Synthetic seed production and physio-biochemical studies in Cassia angustifolia Vahl. — a medicinal plant

Abstract

Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 · 2H2O were found most suitable for encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medium supplemented with thidiazuron (5.0 μM) + indole-3-acetic acid (1.0 μM) produced maximum number of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response. Encapsulated nodal segments demonstrated successful regeneration after different period (1-6 weeks) of cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots when cultured on half strength Murashige and Skoog medium supplemented with 1.0 μM indole-3-butyric acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were successfully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.

Full text

Acta Biologica Hungarica 65(3), pp. 355–367 (2014)
DOI: 10.1556/ABiol.65.2014.3.11
0236-5383/$ 20.00 © 2014 Akadémiai Kiadó, Budapest
SYNTHETIC SEED PRODUCTION
AND PHYSIO-BIOCHEMICAL STUDIES IN
CASSIA ANGUSTIFOLIA VAHL. – A MEDICINAL PLANT
N. A. W. BukhAri, irAm Siddique, * k. PerveeN,
I. SIddIquI and M. S. AlwAhIbI
Department of Botany and Microbiology, College of Science, Female Centre for Scientic and
Medical Colleges, King Saud University, P.O. Box 22452, Riyadh 11495, Saudi Arabia
(Received: January 9, 2014, accepted: March 10, 2014)
Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of
cloned plantlets. Synthetic seeds were produced by encapsulating nodal segments of C. angustifolia in
calcium alginate gel. 3% (w/v) sodium alginate and 100 mM CaCl2 ∙ 2H2O were found most suitable for
encapsulation of nodal segments. Synthetic seeds cultured on half strength Murashige and Skoog medi-
um supplemented with thidiazuron (5.0 µM) + indole-3-acetic acid (1.0 µM) produced maximum number
of shoots (10.9 ± 0.78) after 8 weeks of culture exhibiting (78%) in vitro conversion response.
Encapsulated nodal segments demonstrated successful regeneration after different period (1–6 weeks) of
cold storage at 4 °C. The synthetic seeds stored at 4 °C for a period of 4 weeks resulted in maximum
conversion frequency (93%) after 8 weeks when placed back to regeneration medium. The isolated shoots
when cultured on half strength Murashige and Skoog medium supplemented with 1.0 µM indole-3-butyr-
ic acid (IBA), produced healthy roots and plantlets with well-developed shoot and roots were success-
fully hardened off in plastic pots containing sterile soilrite inside the growth chamber and gradually
transferred to greenhouse where they grew well with 85% survival rate. Growth performance of 2 months
old in vitro-raised plant was compared with in vivo seedlings of the same age. Changes in the content of
photosynthetic pigments, net photosynthetic rate (PN), superoxide dismutase and catalase activity in
C. angustifolia indicated the adaptation of micropropagated plants to ex vitro conditions.
Keywords: Antioxidant enzymes – encapsulation – rooting – synthetic seeds – Thidiazuron
INTRODUCTION
Cassia angustifolia commonly known as Senna is a medicinally valuable drought
resistant shrub of the family Fabaceae and mainly grown as a cash crop in various
parts of the world. Senna is cultivated in Somalia, Arabian Peninsula and near the
Nile river [19]. The leaves and pods contain sennosides A, B, C, D, kampferol, anth-
raquinone, essential oil, isohamnentin and emodin. These are employed in the treat-
ment of amoebic dysentery as an anthelmintic and as a mild liver stimulant. Cassia is
a powerful cathartic used in the treatment of constipation, working through a stimula-
* Corresponding author; e-mail address: siddiqueiram@gmail.com

