Journal of Arid Environments (2003) 54: 133–147

doi:10.1006/jare.2001.0884

Tissue culture technology for the conservation

and propagation of certain native plants

C. Sudhersan, M. AboEl-Nil & J. Hussain

Biotechnology Department, Food Resources Division, Kuwait Institute
for Scientific Research, P.O. Box 24885, Safat 13109, Kuwait

While the contribution of native plants to the ecological balance and to the
preservation of the environment has been recognized, development of methods
for tissue culture of native plants for propagation and conservation is lagging
behind. Tissue culture technology can be utilized for the conservation and
mass propagation of selected native perennial plants that cannot be propagated
on a large-scale by means of seeds and cuttings. Certain genotypes of Rhan-
terium epapposum, Ochradenus baccatus, Nitraria retusa and Lysium shawii were
selected, and tissue culture technology was developed because of their potential
for use in urban landscaping and in desert revegetation.

 2003 Published by Elsevier Science Ltd.

Keywords: biodiversity; conservation; propagation; desertification; cytokinins;

Kuwait

Introduction and literature review

Native plants are key components of the global biological diversity, and are highly
adapted to the local environmental and climatic conditions. Native plant communities
provide a beautiful and healthy environment. Growing native plants will preserve the
ecological balance, and perpetuate the relationship between the native plants and the
many other organisms that depend upon them for their survival. They also provide food
and shelter to wild bees, insects, birds and animals. In addition, native plants require less
water, care and maintenance than the exotic species that are being introduced.
Geographically, Kuwait is a desert area with low rainfall, and harsh environmental and
climatic conditions. Kuwait’s desert ecosystem has been disrupted at an unprecedented
rate (AboEl-Nil, 1997). Native plants are a part of the desert ecosystem that has been
damaged by overgrazing, cutting down the woody plants for fuel, abuse of off-road
vehicles, urbanization, mining, pollution and activities of the Gulf War. The heavy
pollution with crude oil is a serious threat to the wildlife (Karrar et al., 1991). Several
native desert plants are being threatened and are facing the danger of extinction for the
above-mentioned reasons.
Tissue culture technology has often been successfully utilized in propagation of plants
with poor and uncertain responses to conventional methods of propagation, to preserve
endangered species. For many years, interest in the application of tissue culture for the

Kuwait Institute for Scientific Research publication number KISR 5712.

0140}1963/03/010133#15 $30.00/0  2003 Published by Elsevier Science Ltd.

134 C. SUDHERSAN ET AL.

development of plant production in the Arabian Gulf countries has drawn the attention
of the public as well as the private sector (AboEl-Nil, 1977). Many native plants of
Kuwait and the Arabian Peninsula are threatened, endangered or difficult to propa-
gate. Conservation and mass propagation of native plants can play an important role in
the rehabilitation of Kuwait’s desert (AboEl-Nil, 1997).
Tissue culture technology is of great interest for the collection, multiplication and
storage of plant germplasm (Englemann, 1991). The tissue culture system allows the
propagation of selected genotypes with high multiplication rates in an aseptic, temper-
ature-controlled environment. The miniaturization of explants by this system allows the
reduction of space requirements and, consequently, labor costs for the maintenance of
germplasm collections. Plant materials can be maintained in vitro by using slow-growth
culture media for longer duration at normal room temperature or by cryopreservation
methods.
The native vegetation of Kuwait includes scant perennial woody shrubs, herbs and
spring ephemerals. Four ecosystems have been characterized and differentiated
according to habitat factors viz., sand dunes, saltmarshes, desert plain and desert plateau
(Halwagy & Halwagy, 1974; Halwagy et al., 1982). Recently, Kuwait’s desert ecosystem
has been greatly affected by the activities of the Gulf War and crude oil pollution
due to the burning of the oil fields. Certain native plant species have shown tolerance
over these adverse soil-stress conditions, while the majority of them failed to survive.
Many native plants have potential for use as animal fodder and sand protector from
erosion, and in phytoremediation and ornamental landscaping. Such perennial native
plant genotypes need to be preserved and propagated on a large scale for the rehabilita-
tion and restoration of Kuwait’s desert ecosystem.
We have selected four native perennial plant species, Rhanterium epapposum (arfaj),
Lycium shawii (aswaj), Ochradenus baccatus (qirdhi) and Nitraria retusa (ghardah) for
the tissue culture studies because of their ability to tolerate extreme adverse conditions,
such as oil pollution, drought, and salt, in addition to their possible potential use in urban
landscaping.

