Introduction
Date Palm (Phoenix dactylifera L.) is one of the most important
horticultural agricultural crops in the countries of the Middle East, where the
weather and soils are compatible with the agricultural needs of the palm plant
(Jain 2012). The Kingdom of Saudi Arabia is considered the second largest
producer of high-quality dates in the world (Assirey
2015). Although there are about 150 million palm trees planted around the world
(Al-Khayri et al. 2018), this number has
recently been subject to annual decreases due to environmental factors such as
desertification and salinization accompanied by insect problems and
physiological disease. Furthermore, ineffectiveness of sexual reproduction by
seed and limitations in asexual reproduction by offshoot cuttings
(macro-propagation) are limiting the replanting or expansion of date palm
plantations (Al-Mayahi 2015).
To improve the rapid propagation
of palms and avoid the negative aspects of using propagation with seeds or stem
cuttings, plant breeders are resorting to using modern techniques of in vitro
culture. Tissue culture techniques using the planting of various explants such
as leaf, shoot tip, or somatic embryos are an effective alternative method
compared to conventional methods of propagation (Brar and Khush 1994; Aldhebiani et al. 2018). Without success in obtaining an
effective plant propagation protocol in vitro, the development in the
use of other biotechnology techniques such as regeneration of transgenic plants
and in vitro conservation of plant such as cryopreservation will be
limited (Idowu et al. 2009). Accordingly, scientific research in this
field is aimed at developing effective methods with a high success rate in the effectiveness of propagation and
homogeneity (Rai et al. 2012; Reetika
et al. 2019) and genetic stability of micropropagated plantlets
(Eshraghi et al. 2005).
Somatic embryogenesis has been advocated for rapid
propagation in vitro and has proven to be effective for large-scale
propagation of date palm and in vitro raised plants have been
demonstrated to be genetically similar to mother plants (Faqir et al.
2019). This technique however, does carry some risk as it may be subject to
failure due to the physiological disturbances experienced by the explants
during culture (Mazri and Meziani 2015).
The most well-known problems indicated by previous research are tissue browning
which ofter leads to explant death (Mazri and
Meziani 2015), hyperhydricity due to acumulation of water in the
cultured explants (McCubbin and Zaid 2006)
and precocious rooting which leads to a decrease in the multiplication
efficiency of shoot buds (Khateeb 2008).
In order to prevent browning, activated charcoal, PVP, ascorbic acid and citric
acid have applied and added to the medium (Boufis et al. 2014).
Mazri and Meziani (2013) and have
indicated that increasing PGRs, amonium concentration and using liquid medium
may lead to increasing hyperhydricity.
Date palm somatic embryo (SE) culture goes through the standard SE
protocol of embryogenic callus induction, somatic embryo formation, somatic
embryo growth, maturation and conversion (germination). Each of the steps in
this process are often affected by many factors such as genotypes (Parrott et
al. 1989; Kumar et al. 2020), plant growth regulator (PGRs)
especially the combination ratio of auxin and cytokinin (Fatima and Anis 2012; Boufis et al. 2014; Abdolvand et al.
2014; Zayed et al. 2020), texture of the culture media (Al-Khayri 2011; Al-Khayri
and Naik 2017; Salama 2019), time periods of mother plant material collection
and size of explant (Moon et al.
2005) and intensity and quality of light
(Meziani et al. 2015). The objective of the current
study was to investigate the role of different concentrations and combinations
of 2, 4-D, 2iP, NAA, IBA and kinetin to induce callus
and somatic embryogenesis formation from shoot tip explants. We also evaluated
somatic embryos from embryogenic lines established
from two different date palm cultivars Safawi and Magdoul with respect to their ability
to germinate and be converted into plantlets. Establishment of an optimized
protocol for micropropagation of date palm will contribute to the success of
both genetic transformation and cryopreservation experiments which will the
subject of our future research.
Material and Methods
Plant material preparation
and disinfection
Two high quality popularly grown Saudi Arabian date
palm (Phoenix dactylifera
L.) cultivars Safawi and Magdoul,
according to (Lieb et
al. 2019; Steingass
et al. 2020), were chosen
and applied in the current study. The study was conducted from March. 2018 until
the end of Sept. 2019 year at Tissue Culture Units, Biological Sciences Dep., Fac. of Science,
University of Jeddah, SA with cooperation with T issue Culture and Biotechnology Labs., Maryout Research Station, Desert
Research Center, Ministry of Agriculture, Cairo, Egypt.
Offshoots (cuttings) of mother trees were washed with
distilled water (DW), and stored
until processed further. An offshoot was taken and shoot tip explants (8–10 cm in length)
exposed and excised using a sharp knife. Shoot tips was surface sterilized
using following procedure: 1) immersion in 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) immersion in 0.2% HgCl2 solution for 5 min
and rinsed with SDW three times, 3) shoot tip explants were then divided into 4
sections. All steps of the disinfection procedure were performed in a Laminar
Air Flow ''Hood'' and aseptic conditions were applied accordingly (Aldhebiani et al. 2018).
