CHAPTER V: DATE PALM PROPAGATION

by A. Zaid and P.F. de Wet
Date Production Support
Programme

1. Introduction

Although economically important, palms are a much neglected plant group in
terms of understanding development and propagation potential thereof.
Furthermore, progress in the field of breeding, genetics, crop improvement, and
expansion of commercial plantings for palm has been restricted by the habit and
long-lived nature of these monocotyledonous trees. Most palms can only be
propagated by seeds, i.e., Coconut and Oil palm.

There are three techniques to propagate date palm: Seed propagation, offshoot
propagation (traditional methods), and the recently developed tissue culture
techniques. This chapter will highlight each of these techniques.

2. Seed propagation

Seed propagation, also called sexual propagation, although useful for
breeding purposes, is not a proper method of date palm vegetative propagation,
and should be discouraged. Reasons in favour of discouraging seed propagation,
are the following:

* Date palm is a dioecious species and consequently half of the progeny
will be males and half will be females, with no certain way to determine at an
early stage the sex of the progeny, nor fruit or pollen quality prior to
flowering (often only seven years later);

* Female plants originating from seedlings usually produce late maturing
fruits of variable and generally inferior quality compared to established
clonal palms. In a seedling plantation it is rare that more than 10 percent of
the palms produce fruit of satisfactory quality;

* Date palms are heterozygous, and thus there will be much variation within
the progeny, and desirable characteristics of the parent palm may be lost. In
other words, it is not true to type propagation and no two seedling palms are
alike;

* Seedlings differ considerably with regard to production potential, fruit
quality and harvesting time, making them very difficult to market as one
harvest;

* The above reasons result in waste of time, space and
money.

Thus, seed propagation is by far the easiest and quickest method of
propagation. However, it is not a true to type propagation technique and no two
seedlings will be alike. Because of its diversity, the seed approach could only
be useful for breeding purposes. When conditions are known to be unfavourable
for date fruit production (case of marginal areas), the planting of date seeds,
for future selection on fruit quality, is the most economical way of selecting
clones that have some desirable characters such as rain and/or salt tolerance
(Figure 34).

Taking the above into consideration, and also because of the many reasons
listed below, date growers are encouraged to use tissue culture-derived material
of known varieties with high date quality and marketing potential.

3. Offshoot propagation

Offshoot propagation, also called asexual or vegetative propagation, offers
the following advantages:

(i) Offshoot plants are true to type to the parent palm. The offshoots
develop from axillary buds on the trunk of the mother plant and consequently
the fruit produced will be of the same quality as the mother palm and ensures
uniformity of produce.

(ii) The offshoot plant will bear fruits 2 - 3 years earlier than
seedlings. The life span of the date palm is divided into two distinct
developmental phases: vegetative, in which buds forming in the leaf axils
develop into offshoots; and generative, in which buds form inflorescences and
offshoots cease. From the time that the axillary bud of a leaf has
differentiated into an offshoot until the time it grows outwards, takes up to
three years (18 to 36 months), with another three to four years before it
reaches the desired size for its separation and planting (Hilgeman,
1954).

Offshoots are mainly produced in a limited number (20 to 30 at most) during
the early life of the palm (10 to 15 years from the date of its planting)
depending on the variety and on prior fertilisation treatment, irrigation and
earthing up around the trunks, (Nixon and Carpenter, 1978). Although 20 to 30
offshoots are produced by a palm, only three or four offshoots are suitable for
planting out in one year and must still go into the nursery for 1 to 2 years
before field planting. Zahidi, Berim and Hayani varieties are known to produce
large numbers of offshoots, while Mektoum and Barhee varieties produce
relatively low numbers of offshoots.

Offshoots are recognised by their curved form while seedlings have a straight
form. Another way to differentiate between the two is that seedlings have roots
all around their base with no connecting point to the palm, while an offshoot
does not have any roots on the side where it was connected to the mother plant.
Furthermore, an offshoot always has a mark on one side which is a result of
detachment from its parent palm.

To obtain a high survival rate of transplanted offshoots, the following steps
are recommended:

Offshoot selection

The offshoot selected for removal must be disease and pest free and at least
three to five years old with a base diameter between 20 and 35 cm (Table 32),
weighing over 10 kg but not more than 25 kg because of handling difficulties.
Signs of mature offshoots are the availability of theirown roots, first
fructification and the production of a second generation of offshoots (Nixon and
Carpenter, 1978).

Small offshoots weighing 5 kg and less, if needed, could also be used, but
their survival potential will be much lower than that of larger offshoots. They
should initially be looked after, for at least two years, in a nursery, or mist
bed in a greenhouse or a shade net structure (Reuveni et al., 1972). Fungi are
usually a serious problem in a mist bed, and the offshoots must be treated twice
a month with a large spectrum fungicide.

TABLE 32
Relationship between diameter and weight of the
offshoot

Base diameter of the offshoot (cm)

Approximate weight (Kg)

12 - 15

4-8

15 - 20

8 - 15

25 - 35

22 - 35

The best time for the removal of offshoots and transplanting into the nursery
for rooting (never directly into the field) is after the soil begins to
warm up in the late spring and early summer (September/October in Southern
hemisphere and March/April in the Northern hemisphere). February/March and
September/October are then the most suitable period for field planting,
respectively.

Offshoot rooting

Two types of offshoots occur on a date palm tree: the lower and older ones,
and the upper and younger ones. It is believed that low offshoots are more
active physiologically than high ones; they probably grow faster (the number of
leaves produced increases with age). In fact, the high offshoots have less
carbohydrates than low offshoots, resulting in low roots production and
consequently low survival rate. It is also suspected that high offshoots develop
when no fruit is on the palm.

Early offshoot removal is desirable because:

(1) removal allows easy access to the palm,

(2) removal improves the development and fruit production of the parent
tree, and

(3) planting young offshoots is advantageous as they will in turn produce a
greater number of offshoots than older ones.

Numerous factors to consider when rooting offshoots include: the size of an
offshoot (often expressed in weight), type (upper or lower), origin of the
offshoot, the method of removal and preparation for planting, as well as
treatment of an offshoot after planting (Nixon and Carpenter, 1978).

To promote rooting, the base of the offshoot should be in contact with moist
soil for at least twelve months before removal. Production of high offshoots is
primarily of a varietal character but also in some cases related to a damp
climate. For these high offshoots, boxes or plastic bags/Hessian material could
be fastened around the base of the offshoot. Another technique is to leave them
on the mother palm until they mature. They are then removed and rooted in a
nursery (Figure 35a and 35b).

Offshoot pruning

When the aim is the production of offshoots, no green leaves should be
removed from an offshoot until it is cut from the mother palm, since the growth
of an offshoot is in proportion to its leaf area. When larger offshoots are
selected for the following year's cutting, all their leaves must be retained
until the offshoots are removed. When leaves interfere with cultivation, they
may be tied together.