Rhanterium epapposum Oliv.

This species belongs to the botanical family Asteraceae (Compositae), and it grows as
a perennial woody shrub mixed with other perennials and annuals. It dominates its
steppe with a frequency of about 50% and coverage of 1–25% (Halwagy & Halwagy,
1974). It is a major component of the herbage for the grazing animals in Kuwait
(Al-Rawi, 1987; Batanouny, 1990; Taha et al., 1990). The regeneration of Rhanterium
from seed has not been observed, although this must be the natural method of propaga-
tion (Halwagy et al., 1982). Seed germination under simulated environmental condi-
tions ranged from 0 to 4)69% (Taha et al., 1988). It is difficult to propagate and
establish this species by vegetative methods. Recently a tissue culture protocol for
mass propagation of this species has been developed for the first time (AboEl-Nil,
1997).

Ochradenus baccatus Del.

This is a branched perennial deciduous shrub that belongs to the botanical family
Residaceae. In nature, this plant grows usually in a unique and peculiar habitat of rocky
terrain with shallow-course sandy slopes up to 1)5 m in height. It is a dioecious species
which produces male and female inflorescence in separate plants (Daoud & Al-Rawi,
1985; Mandavillae, 1990). This species is reported to be very rare in Kuwait (Shuaib,
1995). In vitro protocols for mass propagation of this species via somatic embryogenesis
 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 135

(Sudhersan et al., 1999), organogenesis and nodule multiplication methods (Sudhersan

et al., unpublished report) have been developed.

Lycium shawii Roem et Scult.

This is a thorny perennial shrub that belongs to the botanical family Solanaceae. It grows
along sandy stone ridges. It produces whitish to purple flowers during March to April in
its natural environment and throughout the year in irrigated soil (Sudhersan’s personal
observation). The fruits of this species are globular, many seeded, red to orange berries
which are edible and somewhat sweet. It provides honey for wild bees, and food and
shelter for wild birds and animals. In vitro studies on other species of this genus, Lycium,
have been reported (Xy et al., 1991; Harsh, 1989; Liu, 1991; Park et al., 1993; Kim et al.,
1993). Tissue culture protocols for the mass propagation of Lycium shawii through
somatic embryogenesis and organogenesis methods were developed for the first time
(Sudhersan et al., unpublished report).

Nitraria retusa ( Forsk.) Aschers.

This is one of the native perennial species that belong to the botanical family Zygophyl-
laceae. It grows along shallow sand hummocks on saline grounds near the coastal areas.
It is a thorny shrub with fleshy, grayish, heart-shaped leaves. It is a salt-tolerant and
drought-resistant species which produces fleshy red fruits. The fruits are tasty, and
a refreshing juice may be extract from them. Many wildlife forms feed on the fruits of
this plant. The natural propagation of this species is through seeds. This species is under
severe pressure from grazing animals and harsh climatic conditions. Tissue culture
technology for the mass propagation and conservation of this species has been de-
veloped for the first time (Sudhersan et al., unpublished report).
Literature study indicated that there are no previously published reports on tissue
culture technology developments for these four native plant species. Hence, we under-
took detailed investigations of the possibility of mass propagation via tissue culture
technology of these important native perennial plant species.

Material and methods

Plant materials

Shoots with terminal and axillary buds from the healthy plants of selected genotypes
of Rhanterium epapposum, Ochradenus baccatus, Lycium shawii and Nitraria retusa
growing in the desert of Kuwait were collected and utilized as plant materials for the
study.

