Annals of Agricultural Science (2016) 61(1), 125–133

HO ST E D BYFaculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

Floral reversion of mature inflorescence of date

palm in vitro

* Corresponding author.

E-mail address: im_zi@yahoo.com (E.M.M. Zayed).

Peer review under responsibility of Faculty of Agriculture, Ain-Shams

University.

http://dx.doi.org/10.1016/j.aoas.2016.01.0030570-1783 � 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Eman M.M. Zayed a,*, Amal F.M. Zein El Din a, Hossam H. Manaf b,

Ola H. Abdelbar b

aThe Central Laboratory for Date Palm Researches and Development, Agriculture Research Center, Cairo, EgyptbAgriculture Botany Department, Faculty of Agriculture, Ain Shams University, Egypt

Received 3 December 2015; accepted 10 January 2016Available online 6 February 2016

KEYWORDS

In vitro;

Mature female inflorescence;

Floral reversion;

Date palm;

ABA;

PBZ

Abstract Regeneration plants of date palm (Phoenix dactylifera L.) cultivar Siwi (semi-dry cv.) via

reversion of mature female flower were studied. Mature female flowers were cultured on MS med-

ium supplemented with different concentrations of paclobutrazol (PBZ) and abscisic acid (ABA)

(0.0, 0.5, 1.0 and 2.0 mg l�1). Callus induction was achieved by all treatments from 8 to 16 weeks

of culture. The highest formation of callus performed on MS medium containing 2.0 mg l�1

PBZ, which also gave the highest values of total soluble sugars and their fractions. This callus

was initiated from different zones: the basal part of the flower, the surface of the perianth segments,

carpels and the receptacle. Meanwhile, direct shoot and somatic embryos were only obtained by cul-

tured explants on different concentrations of ABA which contained the highest concentration of

total soluble sugars and their fractions. Shoots initiated from some meristematic cells occurred

between the basal part of the petal and carpel. This protocol provides a simple and rapid way to

regenerate date palm through reversion the mature female flower to the vegetative growth.� 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams

University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

The date palm, Phoenix dactylifera L., is one of the most eco-

nomically important plants in arid and hot regions, especiallyin the Middle East and southern Mediterranean countries.This palm is propagated from off-shoots, which are limited

in quantity in the whole life of tree. In addition seed

propagated palms show variable field performance becauseof their genetic heterogeneity (Fki et al., 2011).

Available techniques of rapid multiplication of date palmhave contributed immensely to meet the increased demand ofdate palm fruits worldwide (Jain et al., 2011). In vitro micro-

propagation thus soon became an essential and effective meansto ensure the renewal and the extension of palm plantations(Smith and Aynsley, 1995).

Using inflorescence tissue remains one of the methods to

micropropagate date palm (Bhaskaran and Smith, 1992;Abahmane, 2011; Zayed, 2011; Kriaa et al., 2012; Zayed andAbdelbar, 2015). Also, these plant materials are abundantly

available every year and can be used as cheap and potent

126 E.M.M. Zayed et al.

micropropagation sources, especially for the critical endan-gered species of flowering plant in Arecaceae family (Zayed,2011). Moreover, using mature female inflorescences can been

isolated easily without touching the vegetative tissue and men-acing the mother plant. This is because, at the mature stage,they are fully emerged and separated from the apical bud tis-

sues, so through collection would not present any menace tothe mother plant, (Kriaa et al., 2012).

If the leafy shoots were formed after reaching flowering

state this is known reversion phenomenon. This occurredunder certain environmental conditions (Battey and Lyndon,1990; Pouteau et al., 1997).

Levels of endogenous plant growth regulators play critical

role in creating variations in morphogenetic potentials as hasbeen manifested in different plant genotypes. Meanwhile,exogenous application of PGRs and occurrence of competent

cells of the explant require stimulating induction of organ dif-ferentiation (Bhaskaran and Smith, 1990; George et al., 2008).A suitable nutrient media are considered to be one of the most

crucial phases in in vitro growth and differentiation of excisedplant tissue (Williams et al., 1985; Johnson, 1996).

