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Commercial production of tissue culture date palm (Phoenix dactylifera L.) by inflorescence technique

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Abstract

Date Palm (Phoenix dactylifera L.) is a salt and drought tolerant fruit crop mainly cultivated in Arabian countries. Shoot tip explants of offshoots were used traditionally for various micropropagation protocols on research or commercial levels. However, its main disadvantage was the scarification of the entire plant. Subsequently, this hinders the micropropagation of male and female recalcitrant individuals with no offshoots or the interesting cultivars with a limited population. Consequently, current study is investigating the factors affecting commercial production of tissue cultured palms with cost-effectively and short-production cycle, which may also strongly enhance the transformation protocols. The innovative way by which the 15 cm long immature inflorescence was excised before emergence between fronds is reported herewith this study for the first time. Established spikelet explants were able within 2-3 months only without any callus phase to produce shining globular structures instead of immature florets. These embryogenic structures couldn't develop further without maturation process for 1-2 months under full darkness as well. Subsequently, well-matured embryogenic structures were shifted to the subsequent differentiation medium under illumination conditions. After then, green shoots and multiple somatic embryos have been subjected to the multiplication stage, then rooting and eventually successfully transplanted in the greenhouse. All used nutrient media and their sequential usage is reported in this study by which it became possible from one inflorescence to produce 10000 plantlets in rooting stage right now of Gulistan Pakistani cultivar without any bad consequences on the mother tree.
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Personal non-commercial use only. JGEB copyright © 2010. All rights reserved
ORIGINAL ARTICLE JGEB Vol. 8, No. 2, 2010
ISSN:1687-157X
Commercial Production of Tissue Culture Date Palm (Phoenix dactylifera L.) by
Inorescence Technique
Adel Ahmed Abul-Soad and Shaimaa Mohamed Mahdi
9 Cairo Univ. St., Orman, Horticulture Research Institute,Agricultural Research Center, Cairo, Egypt.
ABSTRACT
Date Palm (Phoenix dactylifera L.) is a salt and drought tolerant fruit crop mainly cultivated in Arabian countries. Shoot
tip explants of offshoots were used traditionally for various micropropagation protocols on research or commercial levels.
However, its main disadvantage was the scarication of the entire plant. Subsequently, this hinders the micropropagation
of male and female recalcitrant individuals with no offshoots or the interesting cultivars with a limited population.
Consequently, current study is investigating the factors affecting commercial production of tissue cultured palms with
cost-effectively and short-production cycle, which may also strongly enhance the transformation protocols. The innovative
way by which the 15 cm long immature inorescence was excised before emergence between fronds is reported herewith
this study for the rst time. Established spikelet explants were able within 2-3 months only without any callus phase
to produce shining globular structures instead of immature orets. These embryogenic structures couldn’t develop
further without maturation process for 1-2 months under full darkness as well. Subsequently, well-matured embryogenic
structures were shifted to the subsequent differentiation medium under illumination conditions. After then, green shoots
and multiple somatic embryos have been subjected to the multiplication stage, then rooting and eventually successfully
transplanted in the greenhouse. All used nutrient media and their sequential usage is reported in this study by which
it became possible from one inorescence to produce 10000 plantlets in rooting stage right now of Gulistan Pakistani
cultivar without any bad consequences on the mother tree.
Journal of Genetic Engineering and Biotechnology, 2010, 8(2): 39-44
Key Words: Date palm, inorescence, micropropagation. Corresponding Author: Adel Ahmed Abul-Soad
E-mail: adelaboelsoaud@gmail.com
INTRODUCTION
Date Palm (Phoenix dactylifera L.) is a dioecious,
perennial monocot plant species of the Arecaceae family.
It is one of the oldest fruit crops mainly cultivated in North
Africa and Middle East countries. Shoot tip explants of the
offshoots traditionally used for various micropropagation
protocols of date palm on the research and commercial
levels together (Abul-Soad et al. 2002a,b, Abul-Soad
et al. 1999, 2004, El-Hadrami and Baaziz 1995 and
Tisserat 1984b). Furthermore, other possible explants
were tested and proved un-successful (Tisserat 1984a).