Induction of calli
Shoot tip explants
were cultured on induction medium (M1) including: 1) 4.4 mg L-1 MS (Murashige and Skoog 1962), 2) 170 mg L-1 Na H2 PO4,
3) 125 mgL-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, 11) 7.0 g L-1 agar, 12) 2.5 g L-1 activated charcoal and 13) 2.0 g L-1 gelrite, supplemented with different concentrations (10,
25, 50, 75 and 100 mg L-1) of 2, 4-D alone or in combination with (2.0, 3.0, 5.0, 6.0 and
8.0 mg
L-1) of 2iP in order to optimize the best callus
induction media. Four replicates of five of petri dishes (20 total) were set-up for each
treatment and each petri dish cultured with 5 explants. To stimulate induction and growth of callus, the
cultures were incubated in total darkness in the growth room at 25 ± 2°C for 12
months (period between August 2018 to July 2019), then callus induction
percentage and RWC (%) was assessed accordingly (Elmeer and Hennerty 2008) and data was analzed as shown in Table (1).
Somatic embryo
formation
Following on from callus
induction 5–10 mg fresh weight of
healthy callus was cultured on MS basal medium containing
NAA at various rates (0.5, 1.0, 2.0,
3.0 and 4.0 mg L-1) either alone or in combination
with various concentrations of 2iP (1.0, 2.0, 4.0, 6.0 and 8.0 mg L-1), each treatment comprised of
five jars each with four callus pieces and was replicated 10 times. The cultured jars were grown in a growth room for 10
weeks at 25 ± 2oC under cool white fluorescent lamps with an intensity 70 μmol m−2
s−1 for 16 h
photoperiod. After ten further weeks,
data were recorded as number and percentage of somatic embryogenesis
formation and analyzed (Table 2).
In vitro
germination of somatic embryogenesis
The
induction of somatic embryogenesis was
carried out on with MS medium containing
different concentrations of 2iP at (2.0, 4.0 and 6.0 mg L-1) alone
or in combination with kinetin (2.0 and 3.0 mg L-1) and IBA (0.5
and 1.0 mg L-1). The experiment was designed and cultured in the same
conditions as described in the previous stage. After 2 months the number of shoots
formed and shoots length were recorded and analyzed (Table 3).
Rooting and acclimatization of regenerated plantlets
To obtain shoot elongation and root formation from in vitro
germination of somatic embryos, MS culture was used supplemented with different
concentration of NAA (1.0, 1.5, 2.0 and 2.5 mg L-1) alone or in combination with IBA (0.5, 1.0 and 1.5 mg L-1) in the presence of 2.5 g L-1 activated charcoal. Each was treatment replicated five times
and each replicate was represented by three jars with each jar containing two
plantlets. The cultures were kept in the same conditions
as described for the somatic embryogenesis
formation stage. Four weeks later, the following measurements were
recorded: percentage of plantlets with root formation (%), root length (cm) and
number of roots per plantlet (no.).
Acclimation
of in vitro
date palm plantlets followed the following procedure:
1) covers of cultured jars were
opened, healthy date palm plantlets (5–8 cm long) gently removed and washed
with tap water to remove any residual of traces the medium attached to the
roots, 2) plantlets directly rinsed for three min in 1.0 mg L-1
Ridomil solution as a fungicide, 3) plantlets transferred to plastic pots
filled with peat moss and sand (1:1 v/v), 4) Pots were covered with two layers of
polyethylene, the lower layer was transparent while the upper layer was opaque,
and placed in a greenhouse at 27°C under 16 h with cool-white fluorescence
tubular lamp 50 μmol m−2 s−1and watered
with freshly modifed Hoagland nutrient solution (Cramer et al. 1986), 5) after
two weeks, the opaque covers were removed, and the transparent cover was
punctured to reduce the humidity, 6) after a further four weeks the transparent
cover was removed and ten weeks later percentage of survived transplants (%)
was recorded.
Statistical analysis
All experiments were arranged in a Randomized Complete
Block Design (RCBD). Analysis of variance
(ANOVA) and the calculation of LSD or Duncan's Mulitple Range test (0.005) was
undertaken using CoStat software programme, version no. 6.4. All values were reported as means ± standard error
according to Snedecor and Cochran (1989).
Results
Effect of PGRs
With the exception of the
medium with 10 mg L-1 2, 4-D
0.0 mg L-1 2iP treatment all media hormone combinations induced
callus in both cultivars (Safawi and Magdoul) of date palm (Table 1). However
not all hormone combinations were equally effective and the highest induction
rates (89.30%; 91.50%) were achieved with 25 mg
L-1 2, 4-D and 5 mg L-1
2iP for cv. Safawi and 10 mg L-1
2, 4-D and 8 mg L-1 2iP for cv. Magdoul,
respectively, after incubation for 12 months under dark regimes compared
with other treatments (Fig. 1). These hormone combinations were
also amongst the highest for Relative Water Content and Shoot Induction. Also,
the optimum rate (+++) of shoot regeneration was recorded of cv. Safawi at 25
mg L-1 2, 4-D with 5 or 3 mg L-1 2iP and 10 mg L-1
2, 4-D with 8 mg L-1 2iP, while in cv. Magdoul 10
mg L-1 2, 4-D with 6 or 8 mg L-1 2iP and 100 mg L-1
2, 4-D with 0.0 mg L-1 2iP were more efficiency
(+++) comparing with other treatments (Table 1). In contrast, MS medium
supplemented with 75 mg L-1 2, 4-D and 2.0 mg L-1
2iP were less effective of callus induction percentage (17.05%; 14.35%) in cvs.
