Introduction
The date palm (Phoenix dactylifera L.) is
considered as one of the earliest cultivated fruit trees and is now cultivated
across the world. Date palm has a high nutritional value and it is a staple
food source especially for many inhabitants of the Arabian Gulf include Saudi
Arabia. The quality of date palm varietal clones fruits varies and as a
consequence price varies according to the variety. Many producing countries,
including Saudi Arabia, are keen to keep distinct variety homogenetiy and avoid
clonal deterioration and variety extinction (El-Juhany
2010). The number of palm trees around
the world has reached about 150 million trees (Al-Khayri et al. 2018)
but is subject an annual decline due to environmental conditions including
desertification and salinization and biotic stress from insects and pathogens. Such factors are a major constraint on the conservation
of date germplasm and consequently lead to a threat to locally maintained
germplasm (Khan et al. 2012). Date palm is a dioecious fruit tree that
is vegetatively propagated through offshoots, and its germplasm cannot be
stored or handled easily using conventional means. Furthermore a deterioration
in the productivity of date palm plants due to the use of traditional methods
of propagation has been reported (Rajmohan 2011).
It is inevitable that with the loss of date palm cultivars, the genetic
diversity will decrease and threaten the future productivity.
In order to limit the loss of genetic diversity in many
crops, plant breeders have resorted to preserving germplasm either in
seed-banks or tissue culture clone banks as a source of diversity for use in plant
breeding programes. In vitro methods to propagate the date palm
have been developed which may also be employed to preserve germplasm (Taha et
al. 2003). By combining the benefits of the vegetative propagation system
with the capability of long-term storage, synthetic seeds have been created which
have great potential in date palm agriculture (Bekheet et al. 2002). Conservation, diversity and ecological restoration are
terms that have had relevance in recent times (Duarte
et al. 2018). For date palm ex situ conservation of genetic
material under field conditions suffers from such several disadvantages
including extensive labour cost andl the risk of losses due to pathological and
environmental threats (Shatnawi 2013). Preserving
germplasm as seeds through seed banks is normally the first resort for the in situ conservation for many plants
(Bonner 1990; Towill 2005) but is nt very applicable to Date palm. Although
conventional methods of storing seeds is an inexpensive method for moste plants
for conserving germplasm, but in the case of palm there are several drawbacks
to using this method. Firstly there is decline in vitality of seeds with
increasing preservation period and most importantly seeds are very heterozygous
and therefore, not suitable for maintaing true-to-type palm genotypes (Bekheet et
al. 2007). As a result, those working in this field are beginning to use
modern technology methods, such as cryopreservation of clonal material for the
preservation the germplasm of palm trees and Soliman (2013) indicated that
crystorage is probably the priniciple long-term in vitro conservation method to be used for such biological
materials.
Cryopreservation of different plant explants has been
used for storage of genetic material under low temperature (−196°C) for
long periods (Panis and Lambardi 2006).
The major advantages of this technique
are: 1) it is safe, 2) cells retain their viability and vitality, 3)
cryopreserved materials remain genetically stable, 4) metabolic process and
biological deterioration are considerably slowed or even halted, 5) the process
facilitates the exchange of germplasm between countries and regions and 6) it
helps in the reduction or eradication of viruses from plant tissues (Kohmura et al. 1992; Feng et al.
2011). Cryopreservation of plant materials requires optimization of the such
as: 1) size of explant, 2) concentration of cryoprotective amendments, 3)
sample water content, 4) rate of freezing, 5) rate of thawing, 6) fitness of
the explants, 7) materials, 8) culture conditions, 9) nature of liquid nitrogen
solidifying agents, 10) dehydration processes and 11) cooling rate (Alansi et al. 2019). Therefore,
establishment of a successful protocol for cryopresrvation can be a difficult
and laborious process that requires the manipulation of all these factors to
achieve success of the cryopresrvation technique. There are relatively few accounts
of research in the literature on germplasm conservation of date palm somatic
embryos by in vitro encapsulation/dehydration. The current work
describes the results of a study implemented to develop an effective and simple protocol with maximum
viability for long term in vitro
conservation of date palm via the cryopreservation of somatic embryos.
