An offshoots. Propagation by seeds cannot be used

overview of organogenesis of date palm true to type plant.

: Muhammad Umar, Muhammad Yaqoob Aslam

college university Faisalabad



                     Somatic embryogenesis and
organogenesis are the two pathways of choice for quick and across-the-board
propagation of date palm. They have been positively used for the
micropropagation of best genotypes and have proved their value for the
commercial production of many cultivars. Nevertheless, regeneration through
somatic embryogenesis or organogenesis in date palm is static difficult to
achieve for recalcitrant genotypes and is often in a weak position by certain
physiological disorders. In the review, we report the results of a sum of
studies carried out on date palm micropropagation. It also defines different
factors that influence each stage of somatic embryogenesis and organogenesis
and the main problems come across during these two micropropagation processes.



Phoenix dactylifera L.; Organogenesis; Somatic embryogenesis


        Date palm (Phoenix dactylifera L.)
is one of the most vital fruit crops cultured in arid and semi-arid regions. It
is circulated throughout the Middle East, North Africa, South Sahel, areas of
East and South Africa, Europe and USA (Mazri et al ., 2015), with nearly 150
million trees worldwide (Mazri et al ., 2015). Date palm is refined for its
high yield and the high nutrient value of its fruit, for preserving ecosystems
threatened by desertification and creating suitable microclimate for
agriculture under arid environments. In addition, date palm cultivation
generates considerable chances for rural employment, provides a chief source of
income for farmers and confirms livelihood and food security of the rural areas
(Mazri et al ., 2015).Date palm can be multiplied sexually by seeds or
asexually by offshoots. Propagation by seeds cannot be used for the commercial
production of best genotypes due to its heterozygous character (Tisserat, B. 1982), and because of the
considerable difference between seedlings and vegetatively propagated plants in
expressions of production potential, fruit maturation and value, and harvesting
time. Propagation by offshoots is a slow procedure that is hampered by the limited
number of offshoots produced by a single date palm tree, the low survival rate
and the risk of transferring diseases. Propagation of date palm concluded in
vitro techniques presents a competent alternative for the orthodox methods.
Indeed, date palm micropropagation allows fast and
large-scale proliferation of uniform and healthy plants, with neither seasonal
effects nor the risk of distribution diseases and pests during plant material
exchange (Quiroz-Figueroa et al ., 2006).

Aim of this review is to summarize the literature on date palm micro propagation through somatic embryogenesis and
organogenesis, and highlight the main factors affecting each stage of these two
micropropagation methods. Beside this, the main problems come across during
date palm micropropagation are defined.



Date Palm Propagation Methods:-

Available techniques of rapid multiplication of date palm have
contributed hugely increased demand of date palm fruits worldwide (Jain et al.,
2011). Traditionally, date palm is proliferated by both sexually through seeds
and vegetatively by off shoots that produced from axillary buds located on the
base of the trunk during the juvenile phase in date palm tree. It is very slow
for off shoots to progress and that hampers vegetative propagation of date palm
plant. So far, there is no obtainable technique to speed up in increasing the
off shoot quantities as well as reduce the time in developing them. Use of off
shoots preserve true-to-type character of reproduced genotypes. Moreover,
sexual propagation of date palm is unsuitable for commercial production of
true-to-type value-added genotypes. It is due to heterozygous character of date
palm seedlings and also their dioecious nature (Jain, 2007a). In addition, half
of this progeny is collected of male trees which not distinguished before
flowering stage. The female plants produce variable fruits and commonly of
inferior quality (Eke et al., 2005). Additionally, seed propagation method has
another drawback that the growth and maturation of seedlings is extremely low,
and this is a reason, date palm seedling may begin to fruit after 8-10 years of
plantation. Though offshoot propagation is a true-to-type technique, it is not
commercially practical for the following causes:

 Offshoot production is restricted
to a relatively short vegetative phase of about 10 to 15 years;

Only a limited number of offshoots 
are formed during this phase (20 to 30 offshoots, depending on variety);

Some varieties harvest more offshoots than others (some do not produce
offshoots at all);  

Offshoot survival ratio is low;

The use of offshoots improves the spread of date palm diseases and pests;
Offshoot propagation is difficult, lengthy, and therefore expensive.