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tion of intestinal peristalsis. Besides being an excellent laxative, the senna is used as
a febrifuge in splenic enlargements, jaundice, tumors, bronchitis and leprosy [7].
C. angustifolia is exploited heavily from wild conditions by pharmaceutical com-
panies and local tribes for medicinal purposes. Conventionally, it is propagated by
seeds. However, low germination percentage and poor viability restricts its propaga-
tion on large scale. To cope with the heavy demand, large scale cultivation of the plant
is essential, however, high seed mortality and susceptibility of the crop to frost [4]
creates a hurdle which cannot be cleared with the conventional methods alone. Tissue
culture technology has been successfully utilized in overcoming such problems [27].
Synthetic seed technology is a potential tool for a more efcient and cost effective
rapid in vitro propagation system. This technology has developed considerably in
recent years for ex situ conservation of the germplasm of elite and important medici-
nal plant species. The technology offers excellent scope for propagation of rare
hybrids, elite genotypes and genetically engineered plants for which the seeds are
either very expensive or are not available. During the last few years, considerable
efforts have been made for in vitro regeneration of this medicinally valuable species
from different explants [2–4, 33–34, 36–38]. The in vitro growth conditions can result
in the formation of plantlets of abnormal morphology, anatomy and physiology which
resulted in high percentage of plantlets death because of sudden changes after their
ex vitro transfer [11, 17, 28]. The physiological status of in vitro grown plantlets dur-
ing acclimatization is an important factor determining success rates [39] and is a
crucial step for many species, requiring time and expensive installation that restrict
the commercial application of the micropropagation processes. Sudden changes in
environmental conditions during acclimatization of tissue culture raised plants gener-
ate stress through the formation of reactive oxygen species (ROS) [9, 29]. These
include superoxide radical (O2∙–), singlet oxygen (1O2), hydrogen peroxide (H2O2)
and hydroxyl radical (OH˙), which cause tissue injury [18]. To combat the danger
posed by the presence of ROS, plant cells have evolved defense antioxidant mecha-
nism. To scavenge these ROS, different mechanisms, both enzymatic and non enzy-
matic are present in plants [20]. Among the enzymatics, superoxide dismutase SOD
and catalase CAT are efcient antioxidant enzymes [30] and their joint action pre-
vents cellular damages caused by O2, H2O2 and OH–. Changes in CAT and SOD
activity in response to adverse environmental conditions have been reported by
Sgherri and Navari-Izzo [31].
To our best knowledge, no report is available on the development of synthetic seed
system for clonal propagation of C. angustifolia using nodal segment. However, to
date, there have not been any reports either published on physiological and biochem-
ical studies of this species. The present investigation reports on the optimized param-
eters for the production and conservation of synthetic seeds to study their conversion
storage under in vitro conditions and to optimize the various physiological and anti-
oxidative enzymes activity during acclimatization of micropropagated plants.

Synthetic seed production by Cassia angustifolia 357
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MATERIALS AND METHODS
Explant source
Nodal segments (1–2 cm) collected from 2-year-old mature plant of C. angustifolia
growing in the botanical garden of the King Saud University, Riyadh were used to
initiate in vitro cultures.
Surface sterilization
Nodal explants were washed thoroughly under running tap water for at least 30 min,
to remove adherent particles, treated with 5% (v/v) laboleneTM (Qualigens, Mumbai,
India) for 20 min and nally rinsed with sterilized distilled water. The explants were
then surface sterilized with freshly prepared 0.1% (w/v) HgCl2 (Qualigens, Mumbai,
India) solution for 5 min followed by ve rinses with sterile distilled water to remove
any traces of sterilant.
Encapsulation
Sodium alginate (Qualigens, Mumbai, India) at different concentrations (2, 3, 4 and
5%) (w/v) was added to Murashige and Skoog [26] liquid medium. For complexation
25, 50, 75, 100 and 125 mM calcium chloride (CaCl2 ∙ 2H2O) solution was prepared
using MS liquid medium. The pH of the gel matrix and the complexing agent was
adjusted to 5.8 prior to autoclaving at 121 °C for 20 min. Encapsulation was accom-
plished by mixing the nodal segments with sodium alginate solution and with a
pipette by droping them into CaCl2 ∙ 2H2O solution. The droplets containing the
explants were held for at least 20 min, to achieve polymerization of the sodium algi-
nate. The calcium alginate beads containing the nodal segments were retrieved from
the solution and rinsed twice with autoclaved distilled water to remove the traces of
CaCl2 ∙ 2H2O and thereafter cultured on different medium.
Growth media and culture conditions
The encapsulated nodal segments were planted onto Petri dishes containing
Murashige and Skoog (MS), half-strength MS and half-strength MS medium sup-
plemented with different concentrations of thidiazuron (TDZ) (0.5, 2.5, 5.0, 7.5 and
10.0 μM) and an auxin indole-3- acetic acid (IAA) (1.0 μM). After sprouting of
shoots, the encapsulated nodal segments were transferred to 100 ml culture ask
containing the same medium as the one on which they developed shoots. The medium
was supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar and pH was adjusted