When a date palm is crowded with offshoots, only 5 to 6 larger offshoots
could be left, considering the tree's equilibrium, and the other smaller ones
could either be totally removed if not needed in the future, or have their
leaves cut back close to the bud to retard their growth.

Offshoots removal

After 3 to 5 years of attachment to the parent palm, depending on the
variety, offshoots will form their own roots and start producing a second
generation of offshoots. Only at this stage are they ready to be removed (Nixon,
1966; Nixon and Carpenter, 1978).

Care and skill, acquired only by experience, is important in order to cut and
remove an offshoot properly from its mother palm. The operation, usually carried
out by two skilled labourers, starts by irrigation several days before cutting.
Soil is then dug away from the offshoot(s) using a sharp, straight-blade shovel
(a ball of earth, 5 to 8 cm thick, must be left attached to the roots of the
offshoot, with the connection exposed on each side). Roots should at no time be
cut closer than necessary, since most of the cut roots die and new roots just
emerging are susceptible to injuries (Nixon and Carpenter, 1978).

A specially designed chisel is recommended to cut offshoots. It is a
rectangular cutting blade made of tempered steel, which is welded to a solid
iron handle. One side of the blade is fl at and the other bevelled so as to form
a sharp cutting edge. The following chisel dimensions could be suggested: Blade:
11 cm wide, 22 cm long and 2,5 cm thick; Handle: 120 cm long and 3 cm thick
(Figure 36).

Lower leaves must be cut off and the remaining ones tied together in order to
facilitate handling. Once the loose fibre and old leaf bases are cut away and
the connection between the offshoot and the mother-palm is located, the first
cut is made to the side of the base of the offshoot close to the main trunk. The
fl at side of the chisel is put towards the weak point of the offshoot and the
bevelled side towards the mother palm. Injury must be avoided at all times, the
offshoot's tender heart should never be damaged and the cutting operation must
be only from one side to obtain a smooth cut surface.

After completion of the removal of the offshoot, the old leaf stubs and lower
leaves are cut off close to the fi bre and the basal part left bare of leaves.
Ten or twelve leaves around the bud are retained and tied close together 6 to 8
cm above the bud with heavy twine or wire. The terminal parts of these leaves
extending beyond the tie (20 cm above the tip - centre of the offshoot) are also
cut off (Figure 37). It is advised that the cut surfaces of both the offshoot
and the mother palm be covered with a copper sulphate product in order to avoid
infection by Diplodia and other parasites.

Survival of cut-offshoots depends to a large extent on the variety. Medjool's
offshoot is far more difficult to establish than Deglet Nour or Zahidi.

In places such as Fezzan (Libya), some areas of Iraq and Saudi Arabia, and
Hadramaout (Yemen), offshoots are not at all removed and continue to grow
outwards from the original mother palm, producing large clumps consisting of
hundreds of shoots, none of which produces a trunk and of course with no
significant yield (Dowson, 1982).

Planting offshoots

It is advisable that an offshoot never be planted into the field directly
after removal from the mother plant. A rooting period of one to two years in a
nursery is essential in order to ensure an optimum survival rate and to avoid
uneven development of the plantation.

In most soils, the early and rapid growth of the offshoot is better when the
holes are prepared one to two months before planting. The size of the hole
should be one m³ and the holes should be filled with a mixture of topsoil and 10
to 15 kg of manure of high quality (with very little unmatured matter) and NPK
fertilisers. The filled holes should be irrigated several times to promote the
decomposition of the manure and also to allow the mixed soil to settle in the
hole. Well-rotted manure can be used in holes prepared and irrigated shortly
before planting, but extreme care must be taken to put the manure (and
fertilisers) deep enough to form a layer of soil of at least 15 to 20 cm thick
between the manure and the base of the offshoot.

The leaf base of the offshoot should be clearly above the soil level. It is
important to plant the offshoot to the depth of its greatest diameter in order
to avoid the rotting of the base (if it is too low) and to prevent the water
reaching the loose fibre near the bud which causes its desiccation (if it is too
high). The plant water basin, of 1.5 to 1.8 m in diameter and 20 to 30 cm deep,
should be prepared around the offshoot (Figure 41).

The soil near the newly planted offshoots should be kept moist at all times
by light and frequent irrigation. The irrigation frequency is dependent on the
type of soil. Very sandy soils require daily irrigation during the first summer.
Heavy soils require irrigation only once a week; while in most soils irrigation
is required every second or third day. During the first six weeks (or till the
appearance of new growth) the date grower should always inspect his/her planted
offshoots to make sure that the surface soil does not dry and shrink away from
the offshoot. A mulch of hay or straw around the offshoot will enhance moisture
contention, weed control and finally improve humus in the basin (Figure 38).

Young offshoots and tissue culture-derived plants should be protected from
harsh climatic conditions (sun and wind during the first summer and cold the
following winter) and against some animals (rabbits, etc.). The use of shade
net/hessian wrapping or a tent of date leaves is recommended (Figure 39). The
top is to be left open so that new growth may push through.

Under Namibian conditions (Southern hemisphere), there are two appropriate
periods for planting: February/March and September/October. The first period is
preferable since it allows a longer time for the offshoot to establish itself
before the arrival of the next year's hot summer temperatures, although it
passes through the cold months of winter (June, July and August) while the plant
is still in its initial establishment phase. The second period
(September/October) avoids the cold temperatures and later receives warm
temperatures that allow an active growth followed by the hot summer (December,
January).

To summarise, offshoot propagation is true to type but it is not very
practical from a mass propagation point of view, and consequently does not
satisfy the large needs of plant material. The following reasons illustrate this
handicap:

- Offshoot production is limited to a certain period in the palm's life
span (a short vegetative phase of about 10 to 15 years);

- During this short phase, only a limited number of offshoots are produced
(20 to 30 offshoots, at most, depending on the variety);

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

- A mature specimen with no offshoots will be lost if not propagated
through another technique;

- Depending on the care given, a low planting survival rate is frequently
obtained when using offshoots;

- The use of offshoots will enhance the spread of date palm diseases and
pests;

- Offshoot propagation is difficult, laborious, and therefore
expensive.

In comparison to the seed propagation technique, offshoots which are axillary
vegetative buds, will offer the following two advantages:

- The fruits produced will be of the same quality as the mother palm and
ensure uniformity of produce (true to type).

- The offshoot will bear fruit earlier than seedlings (by 2-3
years).