However, the inorescence explants proved promising
and the required alternative explant for micropropagation
of elite cultivars and rare male and female individuals
of date palm (Abahmane et al. 1999, Abul-Soad 2003,
07a, Abul-Soad et al. 2004, 05, 07, 08, Bhaskaran and
Smith 1992, El-Korchi 2007 and Feki and Drira 2007).
Nevertheless, no any data was reported for commercial
production of date palm based on the inorescence
explant. Handling pilot production is not easy task and
many factors should be taken. On the practical side, the
excision of immature inorescence from the mother
tree, composition of starting medium during initial stage,
sequential handling of different nutrient media remained
the most decisive three factors could interfere the real
success of inorescence technique. Current study is
investigating the above mentioned factors and discussing
the commercial production business. A unique commercial
trial has been applied at Date Palm Research Institute,
Khairpur, Pakistan. The success have been done by Arab
scientist is strongly recommended to be applied in one
or more of Arab countries. It is expected that by which
a huge number of elite Arabian cultivars that t good for
processing industry (Abul-Soad 2007b) in addition to
recalcitrant male and female individuals can be produced
in short time and less effort. Furthermore, an example of
how and why micropropagation by inorescence technique
is being used will be presented.
MATERIALS AND METHODS
This work was carried out in the Biotechnology Lab.
of Date Palm Research Institute, Shah Abdul Latif
40
Adel Ahmed and Shaimaa Mohamed
University, Khairpur, Sindh, Pakistan in 2008 - 2010. The
protocol was done as under:
1. The immature inorescence was excised from the
mother tree of Gulistan cv. at D. I. Khan area, Pakistan
in early spring.
2. The excised inorescence was kept in clean plastic
cover and handled carefully from an open eld to the
laboratory. In current case of far away places of source
materials (spathes), it could be conserved for 1-2 days
in ice box. It is observed that this process hasn’t adverse
impact on the survivability of transferred spathes from
D. I. Khan area which is far 600 Kilometers away from
Khairpur.
3. Once the whole inorescence (spathe) has been brought
to the lab, the real work was quickly started and the
whole inorescence was subjected to the surface
sterilization procedure.
4. The intact spathe was dipped into fungicide solution for
30 seconds only without any shaking. The 2 grams l-1
Topsin M 70 (the systematic fungicide) solution was
used.
5. The spathe was carefully handled while washing it
under current tap water for 30-60 seconds only. Water
stream should be focused on the basal part of the spathe
to remove the little dust.
6. 30 % Sodium Hypochlorite (NaOCl) solution (16%)
was used for only 1-2 minutes.
7. The spathe was washed with sterilized distilled water
for 30-60 seconds one time without shaking.
8. Once the spathe is surface sterilized, the outer protective
sheath was longitudinally cut from the middle like T
letter from one side only. Cut may be done in the central-
swelled portion of the spathe due to its softness.
9. Spikelet explants were cut from their base and cultured
intact if they are 3-4 cm in length. Longer spikelet
explants were cut to pieces, each 2-3 cm and laid in
such a way that the entire explant is in contact with the
surface of nutrient medium. Each of which possessed
2-4 immature orets.
10. All cultured explants were incubated in a controlled
growth room at 25 ± 2 ºC under full darkness. Incubated
explants were re-cultured 1-2 times, each was about 3-4
weeks on same starting medium as described in Table (1).
11. Well-responded explants were transferred into
maturation medium for 1-2 re-cultures.
12. Matured and early-differentiated explants under
darkness were shifted onto differentiation medium
under illumination conditions for 1-2 re-cultures.
13. Subsequently the differentiated cultures were shifted
to the multiplication stage or may be the rooting stage
directly.
14. Well-rooted plantlets were subjected to the in vitro
hardening by culturing onto low-nutrients medium
along with more ventilation.
15. Date palm plantlets were successfully transplanted in
the greenhouse.