358 N. A. w. bukhArI et al.
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to 5.8 prior to autoclaving at 121 °C for 20 min. Cultures were maintained at
24 ± 2 °C under 16/8 h light : dark conditions with a photosynthetic photon ux den-
sity (PPFD) of 50 μmol m–2 s–1 provided by cool white uorescent tubes.
Low temperature storage
A set of encapsulated nodal segments were transferred to Petri dishes containing
water and agar medium and stored in a refrigerator at 4 °C. Seven different low tem-
perature exposure times (0, 1, 2, 3, 4, 5, and 6 weeks) were evaluated for regenera-
tion. After each storage period, encapsulated nodal segment were cultured on half-
strength MS medium supplemented with 5.0 μM TDZ and 1.0 μM IAA for conver-
sion into plantlets. The percentage of encapsulated nodal segments forming shoots
and roots were recorded after 8 weeks of culture to regeneration medium.
Rooting and acclimatization
For rooting, the in vitro-regenerated shoots (3–4 cm) were harvested from proliferat-
ing cultures and transferred to 1/2 MS medium amended with IBA (1.0 μM). After
rooting, rooted shoots were washed gently under running tap water to remove the
nutrient medium and subsequently transferred to plastic cups containing sterile soil-
rite (Keltech Energies Ltd., Bangalore, India). Potted plantlets were covered with
transparent polythene bags to ensure high humidity and watered every 3 days with
half-strength MS solution for 2 weeks. After 4 weeks, acclimatized plants were trans-
ferred to pots containing normal garden soil and maintained in a greenhouse under
normal day-length conditions.
Physiological and biochemical studies
Plant growth was recorded by total shoot and root length, shoot, root fresh and dry
mass and number of leaves per plant determined after 2 months of transplanting of
in vitro-propagated plants and compared with control plants of same age.
Relative water content (RWC) of micropropagated plants were determined after
2 months of transplanting and compared with control plants of same age by using the
formula
RWC (%) = (FM – DM) × 100/(SM–DM)
where FM = fresh mass, DM = dry mass and SM = saturated mass.
A set of tissue culture raised healthy plantlets were transplanted in sterile soilrite
and placed in culture room at 24 ± 2 °C and 16/8 h photoperiod at 55–60% relative
humidity under controlled conditions. Leaf samples were taken at transplantation day

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(0), and after 7, 14, 21 and 28 d and stored in liquid nitrogen for physiological and
biochemical studies.
PN was measured on fully expanded leaves using portable Infra Red Gas Analyzer
(IRGA, LI-COR 6400, Lincoln, USA) on the basis of net exchange of CO2 between
leaf and atmosphere by enclosing the leaf in the leaf chamber, and monitoring the rate
at which the CO2 concentration changed over a short time intervals. The net photo-
synthetic rate was expressed as µmol CO2 m–2 s–1.
The chlorophylls (chl) a and b and carotenoid contents were estimated by the
method of Mckinney [23] and McLachlan and Zalik [24], respectively.
Super oxide dismutase (SOD)
SOD (superoxide: superoxide oxidoreductase, EC 1.15.1.1) activity was measured by
the method of Dhinsa et al. [12] with slight modications in concentrations. SOD
activity in the supernatant was assayed by its ability to inhibit the photochemical
reduction. The assay mixture consisting of 1.5 ml reaction buffer, 0.2 ml of methio-
nine, 0.1 ml enzyme extract with equal amount of 1 M Na2CO3, 2.25 mM NBT solu-
tion, 3 mM EDTA, 60 µM riboavin and 1.0 ml of DDW was taken in test tubes
which were incubated under the light of 15 W uorescent lamp for 10 min at
25/28 °C. Blank A containing all the above substances of reaction mixture along with
the enzyme extract, was placed in the dark. Blank B containing all the above sub-
stances of reaction mixture except enzyme was placed in light along with the sample.
The reaction was terminated by switching off the light, and the tubes were covered
with a black cloth. The non-irradiated reaction mixture containing enzyme extract did
not develop light blue colour. Absorbance of samples along with blank B was read at
560 nm against the blank A. The difference of %reduction in the colour between
blank B and sample was then calculated. 50% reduction in colour was considered as
one unit of enzyme activity and the activity was expressed in Enzyme Units (EU)
mg–1 protein.
Catalase (CAT)
Catalase (H2O2:H2O2 oxidoreductase: EC 1.11.1.6) activity in the leaves of regener-
ated plantlets was determined by the method of Aebi [1] with slight modications
in concentrations. Reaction mixture containing 0.5 M Potassium phosphate buffer,
3 mM EDTA, 0.1 ml enzyme extract, and 3 mM H2O2. The reaction was allowed to
run for 5 min. CAT activity was determined by monitoring the disappearance of H2O2
by measuring a decrease in absorbance at 240 nm. CAT activity was calculated by
using extinction coefcient (Σ) 0.036 mM–1 cm–1 and expressed in enzyme units (EU)
mg–1 protein min–1. One unit of enzyme determines the amount necessary to decom-
pose 1 μmol of H2O2 per min at 25 °C.