4. Tissue culture propagation

Palms are a much neglected plant group in terms of understanding their
development and vegetative propagation potential. Yet, they are economically
important in tropical and subtropical regions. The rapid propagation of date
palm as well as propagation from a mature specimen, is impossible due to the
limited number of offshoots produced and the fact that offshoot production is
limited to a certain period in the palm's life span. As mentioned above, seed
propagation of date clones and cultivars is impractical.

The application of tissue culture techniques for date palm, also called in
vitro propagation, has many advantages (in comparison to the above two
techniques) and enables the following:

- Propagation of healthy selected female cultivars (disease and pest-free),
Bayoud resistant cultivars, or males having superior pollen with useful
metaxenia characteristics which can easily and rapidly be propagated;

- Large scale multiplication;

- No seasonal effect on plants because they can be multiplied under
controlled conditions in the laboratory throughout the year;

- Production of genetically uniform plants;

- Clones to be propagated from elite cultivars already in existence, or
from the F1 hybrids of previous selections, and seed-only originated
palms;

- Ensure an easy and fast exchange of plant material between different
regions of a country or between countries without any risk of the spread of
diseases and pests; and

- Economically reliable when large production is required.

The success of propagating monocotyledons in vitro has been limited to
relatively few herbaceous species. Similarly, most dicotyledons, successfully
tissue cultured, have also been the herbaceous types. It has been postulated
that in woody plants, the ability to regenerate plantlets using tissue culture
techniques was lower in comparison to herbaceous plants. In palms, until twenty
years ago, little success was achieved in inducing and maintaining good callus.
Plant tissue culture techniques have been employed to clone a wide range of
plants and economically important palms e.g., coconut, oil and date palms
(Cheikh et al., 1989).

In reviewing date palm tissue culture, the classification followed will be
that of behaviour and relevant techniques of tissue culture as a whole from a
perspective of their eventual applications to date palm (Zaid and Djerbi, 1984;
Zaid, 1985; 1986a; 1986b).

This review also explains the background to the cloning methods applied to
the date palm and explores the wide range of results obtained with embryo
culture, meristematic tissues (shoot tips and buds) and highly differentiated
somatic tissues (leaf, stem, inflorescence and root sections).

4.1 Embryo culture

Embryo culture involves excising an embryo-aseptically from the seed and
planting it in a sterile nutrient medium (Hoded, 1977). Embryo culture is
suggested to have several potential applications in plant research. It is used
to save embryos that fail to develop naturally in the fruit or seed, or grow out
embryos from interspecifi c hybridisation where defective endosperms are common
(Johnston and Stern, 1957). Embryo culture may also be used to reduce lengthy
dormancy periods due to physical and/or chemical inhibitors present in the fruit
or seed (Hoded, 1977). Excised embryos cultured in vitro, free from these
inhibitors, usually germinate immediately. Isolated embryos were also chosen as
explant material in metabolic studies (Raghavan, 1976). The culture of isolated
embryo segments may be useful to study the development of the primary meristems,
organogenesis and the interactions between different organs (Rabéchault and Gas,
1974). The culture of embryo outside the seed was first performed with crucifers
(Haning, 1904). It has since become a routine procedure.

With regard to date and other palms, callus initiation and embryoid induction
was first observed by Rabéchault (1962) working with oil palm embryos. Reuveni
(1979) reported that callus and roots developed from the date palm embryo
cotyledonary sheath tissue in media containing naphthalene acetic acid (NAA).
This callus continued to proliferate and to differentiate roots when subcultured
if a piece of the cotyledonary sheath was present. Ammar and Benbadis (1977)
established organogenic callus from date palm cotyledonary sheath of zygotic
embryo germinated in vitro.

Reynolds and Murashige (1979) cultured embryo explants of Chamaedores
costaricana Oerst, Howeia forsteriana Becc., and Phoenix
dactylifera L. in vitro. Green date palm fruits, harvested two to
three months after pollination were planted in a medium enriched with
2,4-dichlorophenoxy acetic acid (2,4-D), and a creamy-coloured grainy callus was
subsequently developed. Transfer of this callus to an auxin-free medium resulted
in the development of numerous asexual embryos. Mature zygotic embryos cultured
in nutrient media containing charcoal with high auxins levels, 10 and 100
mg/1NAA, also produced nodular callus (Tisserat, 1979). Repeated culture
resulted in the formation of plantlets. Tisserat and DeMason (1980), described
plantlet formation from date palm tissue cultures. The morphological development
of asexual embryos from callus closely paralleled excised zygotic embryo
germination in vitro (Figure 40).

Zaid and Tisserat (1984) performed a survey study to determine excised embryo
callus production. In the Arecaceae, embryo excised from mature seeds of 38
species were cultured on modified Murashige and Skoog (MS) medium containing
3g/L-¹ activated charcoal; with 100 mg L-¹, 2,4-D and 3 mg
L-¹ N6- (2 - isopentyl) adenine (2-iP). Embryo cultures from 18 of
these species produced prolific callus after repeated reculturing for six
months. Zaid (1987) also cultured embryos of date palm to follow up their
development. The sequence of germination is shown in Figure 41.

4.2 Culture of date palm meristematic tissues

When comparing shoot-tips and lateral buds in vitro versus culturing
other explant sources, the following advantages become apparent:

1. Shoot-tips and lateral buds are protected by bud scales and leaves, and
are usually easier to surface-sterilise than root or stem explants (Morel,
1960).

2. By culturing shoot-tips or buds, an entire shoot is already present,
thus only root induction is required to produce a whole plantlet (Morel, 1965;
Williams, 1974).

3. The cells of the shoot-tips and buds are more uniformly diploid than
those derived from less meristematic regions (Murashige, 1975). Presumably,
plantlets derived from naturally meristematic regions are likely to be clonal
and generate faster than other explant sources.

A distinction is made between bud and apical meristem cultures. Lateral bud
culture involves the growth of an entire rudimentary vegetative shoot. Apical
meristem culture, ideally involves only the excision and growth of apical dome
of the shoot usually less than 0.1 mm in diameter and 0.25 mm in length,
sometimes with, though preferably without, a few leaf primordia attached
(Cutter, 1965). In contrast to culturing herbaceous angiosperm shoot apices, few
woody angiosperm shoot-tips have been established in vitro (De Fossard,
1976).

4.2.1 In vitro culture of date palm shoot-tips

Schroeder (1970) and Staritsky (1970) employing date and oil palms
respectively, cultured shoot-tips in vitro with some success. However,
most of excised shoot-tips either failed to grow or showed no root
differentiation.

Reuveni et al.(1972) found that growing tip cultures of date palm responded
irregularly to growth regulators, but optimal leaf development occurred when
media contained 0.1 mg/1 NAA and 0.01 mg/1 kinetin. Callus occasionally formed
at the cut surface of the tip, particularly in dim light, when low
concentrations of auxin and/or cytokinin were present.

