In vitro Phoenix dactylifera

Journal of Applied Sciences Research, 3(9): 859-866, 2007
© 2007, INSInet Publication
Application of Cryopreservation Technique for In vitro Grown
Date Palm (Phoenix dactylifera L.)Cultures
Bekheet S.A., Taha, H.S., Saker, M.M. and Solliman, M.E.
Department of Plant Biotechnology, National Research Centre, Dokki - 12622, Cairo, Egypt.
Abstrct: A very simple cryopreservation method with potential application to a wide range of date palm
cultivars is described. Undifferentiated tissue cultures were successfully cryopreserved by freezing methods
subsequently regenerated plantlets. Nodular cultures were initiated by culturing of shoot tip explants (excised
from offshoots) on MS-medium contained 10 mg/l dichlorophenoxyacetic acid (2,4-D) + 3mg/l
dimethylaminopurine (2iP). The potential of dehydration caused by air drying to cryopreservation of date
palm tissue cultures through direct immersion in liquid nitrogen was subjected. Cultures of about 65% water
content resulted from 20 min air drying period registered highest percentage of survival and in vitro
conversion to plantlets. Among different types of sugars (fructose, glucose, sorbitol and sucrose) used as
osmotic agents in preculture medium, sucrose was the best for the survival of cryopreserved date palm tissue
cultures. The highest percentage of survival (80 %) and conversion to plantlets (75%) were observed with
1 M sucrose. To determine the potential of vitrification on freezing tolerance, cultures were exposed to a
vitrification solution for 20-100 min. The maximum rate of survival was obtained with cultures exposed for
80 min at 0 <C followed by 40 min at 25<C. Random Amplified Polymorphic DNA (RAPD) technique has
been used to study the genetic stability of cryopreserved tissue cultures of date palm. According to RAPD
analysis, plantlets derived from cryopreserved cultures were identical to that derived from nontreated cultures
and both were similar with the field grown plants. Finally, complete plantlets from the cryostored cultures
were successfully adapted to free living conditions after phase of acclimatization procedures.
Key words: Date palm, cryopreservation, vitrification, RAPD analysis.
space and mantenance [24]. One of the principle long-term
in vitro conservation methods is cryostorage.
Cryopreservation is generally understood as storage
between -79 and -196<C, the low extreme being the
temperature of liquid nitrogen. The major advantage of
plant materials at such temperature is that both
metabolic process and biological deterioation are
considerably slowed or even halted [11]. In addition, it is
believed that cryopreserved materials remains genetically
stable, thus affording on advantage over conventional
conservation methods [30,31]. Successful cryopreservation
requires the optimization of numerous variables
including the size of specimen, the correct type and
concentration of cryoprotectant, sample water content
and rate of freezing and thawing. In the concern of
using of cryostorage methods, the survival after
cryopreservation of plant tissues can be increased
by pretreatments such
cold
acclimation [15,21], air
[28]
drying and preculture on a medium supplemented with
abscisic acid [6] or Me 2SO [12,14]. In this respect, studies
INTRODUCTION
Plant germplasm preservation is an integral part of
any plant breeding program. The most efficient and
economical way of germplasm storage is in the form of
seeds. However, this kind of storage is not always
feasible because: 1)some plants do not produce seeds
and thus they are only propagated vegetatively, 2)seeds
remain viable only for a limited duration, 3) some seeds
are very heterozygous and, therefore, not suitable for
maintaing true-to-type genotypes and, 4) seeds
of certain species deterioate rapidly due to seed-born
pathogens.