Safawi and Magdoul, repectively. On the other hand, less relative water content
percentage (36.28%; 30.05%) was recorded under MS medium supplemented with 25
mg L-1 2, 4-D and 50 mg L-1 in the absent of 2iP of cvs.
Safawi and Magdoul, repectively. It is noted that no shoot regeneration growths
were recorded either when 2-4-D applied alone at 10, 25, 50 and 75 mg L-1
without adding hormone 2iP in both cultivars, or when it applied at 100 mg L-1
with different concentration of 2iP in cv. Safawi.
Somatic embryogenesis
The efficiency of somatic embryogenesis from callus
samples explants was evaluated on MS basal medium containing different
concentrations of 2iP alone or incombination with different concentration of
NAA as described above in material and methods section. The
results indiacted the somatic embryo (SE) formation was achieved in all media
hormone combinations but ranged from 28% to 96% (Table 2). There was a strong
correlation between the proportion of callus forming SE’s and the number of
SE’s induced. The best hormone combinations differed between the 2 varieties
with 6 mg L-1 2iP
+ 1 mg L-1 NAA being the best for cv. Safawi and 6
mg L-1 2iP + 3
mg L-1 NAA being best for cv. Magdoul (Fig. 2).
Somatic embryos germination and development of plantlets
All media hormone combinations showed the ability to promote
SE germination (%) and the production of shoots (no.) and length of shoot (cm),
the value ranged from 32% to 92%, 1.50 to 12.6 and 0.9 cm to 3.89 cm,
repectively (Table 3). There was a
strong correlation between germination % and the mean number of shoots produced
and the shoot length such that the best media also gave the highest number of
shoots and the tallest plantlets. For the cv. Safawi the best medium contained 6 mg L-1 2iP + 3 mg L-1 Kin + 0.5 mg L-1 IBA,
whilst for cv.
Table 1: The effect of different combination of 2,4-D and 2iP as
a growth regulators on callus induction (%), relative water content (%) and adventitious shoot
regeneration response of the date palm cvs. Safawi and Magdoul using shoot tip section explants after 12 months
|
Shoot regeneration |
Relative water content (%) |
Callus induction (%) |
Growth regulators (mg L-1) |
||||
|
Magdoul |
Safawi |
Magdoul |
Safawi |
Magdoul |
Safawi |
2iP |
2,4-D |
|
- |
- |
0.0 ± 0.0 |
0.0 ± 0.0 |
0.0 ± 0.0 |
0.0 ± 0.0 |
0.0 |
10.0 |
|
- |
- |
36.28 ± 0.43f |
25.38 ± 0.92h |
16.53 ± 0.36i |
20.40 ± 0.27j |
0.0 |
25.0 |
|
- |
- |
30.05 ± 0.22f |
28.42 ± 0.57g |
12.45 ± 0.22i |
25.30 ± 0.23j |
0.0 |
50.0 |
|
+ |
- |
42.25 ± 0.19e |
38.09 ± 0.72f |
25.15 ± 0.57g |
45.30 ± 0.11h |
0.0 |
75.0 |
|
+++ |
+ |
74.18 ± 0.67b |
41.21 ± 0.77e |
79.04 ± 0.62b |
65.43 ± 0.32f |
0.0 |
100.0 |
|
++ |
++ |
50.40 ± 0.49d |
46.05 ± 0.59e |
40.18 ± 0.56e |
55.22 ± 0.74g |
2.0 |
10.0 |
|
++ |
+ |
52.45 ± 0.81d |
48.30 ± 0.29e |
45.20 ± 0.59e |
57.02 ± 0.44g |
3.0 |
10.0 |
|
++ |
+ |
54.55 ± 0.19d |
52.21 ± 0.50d |
65.50 ± 0.74c |
62.09 ± 0.84f |
5.0 |
10.0 |
|
+++ |
++ |
77.05 ± 0.83b |
54.10 ± 0.47d |
83.22 ± 0.38b |
79.10 ± 0.48b |
6.0 |
10.0 |
|
+++ |