Sterilization and explant preparation

Shoot cuttings pre-washed with soapy water were surface-sterilized with 20% commer-
cial Chlorox] containing 1% sodium hypochlorite with 1 drop of Tween 20] for
15 min and ultrasonic vibration for 1 min. After rinsing four times with sterile distilled
water, shoot buds were treated with 0)1% mercuric chloride solution for 3 min. and
washed thoroughly in sterile distilled water. After the surface sterilization, leaf, stem
nodal and internodal segments, and shoot tips with two to three leaf primordia were
isolated using a sterile surgical knife and forceps were used as explants.
136 C. SUDHERSAN ET AL.

Table 1. Composition of organic compounds added to the MS basal media used for
native plant tissue culture

Chemical Concentration
(mg l!1)

Thiamine HCl 5
Pyridoxine HCl 2)5
Nicotinic acid 5
Calcium panthothenate 2)5
Glycine 3
Myo inositol 125
Glutamine 100
Sucrose 30,000
Gelrite] 1)3

Media and culture procedure

Explants from all of four species were cultured in vitro, to explore the possibilities of
developing protocols for propagation via organogenesis and somatic embryogenesis,
utilizing standard cell and tissue culture methods (Evans et al., 1983; Ammirato et al.,
1990). Since the literature survey showed no published reports on any of the four native
plant species, Murashige & Skoog’s inorganic basal salt media (MS) was used as the
basic medium for all experiments (Murashige & Skoog, 1962). Different organic
additives were added to the basal medium to determine the factors affecting the
morphogenetic response at different stages of the culturing process (Table 1).
Experiments were carried out to control the development of callus initiation, de novo
shoot bud regeneration (organogenesis), somatic embryogenesis, plantlet growth, root-
ing (rhizogenesis) and in vitro storage (Table 2). All media were gelled with 0)13%
Gelrite] (Gellan gum). The pH of the media was adjusted to 5)6 prior to autoclaving.
All media were autoclaved at 1213C for 15 min and stored in a media storage room until
utilized. Explants were placed on the surface of the sterilized media, and kept in
temperature-and light-controlled culture rooms. Each experiment contained 36 explants
per treatment, and the experiments were repeated twice. All cultures were renewed by
subculturing every 15 days, and the contaminated cultures were discarded. Observations

Table 2. Media components used in different stages of native plant tissue

culture to supplement the MS basal medium

Addenda* Culture stages

Embryogenesis Organogenesis Growth & rooting Storage

2,4-D 1–3 mg l!1 — — —

Kinetin 0)1 mg l!1 — — —
BA — 1 mg l!1 — —
NAA — — 0–0)1 —
Silver nitrate — — — 40 mg l!1

*2,4-D"2,4-dichlorophenoxyacetic acid, BA"benzyladenine, NAA"naphthaleneacetic acid.

 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 137

were made once every 10 days and the morphogenetic responses of the explants were
recorded.

Acclimatization

The soil mix prepared by mixing sand, peat moss and humus at a 1:1:1 ratio was
autoclaved at 1213C for 45 min prior to planting of the rooted plantlets. Rooted plantlets
were washed in running tap water to remove the nutrient media to avoid the fungal
attack of the root system. Cleaned plantlets were treated with 0)5% Benlate] solution
and planted in small plastic containers. All the plantlets were acclimatized for 20 days at
controlled temperature prior to greenhouse acclimatization. Plantlets were acclimatized
to the greenhouse environmental conditions before the field experimentation by gradual-
ly decreasing the humidity.

Results

Rhanterium epapposum

Tissue explants from stems and leaves of this species showed high morphogenetic
potential in plantlet regeneration via callus. Explants cultured on MS basal media
containing various organic additives and cytokonins produced organogenic callus after
3 weeks in culture.