Plant cells being inherently totipotent, tissue culture
techniques in conjunction with careful manipulation of
cryobiological methods could be profitably used for the
storage and preservation of important and recalcitrant
plant germplasm. Preservation of plant cells, meristems
and somatic embryos has become an important tool for
the long-term storage of plant species using minimum of
Corresponding Author: Dr. H.S. Taha, Plant Biotechnology Dept., National Research Centre, Dokki, Cairo,
E-mail:[email protected]
859
J. Appl. Sci. Res., 3(9): 859-866, 2007
on cryopreservation of asparagus [28] carrot [7] and
date palm [1] tissues reveal that survival rates after
cryopreservation could be increased by preculturing the
tissues on media that contained high concentrations of
sugar, instead of other cryoprotection procedures.
Moreover, vitrification procedure would eliminate the
need for controlled slow freezing and permit tissues to
be cryopreserved by direct transfer to liquid nitrogen [13].
As the dioecious plant, date palm is generally
propagated vegetatively by offshoots. Thus, its
germplasm cannot be effectively stored in the long-term
using conventional means. In early report [3], developed
a protocol for in vitro medium-term storage of date palm
through slow growth technique. T he present work aims
to develop an effective system for cryopreservation of
tissue cultures of date palm by investigate the role of
dehydration, sugar-rich medium and vitrification as
c ryo p ro te cta nt p re-treatm e nts o n su rv ia vl o f
cryopreserved tissue cultures.
(determined by drying samples at 70<C for 48 hr)
expressed as percentage of fresh weight. The cultures
were then tranferred to cryotubes and prepared for the
storage procedures. To dtermine the role of high
concentration of sugars in decreasing of freezing injury,
fructose, glucose, sorbitol and sucrose in two levels i.e.
0.5 and 1.0 M were added seperatly to preculture
medium. After one week of sugar-treatments, the healthy
cultures of date palm were taken for cryostorage. In the
vitrificatin experiment, segments of nodular cultures of
date palm were treated with vitrification solution
described by Uragami et al.[27]. This solution contained
22 % (w/v) glycerol, 15 % (w/v) ethylene glycol, 15 %
(w/v) propylene glycol and 7 % (w/v) dimethyl
sulfoxide (DMSO) in MS-medium containing 0.5 M
sorbitol and is adjusted to pH 5.8. After 48 hr, the
cultures were transferred into cryotubes contained
vitrification solution and held at 25 or 0<C for 1 hr
before plunged into liquid nitrogen.
M ATERIALS AND M ETHODS
Freezing Procedures and Recovery: The crytubes
(contained date palm cultures) were kept at 0 <C for 2
hr and then they were plunged directly into liquid
nitrogen (-196<C) for 48 hr. For recovery, the cultures
were rapidly thawed in warm water bath (37<C) and then
inoculated on regrowth medium. Four weeks after
inoculation, survival was evaluated by examining the
colors of cultures: the green ones (with increase of the
volume) had survived; the brown ones had died. Also,
the conversion to differentiated cultures was observed.
Induction of Aseptic Nodular Cultures: Date palm
(Phoenix dactylifera L.cv. Zaghlool) offshoots detached
from adult female plants were used as a plant material.
The outer leaves were removed and the tips were kept
in an anti-oxidant solution (citric acid of 150 mg/l
concentration). Shoot apices were then sterilized with
70 % ethanol for 1 min followed by immersion for 20
min in a 2.6 % sodium hypochlorite and throughly
washed with sterile distilled water. External leaves were
removed and shoot tips 1 cm in length were excised
with a small part of submeristematic tissues and then
cultured on Murashige and Skoog [18], (MS)medium
supplemented with 100 mg/l myo-inositol, 40 mg/l
adenine sulfate, 170 mg/l KH 2PO 4 and different
combinations of growth regulators. Cultures were then
incubated in darkness at 25<C and sub cultured every
five weeks. After three subcultures, nodular culture
frequency, culture fresh weights, the percentage of
differentiated cultures were recorded.