RESULTS AND DISCUSSION
The decisive factors and basic steps involved in date palm
micropropagation using inorescence explants could be three
factors. These are the method of immature inorescence
excision, the composition of starting-induction medium of
direct organs and the sequence of different nutrient media during
entire protocol. This study has explained these three factors.
Current innovative protocol recommended excision
process of the spathe while it remains beneath the frond’s
base in such a way that the location of young spathe was
successfully estimated. Since decades and every date-palm
tissue culturist in the world was trying to be away from
touching the tree’s head, otherwise it may be fallen down.
The principal is the ower buds that are going to develop
to inorescences which are emerging in reciprocal position
around the head of the tree mostly above the older 4-5
frond whorls. Then the spathes are emerging between the
next 3-4 frond whorls (Abul-Soad 2003). Two innovative
methods that are not done before were tested in current
study. First method is based on a preliminary estimation
of a frond which covers the spathe and select it. Then, the
adjacent 4-5 fronds were technically peeled away. In case
of wrong estimation, operation should be aborted to avoid
any risk on the tree’s head. Only the skilled person can
carry out a second estimation for same tree to get maximum
1-2 spathes. Trial to get more than 2 spathes wasn’t tested.
The second method proved successful as well, through
pruning of outer mature fronds until to reach the rst outer
spathe. In latter method, the number of excised fronds was
about 36-40 mature fronds which lost the tree a paramount
source of food for ongoing season of emerging spathes
compared to rst method. In this regard, bunch thinning
has obligatory been done to reduce the weight on the tree’s
head. The number of fruit bunches which were reduced
are from 15-20 bunches to be 4 bunches only. However, in
the subsequent season the mother tree entirely recovered
and place of excision was almost disappeared. In both
methods, time of excision, cultivar and climatic conditions
particularly temperature are the variable factors that control
the appropriate excision time, i.e. age of the spathe.
In the rst method, the mother tree from which the spathe
was excised lost only one bunch at that year and entirely
41
Commercial production of tissue culture date palm (Phoenix dactylifera L.) by inorescence technique
recovered in the next year. It is worth mentioning that the
wound must be subjected to a post treatment with fungicide
and pesticide to avoid any further infection with diseases or/
and pests particularly Red Palm Weevil (Palm Cancer). Also,
this experiment was tested for 20 trees at different heights
and repeated for 3 consecutive years with no even a single
failure trial (fallen head).
The second factor is the starting medium to induce the
direct organs Table (1). The entire step-wise protocol
was mentioned above in the materials and methods
part. Therefore, one of the worth benets of using the
inorescence technique is the simple sterilization protocol
to get free contamination explants. Spikelet was the only
explant type of whole inorescence that responded well
to the starting nutrient medium Figure (1a). Other tissue
explants failed even to produce callus when cultured onto
callus induction medium (Abul-Soad 2009). This is why
current protocol is using only the spikelets explants.
The sequence of required nutrient media (Third factor)
along with the time schedule of organogenesis from the
well established initial explants till successfully ex vitro
acclimatized plants was as below:
1. Shining globular creamy structures formation was about
2 months through 1-2 re-cultures (Figure 1a).
2. Maturation of initial structures for 2-3 months through
2-3 re-cultures.
3. Differentiation process for 1-2 months through 1-2
subcultures.
4. Proliferation phase of the individual shoots and the
multiple somatic embryos extended up to 13 subcultures
after differentiation (Figure 1b).
5. Rooting stage which can be extended from few months
to 1-2 years depending on the required number of
plantlets simultaneously its health (Figure 2a).