Material and Methods
Plant material preparation and disinfection: Vegetative offshoots of mother trees of Saudi Arabian date palm (Phoenix dactylifera L.) cv. Magdoul was
collected,
washed with distilled water (DW) and shoot tip explants (8–10 cm in length) were exposed and excised using
a scalpel. Shoot tips was surface
sterilized using the following procedure: 1) Shoot tips were immersed in
freshly prepared 50% Clorox (NaOCl at 5.25%) containing 2 drops of Tween-20 for
30 min followed by washing three times with sterilized distilled water (SDW),
2) then immersed in 0.2% HgCl2 solution for 5 minutes and rinsed
three times with SDW, 3) each shoot tip explant was then divided into 4
sections in preparation for culture on callus induction medium. All steps of
the disinfection procedure were performed in a Laminar Air Flow ''Hood'' and
aseptic procedures were applied according to Soliman
et al. (2010)
Somatic embryogenesis induction: Shoot tip section explants, that were prepared as
mentioned above, were cultured on
callus induction medium (M1) including : 1) 4.4 mg L-1 MS (Murashige and Skoog 1962) 2) 170 mg L-1 Na H2PO4, 3)
125 mg L-1 myo-inositol, 4) 200 mg L-1 glutamine, 5) 100 mg L-1 ascorbic acid, 6)
100 mg L-1 citric acid, 7) 5.0 mg L-1 thiamine-HCl, 8) 1.0 mg L-1 nicotinic acid, 9) 1.0 mg L-1 pyridoxine-HCl, 10) 30 mg L-1 sucrose, and 11) 2.0 g L-1 gelrite, supplemented with the growth
regulator hormones (GRHs) 10.0 mg L-1
2,4-di-chloro-phenoxy-acetic acid (2,4-D), 8.0 mg L-1
2–isopentenyle adenine (2iP) and 2.5 g L-1 activated charcoal according to
(Aldhebiani et al. 2018 ) with some modification. The pH of
the medium was adjusted to pH 5.6 before the addition
of 2.5% phytagel and then sterilized by autoclaving for 15 minutes at
121°C. To
stimulate induction and growth of callus, the cultures were incubated in total
darkness in a growth room at 25 ± 2°C for 12 months.
To
induce somatic embryogenesis, healthy
callus (100–200 mg fresh
weight) obtained from the previous stage was cultured on MS basal medium (MS2) containing
the GRHs 3.0 mg L-1 Naphthalene Acetic Acid (NAA), 6.0 mg L-1
2iP and 2.5 g L-1 activated charcoal. The culture jars were placed in
a growth room for 8–10 weeks at 25 ± 2°C
under cool white fluorescent lamps with an intensity 75 μmol m−2
s−1 and 16
h photoperiod. Sub-culturing of embryogenic callus formation was
performed once every five weeks to generate sufficient embryogenic
callus stocks for cryopreservation experiment.
Preparation of embryogenic callus for cryopreservation: Embryogenic callus was placed
vertically on initiation medium (M3) consisted of MS medium containing the GRHs
2.0 mg L-1
NAA, 3.0 mg L-1 2iP
and 2.5
g L-1 activated charcoal, the pH of the medium was
adjusted to 5.6 before the addition of 2.5%
phytagel. Cultures were incubated for two weeks at 25°C and 16h photoperiod, then
transferred to the same initiation medium (M3) but without any other supplement
of GHRs and only containing 0.5 M
sucrose for one week. Embryogenic callus was excised and used as explants for encapsulation.
Encapsulation and dehydration: Embryogenic callus was
suspended in a calcium-free liquid MS medium with 3% (w/v)
Na-alginate according to (Bose et al. 2017). Drops of the same alginate
solution with contained embryogenic calli were dispensed as drops into
0.1 M calcium chloride. Encapsulated embryogenic calli were precultured
in MS medium with different concentration of sucrose (0.0, 0.5, 0.75, 1.0 and
1.5 M) free from growth regulators
and incubated at 25 ± 2°C under cool white fluorescent lamps with an intensity 75 μmol m-2 s-1 with a 16 h photoperiod for 24
hours following by incubation at 4°C in the dark for 24 h.