Randomly Amplified Polymorphic Dna (Rapd)
Analysis: DNA isolation was performed using the Cetyl
Trimethyl Ammonium Bromide (CTAB) method
described by Doyle and Doyle [9]. Half gram of fresh
sample was ground to powder in liquid nitrogen with a
prechilled pestle and mortar, suspended in 5 ml
preheated CTAB buffer, and incubated at 65°C for 1 hr
with occasional shaking. The suspension was then mixed
with 1/3 volume of chloroform, mixed gently,
centrifuged and the upper phase was transferred to a
new sterilized tube. Extraction was repeated with an
equal volume of chloroform. The aqueous layer was
transferred to a new tube, 2/3 volume of isopropanol
was added and nucleic acids were either spooled using
a Pasteur pipette or sedimentated by centrifugation. The
pellet was washed carefully twice with 70% ethanol,
dried at room temperature and resuspended in 0.5 ml TE
buffer. The enzyme, RNAse A (20ìg ) was added to the
resuspended mixture to dugest any contaminating of
RNA and the tube was incubated at 37 <C for 30 min.
To remove the enzyme and other contaminating protein,
Cryopreservation: Small pieces (1 cm³ size) of the
nodular cultures of date palm were aseptically taken and
used for cryopreservation experiments. The potential of
dehydration of cultures caused by air drying,
preculturing on sugar-rich medium and vitrification were
evaluated for the very low temperature storage of date
palm tissue cultures. For air drying treatment, the
cultures were placed on a dry sterile filter paper and
exposed to the air flow of the laminar air-flow cabinet
for 10-50 minutes. W ater content of the cultures
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J. Appl. Sci. Res., 3(9): 859-866, 2007
phenol/chloroform extraction was performed.
The polymerase chain reaction (PCR) mixture (25
µl) consisted of 0.8 units of Taq DNA polymerase, 25
pmol dNTPs, and 25 pmol of random primer, and 50 ng
of genomic DNA. The reaction mixture was placed on
a DNA thermal cycler. The PCR programme included an
initial denaturation step at 94°C for 2 mins followed by
45 cycles with 94°C for 1 min for DNA denaturation.
Annealing as mentioned with each primer, extension at
72°C for 30 seconds and final extension at 72 °C for 10
minutes
were carried out. The amplified DNA
fragments were separated on 2% agarose gel and
stained with ethidium bromide. Four 10-mer primers
(Operon technologies Inc., Alameda, California)
randomly selected were used in RAPD analysis (Table
1). A 100 bp DNA ladder (Promega) was used as a
marker with molecular size of 1000, 900, 800, 700, 600,
500, 400, 300, 200 and 100 bp. The amplified pattern
was visualized on a U V transilluminator and
photographed.
and rooted in vitro using medium contained 1 mg /l
indole-3-acetic acid (IAA) and 0.2% activated charcoal.
Healthy plantlets were washed with tap water and
disinfected by soaking in benlate solution (1g/l ) for 20
min. Then, the obtained plantlets were transplanted into
plastic pots contained peatmoss and vermiculite (1:1).
The pots were covered with clear polyethylene bags
which were sprayed with water to maintain a high
relative humidity. Gradually, humidity was reduced
and covers were completely removed within four
weeks of transplanting.
Culture Conditions and Statistical Analysis: Tissue
culture media were solidfied with 0.7 % agar and
adjusted to pH 5.8 before autoclaving at 121<C and
1.5 Ib/M² for 25 min. Cultures were normally incubated
at 25 <C and 16 hr photoperiod. Each experiment was
setup as a separate completely randomized design with
20 replicates / treatment. Data were statistically analyzed
using standard error (SE) according to the method
described by Snedecor and Cochran.
Table 1:
Primer
K1
K2
K4
K5
RESULTS AND DISCUSSIONS
Morphogenesis of date palm tissue cultures. The
effect of various combinations of phytohormones added
to culture medium on differentiation of date palm tisuue
cultures are summarized in Table (2). The results reveal
that morphogenetic responses vary depending on type
and concentration of the used phytohormones.
Primers used and their annealing temperature.