Organogenesis

A high percentage of tissue explants showed callus regeneration in MS media containing

low concentrations of Benzyl adenine (BA) alone or in combination with auxin. Two
types of callii: nodular (Fig. 1(a)) and compact (Fig. 1(b)) were observed in this species.
Both types of callii, when transferred to growth-regulator-free MS media containing
20 mg l!1 silver nitrate, produced numerous shoot buds. Multiplication of this or-
ganogenic callus was continuous in the same media. Shoot bud elongation was achieved
on media containing 0)1–1 mg l!1 gibberellin3 (GA3) alone or in combination with
a cytokinin. Shoot buds (Fig. 1(e)) elongated into 1)5–2 cm on this medium after 15
days. Further growth and elongation was observed when the shoot buds were transferred
to growth-regulator-free media. Prolonged cultures on the elongation media showed
thin, weak stems with a glassy appearance due to the effect of GA3. Elongated
shoots produced adventitious roots when isolated and cultured in growth-regulator-free
MS media or with a low concentration of naphthaleneacetic acid (NAA) or indolbuteric
acid (IBA).

Somatic embryogenesis

Stem and leaf explants cultured in MS media containing 1–5 mg l!1 2,4-
dichlorophenoxyacetic acid (2,4-D) alone or in combination with 0)1 mg l!1 kinetin
produced embryogenic callus (Fig. 1(c)). These embryogenic callii, when transferred
to the growth-regulator-free media or with low concentrations of cytokinin,
produced somatic embryos. None of the embryos produced shoots. All the
embryos produced primary roots, and the cotyledon portion produced organogenic
callus (Fig. 1(d)) in growth-regulator-free media or with low concentrations of
a cytokinin.
138 C. SUDHERSAN ET AL.

Figure 1. Morphogenesis of Rhanterium epapposum in vitro. (a) nodular callus, (b) compact
callus with shoot buds, (c) embryogeneic callus, (d) somatic embryo forming callus, (e) isolated
shoot buds, (f ) rooted plantlet, (g) acclimatized plantlets.

Acclimatization

Rooted plantlets (Fig. 1(f )) developed via organogenesis from callus or via axillary bud
break were successfully acclimatized to the greenhouse environmental conditions ( Fig.
1(g). More than 70% of the plants in this species survived during the acclimatization
procedure.

Ochradenus baccatus

Results obtained from various in vitro experiments on this species showed high
morphogenetic regeneration potential. Different types of tissue explants, such
 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 139

as leaf, stem, inflorescence axis and root, responded positively by regenerating

plantlets.

Axillary bud multiplication

Stem nodal explants cultured on MS medium containing 1 mg l!1 of BA produced

numerous adventitious and axillary shoot buds. These shoot buds multiplied and
elongated into plantlets in growth-regulator-free medium. Adventitious roots were also
developed from the elongated shoots on the same medium after 45 days. Low concen-
tration of NAA also induced adventitious root initiation.

Regeneration via nodules

The whole-leaf explants or leaf segments produced numerous meristematic nodules on

the entire surface of the leaf (Fig. 2(e)) and the cut end of the petiole in the presence of
1–5 mg l!1 of any cytokinin. These nodules were round to oval in shape. All the
meristematic nodules produced adventitious shoot buds (Fig. 2(f )) on the same media
after 30 days. Shoot elongation and rooting were achieved on growth-regulator-free media.

Organogenesis in callus cultures

All the tissue explants produced organogenic nodular callus (Fig. 2(a)) on MS media
containing low concentrations of a cytokinin. Numerous shoot buds (Fig. 2(b))
appeared on the surface of the callus on the same media after 45 days. The shoot buds,
when transferred to growth-regulator- free medium, elongated and produced adventi-
tious roots.

Somatic embryogenesis

Tissue explants planted in MS media containing 1–3 mg l!1 2,4-D induced em-
bryogenic callus (Fig. 2(c)). These embryogenic callii produced embryos on growth-
regulator-free medium and germinated into plantlets (Fig. 2(d)). Embryogenic callii,
when transferred to media containing a cytokinin, formed multiplying masses that then
produced several shoot buds without roots.