Generally this callus was very short lived and its subculture was
unsuccessful (Reuveni and Kipnis, 1974).

El Hannawy and Wally (1978) observed some bud differentiation in date palm
cultures. They reported that by adding 200 mg/1 "fermentol" to MS medium
containing 1.0 mg/1 auxin and kinetin, and using an incubation temperature of
25°C, 60 % bud differentiation occurred. Scharma et al. (1980), using date palm
shoot-tips, reported limited success in their development due to the browning of
the tissue and media. Tisserat (1979), culturing date palm shoot-tips,found that
a high auxin concentration of 10 and 100 mg/1 NAA and 2,4-D caused a reduction
in the culture weight, and inhibition of shoot growth, but promoted the
formation of yellow-white nodular callus. These nodules were precursors to
asexual embryos. Transfer of callus to nutrient medium containing lower levels
of auxins such a 0.1 mg/1 NAA or 2,4-D allows shoot development from tips to
occur. Male and female shoot-tips were found to grow equally well. Root
initiation was infrequent and did not appear to be related to the nutrient
medium composition.

Zaid and Tisserat (1983a) cultured date palm shoot tip explants from adult
palms, offshoots, seedlings and asexual plantlets on modified MS nutrient media
containing 10 mg/1 NAA. Differential morphogenetic responses were obtained
dependent on the explant type and parent source (Table 33). The same authors
also determined the action of several auxins and cytokinins on development of
date seedling shoot-tips and apical meristems (Table 34). Shoot-tip explants
consisted of the apical dome with two to four leaf primordia, and varied in size
from 0.5 to 1 mm². Meristems and tips were cultured on modified MS medium
containing 3 mg L-¹ activated charcoal, 0.1-300 mg L-¹
NAA, 2.4 - D, indoleacetic acid (IAA), indolebutyric acid (IBA), 4 -
chorophenoxyacetic acid and 2iP. Best consistent shoot regeneration occurred on
nutrient media containing 10 mg L-¹ NAA. These shoots were recultured
on nutrient media, devoid of charcoal, containing 10 mg L-¹ NAA or
kinetin to obtain rooting and enhanced shoot development. Best rooting was
achieved with 0.1 mg L-¹ NAA with 63% of the shoots initiating
adventitious roots after the first culture passage. Axillary bud outgrowths were
occasionally obtained from shoots cultured on media containing 0.01 and 0.1 mg
L-¹NAA only.

TABLE 33
Morphogenesis obtained from shoot tip cultures derived
from various date explant sources

Explant sources (*)

Survival/
treatment (%)

Shoot growth/
culture (%)

Shoot length/
culture (%)

Leaves/
culture

Rooting/
culture (%)

Adult palm

70

85

2.12 ±.71

1.5 ±.5

0

Juvenille offshoot

78

80

2.75 ±.69

2.5 ±.6

0

Seedling

85

100

2.35 ±.65

2.0 ± 0.0

60

Asexual plantlet

95

100

1.67 ±.39

2.2 ±.4

80

(*) 15-20 cultures employed per treatment; results taken 8 weeks after
planting.

TABLE 34
Influence of growth regulators on the growth of date
palm shoot tips

Test levels (mg/l)

Shoot growth (%)
Growth Regulator
Type

NAA

2,4-D

IAA

Kinetin

BA

2iP

0.0

58

50

42

80

58

80

0.1

58

-

-

60

67

60

0.3

67

47

33

40

58

40

1.0

58

53

42

53

50

53

3.0

67

53

33

66

58

66

10.0

75

53

33

73

33

73

30.0

-

67

50

-

33

-

100.0

75

13

-

60

42

60

300.0

58

6

25

53

42

53

4.2.2 In vitro culture of date palm buds

Most buds of date palm were reported to die within the first 30-50 days after
planting in vitro (Reuveni and Kipnis, 1972; Schroeder, 1970). Only the
largest and most distinctly differentiated buds grew. These buds exhibited leaf
expansion and produced additional leaves. Tisserat (1979) and Zaid (1981) also
investigated the conditions for bud development, and found that in nutrient
medium shoot-tips and lateral buds grew equally well on the same medium. Callus
cultures have been initiated from axillary buds of 2 to 4 year old date palm
offshoots. Zaid and Tisserat (1983b) found that subcultured lateral buds callus
on nutrient media devoid of charcoal and supplemented with 0.1 mg/1 NAA,
produced adventitious plantlets. Tisserat and DeMason (1980) found that on a
medium devoid of 2,4-D and 2-iP, sectioned buds callus consisted of two distinct
types of tissues; a loose friable tissue and compact aggregates. The friable
portion of the callus was composed of large non-meristematic cells and
disorganised clumps which were highly vaculated and ranged in diameter from
20-40 µm. This tissue was not involved in embryo formation and was generally
found surrounding the aggregate clumps which consisted of densely cytoplamic
cells containing few vacuoles and usually were 8-20 µm in diameter. The
formation of vascular bundles within the asexual plantlet at the 8- week old
stage corresponded to that found in the zygotic seedling.

Starting from the bottom of young leaves, soft tissues, shoot tips or
axillary buds of date palm offshoots (Figure 42),and using MS half strength or
Beauchesne medium supplemented by various auxins at a low concentration, buds
were obtained after six months of in vitro culture (Beauchesne et al.,
1986) (Figure 43).

Early rooting of date palm tissues reduce bud multiplication and is,
occasionally, responsible for the inhibition of bud initiation. In order to
solve this problem, the bottom of young leaves of date palm offshoots were
cultured on eight different nutrient media with different levels of growth
regulators (Anjarne and Zaid, 1993). High level of auxins, especially NAA,
allowed root initiation. These roots showed a rapid growth after subculturing on
a medium containing a lower level of auxins. Furthermore, organogenesis was
inhibited on media with a low concentration of auxins.

Vitrification phenomenon of date palm tissues is a handicap for the
successful in vitro multiplication of some date varieties and selected
clones. In order to overcome such a problem, four culture media with different
ammonium/total nitrogen ratio were tested, and bottom young leaves from
offshoots of AGUELLID variety were used (Bougerfaoui and Zaid, 1993). It was
found that ammonium plays an important role in the vitrification process. High
levels of ammonium nitrate were found to enhance rapid growth and consequently
tissue vitrification (46 to 53 % of cultures); while this phenomenon is reduced
to 14 - 19 % in media with low levels of ammonium nitrate.

4.3 Culture of highly differentiated date somatic tissues in
vitro

4.3.1 Leaf cultures

Callus developed from a seedling date palm leaf (Schroeder, 1970), and gave
rise to roots several months later. Similar results were obtained by Reuveni and
Kipnis (1974). In their study, primordial leaves survived in culture and
expanded, especially in the presence of light. The addition of plant growth
regulators at concentrations of 0.1 mg/l and above was injurious to cultured
leaves.