Sequence 5’- 3’
Annealing Tm °C / Sec
TGGCGACCTG
36
GAGGCGTCGC
TCGTTCCGCC
CACCTTTCCC
The auxin i.e., 2, 4-D was more effective on in
vitro morhogenesis of date palm copmared with
naphthaleneacetic acid (NAA). Also, the cytokinin i.e.,
2iP in combination with auxins used strongly enhanced
the differentiation and growth of date palm tissue
cultures. In this respect, the highest percentage of
nodular cultures (80 %) as well as the highest value of
Plantlets Development and Acclimatization: Shoots
were proliferated
from
nodular
cultures using
phytohormone-free medium. The shoots were elongated
Table 2: Effect of different growth regulators added to MS-medium on in vitro morphogenesis of date palm.
NO
MS-medium
Nodular culture
Culture fresh
Differentiated
supplemented with :
frequency(%)
weight (g)
cultures(%)
1
1 mg/l NAA
20
0.70 ± 0.20
_
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2
2 mg/l NAA
30
0.90 ± 0.19
10
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3
5 mg/l 2,4-D
50
1.11 ± 0.33
20
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4
10 mg/l 2,4-D
60
1.20 ± 0.25
20
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------5
1mg/l NAA+3 mg/l 2iP
25
1.00 ± 0.18
_
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6
2mg/l NAA+3 mg/l 2iP
35
1.15 ± 0.22
15
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7
5 mg/l2,4-D+3 mg/l 2iP
70
1.30 ± 0.40
60
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8
10 mg/l2,4-D+3 mg/l 2iP
80
1.48 ± 0.33
55
Each treatment is the average of 20 replicates ± SE.
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J. Appl. Sci. Res., 3(9): 859-866, 2007
65.80% water content which caused by air drying for 20
min. Results also reveal that high proportion of date
palm cryopreserved cultures were irreversibly dameged
when their moisture content dropped below 60%. Our
results are in line with those of Uragami et al.[28] on
Asparagus officinalis. They reported that the axillary
buds tolerated to dehydration remained a life after
exposure to liquid nitrogen. In a study on
cryopreservation of somatic embryoids of date palm,
Mycock et al. [19] mentioned that drying samples down to
the range of 0.7-1.2 g.g-¹ allowed for a 32% survival
rate. On the other hand, a reduction of the moisture
content of banana meristem cultures by exposure
of the meristematic clumps to sterile air flow did
not result in an increased survival
rate
of
cryopreseved cultures [20].
Fig. 1: A- Nodular cultures of date palm proliferated on
MS-medium contained 10 mg/l 2,4-D + 3 mg/l
2iP
BSho o t
cultures
regenerated
form
cryopreserved cultures on recovery medium
C- Successful acclimatization for transplanting
of plantlets to free-living conditions.
Effect of Exposure to Sugars on Cryopreservation:
Although an effect of water content is one possibility,
the precise mode of action of sugar preculture in
enhancing freeze resistance is not exactly known [20].