Acclimatization

Rooted plants (Fig. 2(g)) obtained from organogenesis, axillary bud multiplication,
nodule culture and somatic embryogenesis were successfully acclimatized in the green-
house. More than 95% of the plantlets survived during the acclimatization procedure.
Acclimatized plantlets showed normal growth and development both in the greenhouse
and in the field (Fig. 2(h)).

Lycium shawii

Experiments on this species also showed a high percentage of morphogenetic regenera-

tion potential in vitro. Plantlets were developed via shoot tip and axillary bud multiplica-
tion, organogenesis via callus, and somatic embryogenesis methods.

Shoot tip and axillary bud multiplication

Shoot tip and stem nodal explants cultured on MS medium without any growth
regulators showed shoot development and axillary bud elongation, respectively.
140 C. SUDHERSAN ET AL.

Figure 2. Morphogenesis of Ochradenus baccatus in vitro. (a) nodular callus, (b) shoot buds,
(c) embryogenic callus, (d) Stages of somatic embryo germination, (e) Leaf explant with meri-
stematic nodules, (f ) leaf explant with shoot buds, (g) rooted plantlet, (h) acclimatized plantlet.

Elongated shoots (Fig. 3(f )) produced adventitious roots (Fig. 3(g)) in the same media.
Each shoot bud produced an average of 8–10 nodes within one month. More than
90% of the explants showed morphogenetic responses on growth-regulator-free
MS medium.

Organogenesis

Stem and leaf explants cultured on MS medium containing 1 mg l!1 BA produced

organogenic callus. Two types of callii, nodular and compact, were produced on the
same medium. Each type of callus produced its own type and multiplied. After three to
four weeks in culture, the compact callus produced several greenish shoot primordia
(Fig. 3(d)), and nodular callus (Fig. 3(e)) produced numerous shoot buds. In
 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 141

Figure 3. Morphogenesis of Lycium shawii in vitro. (a) embryogenic callus, (b) somatic embryos,
(c) stages of embryo germination, (d) compact callus with shoot buds; (e) nodular callus,
(f ) isolated plantlets in the elongation media, (g) rooted plantlet, (h) acclimatized plantlet.

growth-regulator-free MS medium, shoot buds developed into plantlets. Isolated plan-

tlets, when subcultured on the same medium, produced adventitious roots.

Somatic embryogenesis

Explants cultured on MS media containing 1–3 mg l!1 2,4-D alone or with 0)1 mg l!1
Kinetine] (K) induced embryogenic callus (Fig. 3(a)). Somatic embryogenesis and
142 C. SUDHERSAN ET AL.

embryo germination (Fig. 3(b,c)) occurred when the callus transferred to growth-
regulator-free medium. Numerous rooted plantlets were obtained within a short time by
this method. On MS basal medium with a cytokinin, embryogenic callus developed
multiplying mass clumps and produced shoots without roots.

Acclimatization

Rooted plantlets that were obtained through different morphogentic routes were
successfully acclimatized and transferred to the greenhouse (Fig. 3(h)) and field envir-
onmental conditions. The survival rate during the acclimatization of the rooted plantlets
reached 100%.

Nitraria retusa

Several experiments that were carried out on this species, indicated that it is recalci-
trant to organogenesis and somatic embryogenesis differentiation. Axillary bud
multiplication was achieved after prolonged subculturing on MS medium containing
1 mg l!1 BA.

Organogenesis

Stem and leaf explants produced callus on MS medium containing low concentrations
of a cytokinin, alone or in combination with an auxin. Soft, loose, nodular types of callii
were produced on the same media. The soft callus was yellowish, and the nodular callus
greenish. Both types of callii multiplied continuously without producing any shoot buds
or roots in MS media with different growth-regulator combinations.