Eeuwens and Blake (1977) working with date palm leaf found development of
root initials to be enhanced by the presence of a low level of gas and auxins,
and by a reduction in either the level of minerals or sucrose. Phoenix
leaf petiol explant has initiated roots within 6 weeks when subcultured onto a
medium with high levels of auxin (Eeuwens, 1978). Root initiation was not
prevented by the presence of high cytokinin or low sucrose levels, but occurred
more frequently in media containing high sucrose and reduced cytokinin levels.
Poulain et al (1979) obtained some callus at the base of young date palm leaves.
Buds developed at the insertion zone between young leaves and rachis. Roots were
obtained on MS supplemented with a combination of low auxin levels such as 1.2
and 3 mg/l NAA, IBA, and IAA, respectively. Scharma et al (1980) noted callus
from leaf petioles of date palm initiated in media employed by Staritsky (1970)
or using Eeuwens Y/3 mineral formulation (Eeuwens, 1976).

Zaid (1981), working with date palm leaf explants from adult trees,
offshoots, seedlings and asexual plantlets, found that only subcultured leaf
callus from seedling and asexual plantlets produced roots.

4.3.2 Stem culture

Staritsky (1970) and Smith and Thomas (1973), both working with oil palms,
and Eeuwens (1978) with coconut and date palms, obtained a white callus on a few
stem cultures. Further attempts to subculture this callus failed.

Phoenix stem explants reportedly enlarged considerably in size during
the first few weeks of culture (Tisserat, 1979). Repeated culture to fresh media
resulted in the formation of non-friable nodular callus. Plantlets were
developed from this callus. Poulain et al. (1979) working with date palm stem
tissues also successfully initiated callus.

4.3.3 Inflorescence culture

Inflorescences of several species have been cultured in vitro (Nitsh,
1963). Since 1973, several workers attempted to culture palm inflorescences.
Explants of female and male oil palm inflorescences were cultured on a variety
of media and usually developed somewhat normally, but callus was not obtained
(Smith and Thomas, 1973). A high auxin level was speculated to be necessary to
disrupt normal development. This has subsequently been confirmed in date palm
(Eeuwens and Blake, 1977).

Date palm ovules, carpel tissue, parthenogenetic endosperm, and the fruit
stalk blackened within 24 hours after culturing on nutrient media, and
subsequently died (Reuveni and Kipnis, 1974). Also cultures of date palm floral
bud reproductive tissues and especially male anthers, usually turned brown and
died after a few weeks in culture (Tisserat et al., 1974). De Mason and Tisserat
(1980) found that in vitro applications of auxins to media increase the
frequency of visible expanded carpels developing from supposedly date palm male
fl owers.

Vestigial female date carpels on surviving male flowers enlarged and became
quite prominent (Tisserat, 1979). White friable callus usually initiated from
the floral bud strand (Tisseral et al., 1979). In some cases, roots and
embryoids were initiated from explants of Cocos inflorescences rachillae
(Eeuwens, 1978) and from date palm (Tisserat, 1979). Roots have not been
initiated on inflorescence rachis explants which lack leaf or meristem
tissue.

Date palm inflorescence culture was also largely investigated by Drira
(1981). Morphogenetic responses were found dependent on the origin and
physiological stage of the explant.

4.3.4 Root culture

Staristsky (1970) and Schroder (1970) were the first to investigate root
cultures in palms in vitro. Oil palm root and root primordia failed to
develop. Schroder (1970) observed that date palm root pieces in turn developed
secondary rootlets but did not produce shoots. Eeuwens (1978) found that
isolated roots excised from cultured explants of date and coconut palms continue
growth and produce laterals when subcultured on liquid static media. Callus was
also reported to form at the root tip region of young date palm seedlings
(Smith, 1975; Smith and Thomas, 1973). This callus had produced leaves and
shoots. Other investigators (Scharma et al., 1980) reported no growth for
cultured date palm roots. Usually, severe browning and death of root explants
occurred within the first few weeks of culture. However, Zaid and Tisserat
(1983a; 1983b), obtained some callus from seedlings and asexual plantlets roots
when callus failed to exhibit any morphogenic response.

4.4 Browning of tissues and media in date palm tissue
culture

During the course of in vitro growth and development, plant tissues
not only deplete the nutrients that are furnished in the medium, but also
release substances that can accumulate in the cultures. These substances, such
as phenols, may have profound physiological effects on the cultured tissues.
Date palm tissue cultures, like those of many other plants, have been commonly
observed to release discolouring substances into the medium which inhibit their
own growth. For date, injury through cutting of tissue is accompanied by
secretion of the substance(s) into the medium. The intact organ, as exemplified
by embryos or whole leaves on tips do not brown and thus grow well in culture
(Reuveni and Kipnis, 1974). Browning of the tissue and the adjacent medium is
assumed to be due to the oxidation of polyphenols and formation of guinones
which are toxic to the tissues (Maier and Metzlier, 1965; Zaid, 1987).

To minimise browning, Murashige (1974) has suggested the pre-soaking of
explants in ascorbic and citric acid solutions and adding them to the culture
medium. Zaid and Tisserat (1983a; 1983b) soaked their date palm explants in an
anti-oxidant solution (150 mg/l citric acid and 100 mg/l ascorbic acid) prior to
the surface sterilisation treatments. Addition of a combination of adsorbents
including citrate, adenine and glutamine, retarded browning in date palm
explants (Rhiss et al., 1979).

Addition of other adsorbents to nutrient media, such as dihydroxynaphtalene,
dimethylsulfoxide, were ineffective against browning in date palm explants
(Zaid, 1984). Apavatjrut and Blake (1977) suggested that browning could be
eliminated by a nutritionally balanced medium. Excision of browning explant
parts during culture was also advocated to prevent this problem (Zaid,
1984).

The use of charcoal is preferred over cysteine and other adsorbents because
the latter are often toxic to the plant tissues at higher concentrations (Zaid,
1984, 1990). Addition of 3 % charcoal has caused substantial root and shoot
growth of date embryos. Constantin et al. (1977) suggested that the growth
regulators required for callus growth and shoot development for tobacco are
adsorbed by charcoal addition. Similarly, Fridborg and Erikson (1975),
postulated that the addition of charcoal to a culture medium drastically alters
the properties of the medium. Hence, growth regulator substances are tested at
high levels (e.g. 10 and 100 mg/l) with charcoal included in the nutrient media
to obtain beneficial effects on tissues (Zaid, 1990; Zaid et al., 1989).