Sugars reduce moisture content slowly due to osmotic
action. As a result of its uptake, it decreases the freezing
point and the amount of freezable water present in the
tissues. The role of fructose, glucose, sorbitol and
sucrose added seperatly to preculture medium on
freezing tolerance of cryopreserved nodular cultures of
date palm was investigated. Survival and conversion
percentages after freezing together with their mositure
contents resulting from treatments are shown in
Table (4). Data presented indicate that most of nonprecultured cultures were killed by freezing. Generally,
increasing of sugars concentration from 0.5 to1.0 M
decreased the survival rate of cryopreserved cultures of
date palm. In spite of addition of other various sugars to
the preculture medium effectively promoted survival,
sucrose was more effective in increasing freezing
tolerance. The highest percentage of survival (80 %) and
conversion to plantlets (75%) were observed with 1 M
sucrose. The results are closed with those of Pains et
al. [20] on banana. They reported that sucrose preculture
improves post-thaw survival of cryopreserved meristem
cultures. They added concentration of 0.4 and 0.5 M
was especially effective, resulting in a regrowth of 25
and 42% respectively. In this connection, Uragami et
al.[28] they mentioned that a considerable amount of
sucrose uptake and its subsquent dissociation into
glucose and fructose during preculture in the presence of
high sucrose levels was demonstrated with axillary buds
of asparagus. This dissociation inside the cell causes a
considerable increase in osmolarity since 1 M sucrose
results in 2 M of monosaccharides. An indirect
growth presented as culture fresh weight were observed
with medium contained 10 mg/l 2,4-D + 3 mg/l 2iP
(Table 2 and Fig. 1-A). However, the highest percentage
of differentiation was noticed when 5 mg/l 2,4-D +3
mg/l 2iP were added to culture medium. From the
obtained results, it clear that the combination of 2,4-D
and 2iP appeared to be the most suitable copmared with
other treatments used. These results are closed with that
of Taha et al.[25]. They used medium contained 10 mg/l
2,4-D + 3mg/l 2iP for induction of embryogenic cultures
and subsquently differentiation of date palm shoot tips
and leaf premordia cultures. The results also are in line
with those reported by Mater [17] and Madhuri et al.[16].
Effect of Drying Explant on Cryopreservation:
Sufficiently dehydrated seeds and small seedlings were
directly immeresed in liquid nitrogen (LN) from room
temperature and stored there without
freezing
injury [2]. Thus desiccation prior to direct immersion in
LN seems to be a practical method for cryopreservation
of plant materials.In this experiment, the effect of
reducing water content of in vitro proliferated nodular
cultures of date palm caused by air drying on survival
and conversion of cryopreserved cultures was
investigated. The obtained results presented in Table (3)
show that survival as well as conversion percentages of
cryopreseved tissue cultures were strongly affected by
their moisture levels. The highest rate of survival and
subsquently conversion to plantlets were observed with
862
J. Appl. Sci. Res., 3(9): 859-866, 2007
Table 3: Effect of drying time on survival and conversion of in vitro crypreserved date palm cultures at -196<C.
Drying time (min)
Water content (%)
Survival (%)
Conversion (%)
0
90.1
10
_
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10
75.9
70
60
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20
65.8
80
65
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------30
60.3
40
30
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------40
57.8
20
10
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------50
50
10
_
Each treatment is average of 20 replicates.
Effects of different sugar types at the concentrations of (0.5 or 1.0M) added to pre-culture medium on survival and conversion of
cryopreserved date palm cultures at -196 <C.
Sugar
Concentration(M)
Water content (%)
Survival(%)
Conversion(%)
None
_
90.20
10
_
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------0.5
75.50
50
40
Fructose
---------------------------------------------------------------------------------------------------------------------------------------------------1.0
70.20
60
50
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------0.5
73.90
50
43
Glucose
---------------------------------------------------------------------------------------------------------------------------------------------------1.0
69.50
65
55
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------0.5
76.00
55
40
Sorbitol
---------------------------------------------------------------------------------------------------------------------------------------------------1.0
71.80
65
58
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------0.5
72.50
70
60
Sucrose
---------------------------------------------------------------------------------------------------------------------------------------------------1.0
65.00
80
75
Each treatment is the average of 20 replicates
Table 4:
Table 5: Effect of exposure to vitrification solution at 25 and 0 <C on survival and conversion of cryopreserved date palm cultures at -196<C.