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Protein content
The total soluble protein content of the leaves of regenerated plants was estimated
following the method of Bradford [8] using Bovine Serum Albumin (BSA, Sigma,
USA) as standard.
Statistical analysis
All the experiments were conducted with a minimum of 20 replicates per treatment.
The experiments were repeated three times. The data was analyzed statistically using
SPSS Ver. 14 (SPSS Inc., Chicago, USA). The signicance of differences among
means were carried out using Duncan’s multiple range test at P = 0.05. The results are
expressed as a means ± SE of three experiments.
RESULTS AND DISCUSSION
Synthetic seed production
The morphology of encapsulated beads in respect to shape, texture and transparency
varied with different concentrations of sodium alginate and CaCl2 ∙ 2H 2O. A 3%
sodium alginate produced clear and uniform beads, while higher concentrations
resulted in the production of hard beads and showed considerable delay in germina-
tion. On the contrary, sodium alginate concentration below 3% was also not suitable
because beads were fragile and difcult to handle (Table 1). Of the various concentra-
tions of CaCl2 ∙ 2H 2O tested, 100 mM was found to be optimum for the production of
uniform synthetic seeds with desired texture. Lower concentration of CaCl2 ∙ 2H 2O
not only prolonged the ion exchange (polymerization) duration but also resulted in
the formation of fragile calcium alginate beads that were difcult to handle (Table 2).
In vitro response of the encapsulated nodal segments inoculated in different con-
centration of TDZ and IAA is summarized in Table 3. Nodal segments encapsulated
in 3% sodium-alginate and 100 mM CaCl2 ∙ 2H 2O exhibited re-growth within 2 weeks
of incubation on MS, 1/2 MS, and 1/2 MS medium augmented with various concen-
trations and combinations of TDZ and IAA. Only few shoots emerged from encapsu-
lated nodal segments in full and half strength MS basal medium. Half strength MS
medium supplemented with TDZ (5.0 µM) and IAA (1.0 µM) gave the maximum
frequency (78%) of conversion of encapsulated nodal segments into plantlets with
maximum (10.9 ± 0.78) shoots after 8 weeks of culture. Though roots were also
formed in this medium but these roots were thin and not sufcient to handle. Our
results showed consistency with the reports [22, 35]. Well-developed roots were
obtained by transferring the individual shoots to rooting media, i.e. 1/2 MS medium
augmented with 1.0 µM IBA. This is in contrast to the reports in Tylophora indica

