In Vitro Cell. Dev. Biol.ÐPlant 37:453±456, July±August 2001 DOI:10.

1079/IVP2001200
q 2001 Society for In Vitro Biology
1054-5476/01 $10.0010.00

OPTIMIZATION OF BIOTIN AND THIAMINE REQUIREMENTS FOR SOMATIC EMBRYOGENESIS OF DATE

PALM (PHOENIX DACTYLIFERA L.)

JAMEEL M. AL-KHAYRI*

Date Palm Research Center, College of Agriculture and Food Sciences, King Faisal University, P.O. Box 400, Al-Hassa 31982, Saudi Arabia

(Received 19 December 2000; accepted 9 March 2001; editor M. E. Horn)

Summary
This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic
embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured
on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l21 combined with thiamine at 0.1, 0.5, 2, or 5 mg l21.
Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine
and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l21 thiamine and 2 mg l21 biotin.
This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l21 thiamine
combined with 1 mg l21 biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil.
This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro
recalcitrance of date palm.
Key words: callus; regeneration; somatic embryogenesis; tissue culture; vitamins.

Introduction found to exert a profound effect on tissue culture systems of certain

Vitamins are nitrogenous substances required in trace amounts to
serve catalytic functions in enzyme systems. Plant cells grown in
vitro are capable of synthesizing essential vitamins in suboptimal
quantities; thus, culture media are often supplemented with
vitamins to enhance growth. Various standard media formulations
and modifications thereof show wide differences in vitamin
composition (Bhojwani and Razdan, 1983). Thiamine (vitamin B1)
is generally considered to be an essential ingredient for plant tissue
cultures and is usually added at 0.1±5 mg l21. Biotin (vitamin H) is
less common in culture media and is usually added at 0.01±
1 mg l21 (Bhojwani and Razdan, 1983; Pierik, 1987). Thiamine is
an important cofactor in carbohydrate metabolism, and biotin is
important in carboxylation reactions. Both thiamine and biotin
biosynthesis pathways utilize the transfer of sulfur from cysteine to
cofactor precursors (Begley et al., 1999).
Although in vitro regeneration systems have been established for
date palm, Phoenix dactylifera L. (Abo El-Nil, 1986a; Zaid, 1986;
Omar and Novak, 1990; Tisserat, 1991; Omar et al., 1992; Al-
Ghamdi, 1993; El Hadrami and Baziz, 1995; Veramendi and
Navarro, 1996; Al-Khayri and Al-Bahrany, 2001), the role of
vitamins in callus induction and plant regeneration has not been
defined. Instead, culture media of existing regeneration systems are
supplemented with arbitrarily selected vitamins at variable
concentrations, including inositol, calcium pantothenate, nicotinic
acid, pyridoxine, thiamine, and biotin (Omar et al., 1992). Specific
media components such as amino acids and vitamins have been
*Author to whom correspondence should be addressed: Email jkhayri@
kfu.edu.sa
species and optimization of such compounds can stimulate
regeneration in recalcitrant cultivars (Benson, 2000). Studies on
vitamin and amino acid requirements of date palm in vitro cultures
are scarce (Abo El-Nil, 1986b). To contribute to overcoming the
recalcitrance of date palm, the present study was conducted to
define the optimum requirements of two key vitamins, thiamine and
biotin, in an effort to enhance growth of embryogenic callus and
subsequent formation of somatic embryos.
Materials and Methods
Explant preparation. The tissue culture system used in this study was
according to a previously described protocol by Al-Khayri and Al-Bahrany
(2001) based on modified procedures described by Tisserat (1991) and Omar
et al. (1992). Outer leaves of 3±4-yr-old offshoots, cv. Khalas, were removed,
exposing the shoot tip region, which was excised and placed in a chilled
antioxidant solution consisting of ascorbic acid and citric acid, 150 mg l21
each, to prevent browning. Shoot tip tissue, about 8 cm long, was surface
disinfected in 70% ethanol for 1 min, followed by 15 min in 1.6% (w/v)
sodium hypochlorite (30% v/v Clorox, commercial bleach) containing a few
drops of Tween 20 (Sigma Chemical Co., St. Louis, MO). The tissue was
rinsed with sterile distilled water four times and placed in sterile antioxidant
solution. Shoot tip termini, sectioned longitudinally into four 5-mm long
pieces, and leaf primordia explants were cultured individually on culture
initiation medium.
Culture initiation and callus proliferation. The culture medium
consisted of MS (Murashige and Skoog, 1962) basal salt medium containing
(per liter) 170 mg NaH2PO4, 125 mg myo-inositol, 200 mg glutamine, 1 mg
nicotinic acid, 1 mg pyridoxine±HCl, 5 mg thiamine, 30 g sucrose, and 7 g
agar (purified Agar-agar/Gum agar) (Sigma). Media were adjusted to pH 5.7
with 1 N KOH, dispensed in 125-ml flasks (50 ml per flask), capped with
non-absorbent cotton plugs and aluminum foil, and autoclaved for 15 min at
1218C and 1  105 Pa (1.1 kg cm22).
Culture medium was modified depending upon the culture stage. Culture