Somatic embryogenesis

Leaf and stem explants produced friable embryogenic callus (Plate 4C) on MS medium
containing 1–3 mg l!1 2,4-D alone or in combination with 0)1 mg l!1 K. Numerous
round proembryos developed from these friable callii when transferred to growth-
regulator-free MS medium. Proembryos that were developed on media containing 2,4
D alone produced roots without shoots, while those developed on media containing
2,4-D and K multiplied without germination. Embryogenic callus transferred to the MS
liquid media produced somatic embryos which multiplied in the same media but failed
to germinate upon transfer onto a semisolid medium.

Shoot tip and nodal multiplication

Shoot tip and stem nodal explants enlarged and remained without shoot bud initiation
on MS medium without growth regulators and on media with 1 mg l!1 BA. After six
months of subculturing, axillary shoot buds developed on media containing 1 mg l!1 BA
(Fig. 4(a, b)). These buds elongated into rooted plantlets (Fig. 4(d)) in growth-
regulator-free medium or on media containing 0)001–0)1 mg l!1 BA. On the medium
containing 1 mg l!1 BA, axillary shoots multiplied continuously. The plantlets attained
lengths of 10–15 cm with 10–15 nodes within 60 days of subculturing. These nodal
segments from elongated plantlets were cut and subcultured in shoot elongation media
to develop into rooted plantlets.
 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 143

Figure 4. Morphogenesis of Nitraria retusa in vitro. (a) shoot tip producing axillary buds,
(b) shoot multiplication and elogation, (c) embryogenic callus (d) growth response of shoot tips in
different concentrations of BA, (e) rooted plantlet.

Acclimatization

Rooted plantlets (Fig. 4(e)) were acclimatized successfully and maintained in the
greenhouse till transferred into the permanent field plantation. More than 75% of the
plantlets survived during the acclimatization process.

In vitro conservation

Experiments were carried out to slow down the growth and to maintain the germplasm
by transferring in vitro plantlets onto MS media containing 30–40 mg l!1 silver nitrate.
Media containing 40 mg l!1 silver nitrate slowed down the growth and elongation of the
plantlets in all four native plants studied. The plantlets were miniature in size when
compared to the normal plantlets and stayed more than one year without a single
subculture.

Discussion

Native plants play an important role in the conservation of aridland biological diversity.
Recently, the scientific community is stressing the importance of native plant preserva-
tion, and their utilization of in desert restoration and rehabilitation programs (AboEl-
Nil, 1997). In Kuwait, several native perennial plant species are threatened due to
various environmental factors. In order to preserve and propagate certain species of
native plants, different regeneration methods of tissue culture technology have been
developed (Table 3) for Rhanterium epapposum, Ochradenus baccatus, Lycium shawii and
144 C. SUDHERSAN ET AL.

Table 3. Regeneration protocol developed for different native plant species

Plant Method of regeneration

Rhanterium epapposum Organogenesis via callus and axillary bud break

Ochradenus baccatus Organogenesis via callus, axillary bud break and
somatic embryogenesis
Lycium shawii Organogenesis, via callus axillary bud break and
somatic embryogenesis
Nitraria retusa Axillary bud multiplication