4.5 Cryopreservation of date palm shoot tips

Studies on the cryopreservation of date palm for germplasm collections were
initiated by Towill et al., (1989). Shoot-tips were excised from 2 month-old
seedlings derived from the cultivar "Medjool", precultured for 2 days and then
cooled to liquid nitrogen (LN) temperatures using procedures described for
potato and mint species. Viability of treated shoot-tips was assessed by growth
in vitro. Dimethylsulfoxide (DMSO) in concentrations up to 10 % was not
toxic, although growth was slower than untreated shoot-tips. Several
combinations of DMSO and sucrose were effective in obtaining survival after LN
exposure. In most cases, the LN-treated shoot tips developed directly into a
shoot without callus formation (Towill et al., 1989).

4.6 Organogenesis and asexual embryogenesis

Date palm plantlets may be produced through either; asexual embryogenesis,
i.e. initiation and germination of somatic embryos from callus; or
organogenesis, i.e. rooting and division of shoot tips and lateral buds.

Organogenesis technique, based on meristematic tissues potentiality, avoids
callus formation and does not use 2,4-D. Growth substances included in the media
are used as low as possible.

Organogenesis technique consists of 4 steps: Initiation of meristematic buds
(also called the starting step), multiplication (Figure 42), elongation and
rooting (+ swelling step). The success of such a technique is tremendously
dependent on the success of the first step (initiation); Furthermore, various
problems met at other levels have their origin at the initiation phase. These
technical problems could be summarised as follows:

* At the initiation phase

- Physiological stage of the offshoot, weight, age, signifi cation
degree, period of introduction.

- Initiation: too long.

- Bacterial contamination.

- Browning phenomenon.

- Varietal response to the technique/Lack of reactions of some clones and
varieties.

- Yield of the technique/offshoot.

- Precocious root development.

- Lack of results repetition.

* At the multiplication phase

- Low and irregular multiplication rate.
- Decrease of regeneration
capacity (precocious rooting).
- Loss of totipotency for some
varieties.

* Rooting and elongation

- Low effi cient rooting.

* At the acclimatisation phase

- Low rate of survival.

Asexual (also called somatic) embryogenesis, is based on the callus
production and multiplication, followed by the germination and elongation of
somatic embryos. Up to now, this technique had shown to be genotype independent
with a high rate of multiplication and a high survival rate upon transfer to
soil.

4.7 True-to-Typeness

There is always a dispute amongst date growers, technicians and scientists
about the true-to-typeness of plants produced in vitro. It is worth
mentioning that tissue culture-derived plants of many species are subject to
somaclonal variation in particular, and to genetic variations in general. Unlike
epigenetic variations, which are at physiological level with non-heritable
effect, genetic variations are affecting the genome and consequently are
heritable (Pierik, 1987; Zaid, 1987; 1990).

According to these authors, factors causing variations in plant tissue
culture are:

- Technique used for propagation;
- Nature of plant mother
(chimera);
- Type of growth regulators used;
- Type of explant used
(ploidy gradients: apex to root);
- Age of culture (> one year);
-
Medium composition; and
- Incubation conditions.

Most of the commercial laboratories are doing their best to ensure the true
to typeness of the produced date plant material. Various techniques are used to
produce and certify the conformity of the plants (Histo-cytology:Figure 44),
iso-enzyme, RFLP (Figure 45), RAPD techniques). In most cases, fi nger printing
is the technique actually used, but according to our experience we feel that the
fi eld response is the only reliable way to confi rm if the palms derived from
tissue culture are true to type to the plant mother.

Up to now, only two cases of variation with Medjool and Barhee have come to
our attention. Out of 2000 Barhee palms derived from asexual embryogenesis, only
2 are showing an abnormal vegetative growth (a ration of 0.1 %). These palms are
marked and their fruits will be compared to the mother variety (Figures 46, 47
and 48).

4.8 Commercial production

Various laboratories in the world have made attempts to propagate date palm
by tissue culture techniques. According to the knowledge of the authors, success
has been achieved at only a few international laboratories (Table 35).

Some of these laboratories are recent (2 to 3 years), while others have been
functioning for approximately 15 years. There are 9 functional laboratories
known to the authors. These are found in England (1), France (2), Israel (1),
Morocco (1), Namibia (1), UAE (1), Oman (1), and India (1). Information about
the last two laboratories is not available.

The commercial laboratory of the "Domaine Agricole El Bassatine" (Morocco),
which since its start had produced ± 500,000 plants, is reserving all its
production for national use. No signifi cant sale outside Morocco has been
implemented because all the production is destined to rehabilitate the Moroccan
Date plantations destroyed by the Bayoud disease.

The remaining laboratories (England, France, Israel and Namibia) are
potential sources of date plant material. Most of these laboratories' efforts
were focused on the Medjool (and Barhee recently) variety with an average sale
price (FOB) of about 20 to 23 US$ per plant. Delivered plants have only juvenile
leaves and still need to be hardened-off by the buyer before fi eld planting
(Figure 49). Note that the selling price depends on the variety, the number of
plants ordered and the growth stage at delivery.

TABLE 35
List of international date palm commercial
laboratories(*)

Country

Company

Address

UNITED KINGDOM

- DATE PALM DEVELOPMENTS

Baltonsborough, Somerset
BA6. 8QG, United Kingdom
Tel: (+44) 1458
850576
Fax: (+44) 1458 851104

France

- MARIONNET G.F.A.

21 Rue de Courmemin 41230 Soings - France
Tel: (+33) 254 987
103
Fax: (+33) 254 987 523

- PALMDAT - France

"Laboratoire de Physiologie Végétale"
"Recherche et
Développement"
Marolles 37460, Genille,
France
Tel: (+33) 247
5952 52
Fax: (+33) 247 59 59 18

ISRAEL

- RAHAN MERISTEM

Propagation Nurseries
Kibbutz Rosh Hanikra
Western Galilee 22825,
Israel
Tel: (+972) 4 985 7100
Fax: (+972) 4 982 4333

MOROCCO

- DOMAINE AGRICOLE EL BASSATINE

B.P. 299 Meknes, Morocco
Tel: (+212) 5 50 0493
Fax: (+212) 5 50
0730

NAMIBIA

- PALMDAT NAMIBIA

P.O. Box 20519
Windhoek, Namibia
Tel: (+26461) 230480
Fax:
(+26461) 250889

UNITED ARAB EMIRATES

UNITED ARAB EMIRATES UNIVERSITY - DATE PALM DEVELOPMENT
RESEARCHUNIT

P.O. Box 81908-Al-Ain
Tel: (+9713) 8732334
Fax: (+9713)
7832472

Others in Middle East (Oman) and in India

- No information available

Total Laboratories

9

(*) There is no order of importance in the list, which should also not be
considered as exhaustive. Countries were classifi ed in an alphabetical
order.