453
454 AL-KHAYRI

TABLE 1

ANALYSIS OF VARIANCE OF THE EFFECT OF THIAMINE AND BIOTIN ON DATE PALM CALLUS GROWTH AND SOMATIC EMBRYOGENESIS OF
DATE PALM

Callus weight Embryo number Embryo length

Source df SS F value P.F SS F value P.F SS F value P .F
Thiamine 3 4.86 2.96 0.0469 1658.39 6.96 0.0010 44.83 0.62 0.6045
Biotin 3 10.47 6.38 0.0016 5090.73 21.03 0.0001 454.50 6.33 0.0017
Thiamine  biotin 9 7.24 1.47 0.2008 2576.69 3.55 0.0038 599.33 2.78 0.0158

P values less than 0.05 are significant. SS, sum of squares.

initiation medium was supplemented with (per liter) 100 mg 2,4-
dichlorophenoxyacetic acid (2,4-D; 453 mM), 3 mg 2-isopentenyladenine
(2iP) (15 mM), and 1.5 g activated charcoal (acid-washed, neutralized)
(Sigma). These cultures were incubated in darkness at 24 ^ 38C for 12 wk
during which they were transferred at 3-wk intervals. The resultant callus
from each explant was transferred individually to callus proliferation
medium that contained MS basal salt to which was added (per liter) 10 mg
a-naphthaleneacetic acid (NAA; 54 mM), 30 mg 2iP (147 mM), and 1.5 g
activated charcoal. After 3 wk, the callus was transferred to MS basal
medium containing (per liter) 10 mg NAA (54 mM), 6 mg 2iP (30 mM), and
1.5 g activated charcoal. Callus proliferation cultures and subsequent
culture stages were maintained at 24 ^ 38C and a 16-h photoperiod
(50 mmol m22 s21) provided by cool-white fluorescent lamp. After 9 wk
(three subcultures), embryogenic callus was transferred (about 0.5 g per
flask) to MS basal callus maintenance medium containing (per liter) 10 mg
NAA (54 mM) and 1.5 mg 2iP (7 mM).
Callus growth and embryogenesis in response to thiamine and biotin. To
test the effect of vitamins on embryogenic callus proliferation and
subsequent somatic embryogenesis, callus from the maintenance cultures
were transferred to MS hormone-free medium modified to contain thiamine
at 0.1, 0.5, 2, and 5 mg l21 (0.3, 1.5, 6, 15 mM) combined with biotin at 0,
0.1, 1, and 2 mg l21 (0, 0.4, 4, 8 mM). In addition, this medium also
contained vitamins used in the culture initiation medium. Each of the 16
treatments consisted of 10 culture flasks (2 g callus per flask). The cultures
were maintained for 16 wk during which they were subcultured at 4-wk
intervals. After 8 wk of culturing and prior to embryo formation, the calluses
were weighed to determine the effect of the treatments on embryogenic
callus growth. After embryo development occurred, the resultant embryos
were counted and their approximate length was determined. The data were
subjected to analysis of variance (ANOVA) and the means were separated
with a least significant difference (LSD) at 5% significance level. The
experiment was repeated once to confirm observations and data presented
here are from one experiment.
Embryo germination and plant establishment. Embryos were cultured on
hormone-free medium and incubated for 8 wk after which germinated
embryos were transferred to rooting medium consisting of MS basal medium
0.1 mg l21 (0.54 mM) NAA and dispensed in 25  120-mm culture tubes
(15 ml per tube). For acclimatization, the plantlets were placed in beakers
containing enough water to submerge the roots, kept in the culture room
covered with a transparent plastic bag for 7 d and uncovered for an
additional 5 d. Plantlets were transplanted into 5-cm plastic pots containing
potting mix (1 soil:1 peat moss:1 vermiculite) and watered with 100 mg l21
of Peters (20±20±20) fertilizer (Grace-Sierra). Potted plantlets were misted
three times a day for 6 wk, then transferred to a greenhouse for further
development.
vitamin additive, was also observed in tobacco callus cultures
(Linsmaier and Skoog, 1965) and soybean (Ikeda et al., 1976). In
the present study, date palm callus growth was significantly
influenced by the concentrations of thiamine and biotin (Table 1).
Callus weight significantly increased as the concentration of
thiamine increased from 0.1 to 0.5 mg l21, the level with which
maximum callus growth was obtained (Fig. 1A). Further increase in
thiamine concentration to 2 mg l21 caused a slight decrease in
callus weight. As the highest thiamine level, 5 mg l21, significant
reduction in callus proliferation was observed. These results are in
accordance with previous studies on tobacco and rice where callus
growth was promoted and callus browning was suppressed in
response to thiamine (Bergmann and Bergmann, 1968; Inoue and
Maeda, 1980).
Studies on the effect of biotin on callus growth are rare. The
present study has shown a direct relationship between biotin
concentration and date palm callus weight (Fig. 1B). The addition of
0.1 mg l21 biotin caused a slight increase in callus weight, whereas
1 mg l21 significantly stimulated callus proliferation. Further
increase of biotin to 2 mg l21 induced a non-significant increase
in callus weight.
Number of somatic embryos. Little is known about the effect of
thiamine (Inoue and Maeda, 1980) and biotin on tissue rediffer-
entiation and organ formation in callus cultures. In the present
study, the effect of thiamine concentration on embryo formation was
dependent upon biotin concentrations, as indicated by a significant
two-way interaction (Table 1). Embryo number generally increased
as biotin concentration reached 2 mg l21 when used in combination
with 0.1, 0.5, or 2 mg l21 thiamine (Fig. 2A). At a higher thiamine