The plant growth retardants have become the most widely

used group of bioregulators in agricultural and horticulturalpractice. When applied in appropriate concentrations growthretardants modify plant architecture in a typical fashion(Rademacher, 2000).

Recently, Zayed and Abdelbar (2015) reported that, thelevels of endogenous GA3 and IAA, total soluble sugars, reduc-ing sugars and non-reducing sugars were recorded the highest

values in immature female inflorescences of date palm (Phoenixdactylifera L.) cultivar Siwi. Also, exogenous GA3 is frequentlyinhibitory to in vitro growth and development, and chemicals

capable of blocking the biosynthesis or action of endogenousgibberellin may have promoter effects on cultures in which nat-ural gibberellin levels are supra-optimal (George et al., 2008). It

appears that for many species, the somatic embryogenesis isinhibited by the presence of exogenous gibberellins in the callusinduction medium (Ezura and Harberd, 1995; Gomes daCunha and Ferreira, 1996; Hutchinson et al., 1997). The inclu-

sion of certain gibberellin biosynthesis inhibitors such asancymidole, PBZ or uniconazole influenced positively thesomatic embryogenesis and plant regeneration of several spe-

cies (Feng and Wolyn, 1993; Hutchinson et al., 1997;Senaratna et al., 2001; Chen and Chang, 2003).

PBZ inhibits gibberellin biosynthesis and stimulates accu-

mulation of ABA, and changes in cytokinin concentrationswould result in a new endogenous hormone balance. More-over, it has also been shown that PBZ induces large elasticmoduli in cell walls in white spruce treated plants (Wang

and Steffens, 1987; Fletcher et al., 2000; Marshall et al., 2000).ABA has been observed to influence morphogenesis in a

number of plants by modifying the effects of other hormones,

notably cytokinins and gibberellins, but also auxins (Georgeet al., 2008).

Using mature female flower of date palm in vitro has a little

evidence. So, the current study was carried out to elucidateusing mature female inflorescence as a potential strategy toproduce callogenesis, direct embryos and direct shoot instead

of using shoot tips. The most feature of reversion was itsstrong association with growth regulators, hence, this paperalso clarifies the role of PBZ and ABA in success of reversion

process by studying morphological, histological and biochem-ical changes.

Material and methods

The present study was conducted during the period from 2013to 2015 in the Central Lab. for Date Palm Researches and

Development, ARC, Egypt and Agricultural Botany Depart-ment, Faculty of Agriculture, Ain Shams University.

Plant material and tissue culture protocol

Mature female inflorescences excised from adult female treesof date palm cv. Siwi which grow in the field of agriculture

ministry at Abo-Rawash, Giza, Egypt. All mature female inflo-rescences were collected in the end of March that became com-pletely mature and spathe fully emerged. Spathe (40–50 cm in

length) was washed well by using a soap and tap water toremove dust particles. The explants were surface-sterilizedusing ethanol 70% (v/v) for 1 min and then mercuric chlorideat 0.01% (w/v) for 1 h, followed by three independent rinses

each in sterile distilled water under aseptic conditions. Afterthis, the outer sheath and base were removed; then, the inflo-rescence axes were sliced horizontally into 2–3 cm segments

(each segment contained 2–3 flowers), and each explant wascultured horizontally with a good contact with the surface ofthe induction media.

The basic cultured medium consists of a basal MS salts(Murashige and Skoog, 1962) supplemented with sucrose(30 g l�1) and the media were gelled by agar (6 g l�1) and acti-vated charcoal (1.0 g l�1). Different concentrations of PBZ

and ABA (0.0, 0.5, 1.0 and 2.0 mg l�1) were added individually.The pH of the media was adjusted to 5.8 prior to sterilization inthe autoclave and the cultures also were incubated in total dark-

ness at 27 ± 2 �C and transferred to fresh media every 8 weeks.Data were taken during three subcultures as follows:

� The average percentage of the browning degree was scoredvisually according to Pottino (1981) as follows:

1

(�) Negative results

2

(+) Below average results

3

(++) Average results

4

(+++) Good results

� The average percentage of callus formation/explant.� The average percentage of direct shoots formation/explant.