Survival (%)
Conversion (%)
--------------------------------------------------------------------------------------------------------------------------Exposure time (min)
Exposure at 25 <C
Exposure at 0 <C
Exposure at 25 <C
Exposure at 0 <C
0
10
20
_
10
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------20
60
55
40
45
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------40
80
75
60
65
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------60
50
85
40
75
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------80
40
75
30
70
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------100
20
70
10
60
Each treatment is the average of 20 replicates.
effect of sucrose could be due to the accumulation of
endogenous compounds induced by a mild osmotic
stress, that then offer protection against further water
stress and cryopreservation..
successful cryopreservation, it is necessary to avoid
lethal intracellular freezing, which occurs during rapid
cooling in liquid nitrogen. Thus, cultures have to be
sufficiently dehydrated or concentrated to be capable of
vitrifying before being immersed into LN. Another
possible approach could be extensive concentration
Effect of Vitrification on Crypreservation: For
863
J. Appl. Sci. Res., 3(9): 859-866, 2007
using a highly concentrated vitrification solution.
Vitrification
method
is a very simple appears
promising as aroutine method for cryopreservation
tissue cultures. To determine the potential of exposure
to vitrification solution on freezing tolerance of date
palm tissue cultures, samples were treated for 20-100
min at 25 and 0<C before plunge into LN. Exposure to
vitrification solution produced time dependent
survival and conversion percentages was tabulated in
(Table 5). Results reveal that the survival of non-treated
cultures was much lower than those exposed for
different periods. The highest rates of survival were
obtained with cultures treated with vitrification solution
for 80 min at 0<C or 40 min at 25<C, respectively.
Moreover, the highest percentages of cultures convered
to plantlets were observed when cultures were exposed
to 0<C for 60 and 80 min. respectively. From the
obtained results, it is clear that to reduce the injurious
effects of high osmolarity of vitrification solution, low
temperature are needed and to decrease the time of
exposure, high temperature are preferable. The present
results are in line with those reported by Uragami et
al.[27]. In this connection, Kohmura et al.[13] mentioned
that the keys to success the cryopreservation by
vitrification are to carefly control the procedures for
dehydration and cryoprotectant permeation and to
prevent injury by chemical toxicity or excess osmotic
stresses during treatment. Thus, successful vitrification
requires the use of a highly concentrated yet nontoxix
solution of cryoprotectants and the optimum exposure
time to it.
Fig. 2: RAPD profile of in vivo
grown plant
(lane 1), nontreated tissue
cultures (lane
2), cryopreserved
tissue cultures of date
palm (lane 3) and the DNA marker (M) from
left to right using random primers i.e.,
K1, K2, K4 and K5.
primer K5, a band of 600 bp was not present in the
sample of the in vivo grown culutures. It is particularly
important to confirm that cryopreserved cultures of date
palm produce plantlets genetically similar to both
nontreated and plants grown in free-living conditions.
From the obtained results, we can conclude that, no
genetic variability of the frozen-thawed nodular cultures
of date palm. The present results are in line with those
reported by Saker et al. [23]. They mentioned that no
significant variation observed of tissue cultures derived
plantlets. RAPD analysis showed genetic variation in
only 4 % of analalyzed plants (70 regenerants) which
were incubated for 6-12 months under 25 <C. In this
respect, genetic marker analysis has been used to study
the degree of genetic change in plants regenerated in
vitro such as pea [5], sugarbeet[22] and wheat[4].
M olecular Analysis: Due to its high sensitivity and
accuracy in detecting every single base change, DNAbased analysis was applied to study the genetic stability
of plant tissue cultures [29]. RAPD technology has been
successfully used for measuring diversity in plants, and
the patterns of variation observed have been shown to
closely resemble those obtained using more classical
characters [10]. In this investigation, RAPD analysis was
used to determine the genetic stability of treated and
non-treated tissue cultures of date palm to cryopstorage
and testing the similarity of both two types of cultures
to field grown plants. Four randomly selected primers
were used. Only one of them (K1) did not give
reproducible and sufficient amplification products. As
shown in Fig. (2), DNA fragments varied in numbers
and sizes depending on the primers used. The banding
reveals that the three types of cultures were identical to
each other with primers K2 and K4. However, with
Plantlets Development and Acclimatization: In this
part of study, shoot cultures were regenerated form
nodular cultures on recovery medium (Fig.1-B). Because
the development of a good root system on the in vitro
grown plantlets of date palm is considered one of the
most important factor affecting acclimatization to ex
vitro environment[8], shoots derived from cryopreserved
cultures were transferred into charcoal containing
medium for in vitro elongation and rooting. The
plantlets with healthy root system were successfully
transplanted to free-living conditions within short period
of acclimatization (Fig.1-C). In
this
respect,
Tisserat[26]
reported
that,
high survival
rate
864
J. Appl. Sci. Res., 3(9): 859-866, 2007
(nearly 100 % ) could be obtained when date palm
plantlets with 2-3 foliar leaves and of shoot length
greater than 10 cm ( with a well-developed adventitious
root system ) were transplanted in pots containing a
mixture of peatmoss and vermiculite.