6. Acclimatization stage (Figure 2b).
As a live example, the cultures of inorescence were
multiplying well throughout 13 subcultures done at Date
Palm Research Institute, Khairpur, Pakistan during the
period from 2008-2010. The size of production during this
period is shown in Table 2. After the differentiation process,
three types of cultures were obtained which are embryogenic
callus, somatic embryos and green shoots. Somatic
embryos can be generally divided into two categories. First
category is the individual somatic embryos and second
is a cluster of embryos (multiple embryos). The growth
behavior of the individual embryo is to grow vertically to
produce more leaves and roots while the multiple embryo
is usually proliferating to additional shoots and somatic
embryos which suits the multiplication stage. Some of
these were transferred to the multiplication stage. In the
rst subculture of multiplication stage, 10 jars were having
embryogenic callus, 7 jars with multiple embryos and 4
jars of shoots. All of these were transferred onto the forth
medium of proliferation (Table 1). The embryogenic callus
exposed high morphogenetic potentiality to differentiate
to intact somatic embryos. During this process extra callus
formation was occurred till subculture 7 where the callus
jars increased to 16.
zEach culture vessel (350 ml jar or 250×25 mm long tube)
contained a gram of callus, or 10 embryos or shoots in
average. Each long tube contained an intact plantlet with
shoot-root system.
But gradually the new clones of embryogenic callus
decreased and eventually all callus differentiated to
somatic embryos. This high ability of differentiation can
make from this type of callus the typical targeted tissue in
transformation protocols. Hence the all transgenic cells will
have a big chance to proceed to intact viable plantlets. The
long and complicated regeneration protocol of date palm
is the big hinder of date palm transformation of date palm
(Hassan 2007; Saker and Ghareeb 2007). Furthermore, this
organogenetic potentiality can be used as ideal source for
long-term Cryo-storage. It is reported that Cryo-storage has
an advantage of long-term storage without going through
frequent subcultures and somaclonal variation in compared
to in vitro conservation techniques, cryopreservation and
cold-storage for Gene Bank purposes (Jain 2010).
During multiplication stage some shoots were growing up
and reached to an appropriate height for rooting stage (Abul-
Soad et al. 2006). These were subsequently subjected to the
fth medium Table (1). The shoot jars increased from 4 in
subculture 1, then 40 jars and eventually became 1644 jars
in subculture 7. Each jar maintained 20-30 shoots, 5 of them
at least in the size of rooting stage while 1212 plantlets were
in rooting stage during the period from subculture 7 to 13.
It was achieved from a single inorescence of cv. Gulistan,
10000 healthy plantlets with root-shoot system and partially
gradually transplanted into the greenhouse Figure (2b).
It is quite important to mention that during one century the
population of this recalcitrant Gulistan cv. at D. I. Khan
area was 1-2 thousand trees only due to the limitation
of the new offshoots through the traditional propagation
method. Notwithstanding within only 2 years became
possible to produce that number from one inorescence.
This is telling us the feasibility of using the promising
inorescence technique.
42
Adel Ahmed and Shaimaa Mohamed
Table 1: Nutrient media composition for inorescence protocol and its sequence.
Medium Composition (mg l-1)
Salts Additives Auxins Cytokinins
1. Starting Macro of B5z + Micro
of MSy
30000 Suc.x + 2200 Agar + 1400 Gel + Vit.v of MS + 170
KH2PO4 + 100 Glutamine + 40 Ad.u
0.1 2.4-D + 0.1
IAA + 5.0 NAA -
2. Maturation Macro of B5 + Micro
of MS
30000 Suc. + 2200 Agar + 1400 Gel + Vit. of MS + 170 KH2PO4
+ 100 Glutamine + 40 Ad. + 1500.0 ACw 5.0 2,4-D 1.0 2iP
3. Differentiation MS 30000 Suc. + 2200 Agar + 1400 Gel + Vit.of MS + 0.1 NAA 0.1 Kinetin
4. Proliferation MS 30000 Suc.+ 2200 Agar + 1400 Gel + Vit. of MS + 0.1 NAA 0.05 BA
5. Rooting ¾ MS 50000 Suc. + 2200 Agar + 1400 Gel + 0.1 Ca-panthothianate +
Vit. of MS + with and without 3000.0 AC 0.1 NAA
zB5: Gamborg et al. (1968) Nutrient Medium. yMS: Murashige and Skoog Medium (1962). xSuc.: Sucrose. vVit.: Vitamins. wAC: Activated Charcoal. uAd.: Adenine Sulfate.