� The average value of number of direct somatic embryosformation/explant.

� The average percentage of adventitious roots

formation/explant.

Histological studies

Plant samples were harvested during the three sub-cultures toobserve the morphological changes by OPTICA SZM-2 binoc-

ular. The samples were killed and fixed in FAA solution (For-malin, acetic acid and 50% ethyl alcohol, 5:5:90 by volume) for24 h. The schedule of the paraffin method as described by

Floral reversion of mature female inflorescence of date palm in vitro 127

Johansen (1940) was followed. Serial transverse and longitudi-nal sections (6–8 lm) in thickness were made by LEICA rotarymicrotome model RM 2125 RTS and fixed on slides by means

of Haupt’s adhesive. The sections were stained with aSafranin–Fastgreen combination, and then mounted inCanada balsam (Sass, 1951). Observations and photomicro-

graphs were obtained with LEICA light research microscopemodel DM 2500 supplied with a digital camera.

Biochemical analyses

Explants were taken after the first sub-culture from differentmedia for determination of total soluble sugars, total indoles

and total phenols.Determination of total soluble, reducing and non-reducing

sugars was carried out according to the method of Shales andSchales (1945).

The total indoles were determined according to Larsenet al. (1962).

Phenols determination was carried out according to Malik

and Singh (1980).

Statistical analysis

The used designed was in a complete randomized design withthree replicates, using L.S.D test at 5% according to Snedecorand Cochran (1989).

Results

Morphological changes

The ability of mature female inflorescence to form callus wastested during this study. Flower swelling (the first sign of cal-

lusing) could be observed after 4–6 weeks of culturing. Then,callus emergence was noticed from the swelled carpels. Also,it has been the emergence of callus from the sepals and petals.

The first callus formation was only obvious after 12–16 weeksof culture.

However, in the other observed formed structures like

direct organs (shoots and roots) and somatic embryos, theflowers were less swelled. Root like structures were firstlynoticed after 12–14 weeks, while shoot and embryos were

formed after 20–24 weeks from culture.Concerning the browning degree data shown in Table 1

illustrated that there were no significant differences betweendifferent concentrations of PBZ treatment. The highest value

of callus formation (50%) produced from cultured matureflower on medium contained high concentration of PBZ(2 mg l�1) and also the same concentration produced direct

root formation (12.5%). Meanwhile mature flower cannot ableto form any direct shoots or embryos when cultured on mediacontained PBZ.

On the other hand, data resulted that the highest values ofcallus formation (30%) induced from mature flower explantscultured on medium supplemented with 0.5 mg l�1 ABA, whilethe lowest significant value was obtained with 2 mg l�1 ABA.

The highest significant percentage value of direct shoot for-mation (40%) was obtained from cultured media containing2 mg l�1 ABA. On the other hand, cultured media containing

ABA (1 or 2 mg l�1) gave the highest percentage of directembryos (15% for each). Meanwhile, using 0.5 mg l�1 ABAproduced the highest percentage value of direct root (25%).

Histological changes

The mature pistillate flower at the time of culturing was cream

in color, 3 mm in width and 5 mm in length, with two homoge-nous perianth segments. The outer one consists of three freeshort imbricate sepals, while the inner one consists of three lar-

gely imbricated free petals. Both sepals and petals have anouter and inner uniseriate epidermal layers enclosing inbetween a homogenous mesophyll, which is transverse by

small vascular strands. The flowers have three carpels whichgrow and elongate to achieve a plump flask in shape. The tipsof the carpels and the stigmas are exposed at the apex of theflower. The stigma became curved and narrow projection that

protrudes from the imbricate petals (Fig. 1a and b).Since the explants ‘‘mature pistillate inflorescences” were

cultivated on media supplemented with PBZ and ABA,

remarkable morphogenetic transformation was observed.Morphological observations indicated that all tissues of theinflorescence axis ‘‘the rachillae” acquired different degrees

of brown colors (Fig. 1c). After 6–8 weeks from culturing ondifferent media examinations, the mature pistillate flowersappeared to be swollen ‘‘the perianth segments and/or thecarpels”, particularly that flowers which produced callus latter.