13. Kohmura, H., A. Sakai, S. Chokyu and T. Yakuwa,
1992.
Cryopreservation of in vitro cultured
multiple bud clusters of asparagus (Asparagus
officinalis L.cv. Hiroshimagreen [2n=30]) by the
techniques of vitrification. Plant Cell Reports.,
11: 433-437.
14. Kumu, Y., T. Harada and T. Yakuwa, 1983.
Development of a whole plant from a shoot tip of
Asparagus officinalis frozen down to -196 <C.
J.Fac.Agr.Hokkaido Univ., 61: 285-294.
15. Kuo, C. and R.D. Lineberger, 1985. Survival of in
vitro cultured tissues of Jonathan apple exposed to
-196 <C. HortScience., 20: 764-767.
16. Madhuri, S., P. Shankar and N. Sharon, 1998.
Somatic embryogenesis and plant regeneration from
primordial leaf of Phoenix dactylifera L. cv.
Yakubi. Indian J. Experimental Biology., 36: 526529.
17. Mater, A.A., 1986. In vitro propagation of Phoenix
dactylifera L. Date Palm J., 4: 137-152.
18. Murashige, T. and F. Skoog, 1962. A revised
medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant., 15: 473-497.
19. Mycock, D.J., P. Berjak, N.W . Pammenter and
C.W . Vertucci, 1997. Cryopreservation of somatic
embryos of Phoenix dactylifera L. In: Ellis, R.H.,
M. Black, A.J. Murdoch and T.D. Hong (eds).
Basic Applied Aspects of Biology., pp: 75-82.
20. Pains, B., N. Notte, K. Van Nimmen, L.A. W ithers
and R. Swennen, 1996. Cryopreservation of banana
(Musa spp.) meristem cultures after preculture on
sucrose. Plant Science. 121: 95-106.
21. Reed, B.M., 1993. Responses to ABA and cold
a c c lim a tio n a re ge no typ e d e p e nd e nt fo r
cryopreserved blackberry and rasberry meristems.
Cryobiology., 30: 179-184.
22. Sabir, A., H.J. Newbury, G. Todd, J. Catty and
B.V. Ford-Lloyd, 1992. Determination of genetic
stability using isozymes and RFLP in beet plants
regenertated
in vitro. Theor. Appl. Genet.
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23. Saker, M.M., S.A. Bekheet, H .S. Taha, A.S. Fahmy
and H.M. Moursy, 2000. Detection of somaclonal
variation in tissue culture-derived date palm plants
using isozyme analysis and RAPD fingerprints.
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24. Shuji, N., S. Akira, A. Yoshihiko and M.
Tsunetomo, 1992. Cryopreservation of Asparagus
(Asparagus officinalis L.) embryogenic cells and
subsequent plant regeneration by a simple freezing
method.Cryo-Letters., 13: 379-388.
25. Taha, H.S., S.A. Bekheet and M.K. El-Bahr, 2003.
Alternative approach For micropropagation of the
date palm c.v. Zaghlool. Arab J. Biotech., 6: 103112.
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