OPTIMIZATION OF BIOTIN AND THIAMINE REQUIREMENTS FOR SOMATIC EMBRYOGENESIS OF DATEPALM (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

Vitamins are nitrogenous substances required in trace amounts to

serve catalytic functions in enzyme systems. Plant cells grown invitro 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

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

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 wasaccording to a previously described protocol by Al-Khayri and Al-Bahrany(2001) based on modified procedures described by Tisserat (1991) and Omaret 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 chilledantioxidant solution consisting of ascorbic acid and citric acid, 150 mg l21

each, to prevent browning. Shoot tip tissue, about 8 cm long, was surfacedisinfected 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 fewdrops of Tween 20 (Sigma Chemical Co., St. Louis, MO). The tissue wasrinsed with sterile distilled water four times and placed in sterile antioxidantsolution. Shoot tip termini, sectioned longitudinally into four 5-mm longpieces, and leaf primordia explants were cultured individually on cultureinitiation medium.

Culture initiation and callus proliferation. The culture mediumconsisted of MS (Murashige and Skoog, 1962) basal salt medium containing(per liter) 170 mg NaH2PO4, 125 mg myo-inositol, 200 mg glutamine, 1 mgnicotinic acid, 1 mg pyridoxine±HCl, 5 mg thiamine, 30 g sucrose, and 7 gagar (purified Agar-agar/Gum agar) (Sigma). Media were adjusted to pH 5.7with 1 N KOH, dispensed in 125-ml flasks (50 ml per flask), capped withnon-absorbent cotton plugs and aluminum foil, and autoclaved for 15 min at1218C and 1 � 105 Pa (1.1 kg cm22).

Culture medium was modified depending upon the culture stage. Culture

453

In Vitro Cell. Dev. Biol.ÐPlant 37:453±456, July±August 2001 DOI:10.1079/IVP2001200q 2001 Society for In Vitro Biology1054-5476/01 $10.0010.00

*Author to whom correspondence should be addressed: Email jkhayri@

kfu.edu.sa

454 AL-KHAYRI

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 wkduring which they were transferred at 3-wk intervals. The resultant callusfrom each explant was transferred individually to callus proliferationmedium that contained MS basal salt to which was added (per liter) 10 mga-naphthaleneacetic acid (NAA; 54 mM), 30 mg 2iP (147 mM), and 1.5 gactivated charcoal. After 3 wk, the callus was transferred to MS basalmedium containing (per liter) 10 mg NAA (54 mM), 6 mg 2iP (30 mM), and1.5 g activated charcoal. Callus proliferation cultures and subsequentculture 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 perflask) to MS basal callus maintenance medium containing (per liter) 10 mgNAA (54 mM) and 1.5 mg 2iP (7 mM).

Callus growth and embryogenesis in response to thiamine and biotin. Totest the effect of vitamins on embryogenic callus proliferation andsubsequent somatic embryogenesis, callus from the maintenance cultureswere transferred to MS hormone-free medium modified to contain thiamineat 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 alsocontained vitamins used in the culture initiation medium. Each of the 16treatments consisted of 10 culture flasks (2 g callus per flask). The cultureswere maintained for 16 wk during which they were subcultured at 4-wkintervals. After 8 wk of culturing and prior to embryo formation, the calluseswere weighed to determine the effect of the treatments on embryogeniccallus growth. After embryo development occurred, the resultant embryoswere counted and their approximate length was determined. The data weresubjected to analysis of variance (ANOVA) and the means were separatedwith a least significant difference (LSD) at 5% significance level. Theexperiment was repeated once to confirm observations and data presentedhere are from one experiment.

Embryo germination and plant establishment. Embryos were cultured onhormone-free medium and incubated for 8 wk after which germinatedembryos were transferred to rooting medium consisting of MS basal medium0.1 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 beakerscontaining enough water to submerge the roots, kept in the culture roomcovered with a transparent plastic bag for 7 d and uncovered for anadditional 5 d. Plantlets were transplanted into 5-cm plastic pots containingpotting 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 mistedthree times a day for 6 wk, then transferred to a greenhouse for furtherdevelopment.

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

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

TABLE 1

ANALYSIS OF VARIANCE OF THE EFFECT OF THIAMINE AND BIOTIN ON DATE PALM CALLUS GROWTH AND SOMATIC EMBRYOGENESIS OFDATE 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 0.6045Biotin 3 10.47 6.38 0.0016 5090.73 21.03 0.0001 454.50 6.33 0.0017Thiamine � 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.

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 biotincombination.

455OPTIMIZATION OF BIOTIN AND THIAMINE REQUIREMENTS

level, 5 mg l21, 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.

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 technicalassistance.

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