In vitro propagation of date palm:-

              Use of in vitro techniques such as somatic
embryogenesis and organogenesis is highly proper for large-scale plant
multiplication of vegetatively proliferated crops. Success of these techniques
is highly genotypic dependent, though, have successfully been practical for
plant propagation in wide ranging crops including date palm (Jain, 2007a).
Micropropagation by direct organogenesis is commonly used for rapid clonal
propagation of best genetic material of date palm plant (Khierallah and Bader,
2007). Performance of micropropagated date palm appears to be better than
conventionally grown plants in terms of harvest, early flowering time, and relatively
uniform in fruit value and physical properties. Aaouine reported plant redevelopment
from 30 genotypes of date palm by direct shoot organogenesis. The major concern
with this method is somaclonal variation that is dependent on different factors
including genotype, explants, plant growth regulators (Jain, 2007a). Moreover,
it is highly necessary to maintain genetic fidelity of regenerated plants,
which can be studied by many molecular markers Micropropagation has a benefit
of using low concentrations of plant growth regulators, as a result callus
phase is avoided. Direct regeneration of vegetative buds reduces the risk of
somaclonal variation among regenerants. Duration of culture period is limited
by numerous subcultures for maintaining and given that shoot cultures for
plantlet production. However, the highest number of subcultures must be determined
before starting the fresh cultures from the mother plants. This is done to
prevent or reduce somaclonal variation. 
Currently, only a few laboratories use this technique to produce
commercially in vitro date palm plants, mainly in Morocco, Saudi Arabia and
United Arab Emirates. Micropropagation technique has been used commercially
in  selected  date palm cultivars described advantages and
limitations of  date palm
micropropagation; major advantages are year round availability of plants,
quality control, rapid production of plants of elite cultivars, and cold
storage of elite genetic material.

Advantages and disadvantages of somatic
embryogenesis (Jain, 2007b)

Advantages of somatic embryogenesis:-

Somatic embryos originate
from a single cell and minimize or eliminate chimera depending on the plant

Somatic embryo cell
suspension is ideal for mutation induction due to production of direct mutant
somatic embryos.

 Somatic embryos behave like a zygotic embryo
in germination.

 Single somatic embryo can be encapsulated to
develop into a somatic seed that could germinate like a normal seed. This
aspect still requires further research for use at a commercial scale.

Most suitable approach in
woody species for plant regeneration. Somatic embryos can be produced in a
bioreactor which could be automated for largescale production of somatic

Somatic embryos are
suitable for long term storage by cryopreservation Disadvantages of somatic embryogenesis:-

Somatic embryogenesis is
highly genotypic dependent and therefore culture medium modification may be
needed for different genotype.

Germination rate of
somatic embryos is very poor in most of the crops.

Somatic embryogenic
cultures can lose their property if they are not sub-cultured regularly on the
fresh culture medium, and that raises the chance of getting genetic

Organogenesis of Date Palm


Explant selection:-

                                   The choice
of an explant and its disinfection process can affect the success of
micropropagation including the date palm. Shoot tips and adventitious shoots in
lateral buds contain more meristematic tissues than other organs, and therefore
are frequently used in date palm tissue cultures (Mazri and Meziani, 2015). A
successful regeneration of many date palm genotypes has been achieved when
shoot tips were used as explants: “Jihel” and “Iklane”, “Mordarsing” and
“Khanizi” , “Nabout” and “Khasab” (Al-Khayri, 2007), and “Khalasah”, “Zardai”,
“Barhee”, “Zart”, “Muzati” and “Shishi”. Date palm tissue culturing can also be
achieved by using explants derived from inflorescences, as was reported for
“Barhee” and “Gulistan”. Reynolds and Murashige (1979) induced somatic
embryogenesis from zygotic embryos obtained from green fruits that were
harvested 2-3 months after pollination. Pinker also used zygotic embryos to
induce somatic embryo genesis in “Khistawi”, “Zahdi”, “Barban”, “Asabe” and
“Elarous”. Somatic embryos are useful for the micropropagation and large-scale
production of date palm plants and may also be used to obtain true-to-type