Dehydration of preculture
encapsulated embryogenic callus: To measure the optimum drying time, the encapsulated embryogenic calli
were placed on sterilized filter paper and exposed to continuous air in a
laminar flow cabinet for periods ranging between 0.0 to 10 h at 25 ± 2°C. Samples were weighed every 2 h to obtain the
dessication curves for each osmoticum treatment. Samples were also taken and
tested for their liquid nitrogen tolerance where beads were placed in 2.0 mL
sterile cryovials and directly rinsed into liquid nitrogen. Percentage of beads moisture content (MC%) were
calculated using following equation: MC% = 100*[(Fresh weight - Dry weight (/ Fresh weight].
Also, Liquid nitrogen tolerance for each drying time at 2 h intervals was
tested.
Freezing progress: Beads containing embryogenic
calli were incubated in darkness at 4°C for one day and then transferred to liquid nitrogen
(at -196°C) directly and storage for different periods (1, 2, 3, 4, 5 and 6
weeks).
Thawing and regrowth after
cryopreservation: Beads
following cryopreservation and non-cryopreserved controls, were transferred to
fresh medium, as mentioned below, for assessment of recovery growth. Cryopreserved
samples were taken after 1, 2, 3, 4, 5 and 6 weeks from cryopreservation to
assess the effect of different preservation periods on vitality or survival of embryogenic callus. Cryopreserved vials containing beads with embryo calli
were rapidly thawed at 37°C for 3 minutes using a water bath, then beads were
removed and washed with liquid MS medium. Encapsulated non-cryopreserved (-LN) and encapsulated cryopreserved
(+LN) beads were cultured on MS2 as described above for somatic embryogenesis
germination and then transferred to MS medium (MS4) containing the GRHs 6.0 mg L-1 2iP, 2.0 mg
L-1 kinetin (Kin), 1.0 mg L-1 Indole Butyric Acid (IBA) and 2.5 g L-1 activated charcoal, and incubated in the dark at 25°C in the growth room for 3 days and then exposed to light with a 16 h
photoperiod. Survival rates
were recorded after 35 days and cultures
were kept for observation of recovery growth and development of plantlets.
Statistical analysis
The experiments were designed in a completely randomized design (CRD). Analysis of variance (ANOVA) and the calculation
of LSD or Duncan's Mulitple Range test (0.05) was undertaken using SAS, 2000
software programme, All values were
reported as means ± standard error according to Snedecor
and Cochran (1989).
Results
Effect of preculture treatments
Callus and somatic embryogenesis were successfully
initiated on MS basal medium M1 (Fig. 1a) and M2 (Fig. 1b), supplemented with GRHs 10.0 mg L-1
2,4-D, 8.0 mg L-1 2iP
; 3.0 mg L-1 NAA , 6.0 mg L-1
2iP , repectively. Then embryogenic callus was cultured on initiation medium (M3) consisting of MS medium along
with GRHs 2.0 mg L-1 NAA and 3.0 mg L-1
2iP, in order to improve the quality of the
callus to obtain friable call a key step for encapsulated embryogenic callus.
The survival rate of encapsulated embryogenic callus after pre-growth varied
depending on the sucrose content in
the preculture medium and the duration of the pre-culture. The results indicated that high survival rates (98.2, 91.4 and 78.5%) were obtained after
pre-culture of the embryogenic callus with 1.0 and 1.5 M sucrose at 1, 2 and 1 day, respectively. The highest survival rates (100%) of the embryogenic callus
was observed when encapsulated embryogenic callI were
pre-grown for 3 and 4 d in media with a sucrose concentration of 0.75 and 0.5 M, respectively (Table 1). The addition of 0.75, 1.0 and 1.5 M sucrose after 9, 7
and 4 days to the pre-culture medium inhibited the survival rates. However, low survival rate
(12.7, 14.5 and 17.5%) was obtained at 0.75, 1.0 and 1.5 M sucrose after 8, 6 and 3 days, respectively. Based on our the results, the lowest concentration
tested i.e., 0.75 M sucrose for 3 days was considered to
be optimal for pre-culture of
encapsulated-dehydrated embryogenic calli of date palm cv. Magdoul.