5. Acclimatisation and hardening-off of tissue
culture-produced date plants

5.1 Introduction

Although in vitro mass plant propagation has become commercially
feasible, many problems hinder its application to economically important
crops.

One of the major obstacles concerning the practical application of plant
tissue culture to mass propagation has been the diffi culty of successful
transfer of plantlets from in vitro conditions to a soil medium. Losses
from 50 to 90 % of in vitro propagated plantlets of many species have
been encountered at the time of transfer to soil (Zaid and Hughes, 1989a;
1989b). This isunfortunate because the ultimate success of plant tissue culture
as a commercial means of plant propagation depends on the ability to transfer
plantlets out of culture, on a large scale, at low cost and with a high survival
rate.

It is appropriate at this level to differentiate between the acclimatisation
of date palm vitro plants at the laboratory's glasshouse and their
hardening-off at the farmer's nursery.

5.2 Acclimatisation

Acclimatisation presents challenges at least equal to those posed by the
initiation of cultures because it marks the end of artifi cial control and the
beginning of autonomous plant growth. Approximately 20 years ago it was stated
that research concerning the preparation of in vitro plantlets for
transfer to soil had been neglected (Murashige, 1974). Since that time many
scientists have become interested in the effects that the transfer process has
on tissue cultured plantlets (Zaid and Hughes, 1995a; 1995b).

The culture of date tissue in vitro with almost 100 % relative
humidity within the culture vessel can lead to various abnormalities in the
plant structure (Zaid and Hughes, 1989c). Plants of many species produced in
vitro often show morphological, structural, physiological and biochemical
differences from those produced conventionally. These include reduced
epicuticular wax deposits (Figure 50), altered leaf anatomy (Figure 51),
excessive water loss and stomatal abnormalities compared to greenhouse grown
plants (Zaid, 1995; Zaid and Hughes, 1995c).

It is worth mentioning that loss of viability is attributed to poor control
of water loss from the date plants and their heterotrophic nature.

Stomatal development and frequency can be affected by water availability,
light intensity, temperature, humidity and osmotic concentration of the culture
medium (Zaid and Hughes, 1995b).

Even when gradual hardening off has been used, poor survival and slow growth
of date plantlets have commonly been reported. Such a low survival rate (that
sometimes reaches below 50 %) is caused by several factors which are mainly
young physiological stage of plantlets to transfer, inadequate root system,
unsatisfactory irrigation schedule, and lack of technical care at the in
vitro laboratory stage.

Several techniques have been used to acclimatise date plantlets and improve
their survival during establishment under greenhouse conditions. The
effectiveness of these methods depended upon ambient conditions, and most
methods have involved environmental modifications. Mentioned below are the three
most important factors to be taken into account by the manager of a date palm
propagation laboratory in order to ensure a high survival rate and fast growing
situation of date palm tissue culture-derived plantlets:

5.2.1 Physiological stage

Date palm plantlets are ready for transplanting only when they gain the
following characteristics:

- Two to three healthy and enlarged leaves with no curling phenomenon;

- A shoot length of at least 10 to 15 cm from stem base to the highest
point of the leaves;

- A shoot base with an onion bulb-like form (also called pear-shaped
crown);

- A well developed root system with an average of 5 cm in length.
Adventitious rooting is obtained by trimming the primary roots to 1 - 1.5cm in
length and reculturing the plant to an agar nutrient medium containing
0.01/0.1 mg/l NAA without charcoal; and

- Well acclimatised plant as a fi nal product (Figure 52).

Plants are then rinsed in distilled water to remove adhering agar and
residual sucrose. A spray with Benlate solution at 0.5 % (or any wide spectrum
fungicide) is important since it protects the plant from fungal attack.

In order to achieve the above, and consequently produce a well
pre-acclimatised date plant that will survive the transplanting stress, it is
recommended that the following be ensured:

- Do not transplant any plant until it gains the previously mentioned
characteristics;
- Enhance a root-elongation process by using auxins at the
last in vitro stage;
- Increase the light intensity during the last
4 to 6 weeks; and
- Create an artifi cial osmotic stress (at the nutrient
medium level).

5.2.2 Transplanting to soil medium

The transplanting operation should be done as quickly as possible to avoid
plant dehydration and avoid root damage as far as possible. The soil medium must
always be sterile and usually consisting of 1 peat: 1 vermiculite (v/v) mixture.
Sterile sand with a large grain size could also be added to improve drainage.
Bark is to be avoided because it dries out rapidly and causes a water stress
situation. To summarise, the substrate should be a well drained one, yet with
good water retention capacity. The adequate pH to work with should be about
6.5.

Plastic pots (7.5 - 12.5 cm), jiffy peat pots or trays (25 plants; in case of
commercial production) are often used for date palm transplanting.

5.2.3 Environmental conditions

Plants are immediately irrigated with 50 % Hoagland's solution or 10 % MS
solution before their incubation into a micro tunnel located in an
environmentally controlled glasshouse (or a large plastic tunnel).

These environmental conditions will ensure a high relative humidity (90 - 95
%) and a constant temperature ± 25 - 26°C day time and 21 - 22°C during the
night. Bottom heating of the micro tunnel (± 23°C) was found to be very
helpful.

To ensure a high survival rate, date palm tissue culture-derived plants
should be adapted to gradually decreasing humidity and gradually increasing
light. The light intensity is important during the first 3 to 4 weeks in the
glasshouse (around 10,000 lux) with a 16 hr photo period. Benlate is to be
applied to the foliage once a week, and irrigation using 10% MS solution (or 50
% Hoagland) every 3rd or 4th day depending on the hygrometry level of the micro
tunnel.

Four to six weeks later, the plastic of the micro tunnel is gradually opened
in order to decrease humidity and prepare the plants to adapt to the large
glasshouse (or tunnel) conditions which preferably should have a fog system.
Plantlets are now ready to be transplanted to larger plastic bags.

It is worth mentioning that at all stages, water should never be sprayed form
the top of the plant. Plants could stay in the glass house (or a tunnel) for a
period between 3 to 4 months before their transfer to a less environmentally
controlled nursery, which is usually at the farmer's level, for their further
hardening-off process.

5.3 Hardening-off

Plantlets received from a laboratory are usually about 35 to 45 cm long with
4 to 5 leaves among which are 0 to 2 pinnae leaves (called also permanent
leaves). The plant must have a thick shoot system and the base must be similar
instate to that of a large onion bulb (pear-shaped). As stated above, the plant
must have a well developed root system.

Transportation of these plants must be realised in a proper manner and plants
must preferably not be stacked on top of each other to avoid stem breakage
and/or leaf damage. Transport must preferably also be in one stage and if
plants/truck are to stay over somewhere, it must be in a shaded area; watering
should not be neglected if transport takes up several days.