Explant disinfection and preparation:-

The main disinfecting agent that has been used for shoot tips is sodium
hypochlorite (NaOCl) at a concentration range from 5% to 25% and for spikelets,
mercuric chloride (HgCl2) at 0.1% concentration. In addition, the use of
antioxidants such as 150 mg/l ascorbic acid (for 30 min), 4%
polyvinylpyrrolidone (Aslam and Khan, 2009), citric acid at a concentration of
150 mg/l with 150 mg/l ascorbic acid (soaked overnight), or anhydrous caffeine
are widely used during shoot tip explant disinfection (Khierallah et al., 2007).
Khan and Tabassum (2012) used an effective protocol to eliminate infection from
shoot tips: treatment with 5% (w/v) NaOCl containing one drop of a surfactant
(Tween-20/100 ml), stirred gently for 30 min, rinsed three times in sterile
distilled water (SDW; 5 min each rinse), surface disinfested with 0.2% (w/v)
HgCl2 for 10 min and then rinsed three times with SDW. Leaf primordia of 6 cm
long shoot tips were removed and used as explants and 2 cm long shoot tips with
2-4 intact primordial leaves also served as explants. A similar protocol has been
used by Othmani for leaves adjacent to the apex of axillary shoots of cv.
“Boufeggous”. Fki first washed young leaves with tap water, and surface
sterilized them with 0.01% HgCl2 for 1 h, rinsed three times with SDW, then cut
them into 5-10, 10-15 and 15-20 mm long explants. Ledo described a disinfection
procedure for zygotic embryos from mature (wine-colored, -2.17 g) and immature
(green, -1.68 g) fruits from “açai” palm, an Euterpe species of palm tree
cultivated for its fruit. After being washed under running tap water, fruits
were immersed in 40EC water, and seeds were excised on a laminar flow bench,
immersed in 70% ethanol for 2 min, then in 2% NaOCl for 20 min under agitation,
and finally washed four times with SDW (Khokhar, M. I. et al ., 2017).

Adventitious bud initiation:-

The formation of adventitious buds on date palm explants depends on many
factors such as media components, genotype, and time period of plant material
collection. Various culture media were suggested for adventitious bud
formation, depending on the cultivar. From offshoot-derived explants,
Beauchesne et al. suggested half-strength Murashige and Skoog (MS) medium
supplemented with 1-5 mg/L 2-naphthoxyacetic acid (NOA), 1 mg/L NAA, 1 mg/L
indole-3acetic acid (IAA) and 0.1-3 mg/L 6-(dimethylallylamino) purine (2iP).
Khierallah and Bader recommended MS medium supplemented with 2 mg/L 2ip, 1 mg/L
BAP, 1 mg/L NAA and 1 mg/L NOA for cv. Maktoom. Al-Mayahi suggested MS medium
supplemented with 1 mg/L BAP and 0.5 mg/L thidiazuron (TDZ) for cv.
Hillawi. For cv. Zaghlool, Bekheet used MS medium supplemented with 2 mg/L 2ip
and 1 mg/L NAA while Hussain et al. used MS medium supplemented with 4 mg/L IBA
and 1 mg/L BAP for cvs. Asil, Hussaini and Zaidi. According to Al-Khateeb, low
PGRs concentrations promote the formation of buds while high concentrations
induce abnormal growth without bud formation. Studies on adventitious bud
formation from inflorescence explants are very scarce. Loutfi and chlyah
indicated that shoot primordia is formed mostly on Greshoff and Doy medium
supplemented with 0.5 mg/L NAA, 2 mg/L BAP and 1 mg/L 2iP. In a recent review
of the literature, Abahmane reported that the combination of one auxin and two
cytokinins is effective for bud formation on inflorescence explants. As regard
to the period of offshoot removal, Beauchesne et al. suggested a period
starting from the end of dates harvesting and lasting until the beginning of
flowering. Aissam reported that the explants taken between October and February
show the highest buds formation rate, whereas Zaid et al. reported that the
best period for the in vitro culturing of offshoot-derived explants is from the
onset of flowering . Shoot bud multiplication Many factors influence shoot bud
multiplication in date palm, especially the basal formulation of the culture
medium, the genotype and PGRs. Abahmane mentioned that the main basal
formulation used is MS at full or half-strength, supplemented with PGRs at low
concentrations as compared with the bud initiation stage. Zaid et al. reported
that for shoot bud multiplication, NAA, NOA, IAA, BAP and kinetin might be used
at 0.5-5 mg/L. Beauchesne et al. suggested half-strength MS medium supplemented
with 2 mg/L NOA, 1 mg/L NAA, 1 mg/L IAA, 0.5 mg/L BAP, 1 mg/L 2iP and 1-5 mg/L
kinetin. For cultivar Khalas, Aslam and Khan used 7.84 µM BAP for high shoot
bud multiplication. Khierallah and Bader recommended MS medium with a
combination of 1 mg/L NAA, 1 mg/L NOA, 4 mg/L 2iP and 2 mg/L BAP for date palm
cv. Maktoom while Khan and Bi Bi found that MS medium containing 0.5 mg/L BAP
and 0.5 mg/L kinetin yields the highest number of shoots per explant in cv.
Dhakki. In a previous work on cv. Najda, we found that the best medium for
shoot bud multiplication was half-strength MS medium supplemented with 0.5 mg/L
NOA and 0.5 mg/L kinetin, which yielded an average of 23.5 shoot bud per
explant after 3 months of multiplication. Mazri recommended MS medium
containing 2.5 ?M IBA and 2.5 ?M BAP for cv. 16-bis (22.3 shoot buds per explant)
while he recommended half-strength MS medium supplemented with 3 ?M IBA and 3
?M BAP for cv. Boufeggous, which showed 22.9 shoot buds per explant. Al-Mayahi
suggested MS medium containing 1 mg/L BAP and 0.5 mg/L TDZ for cv.
Hillawi, which resulted in an average of 18.2 buds per culture. Other factors
such as the medium texture, cultivation in bioreactors, explant size and
density and carbon source were also reported to affect shoot bud multiplication
of date palm.