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Table 1
Effect of sodium alginate concentration on conversion of encapsulated nodal segments
of C. angustifolia after 8 weeks of culture on half strength MS medium
Sodium alginate (% w/v) % conversion response into plantlets
2Fragile beads
3 39
4 32
5 29
Different concentration of sodium alginate added to MS medium.
Table 2
Effect of different concentration of CaCl2 ∙ 2H 2O in the presence of optimal sodium
alginate concentration on conversion of encapsulated nodal segments of C. angustifolia
after 8 weeks of culture on half strength MS medium
Sodium alginate
(% w/v) Calcium chloride (mM) % conversion response into plantlets
3 25 Fragile beads
3 50 Fragile beads
3 75 35
3 100 39
3 125 33
Different concentrations of CaCl2 ∙ 2H 2O added to MS medium.
Table 3
Effect of different MS strength and concentrations of TDZ along with IAA on conversion
of encapsulated nodal segments of C. angustifolia after 8 weeks of culture
Treatments (µM) % conversion response
into plantlets Mean number of shoots
MS 22 1.4 ± 0.13f
½ MS 32 3.2 ± 0.20e
½ MS + TDZ (0.5) + IAA (1.0) 42 5.0 ± 0.34cd
½ MS + TDZ (2.5) + IAA (1.0) 57 8.5 ± 0.45b
½ MS + TDZ (5.0) + IAA (1.0) 78 10.9 ± 0.78a
½ MS + TDZ (7.5) + IAA (1.0) 64 8.2 ± 0.43ab
½ MS + TDZ (10.0) + IAA (1.0) 50 6.0 ± 0.19c
Values represent means ± SE. Means followed by the same letter within columns are
not signicantly different (P = 0.05) using Duncan’s multiple range test.

362 N. A. w. bukhArI et al.
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and in Vitex negundo where shoot and root formation took place in the same media
[5, 15]. Higher concentration of TDZ decreased the conversion frequency of encap-
sulated beads into plantlets.
Storage duration
The regeneration frequency was clearly inuenced by storage time. Table 4 shows the
effect of different storage duration of encapsulated nodal segment at 4 °C for 0, 1, 2,
3, 4, 5 and 6 weeks. The synthetic seed stored at 4 °C for a period of 4 weeks result-
ed in maximum conversion frequency (93%) with an induction of 14.8 ± 0.51 shoots
after 8 weeks of culture under in vitro conditions on half strength MS medium sup-
plemented with TDZ (5.0 µM) and IAA (1.0 µM). With an increase in storage time
to more than 4 weeks, the conversion frequency decreased considerably (Table 4).
Decline in conversion response could be attributed to inhibition of tissue respiration
by the alginate matrix, or a loss of moisture due to partial desiccation during storage
as reported earlier [10, 14, 15].
Rooted plantlets with four to ve fully expanded leaves, retrieved from encapsu-
lated nodal segments were transferred to plastic pots lled with sterile soilrite and
covered with transparent polythene bags inside the culture room for 2 weeks. After
one month, these were transferred in earthen pots containing garden soil and main-
tained in greenhouse where they grew normally.
Table 4
Effect of different duration of storage (4 °C) on in vitro regeneration from alginate – encapsulated
nodal segment of C. angustifolia after 8 weeks of culture to half strength MS medium supplemented
with TDZ (5.0 µM) + IAA (1.0 µM)
Storage duration
(weeks)
% conversion
response
into plantlets
Mean number
of shoots
0 78 10.9 ± 0.78c
1 81 11.5 ± 0.67bc
2 85 12.9 ± 0.53b
3 89 13.5 ± 0.42a
4 93 14.8 ± 0.51a
5 86 11.2 ± 0.12c
6 80 9.6 ± 0.28d
Values represent means ± SE. Means followed by the same letter within
columns are not signicantly different (P=0.05) using Duncan’s multi-
ple range test.