These swollen flowers can split easily from the rachillae andfall on the media (Fig. 1d–f). Subsequently, callus was initiatedat the first 8 weeks of culturing on all treated media, and then

obviously increased at the end of the 16 week from culturing.This callus has a translucent appearance with irregular bor-ders. It grows from different zones of the mature pistillate

flowers ex: the surface of the perianth segments (Fig. 1d), thebasal part of the flower at the attachment point with the rachil-lae (Fig. 1e), form the carpels (Fig. 1f) and from the receptacle

(Fig. 1g and h).The well-developed calli consisted of translucent mass of

globules of white color (Fig. 1i). This callus was subculturedon MS medium supplemented with 0.1 mg l�1 NAA and gave

rise to somatic embryos (unpublished data).Obviously, flowers that did not participate in callus initia-

tion were less swelling (Fig. 2a). Shoot initiation was observed

by the histological studies through 12 weeks of culturing onABA media. Through this time, shoots were originated fromthe basal part of the pistillate flower mainly from some meris-

tematic cells occurred between the basal part of the petal andthe carpel tissue (Fig. 2b). These shoot meristem consists of amass of meristematic cells which later differentiated into meris-tematic dome surrounded by leaf primordial (Fig. 2c). Further-

more, these newly shoots will be developed and forming visibleadventitious shoots arising from the flower surface at the endof the 24 week of culturing without callus formation

(Fig. 2d and e).The direct somatic embryo was differentiated from the

flowers without passing of callus formation (Fig. 2f). Mean-

while, adventitious roots formation was differentiated directlyfrom the pistillate flower without any signs of callus formationwhen cultured on ABA medium. It was obviously in this case

that the explant did not swell but became more senescence. Itstissues became necrotic with a brownish color (Fig. 2g).

Table 1 Effect of PBZ and ABA treatments in culture media on regeneration process of mature pistillate flower of date palm cv. Siwi

for the duration of 24–48 week.

Treatments Browning degree Callus formation Direct shoots Direct embryos Direct root

PBZ ABA

mg L�1 %

– – 2.25 25.0 0.0 0.0 10.0

0.5 – 2.50 37.5 0.0 0.0 0.0

1.0 – 2.25 30.0 0.0 0.0 0.0

2.0 – 2.75 50.0 0.0 0.0 12.5

– 0.5 2.25 30.0 0.0 7.50 25.0

– 1.0 2.00 25.0 20.0 15.0 12.5

– 2.0 1.50 20.0 40.0 15.0 0.0

LSD (5%) NS 1.902 9.897 1.258 1.372

128 E.M.M. Zayed et al.

Biochemical changes

Data illustrated in Table 2 showed the levels of sugar fractions(total soluble, reducing and non-reducing sugars), total indolesand phenols in mature flower explants after one sub-culture on

different inductive media. The highest concentrations of PBZ(2 mg l�1) gave the highest significant values of total soluble,reducing and non-reducing sugars (5.148, 2.255 and

2.75 mg g�1 f.wt., respectively) as compared to the other con-centrations of PBZ but also achieved the lowest level of signif-icance as compared to control or ABA treatment at 2 mg l�1.

In contrast using low concentration of PBZ (0.5 mg l�1) ledto significant increase in total indoles and phenols (1.446 and0.874 mg g�1 f.wt., respectively) as compared to control.