Shoot elongation, rooting and plantlet

Shoot elongation and rooting may be achieved either on a medium
containing PGRs or on a PGR-free medium. Beauchesne et al. suggested the use of
half-strength MS medium supplemented with 1 mg/L NAA, 0.5 mg/L BAP, 0.5 mg/L
kinetin and 1-3 mg/L gibberellin for shoot elongation. El Sharabasy et al.
reported that the use of 0.1 mg/L NAA has a better effect on shoot elongation
as compared to IBA and IAA. The use of liquid medium was also reported to
promote shoot elongation. As regard to shoot rooting, Bekheet recommended 1
mg/L NAA, which showed better results than IAA or IBA at the same
concentration. In a previous work on cv. Najda, we compared media with and
without PGRs. Our results showed that shoot elongation is fast in media
supplemented with PGRs, with high root formation rates. However, shoots
cultured on PGR-free media had wider and greener leaves, and exhibited higher
survival rates after acclimatization. This shows that plantlet acclimatization
might be influenced by previous culture conditions. Along this line, it has
been shown that the texture of the elongation-rooting medium influences the
survival rate of plantlets after ex vitro transfer. Indeed, the use of a liquid
medium just before plantlet acclimatization showed lower survival rates as
compared to a semi-solid medium. On the other hand, increasing the level of
sucrose in the elongation-rooting medium increases the survival rate of
plantlets during acclimatization. Other factors such as the nature of the
substrate and the application of gamma aminobutyric acid were reported to
influence plantlet acclimatization (Mazri et al .,2015).


Conclusions and Future Prospects:-

 Micropropagation of date palm either through
somatic embryogenesis or through organogenesis was reported for many cultivars,
and several factors have been revealed to influence these regeneration systems.
Date palm micropropagation presents an efficient way for the large-scale
propagation of genotypes resistant to bayoud, a very dangerous disease caused
by the fungus Fusarium oxysporum f. sp. albedinis, which had decimated more
than 12 million trees during the last century. Plantlets of bayoud-resistant
genotypes are used to rehabilitate palm groves ravaged by this fungus.
Micropropagation also allows the large-scale propagation of cultivars of high
fruit quality, in order to satisfy the high demand of farmers and consumers.
Despite the numerous works published on date palm micropropagation, research is
still needed to optimize culture conditions for the newly selected genotypes
and recalcitrant cultivars, to shorten the time needed to produce plantlets,
and to reduce the incidence of physiological disorders. It is also important to
carry out studies related to the application of somatic embryogenesis to
genetic transformation, synthetic seeds production and cryopreservation of
embryogenic cultures.


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