Synthetic seed production by Cassia angustifolia 363
Acta Biologica Hungarica 65, 2014
Growth performance
Comparative data on some morphological features and relative water content of in
vitro propagated plants and seedlings is summarized in Table 5. Slight reduction in
the morphology of in vitro-propagated plants in terms of shoot, root length and dry
and fresh mass and leaf number were observed in comparison to control plants after
considerable period of establishment in the greenhouse. The plantlets dehydrated
quickly and experienced water stress as soon as they were transferred from the culture
tubes to eld conditions. RWC was decreased due to wiltness of plantlets under low
humidity. However, regenerants slowly recovered from water stress with the passage
of time and obtained higher relative water content than in vivo seedlings. Similar
results have also been reported in pepper plantlets [13, 32].
Photosynthetic pigments and net photosynthetic rate
Photosynthetic parameters including chl a, chl b, carotenoid and PN were evaluated
in regenerated plantlets of C. angustifolia at 0 (control), 7, 14, 21 and 28 d of accli-
matization (Fig. 1). Content of chl a and chl b showed an increasing trend over control
plantlets throughout the study while carotenoid content rst dropped from 0–14 d due
to the sudden changes in environmental conditions and thereafter showed an increas-
ing trend from 14–28 d of acclimatization. An increase in carotenoid level is reported
to be involved in protecting the photosynthetic machinery form photo oxidative dam-
age [6, 16]. PN decreased from 0 to 7 d and thereafter showed an increasing trend with
the formation of new leaves from 7–28 d of acclimatization. Photosynthetically active
Table 5
Comparison of some morphological features between micropropagated plants and
seedlings of C. angustifolia
Parameters Micropropagated plants Seedlings
Root length (cm) 5.84 ± 0.21d6.96 ± 0.34d
Shoot length (cm) 12.31 ± 0.48c15.81 ± 0.67c
Root fresh mass (g) 0.12 ± 0.11gh 0.16 ± 0.13h
Shoot fresh mass (g) 1.36 ± 0.10e1.75 ± 0.14e
Root dry mass (g) 0.05 ± 0.03h0.09 ± 0.02i
Shoot dry mass (g) 0.45 ± 0.01f0.60 ± 0.04f
Leaf dry mass g plant–1 0.25 ± 0.02g0.32 ± 0.01g
Leaf number/plant 20.12 ± 0.86b24.01 ± 0.95b
Relative water content (%) 92.01 ± 1.63a67.53 ± 1.89a
Values represent means ± SE. Means followed by the same letter within columns are
not signicantly different (P = 0.05) using Duncan’s multiple range test.

364 N. A. w. bukhArI et al.
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Fig. 2. Changes in photosynthetic pigments and net photosynthetic rate (PN) of micropropagated plantlets
of C. angustifolia during different days of acclimatization. Bars represents means ± SE. Bars denoted by
the same letter within response variables are not signicantly different (P = 0.05) using Duncan’s multiple
range test
Fig. 1. Changes in superoxide dismutase and catalase activity of micropropagated plantlets of C. angus-
tifolia during different days of acclimatization. Bars represents means ± SE. Bars denoted by the same
letter within response variables are not signicantly different (P = 0.05) using Duncan’s multiple range
test

Synthetic seed production by Cassia angustifolia 365
Acta Biologica Hungarica 65, 2014
in vitro leaves have also been observed in Spathiphyllum oribundum plantlets [40].
After one week, PN showed increasing trend and was found to be associated with the
formation of new leaves [13].
Antioxidative enzyme activities
Acclimatized plantlets of C. angustifolia showed a time dependant increase in both
SOD and CAT activity. SOD activity dropped rst from 0–14 d and thereafter showed
an increasing trend and reached maximum at 28th d of acclimatization (Fig. 2). Higher
SOD activity was therefore associated with better protection against stress induced
oxidative injury. Similar results were reported by Van Huylenbroeck et al. [40] and
Chai et al. [9]. However, CAT activity increased signicantly during 0–28 d of accli-
matization (Fig. 2). Positive changes in CAT activity have often been observed in
relation to mild water stress [21, 25, 35]. Thus increment in SOD and CAT activity
can be considered as the protection against ROS possibly generated during acclima-
tization.
CONCLUSIONS
In conclusion, the present study describes a simple, reproducible and efcient proto-
col for synthetic seed production. This protocol may facilitate conservation, propaga-
tion and mass multiplication. Cold storage of encapsulated nodal segments offers
possibility for germplasm conservation and exchange between laboratories. Changes
observed in physiological and biochemical parameters during acclimatization has
helped to understand better adaptation process of micropropagated plants to ex vitro
conditions.
ACKNOWLEDGEMENT
The authors extend their appreciation to the Deanship of Scientic Research at King Saud University for
funding the work through the research group project No. RGP-VPP-066.
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