Encapsulation/dehydration
in sterile airflow
The modified encapsulation-dehydration method was
studied to determine the optimial dehydration time. There was a significant
impact of sucrose concentration and dehydration duration on the survival and
regrowth of cryopreserved and non-cryopreserved embryogenic calli (Table
2). Minium survival (28.15%) and regrowth (10.08%) for encapsulated
cryopreserved (+LN) calli occurred only when calli were suspended in 1.0 M
sucrose for 3 days with
10 h dehydration time in a sterile airflow, where the beads attained 13.05% MC. In
contrast, pre-treated with 0.5 and 0.75 M sucrose for 4 and 3 days followed by 0–4
and 0–2 h dehydration period recorded the greatest survival (100%) of encapsulated non-cryopreserved (-LN) embryogenic calli where the beads
attained between 66.18–81.50% and 45.00–78.35% MC, respectively (Table 2 and
Fig. 2). While,
the highest regrowth percentage (100–97.18%) of encapsulated
non-cryopreserved (-LN) embryogenic calli were obtained
with 0.5 M for 4d and
0.75 M sucrose for 3d followed by 2 h
dehydration period, where the beads attained 45.05–76.45% MC. Treating encapsulated
cryopreserved embryogenic calli with LN gave significantly lower values
compared with that of control non-cryopreserved treatment (-LN) (Table 2). The
greatest survival (83.25%) and regrowth (79.35%) was obtained when encapsulated
cryopreserved (+LN) embryogenic calli were pretreated with 0.75 M sucrose
for 3 d followed by 4 h dehydration period with 39.50% bead MC.
Table 1: Effect of preculture duration and sucrose concentration on survival
percentage for encapsulated non-cryopreserved (-LN) embryogenic calli of date palm cv. Magdoul
|
Preculture duration (days) |
Sucrose concentration (M) |
|||
|
|
0.5 |
0.75 |
1.0 |
1.5 |
|
0 |
100.0 ±
0.45a |
100.0 ± 0.00a |
100.0 ± 0.00a |
100.0 ± 0.00a |
|
1 |
100.0 ±
0.33a |
100.0 ± 0.00a |
98.24 ± 0.28b |
78.50 ± 0.00b |
|
2 |
100.0
±
0.28a |
100.0
±
0.00a |
91.42 ± 0.35c |
45.82 ± 0.00c |
|
3 |
100.0 ± 0.15a |
100.0 ± 0.00a |
82.50 ± 0.55d |
17.58 ± 0.00d |
|
4 |
100.0 ± 0.44a |
91.54
±
0.14b |
65.25 ± 0.14e |
00.00 ± 0.00e |
|
5 |
96.45 ± 0.29b |
78.26 ± 0.23c |
38.72 ± 0.27f |
00.00 ± 0.00e |
|
6 |
92.82 ± 0.35c |
55.82 ± 0.45d |
14.51 ± 0.31g |
00.00 ± 0.00e |
|
7 |
88.51 ± 0.66d |
29.58 ± 0.56e |
00.00 ± 0.00h |
00.00 ± 0.00e |
|
8 |
72.48 ± 0.23e |
12.70 ± 0.18f |
00.00 ± 0.00h |
00.00 ± 0.00e |
|
9 |
69.52 ± 0.41f |
00.0 ± 0.00g |
00.00 ± 0.00h |
00.00 ± 0.00e |
|
10 |
65.25 ± 0.22g |
00.0 ± 0.00g |
00.00 ± 0.00h |
00.00 ± 0.00e |
Values are means ±
standard error of three replicates. For each cultivar, bars with the same
letters are not significantly different at P