Nitraria retusa because of their importance for desert rehabilitation and possible use in
urban landscaping.
A literature survey indicated no reports on tissue culture technology development of
these species either for conservation or mass propagation. The possibilities of plantlet
regeneration through tissue culture technology has been explored in this study, and
several plantlet regeneration protocols were developed for the first time for these species.
The general approach followed in this research was to test culturing techniques using
low concentrations of auxins and cytokinins individually or in combinations for the
embryogenic or organogenic pathways. The four species studied here showed positive
responses to one MS basal medium containing 1 mg l!1 BA in plantlet regeneration via
organogensis. Among the four species, Lycium shawii and Ochradenus baccatus showed
high rates of morphogenetic potential while the other two species showed recalcitrance
to regeneration through somatic embryogenesis. Though both species produced em-
bryogenic callus and embryos, they failed to germinate in all of the combinations of
media tested.
Vitrification, a common disorder of tissue culture (Ziv, 1993), has been observed in all
four species studied. The prime causes of this disorder were suspected to be the
inappropriate osmotic potential or water potential of the culture medium (Debergh
et al., 1981), high humidity, superfluous nutritional factors, high levels of growth
regulators, low light intensity (Ziv, 1986; Gasper et al., 1987) and ethylene accumulation
in the air space of the culture vessel (Mele et al., 1982). Addition of sucrose did not
rectify this disorder, but lowering the total water potential by increasing the agar
concentration to 1)1% produced normal shoots (Debergh et al., 1981). In the present
study on Rhanterium, an increase in the concentration of sucrose or the gelling agent and
modification of growth-regulator contents, or the addition of charcoal to the MS basal
media did not reduce the vitrification. In addition to the microelements of the MS media,
silver and nickel salts were added to the tissue culture media to promote growth and
differentiation (Chi et al., 1990; Songstad et al., 1991). Silver ions are known to act
as ethylene synthesis inhibitors in the whole plant. Experiments on Rhanterium showed
that the addition of silver nitrate to the media partially eliminated vitrification and
improved culture growth, which indicated that one of the major causes of vitrification in
this species was related to the elevated concentration of ethylene.
It seems that vitrification of cultures in vitro is not due to a single factor, but rather,
multiple factors. In our studies on these four native plant species, we found that partial
desiccation of organogenic callus with shoot buds or embryos and frequent subculturing
(Sudhersan, 1998), addition of silver nitrate (AboEl-Nil, 1997), reduction of growth
regulators, increase in gelling-agent concentration and addition of activated charcoal to
the media enhanced the recovery of shoots from this disorder.
Addition of up to 20 mg l!1 silver nitrate to the in vitro culture media enhanced the
explant’s growth by controlling the ethylene production in the cultures. Cultures
 TISSUE CULTURE TECHNOLOGY FOR NATIVE PLANTS 145

maintained in media without silver nitrate normally grew for the first 15–20 days after
subculturing; then the growth declined gradually and stopped completely due probably
to the increase of phenolic exudates in the medium and the high level of ethylene
production. Frequent subculturing, i.e. every 15–20 days, controlled this problem in
several species, and enhanced the growth and multiplication as well. In our studies on
native plants, the normal subculturing intervals of 15–20 days were increased to 45–60
days by adding silver nitrate to the media. This will be useful in reducing the cost of
plantlet production, which is one of the major concerns in the large-scale application of
plant tissue culture technology for plant propagation. At higher concentrations of silver
nitrate, i.e. up to 50 mg l!1, the cultures showed slow growth, and plantlets were
miniaturized in size. Therefore, inclusion of higher concentrations of silver nitrate to the
culture media could be used for in vitro conservation.
Embryogenesis occurs after primary culture of explants on auxin-containing media
and secondary culture on growth-regulator-free media (Ammirato, 1983; Monier,
1990). The present study, four native plant species confirmed this phenomenon.
Complete protocols for the mass propagation of the four species, which can be used for
the propagation and conservation of native plants of Kuwait, have been developed for
the first time.

Summary

Native plant species are of great importance for the conservation of biological diversity.
Several native plant species are being threatened and face the danger of extinction due to
man’s misuse of their ecosystem and environmental pollution. Certain of Kuwait’s
native plants were selected for mass propagation and conservation using tissue culture
technology for the first time. Different methods of tissue culture technology for
Rhanterium epapposum, Ochradenus baccatus, Lycium shawii and Nitraria retusa were
developed because of their immediate need in mass propagation and conservation
efforts. The technology developed for these species can be utilized for the mass
production of selected genotypes which are tolerant to the adverse soil and climatic
conditions of Kuwait, to revegetate the desert ecosystem for the conservation of aridland
biological diversity.

This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS),
grant no. 96-03-03. This support is hereby acknowledged.

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