+++ |
79.90 ± 0.37a |
56.45 ± 0.18d |
91.50 ± 0.39a |
82.15 ± 0.12b |
8.0 |
10.0 |
|
+ |
++ |
68.18 ± 0.52c |
65.38 ± 0.38c |
72.34 ± 0.85b |
78.25 ± 0.53c |
2.0 |
25.0 |
|
++ |
+++ |
69.25 ± 0.69c |
69.29 ± 0.66b |
74.45 ± 0.58b |
80.34 ± 0.35b |
3.0 |
25.0 |
|
++ |
+++ |
55.40 ± 0.72d |
71.45 ± 0.39a |
78.06 ± 0.36b |
89.30 ± 0.53a |
5.0 |
25.0 |
|
+ |
++ |
58.34 ± 0.39d |
70.05 ± 0.57b |
77.18 ± 0.49b |
77.19 ± 0.66c |
6.0 |
25.0 |
|
+ |
++ |
69.62 ± 0.15c |
68.54 ± 0.91c |
75.02 ± 0.33b |
74.22 ± 0.78d |
8.0 |
25.0 |
|
+ |
+ |
62.55 ± 0.77c |
59.55 ± 0.68d |
76.54 ± 0.55b |
72.06 ± 0.47e |
2.0 |
50.0 |
|
+ |
+ |
60.20 ± 0.59c |
58.30 ± 0.48d |
70.04 ± 0.96b |
66.76 ± 0.23f |
3.0 |
50.0 |
|
+ |
+ |
64.19 ± 0.71c |
61.23 ± 0.55c |
63.20 ± 0.78c |
52.22 ± 0.43g |
5.0 |
50.0 |
|
+ |
+ |
66.05 ± 0.82c |
63.05 ± 0.48c |
61.30 ± 0.28c |
50.19 ± 0.65g |
6.0 |
50.0 |
|
+ |
+ |
67.08 ± 0.60c |
65.20 ± 0.58c |
48.65 ± 0.33e |
44.03 ± 0.67h |
8.0 |
50.0 |
|
+ |
+ |
32.20 ± 0.71f |
28.28 ± 0.82g |
14.35 ± 0.43i |
17.05 ± 0.34k |
2.0 |
75.0 |
|
+ |
+ |
40.25 ± 0.53e |
39.50 ± 0.62f |
19.52 ± 0.35h |
28.33 ± 0.21j |
3.0 |
75.0 |
|
+ |
+ |
44.32 ± 0.61e |
45.03 ± 0.57e |
29.18 ± 0.84g |
32.25 ± 0.72i |
5.0 |
75.0 |
|
+ |
+ |
46.05 ± 0.67e |
47.15 ± 0.45e |
38.54 ± 0.76f |
42.16 ± 0.45h |
6.0 |
75.0 |
|
+ |
+ |
49.30 ± 0.80e |
48.08 ± 0.22e |
43.25 ± 0.87e |
49.15 ± 0.55h |
8.0 |
75.0 |
|
++ |
- |
50.52 ± 0.25d |
28.20 ± 0.75g |
66.15 ± 0.68c |
72.55 ± 0.76d |
2.0 |
100.0 |
|
++ |
- |
60.08 ± 0.61c |
52.04 ± 0.39d |
68.05 ± 0.26c |
59.20 ± 0.35g |
3.0 |
100.0 |
|
++ |
- |
58.05 ± 0.29d |
50.28 ± 0.31d |
62.30 ± 0.63c |
53.40 ± 0.88g |
5.0 |
100.0 |
|
++ |
- |
62.42 ± 0.33c |
58.02 ± 0.94d |
52.22 ± 0.46d |
44.52 ± 0.67h |
6.0 |
100.0 |
|
++ |
- |
69.50 ± 0.49c |
59.55 ± 0.88d |
49.16 ± 0.33e |
43.30 ± 0.38h |
8.0 |
100.0 |
Values are means ± standard error of three
replicates from two experiments. For each cultivar, bars with the same letters
are not significantly different at P
≤ 0.05 level. Shoot regeneration are visually estimated as No
regeneration= -, Poor= +, Good= ++, very good= +++
Table 2: Influence
of plant growth regulators concentrations on percentage of somatic embryos
formation /explant and average number of somatic embryos / 1.0 g FW of
callus of the date
palm cvs. Safawi and Magdoul after 10 weeks
|
Growth regulators (mg L-1) |
Percentage of somatic embryos
formation/explant |
Average number of somatic
embryos/1.0 g FW of callus |
|||
|
2iP |
NAA |
Safawi |
Magdoul |
Safawi |
Magdoul |
|
1.0 |
0.5 |
28.65 ± 0.52j |
32.25 ± 0.85h |
210.75 ± 0.55j |
130.40 ± 0.67j |
|
2.0 |
0.5 |
42.49 ± 0.65h |
39.35 ± 0.37h |
338.25 ± 0.29h |
180.95 ± 0.39i |
|
4.0 |
0.5 |
48.35 ± 0.59h |
42.25 ± 0.59g |
340.20 ± 0.62g |
190.00 ± 0.53i |
|
6.0 |
0.5 |
52.70 ± 0.25g |
52.15 ± 0.27f |
350.00 ± 0.18g |
195.25 ± 0.77i |
|
8.0 |
0.5 |
59.25 ± 0.22g |
55.25 ± 0.55f |
352.88 ± 0.51g |
220.35 ± 0.49h |
|
1.0 |
1.0 |
65.90 ± 0.35f |
45.28 ± 0.68g |
378.15 ± 0.88f |
310.00 ± 0.92g |
|
2.0 |
1.0 |
82.50 ± 0.92d |
62.14 ± 0.33e |