≤ 0.05 level
Table 2: Effect of sucrose concentration
and dehydration duration on survival and regrowth percentages for encapsulated
non-cryopreserved (-LN) and cryopreserved (+LN) embryogenic
calli of date palm cv. Magdoul
|
Sucrose concentration (M) |
Dehydration duration (h) |
Survival% |
Regrowth% |
Moisture content (%) |
||
|
-LN |
+LN |
-LN |
+LN |
|||
|
0.5 |
0 |
100.0
±0.45a |
15.35±0.22k |
100.0±0.43a |
13.25±0.36k |
78.50±0.25a |
|
2 |
100.0
±0.35a |
28.25±0.18j |
100.0±0.52a |
25.38±0.42i |
62.45±0.35c |
|
|
4 |
100.0
± 0.18a |
37.22
± 0.26i |
97.35
± 0.18b |
31.45
± 0.21h |
56.18
± 0.18d |
|
|
6 |
94.35
± 0.28c |
48.45
± 0.33h |
92.25
± 0.32c |
37.95
± 0.34g |
49.25
± 0.55e |
|
|
8 |
91.40
± 0.39e |
61.29
± 0.19f |
88.75
± 0.72d |
60.25
± 0.46e |
45.55
± 0.38f |
|
|
10 |
84.29
± 0.22h |
78.77
±0.42b |
83.30
± 0.22f |
71.85
± 0.29c |
38.55
± 0.25g |
|
|
0.75 |
0 |
100.0 ±
0.15a |
17.15
± 0.51k |
100.0 ±
0.31a |
16.75
± 0.37j |
69.35
± 0.44b |
|
2 |
100.0 ±
0.44a |
62.24
± 0.28e |
97.18
± 0.29b |
58.62
± 0.20f |
45.05
± 0.69e |
|
|
4 |
97.37
± 0.26b |
83.25 ± 0.35a |
96.85
± 0.52b |
79.35 ± 0.25a |
39.50 ±
0.22g |
|
|
6 |
90.18
± 0.62e |
79.88
± 0.26b |
89.50
± 0.43d |
73.85
± 0.15b |
37.25
± 0.15g |
|
|
8 |
86.25 ±
0.52f |
75.35
± 0.42c |
85.37
± 0.45e |
65.35
± 0.30d |
33.75
± 0.36h |
|
|
10 |
71.45
± 0.39j |
69.63
± 0.55d |
69.50
± 0.22h |
61.75
± 0.48e |
31.50
± 0.28h |
|
|
1.0 |
0 |
93.55
± 0.41d |
22.92
± 0.32j |
93.25
± 0.65c |
17.15
± 0.25j |
63.90
± 0.65c |
|
2 |
88.23
± 0.33g |
49.38
± 0.41h |
87.55
± 0.39d |
38.95
± 0.31g |
33.00
± 0.37h |
|
|
4 |
84.35
± 0.25h |
55.62
± 0.72g |
82.15
± 0.52f |
52.28
± 0.44f |
30.80
± 0.42i |
|
|
6 |
76.50
± 0.14i |
32.95
± 0.63i |
75.35
± 0.77g |
28.50
± 0.29i |
25.20
± 0.19j |
|
|
8 |
55.48 ± 0.26k |
19.44 ± 0.40k |
48.65
± 0.15i |
00.00 ± 0.00l |
18.25
± 0.24k |
|
|
10 |
28.15 ± 0.35l |
00.00 ± 0.00l |
10.08 ± 0.32j |
00.00 ± 0.00l |
13.05
± 0.32l |
|
Values are means ±
standard error of three replicates. For each cultivar, bars with the same
letters are not significantly different at P
≤ 0.05 level
M Molar; h Hours; LN
Liquid Nitrogen; % percentage
Survival of desiccated encapsulated embryogenic calli decreased in line with
decreasing bead MC after cryopreservation (Fig. 3). When embryogenic calli had a MC between 31.50 and 45.55% they retained high viability whilst
a reduction of MC content of 18.25% or less led to embryogenic calli death. Our results also, suggested that, pre-culture encapsulated embryogenic calli in medium supplemented with 0.75 M sucrose for 3 d and dehydrated to 39.50% MC followed with exposure to LN was the good optimized
treatment and led to excellent regrowth (Fig. 4).