Table 2: Production capacity of a single inorescence Gulistan cv. after 1, 7, 13 subcultures during the multiplication and rooting stages
Production (culture vesselz)
Sub 1 Sub 7 Sub 13
Callus Embryo Shoot Total Callus Embryo Shoot Total Shoot Plantlet Total
10 7 4 21 16 70 40 126 1644 1212 2856
zEach culture vessel (350 ml jar or 250×25 mm long tube) contained 1 gram of callus, or 10 embryos or shoots in average. Each long tube contained 1 intact plantlet with shoot-
root system.
Figure 1: In A) The initial inorescence explants of 2 months after
culture on the starting medium. Notice direct initiation of the shining
globular structures (pro-embryos) instead of the small orets. In
B) Healthy shoots in the multiplication stage. Each culture vessel
(350 ml3) may contain 20-30 shoots. Note the growth vigor and
length of the proliferated shoots is expressing readiness for rooting.
Figure 2: In A) 40 tubes-racks of in vitro rooting date palm are
reecting mega production. In B) One-month old date palm plantlets
of female inorescence technique were successfully acclimatized.
Note thrive, tall and wide leaves.
43
Commercial production of tissue culture date palm (Phoenix dactylifera L.) by inorescence technique
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... The Date Palm Research Institute (DPRI), Shah Abdul Latif University (SALU), Khairpur-Pakistan has contributed many years of effort in this regard and has successfully micropropagated various local and international date palm cultivars including cv. Dedhi from inflorescence explants [9,10]. The use of inflorescence as source material for initial culture establishment in comparison to the commonly used shoot tip explants is inevitable due to the limited availability of offshoots [10]. ...
... Dedhi from inflorescence explants [9,10]. The use of inflorescence as source material for initial culture establishment in comparison to the commonly used shoot tip explants is inevitable due to the limited availability of offshoots [10]. In addition, inflorescence explants offer a number of advantages over shoot tip explants including no or less browning, lower rates of bacterial contamination and a relatively short production cycle [11]. ...
... The spathes were cut open carefully and the spikes were divided into pieces of about 2-4 cm in length containing 2-4 immature florets. The nutrient medium composition for each of the initiation, maturation, differentiation, multiplication and rooting stage were as reported by [10]. The initial explants were kept at 25 ± 2°C under complete darkness with re-culturing after about 3-4 weeks on the initiation medium (Modified Murashigue and Skoog (MS) medium supplemented with 0.1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.1 Indole-3-acetic acid (IAA) and 5.0 1-Naphthaleneacetic acid (NAA)). ...
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Phoenix dactylifera cv. Dedhi is an excellent soft date palm cultivar of district Khairpur, Sindh-Pakistan with large size fruit, brittle texture and very low tannins at the Khalal stage. The population of this palm cultivar is currently threatened by pest and disease issues, a limited availability of offshoots and by the prevailing monoculture of semi-dry cultivars together with import of exotic elite cultivars into the region. Hence, the current study aimed to determine genetic stability of large-scale in vitro multiplied materials after their transfer to the field. The palms were phenotyped and then genotyped using two Inter-retrotransposons Amplified Polymorphism (IRAP) markers. Thirteen IRAP primers with 24 combinations were screened, of which 14 combinations produced 301 clear and reproducible bands which were all monomorphic. The IRAP banding patterns showed no variation among the tissue culture derived plants tested. No significant variation in fruit characteristics of the tissue culture derived plants was found when compared to mother plant. Using an optimized micropropagation protocol, number of plants unable to form inflorescences was reduced from 36.7% in the year 2016 to 10% in 2018. The study confirms the current micropropagation protocol to be effective for production of true-to-type plants and the applied marker system to be efficient for validating genetic stability of in vitro produced palms.