Data clearly showed that explants cultured in inductionmedia with addition of ABA (2 mg l�1) achieved the highestlevels of sugar concentrations (total, reducing and non-reducing sugars) to rich 10.87, 5.419 and 5.178 mg g�1 f.wt.,

respectively. However, addition of ABA (0.5 or 1.0 mg l�1)to the induction media showed reduction in the levels of sugarconcentration.

Regarding the total indole concentrations, data revealedthat ABA (0.5 mg l�1) was the most effective in increasingindole.

From the obtained data, the most striking observation wecould notice that, the explants that had been cultured in con-trol medium contained the highest significant value of reducing

sugars (7.189 mg g�1 f.wt.).

Discussion

The reversion from the floral stage to the vegetative state is acomplicated process on date palm. This process requires a highcontrol of various physiological and hormonal factors. Fromthese factors, the nature of the explants and plant growth reg-

ulators play a specific role in the reversal of vegetative statefrom the floral stage (Kriaa et al., 2012). Levels of endogenousplant growth regulators also play critical role in creating vari-

ations in morphogenetic potentials as has been manifested indifferent plant genotypes (Bhaskaran and Smith, 1990). Also,exogenous application of PGRs and occurrence of competent

cells of the explant require stimulating induction of organ dif-ferentiation (George et al., 2008).

Paclobutrazol is a growth retarding, potent inhibitor of gib-berellins biosynthesis but it also interferes with the synthesis of

abscisic acid and sterols (Yates et al., 1993). Like other gib-berellins growth retardants (triazoles group) can stimulateembryogenesis, where it is inhibited by GA3 (Hutchinson

et al., 1997).Although the direct role of PBZ is inhibition of the oxida-

tion step of ent-kaurene to ent-kaurenoic acid and thus inhibi-

tion of gibberellin’s (GAs) biosynthesis, it has a secondary roleof promoting the synthesis of cytokinins. This was probablyachieved through the diversion of reactions into the biosyn-thetic pathway of cytokinins, since they have the same interme-

diate precursor (Fletcher et al., 2000). Also, Browning et al.(1992) investigated the effect of PBZ on the translocation ofendogenous IAA in Doyenne du Comice pear cultivar. PBZ

induced changes in growth and productivity of agronomicaland horticultural crops had been described in some studies(Ghosh et al., 2010).

Several studies have reported that the differentiation degreeof the flowers is an important factor defining their ability todevelop (Liu, 1993 and Zayed, 2011). The present studyrecorded obvious swelling in explants treated with PBZ and

this observation matched with Wang and Lin (1992), theyreported that paclobutrazol treatment promoted swelling andincreased cell volume in the primary roots of pea.

Morphological and histological analyses of mature floralexplants indicated that callus initiation started since the secondsubculture. PBZ treatments produce callus and these results

agree with Hamama et al. (2012), who reported that the useof paclobutrazol alone increased callus formation and slightlyimproved the rate of regeneration of Pelargonium species.

Also, Zayed (2011) reported that, the highest value of swellingand callus formation was produced from cultured immatureinflorescence explants of Phoenix dactylefra and Hyophorbev-erschaffeltii on medium containing 0.5 mg l�1 PBZ. Dmitry

et al. (2009) found that, the addition of daminozide stimulatedthe formation of white compact, nodular-organized callus onmost of studied wheat varieties.

On the other hand, PBZ treatments have no effect on thedirect formation of shoots, roots or direct embryogenesis. Inthis respect, Gehlot et al. (1989) reported that PBZ reduced

root and shoot formation in vitro of moth bean. The additionof triazoles to the regeneration medium reduced the formationof green meristematic nodules, which are precursors of shoots

f

a4mm

c4mm

d

s s

p p

cc

c

8 mm

e 6 mm

b

br.Inf.a

perianthC C

ov.