420.15 ± 0.90d |
330.25 ± 0.33g |
|
4.0 |
1.0 |
90.45 ± 0.58c |
66.25 ± 0.69e |
432.70 ± 0.95c |
345.85 ± 0.79g |
|
6.0 |
1.0 |
96.18 ± 0.15a |
72.25 ± 0.49d |
465.25 ± 0.38a |
482.30 ± 0.17d |
|
8.0 |
1.0 |
92.65 ± 0.59b |
70.25 ± 0.22d |
438.40 ± 0.62b |
489.35 ± 0.31d |
|
1.0 |
2.0 |
35.22 ± 0.77i |
38.18 ± 0.19h |
310.18 ± 0.44i |
280.87 ± 0.55h |
|
2.0 |
2.0 |
38.40 ± 0.63i |
40.00 ± 0.25g |
328.00 ± 0.29h |
258.14 ± 0.39h |
|
4.0 |
2.0 |
41.25 ± 0.81h |
51.35 ± 0.50f |
325.14 ± 0.35h |
320.48 ± 0.73g |
|
6.0 |
2.0 |
48.55 ± 0.39h |
52.00 ± 0.58f |
339.85 ± 0.77g |
389.15 ± 0.65f |
|
8.0 |
2.0 |
50.25 ± 0.25g |
55.40 ± 0.90f |
348.45 ± 0.12g |
395.25 ± 0.45f |
|
1.0 |
3.0 |
39.28 ± 0.44i |
68.45 ± 0.88e |
350.00 ± 0.52g |
402.27 ± 0.28f |
|
2.0 |
3.0 |
43.58 ± 0.96h |
74.35 ± 0.42d |
338.25 ± 0.78g |
480.50 ± 0.69d |
|
4.0 |
3.0 |
62.35 ± 0.19f |
88.25 ± 0.17c |
371.25 ± 0.38f |
558.75 ± 0.75b |
|
6.0 |
3.0 |
73.10 ± 0.33e |
92.25 ± 0.56a |
410.22 ± 0.29e |
568.25 ± 0.50a |
|
8.0 |
3.0 |
75.40 ± 0.71e |
90.35 ± 0.25b |
412.75 ± 0.75e |
542.28 ± 0.88c |
|
1.0 |
4.0 |
55.30 ± 0.88g |
67.28 ± 0.91e |
349.55 ± 0.39g |
335.12 ± 0.29g |
|
2.0 |
4.0 |
68.25 ± 0.39f |
69.35 ± 0.15e |
380.65 ± 0.45f |
363.70 ± 0.91g |
|
4.0 |
4.0 |
48.30 ± 0.47h |
55.65 ± 0.72f |
338.25 ± 0.92h |
280.25 ± 0.78h |
|
6.0 |
4.0 |
60.15 ± 0.66f |
68.25 ± 0.49e |
370.25 ± 0.20f |
465.25 ± 0.68e |
|
8.0 |
4.0 |
69.25 ± 0.32f |
69.75 ± 0.33e |
382.39 ± 0.89f |
471.15 ± 0.60e |
Values
are means ± standard error of three replicates from two experiments. For each
cultivar, bars with the same letters are not significantly different at P ≤ 0.05 level
Table 3: Influence of plant growth regulators
concentrations on somatic embryos germination percentage, average number of
shoots formation and average shoots length of the date palm Safawi and Magdoul after 2 months
|
Growth regulators (mg L-1) |
Somatic embryos germination (% )
(germinated/embryo s tested) |
Mean number of shoots formation/(1.0 g callus) |
Mean length of
shoots/explant (cm) |
|||||
|
2iP |
Kin |
IBA |
Safawi |
Magdoul |
Safawi |
Magdoul |
Safawi |
Magdoul |
|
2.0 |
2.0 |
0.0 |
32.35 ± 0.26i |
36.25 ± 0.18g |
1.50 ± 0.29h |
2.75 ± 0.98h |
0.95 ± 0.19f |
1.04 ± 0.28g |
|
4.0 |
2.0 |
0.0 |
38.25 ± 0.59h |
36.85 ± 0.22g |
1.65 ± 0.78h |
2.82 ± 0.73h |
1.05 ± 0.26e |
1.38 ± 0.66g |
|
6.0 |
2.0 |
0.0 |
40.15 ± 0.44g |
55.29 ± 0.75e |
2.25 ± 0.66g |
3.70 ± 0.48g |
1.69 ± 0.34d |
2.84 ± 0.43c |
|
2.0 |
3.0 |
0.0 |
38.28 ± 0.78h |
36.45 ± 0.55g |
1.58 ± 0.57h |
2.78 ± 0.22h |
0.98 ± 0.26f |
1.69 ± 0.36f |
|
4.0 |
3.0 |
0.0 |
38.75 ± 0.33h |
38.65 ± 0.92g |
2.59 ± 0.48g |
3.65 ± 0.67g |
1.45 ± 0.15e |
1.72 ± 0.76e |
|
6.0 |
3.0 |
0.0 |
59.85 ± 0.29e |
69.55 ± 0.63d |
2.85 ± 0.19g |
3.90 ± 0.50g |
1.72 ± 0.66d |
2.95 ± 0.92b |
|
2.0 |
2.0 |
0.5 |
37.38 ± 0.93h |
40.25 ± 0.66f |
2.95 ± 0.33g |
3.45 ± 0.18g |
1.08 ± 0.35e |
1.49 ± 0.47f |
|
4.0 |
2.0 |
0.5 |
40.48 ± 0.49g |
45.39 ± 0.49f |
3.65 ± 0.92f |
5.25 ± 0.55f |
1.63 ± 0.41d |
1.68 ± 0.22f |
|
6.0 |
2.0 |
0.5 |
63.25 ± 0.77d |