Results and Discussion

Callus proliferation. The influence of vitamins on callus

induction and proliferation can be significantly variable among
species. For example, in Bryum coronatum, callusing completely
failed to occur when vitamins were omitted from the culture medium
despite the presence of growth regulators (Kumra and Chopra,
1982). The requirement for exogenous thiamine, the most common
Fig. 1. Callus growth of date palm in response to thiamine (A) and biotin
(B). The means are based on 10 samples per thiamine and biotin
combination.
 OPTIMIZATION OF BIOTIN AND THIAMINE REQUIREMENTS
Fig. 2. Effect of thiamine and biotin interaction on somatic embryogen-
esis of date palm: A, effect on embryo number; B, effect on embryo length.
The means are based on 10 samples per thiamine and biotin combination.
21
level, 5 mg l , the embryo number was unaffected by biotin
concentration. The maximum number of embryos that developed
was on treatments containing 2 mg l21 biotin combined with 0.5 or
2 mg l21 thiamine.
In a study with carrot somatic embryogenesis, biotin-containing
protein, represented by acetyl-CoA carboxylase activity, increased
during embryo development from 2.6 nmol min21 mg protein21
(23.4 nmol min21 (g fresh wt)21) at globular stage to 5.3 nmol
min21 mg protein21 (88.5 nmol min21 (g fresh wt)21) at torpedo
stage (Nikolau et al., 1987), suggesting that exogenous biotin could
influence somatic embryogenesis. Accordingly, the present study
has shown that biotin significantly affected somatic embryogenesis
of date palm. Histological observations made by Inoue and Maeda
(1980) revealed that the stimulatory effect of thiamine on
regeneration occurs by regulating callus texture, since thiamine
induced changes in the inner and outer structures leading to
organogenesis. Based on visual assessment, the texture of date palm
callus in response to thiamine concentration was unaffected;
however, histological studies were not conducted.
Somatic embryo elongation. Similar to the effect on the number
of embryos, embryo size expressed by its length was also affected by
a significant two-way interaction between thiamine and biotin
concentrations, as indicated by the analysis of variance (Table 1).
The embryo lengths associated with various thiamine and biotin
combinations are shown in Fig. 2B. Generally, as the concentration
of biotin increased from 0 to 1 mg l21 embryo elongation increased;
the extent of increase was dependent upon thiamine concentration,
between 0.1 and 2 mg l21. Embryo elongation was unaffected by
the level of biotin at the highest thiamine concentration tested,
5 mg l21. The maximum embryo elongation was obtained with
thiamine at 0.5 or 2 mg l21 combined with 1 mg l21 biotin.
Embryo germination and plant establishment. Embryo germina-
tion and root formation appeared to be unaffected by either biotin or
thiamine concentration supplemented to the regeneration medium.
455
Embryos first developed green shoots that formed within 6 wk of
culturing on the rooting medium. Embryo germination ranged from
44% to 65% and produced plantlets that exhibited normal growth
upon transfer to soil.
In conclusion, this study has shown that vitamin content in the
culture medium is an important factor in date palm tissue culture.
In addition to determining the requirement of thiamine, this study
has highlighted the importance of biotin as a medium additive.
Callus growth and embryo formation was best achieved on a
medium containing 0.5 mg l21 thiamine combined with 2 mg l21
biotin; whereas, embryo elongation was favored by 0.5 mg l21
thiamine combined with 1 mg l21 biotin. Although only two
vitamins were tested, others may also have a similar or a more
profound influence on callus growth and somatic embryogenesis. It
is imperative that future research efforts encompass the evaluation
of other vitamins and their pertinence to genotypic responses.
Acknowledgment
The author wishes to thank Mr. Mohammed A. Abu-Ali for his technical
assistance.
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