%201754-1762_files/image002.png)
Fig. 1: Friable embryogenic callus induction, using MS3 containing 2.0 mg L-1
NAA, 3.0 mg L-1
2iP and 2.5 g L-1 activated charcoalto use it as an explants source for
encapsulation (A). Somatic
embryogenesis formation and plantlet obtained on MS2 supplemented with 3.0 mg L -l NAA, 6.0 mg L-1
2iP and 2.5 g L-1 activated charcoal after 8-10 weeks (B)
%201754-1762_files/image004.jpg)
Fig. 2: Encapsulated embryogenic calli
of date palm cv. Magdoul;
Calcium alginate beads formed by encapsulation of explants using 3% sodium
alginate.
Encapsulated cryopreserved embryogenic calli were
pretreated with 0.75 M sucrose for 3
d followed by (A) 4 h
dehydration period and (B) 2
h dehydration period. ec = embryogenic callus
%201754-1762_files/image006.png)
Fig. 3: Somatic embryogenesis formation recovery from embryogenic calli of date palm cv. Magdoul obtained on MS medium supplemented with 3.0 mg L-1 NAA, 6.0 mg
L-1 2iP
and and 2.5 g L-1 activated charcoal after eight weeks (A). Plantlet
development was obtained on 6.0 mg L-1 2iP, 2.0 mg L-1
kin and 1.0 mg L-1 IBA and
2.5 g L-1 activated charcoal after 35 days (B)
%201754-1762_files/image008.jpg)
Fig. 4: Effect of dehydration duration
on moisture content after preculture
treatments of cryopreserved (+LN) embryogenic calli of date palm cv. Magdoul. Vertical bars indicate ± SE for means
%201754-1762_files/image010.jpg)
Fig. 5: Percentage of regrowth of encapsulated non-cryoprserved (-LN) and cryopreserved
(+LN) embryogenic calli of date palm cv. Magdoul with 0.75 M sucrose for 3 d, following
encapsulation in alginate beads, air drying for 0 to 10 h and exposure in liquid
nitrogen. Vertical bars indicate ± SE for means
%201754-1762_files/image012.jpg)
Fig. 6: The effect of storage duration on percentage of
survival and regrowth of cryopreserved (+LN) embryogenic calli of date palm cv. Madgoul. Vertical bars indicate ± SE for means
Growth recovery after
cryopreservation
The
recovery and subsequent growth of cryopreserved embryogenic calli
occurred directly without the process of callus multiplication. This allows us
to assume that most of the cells of the callus region were largely undamaged
during the process of cryopreservation in LN. The encapsulated cryopreserved embryogenic calli of each pre-treatment were
re-cultured on MS2 medium containing 3.0
mg L-1 NAA, 6.0 mg L-1
2iP and 2.5 g L-1 activated
charcoal for the somatic embryogenesis
development (Fig. 5) and
then the somatic embryos were cultured on MS which contained 6.0 mg L-1
2iP, 2.0 mg L-1 Kin and 1.0 mg L-1 IBA and 2.5 g L-1
activated charcoal for plantlet development (Fig. 5). The greatest survival
percentage (76.35%) and regrowth (72.9%) of the encapsulated cryopreserved embryogenic calli was obtained following the
combination of pre-culture in MS medium containing 0.75 M sucrose for 3 d with air dehydration for 4 hours after one week
in LN storage. While, the survival percentage (74.4%) and regrowth (71.25%) of encapsulated
cryopreserved embryogenic calli were obtained with MS medium plus
0.75 M sucrose for 3 d followed by
dehydration for 4 h after 6 weeks in LN storage (Fig. 6).
Discussion
Encapsulation dehydration technology is a widely used
technique for the preservation of genotypes in situ (Rihan et al. 2017).
This is due to several advantages which include low cost, genetic stability for
longer time, high ability to survive and high cell viability (Farag et al. 2012), however, it requires more handling of alginate beads
and some species do not tolerate the high sucrose concentrations employed (Kaviani 2011).
In order to establish an effective encapsulation
dehydration protocol for date palm (cv. Magdoul), the
production of friable callus was achieved by culturing somatic embryogenic
callus on initiation medium (M3)
consisting of MS medium containing the GRHs 2.0
mg L-1 NAA, 3.0 mg L-1
2iP and 2.5 g L-1 activated charcoal (Fig. 1). Obtaining friable callus has been preferred in several
studies where it was concluded that a high success rate in most plant
biotechnology protocols such as transformation and in situ conservation
was achieved by using friable callus (Utsumi
et al. 2017).
