only resulted in inducing more abundant callus with
higher granular texture, but also eliminated to some extent occurrence of
both browning and necrosis.
2- Malt extract was statistically more effective than two other additives i.e.,
casine hydrolysate and yeast extract for stimulating and improving callus
production and quality, respectively.
Interaction effect:
Data obtained declared that both combinations of (coconut milk at 5%
x shoot tip explant) and (malt extract at 150 mg x shoot tip explant) were the
most preferable ones for all investigated callusing measurements.
2.2. Effect of explant kind :
Data obtained proved the superiority of shoot tip explants over two
other ones (sub-shoot tip and leaf primordium) in stimulating callus production
of best type (more granular texture), but leaf primordium was the inferior in
this respect. Moreover, cotyledonary sheath explant produced smooth callus.
2.3. Effect of explanting Dates:
Data obtained from the conducted four experiments regarding effect of
combinations between 6 lexplanting dates and Z levels of 2,4-D could be
concluded as follows :
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Specific effect:
With regard to specific effect of explanting dates it is quite clear that
excising dates either at March or July were the superior. where both induced
significantly more abundant callus with the most aggregated callus as
compared to four other explanting dates, regardless of lAA level of nutrient
media employed for culturing shoot tip segments i.e., 1, 2.4 and 6 mglliter.
On the contrary explanting date in May ’was the inferior in this respect.
Generally, it could be safely concluded that excising shoot tip explant at
March and July ” especially later” were the most suitable dates while the
reverse was true in Mayas the three callus measurements i.e., produced
amount, type and necrosis were concerned.
As for the effect of 2,4-D rate, it could be noticed that the lower rate
(0.1 mg) was more effective than higher rate (5.0 mg), especially in cases
where culturing media contained 1 or 6 mg lAA per liter.
Interaction effect explanting date x 2,4-D rates :-
from data obtained it could be concluded that the interaction effect of
explanting date x 2,4-D level on the different callus measurements was greatly
influenced by the IAA content in employed culture media. Since, both
combinations between explanting dates at March or July and the lower rate of
2,4-D i.e., 0.1 mglliter were the most desirable ones when culturing was
carried out onto media contained 1 or 2 mg IANliter. While with media of 4
or 6 mg IANliter the combinations of explanting dates at March and July
reflected their superiority with production and type of callus not only when
combined with the lowe.•.rate of 2,4-D (0.1 mg) but also with its higher rate
(5.0 mg). Nevertheless, spreading necrosis was more pronounced in
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developed callus of excised shoot tip at May either combined with the lower
(0.1 mg) or the higher (5.0 mg) 2,4-D per liter. However, differences between
various combinations tended to be decreased with increasing rate of IAA
included in the employed nutrient media.
2.4. Effect of some preculturing treatments :-
Data obtained pointed out that all preculturing treatments (soaking for 2
h. in solution of kinetin, 1M, coconut milk, vitamin mixture, either solely or
some in combination) resulted in a considerable increase in callus production
and its granulation, except kinetin solutions. However, coconut milk 100/0,
followed by (kinetin + vitamin mixture + 1M) and lAA at 8 mglliter were the
superior for both production and granulation (type) of callus, besides soaking
in solution of vitamin mixture with produced mass of callus only.
Meanwhile, other pretreatments were of less importance as compared to
control (soaking in distilled water).
With respect to browning and necrosis both showed less response to
various pretreatments. However, they were wide-spread with soaking in
kinetin solution (4 mglliter), but most treatments showed a relative lower
(insignificant) decrease i.tl their occurrence below control.
3. Establishment of embryogenic callus :-
Various media employed for culturing and repeated reculture of
yellowish aggregated type callus initiated from either shoot tip or axillary bud
of Zaghloul cv. that aimed to establishing embryogenesis revealed the
following:
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1- Charcoal. kinetin and auxins omitted base media was capable and
enhanced callus development for establishing embryogenic callus.
2- Inclusion of kinetin. IAA and malt extract at (0.5.0.1 and 300 mgll) in
charcoal omission base medium resulted also in inducing embryogenic
callus, but extended the number of subcultures needed.
3- However. BAP at 0.1 mgll encouraged embryogenesis process over no
adding. but with 0.5 mg/l forced cultured callus to produce leaves only.
4- Base medium supplemented with IAA + IBA each at 1.0 mgll failed to
show embryogenesis, but stimulate cultured callus to grow and multiply
only.
5- The most preferable medium for establishment embryogenic callus was
that consisting ofM. & S. salts supplemented with IAA + IBA each at 1.0
mgll + vitamin mixture.
4. Histological examination :-
Histological examination throughout asexual embryogenic sequences in
callus tissue initiated from shoot tip segments, revealed the following
developmental stages and their own characteristics :-
1- White glabrous friable callus stage, (4 weeks from culturing origin
explant) shows jhe homogenous tissue with no differentiated
organs/structures.
2- Stage of yellowish aggregated type callus (8 weeks from culturing onto
high auxin media); Two types of tissues are noticed:- l~refers to
multitude compact clumps of smaller cells (known also as embryogenic
compact aggregate~), these clumps are interdispersed among 2nd type i.e.,
loose friable matrix tissue of larger sized cells .
.•
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3~ Dark yellow, large lobed callus stage (after some repeated subcultures
onto medium devoid of Icontaining low auxin). where spherical
proembryos of 2, 4, g ...etc. and multicelled globular proembryos could
be observed.
4~ Stage of dark brown callus with white creamy spots showing the ovate
and bipolar embryoids after further subculture onto medium devoid of I
contained low auxin, as well as cylindrical mature ernbryoids may be also
occurred.
5~ Germination of the somatic asexual embryoid takes place when
embryonic cotyledon starts to elongate and its haustorial end folds away
from medium surface.
5- Germination of new plantlets from embryogenic callus :-
Culturing and sub-culturing of white” creamy colonies onto medium (a)
which consisting of charcoal omitted base medium supplemented in
mglliter with 170 mg NaHzP04, 80 mg adenine sulfate and 6.0 mg IAA
enhanced germination of asexual embryoids into new planlets. Beside on
other sub-culture onto medium (b) resulted in further growth of such
developed plantlets.
6- Direct regeneration :-
6-1- Direct regeneration of clonal explants:-
Data obtained revealed that both shoot tip and axillary bud having the
potentiality to be proliferated directly after culturing onto favourable
media. Since, medium (c) reflected its superiority over the three other
investigated ones in tjhisrespect. While, culturing onto medium (a) did
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not succeed in inducing new plants through direct proliferation. On the
other hand number of regenerated/proliferated new plants per an intact
cultured shoot tip or axillary bud varied obviously from one explant to
anther. Hence, only one vigorous plant was induced per an intact
cultured axillary bud, while with shoot tip the initiated plants were
usually 2 or more of less vigorous.
6-2- Androgenesis ”Tissue cultured pollen grains”:-
Culturing pollen grains on kinetin omitted base medium + 2 ip 3mg +
NAA 100 mg + NaH2P04 170 mg per liter. Scarcely induced new plant
not capable to survive.