... The recent development in the date palm micropropagation protocol proved successful in producing plants and subsequent shifting into the open field conditions (Abul-Soad and Al-Khayri 2018; Abul-Soad and Mahdi 2010; Jatoi et al. 2015;Omar et al. 1992;Zaid et al. 2007). Different kinds of date palm explants for micropropagation have been utilized of which shoot tips of young offshoots have been used in large number of studies (Abul-Soad et al. 2002;Al-Khayri and Naik 2017;Tisserat 1984;Veramendi and Navarro 1997) and juvenile spathes (Abul-Soad 2011; Abul-Soad and Al-Khayri 2018; Abul-Soad and Mahdi 2010;Bhaskaran and Smith 1992;Fki et al. 2003;Jatoi et al. 2015Jatoi et al. , 2019 were found sustainable and productive. However, in in vitro environments numerous genetic and epigenetic differences may be developed in the date palm plantlets produced via tissue culture due to plant growth regulators (PGRs) stress and long production or multiplication cycle (Mirani et al. 2019. ...
... The seed propagation as natural and offshoot transplantation as commercial date palm propagation ways commonly prevail in Pakistan. However, the tissue culture studies on date palm in Pakistan on large scale production is still in its initial stage and only few studies are available reporting successful attempts with considerable number of plants shifting to soil conditions and reaching to fruiting stage (Abul-Soad and Mahdi 2010;Jatoi et al. 2015Jatoi et al. , 2019. ...
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... The need for date palm propagation materials is increasing with an estimate of 1-2 million plants per year in the international market (Jain, 2007). This made the commercial labs outside the date palm origin to work on developing viable somatic embryogenesis protocols (Abul-Soad & Mahdi, 2010). ...
... Cracks in the outer spathe cover occurred during its excision from the parent tree or even during the transfer from field to the laboratory contaminated whole initial In vitro cultures within few days. The spikelets inside the cover sheath were contaminants-free and gave excellent results in tissue culture protocols of date palm (Abul-Soad et al., 2007;Abul-soad et al., 2008;Abul-Soad & Mahdi, 2010). This is what was noticed in all explants during initiation stage, however few explants contaminated with fungus during successive subcultures most probably due to the improper handling during culturing. ...
... Moreover, when plants are obtained from seeds, the ratio between female and male plants is 1:1 and sex can only be determined after flowering, which might take years and is not economically feasible (Simão, 1998). In vitro propagation of date palms has been widely studied in several countries (Taha et al., 2003;Badawy et al., 2005;Eke et al., 2005;Khierallah and Bader, 2007;Othmani et al., 2009;Al-Khayri, 2010;Abul-Soade and Mahdi, 2010;Aslam et al., 2011;Eldin and Ibrahim, 2015), including those that use inflorescence segments as explants (Bhaskaran and Smith, 1992;Zayed et al., 2016;Ribeiro and Teixeira, 2017). Although it has proven to be the ideal technique for the propagation of this species, information and research about it in Brazil are either poor or nonexistent. ...
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... [38]. 27±2°C [33][34][35]. ...
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The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
Biotechnological studies of date palm: Micropropagation of inflorescence, molecular biology and secondary metabolites
  • A A Abul-Soad
Abul-Soad, A. A. 2003a. Biotechnological studies of date palm: Micropropagation of inflorescence, molecular biology and secondary metabolites. Ph.D. diss., Pomology Department, Faculty of Agriculture, Cairo University.
Effect of basal salts and sucrose concentrations on morphogenesis in test tubes of female inflorescene of date palm (Phoenix dactylifera L.) Zaghloul c.v
  • A A Abul-Soad
  • N R El-Sherbini
  • E E Bakr
Abul-Soad, A. A., El-Sherbini, N. R. and Bakr, E. E. 2007b. Effect of basal salts and sucrose concentrations on morphogenesis in test tubes of female inflorescene of date palm (Phoenix dactylifera L.) Zaghloul c.v. Egyptian Journal of Agricultural Research 85(n1B):385-394.
In vitro and ex vitro optimization for rooting and acclimatization of date palm
  • A A Abul-Soad
  • I A Ibrahim
  • N R El-Sherbini
  • E E Bakr
Abul-Soad, A. A., Ibrahim, I. A., El-Sherbini, N. R. and Bakr, E. E. 1999b. 12-14 Sep; In vitro and ex vitro optimization for rooting and acclimatization of date palm. The First International Conference, in Egypt, on Plant Tissue Culture and its Application. pp. 227-241. Egypt.