In the current study drops of
alginate solution containing embryogenic callus was dispensed as drops into
0.1 M calcium chloride where polymerization was
completed and solid beads formed around embryogenic callus. This
protocol was in agreement with a previous study (Bose et al. 2017) where they used different
concentrations of Na-alginate with
concentrations ranging from (1–4%) and found that a concentration of 3% was
most successful in producing uniform beads with regular shape and good coverage
over the callus. Concentrations lower than 3% failed to create a solid cover,
whereas, 4% Na-alginate produced
a very hard cover which was subsequently difficult to rupture. Several other
studies have also successfully used the combination of 3% Na-alginate and 0.1 M CaCl2 for the encapsulation
of cryopreserved embryogenic calli in
several plant species (Saha et
al. 2015; Haque and Ghosh 2016; Kaya et al. 2020). Sodium alginate was also applied for encapsulated cryopreserved embryogenic calli in order to prevent the building of ice inside the cells
during the cryopreservation process (Patra and Gupta 2020).
The key to success in the process of cryopreservation
using the encapsulation/dehydration technique depends largely on avoiding the
formation of ice crystals inside the cells to prevent ice injury to the cells
which will negatively affect survival and regrowth. Ice formation during
cryopreservation can mechanically damage the biological structure of cells
resulting in ice injury (Yang et al. 2019). Factors that can affect the formation of ice crystals
and thus the success of cryopreservation can be applied during the
encapsulation dehydration process and have been found to include: 1) osmoticums
such assucrose concentration, 2) duration of hydration and 3) moisture content
(Soliman 2013). The success in achieving better treatment of sucrose and duration of dehydration will help in the induction of cytoplasmic vitrification (Wilkinson et al. 1998). Different
concentrations of sucrose as an osmoticum and different periods of dehydration
were applied in the current investigation (Table 1) to study the effect of
pre-culture duration and sucrose concentration on survival percentage for encapsulated non-cryopreserved (-LN)
embryogenic calli. The results
indicated that the lowest concentration tested i.e., 0.75 M sucrose for
3 days was optimal for pre-culture of encapsulated-dehydrated embryogenic calli. These results agree with those reported by Farag et al. (2012), who noted that there is an inverse relationship
between increased sucrose concentration and hydration time with the rate of
regrowth and survival. Increasing sucrose to high concentrations in the
pre-culture stage led to sugar accumulation, a decrease in the water content
and induced cytoplasmic vitrification that helped to prevent ice crystalization in apricot (Prunus armeniaca L.) (Soliman
2013). Previous studies (Al-Ababneh et al. 2003; Moges et al. 2004; Baghdadi et al. 2010; Kaya et al. 2020) stated that the highest percentages of survival and
regrowth were achieved when shoot tips were pre-cultured for two or three days
with sucrose ranging from 0.1 to 0.5 M.
Several other studies (Crowe et al. 1987;
Kendall et al. 1993) suggest that maintaining a higher concentration of
sucrose inside the cells may stimulate the expression of some genes responsible
for preventing the degradation of sugars and thus maintain the integrity of
cell membranes and cell walls as well as maintaining the stability of proteins
under frozen conditions. Accumulation of sugar in plant tissues protects cells
against these cryo-injuries and helps to restore vitality during freezing and
allows successful storage at cryogenic temperatures (Yang et al. 2019).