It is recommended that, upon reception of this material by the date grower,
plants are transferred to larger bags (7 to 10 litres capacity) with an adequate
substrate,usually sand (soil), vermiculite and gravel at a ratio of 1:1:1,
respectively. Transplanting should be done properly with no disturbance to the
root system. Original substrate around the roots should stay intact. Plants are
then left in the nursery for approximately 8 to 12 months depending on
surrounding conditions and care given, till most of them reach the 4 pinnae leaf
stage. The date grower is advised to co-ordinate the purchasing and the
hardening-off period, to ensure that planting can betimely implemented (during
February/March for Southern hemisphere and September/October for Northern
Hemisphere).

The nursery size and type are related to the number of plants to be
hardened-off. An average size of 150 m² will be adequate for 1,000 plants. An
ultra-violet resistant shade net of 80 % is recommended during the first 6
months (Figure 53). During the summer time, the top of the nursery should have a
double layer of the shade net for insulation purposes. The nursery should be
well located (close to several trees to benefi t from their shade) but also in a
protected area to avoid sand storms and severe wind. A water tap should be
installed inside the unit for easy irrigation and the unit must be enclosed to
avoid animals getting in and eating the plants.

Irrigation is an important factor and must be implemented once a week in
winter time and at least twice a week during summer. Water should never be
sprayed on top of the plant; soil is to be mounted around the base of the plant
so water can not get into its heart.

Fertilisation is to be applied once per month: apply 5 g of ammonium
sulphate/plant bag (5 % nutrient solution; thus 15 kg deluded per 63 litres
water for 650 plants). Apply 120 ml of solution per plant bag.

Control of diseases and pests is also recommended and the use of Benlate (or
any other large spectrum fungicide) has proven to be highly efficient. Foliar
spray of Benlate is to be applied every 3 to 4 weeks.

Close monitoring is advised as mistakes could be disastrous; It is from our
own experience, that we recommend a close follow-up by the date grower. If all
above recommendations and advicse are respected, the date grower could expect a
survival rate between 90 and 95 % (Figure 54).

In Namibia, a total of 10,007 plants of various date palm varieties were
hardened during 1996 and 1997 in both Naute and Eersbegin project sites (Table
No. 36).

The results obtained are satisfactory and after 8 to 12 months (depending on
the variety and the source), a final survival rate of 92% was obtained (9,177
plants survived and successfully passed the hardening-off operation out of
10,007 plants).

TABLE 36
Hardening-off of date palm tissue culture plant lets:
Survival rate (16/06/1997)

VARIETIES

ORIGINAL NUMBER OF PLANTS ORDERED

ORIGIN OF PLANT MATERIAL

TOTAL SURVIVAL

RATE OF SURVIVAL
(%)

Medjool du Roi

2,922

RSA

2,723

93.10

Medjool Marionnet

2,650

France

2,411

90.90

Kush Zabad

120

UK

106

88.30

Khalas

90

UK

84

93.30

Hilali

90

UK

88

97.70

Nabutsaif

120

UK

116

96.60

Khenezi

135

UK

35

25.90

Barhee

1,965

UK

1,854

94.30

Bou Feggouss

1,225

France

1,225

100

Deglet Nour

120

France

117

97.50

Khadrawy

45

France

01

02.20

Anbara

50

UK

35

70.00

Sukkari

175

UK

162

92.50

Khissab

90

UK

87

96.60

AbuNaringa

120

UK

105

87.70

Lulu

90

France

28

31.10

Total

10,007

9,177

91.70

- Immediately after transplanting, an average percentage a loss of 3.2%
occurred.

- After 8 to 12 months in the nursery, the final survival rate was about
92% (9,177 out of 10,007 plants).

(Source: Date Production Support Programme in Namibia; FAO-UTF/NAM/004/NAM;
1997)

Figure 34. Date palm seedling plantation to select salt
tolerant clones at Guanikontes (Swako-pmund, Namibia)

Figure 35. Rooting of off-shoots:

A - Normal axillary offshoots after their separation from the mother
palm.

B - High offshoots on the palm using plastic bags fi lled with
saw-dust.

Figure 36. Various types of chisel used around the
world

A - American type

B - normal and most common

C - traditional type

(Source: Munier, 1973).

Figure 37. Offshoots pruning methods:

A - as an onion-bulb

B - average pruning

C - short pruning

Figure 38. Basin around a young date palm (1.5 to 1.8 m
diameter and 20 to 30 cm deep) with wheat straw as a mulching.

Figure 40. Comparison of asexual embryo (right) with
excised zygotic embryo (left) at the cotylegon elongation stage

Figure 39. After planting protection against harsh
climatic conditions:

A - Use of hessian wrap for offshoots

B - Protection unit made of wire and shade net for tissue culture
plants

C - Protection tent made of date leaves.

Figure 41. Sequence of germination for
Phoenix dactylifera cultivar Sayer excised embryos cultured on a modifi
ed Murashige and Skoog medium containing 0.3 activated charcoal.

From left to right: early cotyledon elongation stage (1 week old);
emergence of first foliar leaf (3 week old); and established seedling in
vitro (6 week old).

Note that the cotyledon haustorium is much reduced in size in all stages of
seedling develop-

Figure 42. Various types of date palm explants used in
organogenesis technique (mostly the bottom of young meristematic leaves)

Figure 43. Multiple shoot formation of date palm "Tademant"
variety

Figure 44. Cross section of date palm shoot tip

Figure 45. Zymogram of date palm "Black Bousthami"
variety

Figure 46. Medjool palm derived from asexual embryogenesis
showing abnormalities (Eden Expt. Station, Israel, 1996); It looks like Black
Scorch attack

Figure 47. Barhee palm derived from asexual embryogenesis
showing morphological abnormality (Ref. G12-Block2, Naute project,
Namibia)

Figure 48. Large leaf size as an abnormality (Right: Variant
Barhee; left: normal Bar-hee leaf); (Ref B14-Block 2, Naute Project,
Namibia).

Figure 49. Boufegouss variety plants after hardening at the
laboratory's glasshouse

Figure 50. Comparison of leaf epicuticular wax between
greenhouse- grown, tissue culture- derived (Polyethylene glycol-treated and non
treated plants); Note five varieties were tested

Figure 51. Leaf anatomy of a Med-jool date palm. Note the
size of the bulliform cells.

Figure 52. Well acclimatized plants ready to go through the
hardening-off process.

Figure 53. An ultra violet resistant shade net of 80% is
commonly used for date palm nursery (hardening-off at the date grower's
level).

Figure 54. Various stages of growth and development of date
palm tissue culture plants during the hardening-off process.

From right to left: 3 months, 6, 9 and 12 months old.