81.40 ± 0.84b |
4.49 ± 0.85e |
6.87 ± 0.72e |
2.75 ± 0.84b |
2.86 ± 0.46c |
|
2.0 |
3.0 |
0.5 |
46.29 ± 0.58f |
40.69 ± 0.99f |
4.15 ± 0.77e |
5.89 ± 0.33f |
1.15 ± 0.55e |
1.75 ± 0.74e |
|
4.0 |
3.0 |
0.5 |
78.55 ± 0.99c |
69.75 ± 0.29d |
6.25 ± 0.86c |
8.58 ± 0.91d |
1.55 ± 0.29d |
1.89 ± 0.36e |
|
6.0 |
3.0 |
0.5 |
90.45 ± 0.52a |
76.55 ± 0.26c |
8.45 ± 0.39a |
9.75 ± 0.27c |
2.95 ± 0.39a |
3.05 ± 0.73b |
|
2.0 |
2.0 |
1.0 |
55.25 ± 0.26e |
57.45 ± 0.82e |
3.67 ± 0.38f |
5.39 ± 0.49f |
1.38 ± 0.62e |
2.15 ± 0.55d |
|
4.0 |
2.0 |
1.0 |
56.85 ± 0.88e |
59.44 ± 0.78e |
5.90 ± 0.27d |
8.25 ± 0.44d |
2.15 ± 0.91c |
2.93 ± 0.62b |
|
6.0 |
2.0 |
1.0 |
75.45 ± 0.26c |
92.55 ± 0.65a |
6.35 ± 0.45c |
12.65 ± 0.62a |
2.92 ± 0.49a |
3.89 ± 0.39a |
|
2.0 |
3.0 |
1.0 |
57.39 ± 0.49e |
69.15 ± 0.16d |
4.28 ± 0.31e |
5.91 ± 0.15f |
1.25 ± 0.77e |
1.78 ± 0.96e |
|
4.0 |
3.0 |
1.0 |
73.68 ± 0.79c |
79.38 ± 0.39c |
6.69 ± 0.95c |
9.72 ± 0.88c |
2.05 ± 0.82c |
1.92 ± 0.64e |
|
6.0 |
3.0 |
1.0 |
85.15 ± 0.60b |
89.40 ± 0.56b |
7.35 ± 0.44b |
10.40 ± 0.48b |
2.78 ± 0.96b |
3.82 ± 0.38a |
Values
are means ± standard error of three replicates from two experiments. For each
cultivar, bars with the same letters are not significantly different at P ≤ 0.05 level.
%201745-1753_files/image002.png)
Fig. 1: In
vitro callus induction from pieces of
shoot tips of date palm cvs. Safawi and Magdoul. A) Induction of
callus on
MS medium supplemented with 25 mg L-1
2,4-D and
5.0 mg L-1
2iP in the presence
of 2.5 g L-1 activated charcoal of cv. Safawi after 12 months. B) Induction of
callus on
MS medium supplemented with10 mg L-1
2,4-D and 8.0 mg L-1
2iP of cv. Magdoul after 12 months
%201745-1753_files/image004.png)
Fig. 2: Somatic embryogenesis formed
from callus
cultured on MS containing with
6.0 mg L-1 2iP and 1.0 mg L-1 NAA of cvs. Safawi and Magdoul (A)
and 6.0 mg L-1 2iP and 3.0 mg L-1 NAA of cvs. Safawi and Magdoul cultivar in the presence
of 2.5 g L-1 activated charcoal after 10 weeks (B). Somatic embryos germinated on MS medium supplemented with 6.0 mg L-1 2iP, 3.0 mg L-1 Kin and 0.5 mg L-1 IBA of cv. Safawi (C) and 6.0 mg L-1 2iP and 2.0 mg L-1 Kin and 1.0 mg L-1 IBA of cv. Magdoul in the presence of 2.5 g L-1 activated charcoal after 2 months (D)
%201745-1753_files/image006.png)
Fig. 3: Plantlet obtained from a converted somatic
embryo of date palm cvs. Safawi and Magdoul on 6.0 mg L-1 2iP and 2.0 mg L-1 Kin and 1.0 mg L-1 IBA in the presence of 2.5 g L-1 activated charcoal under light after six weeks (A) and after eight weeks (B).
Development of roots obtained on MS medium supplemented with 2.0 mg L-1 NAA and 0.5 mg L-1 IBA of cv. Safawi and 2.5 mg L-1 NAA of cv. Magdoul in the presence of 2.5 g L-1 activated charcoal (C). Plants after acclimatization to free living
conditions (D)Magdoul the best medium
contained 6 mg L-1 2iP + 2 mg L-1 Kin
+ 1 mg L-1 IBA (Fig. 3A and B). The highest value of SE germination (%) and the
production of shoots (no.) and length of shoot (cm) was recorded of cv. Magdoul comparing with cv. Safawi (Table
3).