A degree of dehydration and cellular dehydration
tolerance is required for successful cryopreservation because the cells must
reduce their freezable water in order to avoid ice crystal formation when
exposed to liquid nitrogen (Benson 1999). The results presented here showed that a decrease in the MC of the bead
(dehydration) led to a decrease in survival and regrowth. Maximum tolerance of
non-cryopreserved beads reached a maximum when beads were exposed to 10 h
dehydration whilst cryopreserved embryogenic calli had the highest rate of
survival (83.25 ± 0.35a) and regrowth (79.35 ± 0.25a) under treatment with 0.75 M sucrose and 4 h dehydration with MC of
39.50 ± 0.22 Table (2). These results agree with the earlier work of Wang et
al. (2000) who found that after 10 h of dehydration under laminar-air flow,
the viability of dehydrated cells of grape was 42% at a MC of 16.2%, while 6 h
of dehydration recorded highest viability (78%) with 20.6% MC. Hirai
and Sakai (1999) pointed out the effect of the dehydration process on the
ability of the plant tissues to resist freezing during cryopreservation and
also showed that the dehydration duration not only increased the freezing
tolerance rate but also contributed to improving the regrowth rate of date
palm. Our results also support that of
earlier work by Gupta and Reed (2006), where they were noted that it is
important to optimize the dehydration period, where increasing the dehydration
period led to damage of the encapsulated shoot tip, whilst insufficient
dehydration was associated with ice nucleation and thereby freezing damage (Villouta et al. 2020). With regard to MC previous studies stated that
decreasing bead water content and reduction of the MC of encapsulated explants
to a minimal level is a necessary step for successful cryopreservation and it
leads to an increase in survival rates. However, it was found that the
shrivelling of the cell and its exposure to the condition of hypertension arise
from a lack of water content, which resulted in changes in the plasma membrane
(Palanyandy et al. 2020). Correlation
have been found between dehydration time, low moisture contents, the formation
of the glassy state (vitrification) and survival after cryopreservation
(Martinez and Revilla 1998).
Cryopreserved vials containing beads with embryogenic
calli were rapidly thawed at 37°C for 3 minutes using a water bath, then beads
were removed and washed with liquid MS medium to test growth
recovery. This is in a good agreement
with previous finding on garlic plants (Kim et al. 2004), where they
observed that rapid rewarming resulted in more regrowth than slower warming. In
our study, cryopreserved embryogenic calli of date palm regrew and successfully
developed into plantlets (Fig. 3) with intermediary callus to reduce somaclonal
variation. Takagi (2000) and Fernandes et al.
(2008) also observed that producing plants directly without intermediary
callus minimizes the risk of somaclonal variation. Our results found that the
highest value for growth recovery, whether by calculating the survival rate or the
regrowth was 76.35 and 72.9% respectively after storage in LN for the first
week, but there was an non-significant decrease in those values by increasing
the storage period in LN (Fig. 6). This result may suggest that thawed
cryopreserved embryogenic calli had high viability and exhibited long-term
survival. Previous studies support this contention, as both Nogueira et al. (2013) and Vujović et al. (2011) indicated a
significant decrease in survival rates to reach maximum rates of 20 and 37%
with increased storage time in LN. The growth recovery ratio that was recorded
in our current research is considered one of the best survival rates that has
been reached with date palm plants and this may be due to the optimised
protocol . Recovery and regrowth in other studies of date palm cv. Sagai were
above 30% for all the protocols used such as dehydrated-encapsulation,
vitrification, and vitrification-encapsulation (Alansi
et al. 2019) . It was reported by (Panis et al. 2005; Chen
et al. 2011) the average survival rate also varies between varieties
even when the same cryopreservation treatment conditions were applied in the
experiment.
Conclusion
In conclusion, this study defines the first efficient,
simple protocol for cryopreservation of date palm (cv. Magdoul) germplasm
by encapsulation-dehydration.
Encapsulation plays an important role in the success of cryopreservation, it is
nessary to induce a high level of dehydration tolerance for preservation. It
has been shown that encapsulation and dehydration increased viability of
cryopreserved date palm calli. Also,
encapsulation-dehydration improved the survival of these plants after exposure
to LN. Cryopreservation is now a viable long-term storage technique for date
palm germplasm. This leads to the
preservation of good varieties in the Kingdom of Saudi Arabia for long periods
and the reproduction of them in the laboratory at any time of the year which
will facilitate the clonal production of elite genotypes for future
agricultural use.
Acknowledgements
This work was funded by the University of Jeddah, Saudi Arabia, under grant No. (UJ-11-18-
ICP). The authors, therefore, acknowledge with thanks the University technical
and financial support.
Author Contributions
EMRM, NMSK and HIAS planned the experiments, OAA
and MPF interpreted the results, EMRM and HIAS made the write up and NMSK
statistically analyzed the data and made illustrations, MPF made the language editing.
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