%201745-1753_files/image008.png)
Fig. 4: Effect of MS medium supplemented with different
concentrations of auxins; NAA and IBA on the a) Percentage of shoot forming roots, b) mean number of roots/shoot (no.), c) mean length of roots/shoot (cm) of date palm cvs. Safawi and Magdoul after 4 weeks. Tr.1,
Tr.2, Tr.3, and Tr.4 = 1, 1.5, 2.0 and 2.5 mg L-1 NAA, respectively;
Tr.5 = 1 mg L-1 IAA and 0.5 mg L-1 IBA, Tr.6 = 1.5 mg L-1 IAA
and 0.5 mg L-1 IBA, Tr.7 = 2 mg L-1 IAA
and 0.5 mg L-1 IBA, Tr.8 = 2.5 mg L-1
IAA and 0.5 mg L-1 IBA, Tr.8 = 1 mg L-1 IAA
and 1 mg L-1 IBA, Tr.10 = 1.5 mg L-1 IAA and 1.0 mg L-1 IBA, Tr.11 = 2 mg L-1 IAA and 1.0 mg L-1 IBA, Tr.12 = 2.5 mg L-1 IAA and 1 mg L-1 IBA, Tr.13 = 1 mg L-1 IAA and 1.5 mg L-1 IBA, Tr.14 = 1.5 mg L-1
IAA and 1.5 mg L-1 IBA, Tr.15 = 2 mg L-1 IAA
and 1.5 mg L-1 IBA, Tr.16 = 2.5 mg L-1 IAA and 1.5 mg L-1 IBA
Rooting formation and acclimatization
of regenerated plantlets
In this investigation
isolated single shoots of cvs. Safawi and Magdoul were maintained in the
dark for seven days following with incubation under 16 day/ 8 night photoperiod
for three weeks to induce rooting as clarified in Fig. (3C). All media hormone
combinations were capable of the induction of roots in shoots obtained from SE
but they were not all equally effective with the proportion of shoots forming
roots ranging from 38% to 98% (Fig. 4). For the cv. Safawi the best
medium was 6 mg L-1 NAA + 0.5 mg
L-1 IBA and for cv. Magdoul the best was 2.5 mg L-1 NAA + 0 mg L-1 IBA (Fig. 4). The data showed that the highest rooting percentage
(96.72%) were obtained on MS medium supplemented with 2.0 mg L-1 NAA and 0.5 mg L-1 IBA in the present of 2.5 g L-1 activated charcoal. Also, under this treatment the
highest significant differences were observed of mean number of roots/shoot (6.85) and mean length of roots/shoot (5.94 cm) of cv. Safawi compared with other
treatments.
While, the highest shoot forming roots percentage (98.25%), the highest mean number of roots/shoot (7.45) and mean length of roots/shoot (6.25 cm) of cv. Magdoul was obtained on MS medium supplemented with 2.5 mg L-1 NAA in the present 2.5 g L-1 activated charcoal
compared with other treatments (Fig. 3C).
In vitro plantlets with well-formed
roots were transplanted to a sand: soil media, then
placed back in the same growth chamber, for primary hardened, for 8 weeks and
then transferred to the greenhouse. After 6–8 months new leaves had developed (Fig.
3D). During the acclimatization period, the results indicated that 55–70% of
plantlets successfully survived and all had survived two months later. Plants
with healthy growth were then transferred to the open field and both cvs. Safawi
and Magdoul Plants gave showed a good normal growth in soil and appeared
phenotypically normal.
Discussion
The results clearly demonstrated that 25 mg L-1
2, 4-D with 3.0 mg L-1 2iP and 10 mg L-1
2, 4-D with 8 mg L-1 2iP gave high rates of
callus induction with high relative water content for varieties Safawi and
Magdoul, respectively (Table 1). It is
not surprising that the two varieties had different optimal media hormone
combinations as it has been shown by Arimarsetiowati and Prastowo (2020) that different
responses of induction of callus and formation of embryogenic calli can be
induced by different 2, 4-D and 2iP concentration ratios. Such differences are
attributed to genetic factors among cultivars.
Ozias–Akins and Vasil (1982) showed that low levels of 2, 4-D stimulate the division of cells but increasing the level
beyond 2 mg L-1 can inhibit cell division in wheat. Furthermore,
treating callus cells with high concentrations of 2, 4-D may lead to increased cytoplasm concentration with a
consequent significant reduction in relative water content acting as an induced
osmotic stress (Pan et al. 2010). Under
high 2, 4-D the protein DRT102 which plays
an important role in DNA replication and cell division my be suppressed (Pasternak et al. 2002) and lead to the decreased fresh weight and total
cell number (Ozias–Akins and Vasil
1982). Also, it is proven that adding auxin (e.g., 2, 4-D) to artficial growth medium stimulates the cell to
increase cells division and discourages the process of differentiation. Thus,
this contributes to the orientation of the cell to induce diverse developmental
processes such as somatic embrygenesis and established root induction (Aderkas and Bonga 2000). The difference between
cultivars in optimum induction medium was in agreement with Sané et al. (2012) who also reported a
difference in the rate of callus formation for different cultivars of palm.
Furthermore, Gonzalez et al. (2001) and Rathore et al. (2020)
also reported a strong dependence of callus induction on genotype and induction
media used.
Percentage of
somatic embryos formation/explant and average number
of somatic embryos per 1.0 g FW of callus showed a varied response in the palm cultivars studied.
The presence of 2iP at higher concentration (6.0 mg L-1) in
combination with 1.0 or 3.0 mg L-1 NAA gave the best results. While
under low concentration of 2iP (1.0, 2.0 and 4.0 mg L-1) induction
was significantly lower (Table 2). This
indicated that somatic embryoegensis required a high concentration of
cytokinins and a low or medium concentration of auxin. Similar results have
been recently reported in Digitalis lanata Ehrh (Bhusare et al. 2020).
Removal of 2, 4-D from the somatic embryogensis medium results in a
lowering of amounts of endogenous antioxidants (glutathione, ascorbic acid,
vitamin E), which stimulates somatic embryo development an hypothesis was
purported by Aderkas and Bonga (2000).
The different concentrations of 2iP alone or in
combination with Kin or IBA were highly effective for somatic embryo
germination and plantlet development in both varieties studied (Table 3). In
most plant species, the success of plant tissue culture in vitro depends
on the combinations between auxin and cytokinin. While both are essential to
promote and control cell division, only the cytokinins stimulate the formation
of the shoot system whilst auxins stimulate the formation of the root system (Meziani et al. 2019). The differential
ability of cytokinins in induction of shoots is frequently attributed to
factors such as stability, mobility, and the rate of conjugation and oxidation
of the hormones (D’Onofrio and Morini 2005). In this study significant
differences were found between the cultivars under the same hormone treatments,
for example a concentration of 6.0 mg L-1 2iP plus 3.0 mg L-1
and 0.5 mg L-1 Kin recorded the highest value of somatic embryos
germination % (90.45 ± 0.52) in cv. Safawi, but for cv. Magdoul germination % was
only 76.55 ± 0.26. This can
be explained on the basis of the different genotypes giving rise to differences
in their endogenous hormone contents. Thus the accumulation and sensitivity to
hormone concentrations in media could explain the genotypic variations of
embryogenic potential (Hadrami et al. 1995; Hadi et al. 2015).
The stage of root formation is the last and critically
important stage in the tissue culture procedure and is a key step in
micropropagation because without it plants cannot be weaned and develop in vivo
status (Klerk et al. 1997). To
obtain a high percentage of rooted shoots, many studies indicate the importance
of exogenous auxins such asIAA, IBA and NAA (Abdelaziz
et al. 2019; Kumar et al. 2020). In our study combinations
of NAA and IBA had a positive impact on inducing root, mean number of
roots/shoot and mean length of root/shoot (Fig. 4). The study found thatNAA at
2.5 or 2.0 mg L-1 in combination with low concentrations of IBA at
0.0 or 0.5 mg L-1 was the best concentration to induce roots (96.72 ± 0.91; 98.25 ± 0.84), number of roots/shoot (6.85 ± 0.25; 7.45 ± 0.52) and mean
length of root/shoot (5.94 ± 0.75; 6.25 ± 0.62) of
cvs. Safawi and Magdoul, respectively. Higher
auxin concentrations has been linked to the production of ethylene and degradative
metabolites in tissues which impede root formation. Sanchez et al. (2020) also noted that
rooting is promoted at low auxin concentrations and inhibited at high
(supraoptimal) concentrations with Iraca palm (Carludovica palmata Ruíz & Pavón). It is worth noting that IBA
is more commonly used to induce root compared to IAA and NAA, but in the
current study, NAA has been used because: 1) it is considered best in the case
of plants that have a high auxin-oxidase activity, 2) it is stable and persistent
in the tissue in it is free form, 3) it is taken up faster and 4) low
concentrations have been shown to be efficient to produce roots in other
species (Smulders et al. 1990). Plantlet acclimatization was
successfully achieved with a good survival rate of 55–70% which is similar to
other studies on date palm (Mazri and Meziani 2015). Indeed the survival rate
in previous studies of date palm recorded 50% (Mazri 2015), 60 % (Kurup et
al. 2014), 60–80% (Othmani et al. 2009), 72–84% (Al-Khayri 2010) and
92% (Meziani et al. 2015). This variation in the survival rate (minimum
of 50% and maximum rate of 92%) has not been discussed before, but the
explanation is probably due to variations in genotypes studied, the method of
acclimatization followed and the micropropagation technique used.
Conclusion
The present study provides a new optimised protocol of in vitro propagation of two
high quality popularly grown Saudi Arabian date palm (Phoenix dactylifera L.) This protocol will be useful for continous production of
somatic embryos/plantlets for ex vitro transplantation and genetic
conservation via in vitro germplasm bank for those cultivars. The approach used
here with the systematic variation in hormone combinations is also a sensible
approach for optimization with other elite date palm cultivars.
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,
carried all the experiment lab work and collected the data, OAA wrote the
literature review and interpreted the results, EMRM, HIAS and MPF made
statistically analyzed of the data and made illustrations and MPF made the language editing.
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