300µm

g

c

cp

s

sp

300µm

c

h 100µm i

s s

s

pp

p

c

cc

300µm

Fig. 1 (a) Morphology of mature flower on rachillae of female inflorescence of date palm cv. Siwi, note the flower and the rachillae are in

white creamy color. (b) Longitudinal section of the same flower reveals the development of the floral parts (perianth and carpels). (c) 2–3

weeks after culturing on different media showing the browning color of the explant comparing to (a) swelling explants and callus induction

(d–i), note the flowers separated from the inflorescence axis after swelling appearance. (d) Callus initiated from the perianth surface (white

arrows). (e) Mass of callus initiated from the basal part of the flower (white arrow indicates a mass of callus on the abscission zone). (f)

Black arrows indicate three callus masses arising from the three carpels, note the browning stigmas. (g) Longitudinal section of the flower

after 6 weeks of culturing reveals occurrence of meristematic cells on the receptacle. (h) An enlarged view from (g) (the rectangle shape)

arrow indicates the meristematic cells. (i) Cross section of one flower separated from the inflorescences axis with numerous callus masses

arising from the sepals, petals and carpels. Abbreviations: br., bract; inf.a, inflorescence axis; s, sepals; p, petals; c, carpels, ov., ovule.

Floral reversion of mature female inflorescence of date palm in vitro 129

d

g7mm

a 2mm b

p

c

200µm c

c

ps

Sh.

200µm

e s

p n.l

n.l

4mm

f4mm

Fig. 2 (a) One flower separated from the inflorescences axis after 10 weeks of culturing on ABA. Note the less swelling comparing with

Fig. 1(d–f). (b) Longitudinal section of the same flower reveals that there are meristematic cells (arrow) occurred between the petal and

carpel. (c) Initiation of shoot tip with leaf primordial. (d) Developing of the adventitious shoots from the mature flower. (e) Developing of

new leaves arising from flower, the arrows indicate the degenerating carpels. (f) Initiation of direct embryo arising from the flower. (g)

Formation of direct root arising from the flower. Abbreviations: s, sepals; p, petals; c, carpels; Sh., Shoot; n.l., new leaf.

130 E.M.M. Zayed et al.

in moth bean callus (Sankhla et al., 1991). Different concentra-tions of PBZ cannot able to develop any direct shoots, roots orsomatic embryos from immature female inflorescence explantsof date palm or immature and mature inflorescence of spindle

palm (Zayed, 2011).The present data illustrate that using PBZ in high concen-

tration (2 mg l�1) produced direct root. Similar result is

obtained with Te-chato et al. (2009), who found that high con-centration seemed to promote root formation on orchid.

ABA has been observed to influence morphogenesis in a

number of plants by modifying the effects of other hormones,notably cytokinins and gibberellins, but also auxins (Georgeet al., 2008). Shepard (1980) found that adding ABA to thegrowth medium (0.05–0.2 mg l�1) caused morphogenesis to

occur more rapidly than it otherwise would from potato callus.The present result indicated that, callus was initiated from

the abscission zone after the swollen flowers were fallen. This

result agrees with Altman and Goren (1974), who mentionedthat callus was stimulated by ABA (0.3–2.6 mg l�1) andformed especially on the abscission zone between the petioleand the stem of citrus leaf. ABA has been capable of stimulat-

ing callus growth, on hypocotyl explants of Cryptomeria1 mg l�1 ABA in the culture medium had the same effect as10 mg/l of the cytokinin BAP in stimulating internal callus

(Isikawa, 1974). Also, ABA culture medium promoted carrotcallus induction and proliferation in the tissue (Jimenez andBangerth, 2001).

Histological analysis indicated that shoot primordial initi-ated directly from the basal part of the petals, while carpelsdegenerated. The basal part of the petals was characterizedby zone maintained at a meristematic stage. These zones might

be more important than the other flower organs since they rep-resent a store of dormant tissues than that can be activated byABA treatment. The previous study by Loutfi and Chlyah

Table 2 Effect of PBZ and ABA treatments on the levels of sugars fractions (total soluble sugars, reducing and non-reducing sugars),

total indoles and phenols after the 1st sub-culture of mature pistillate flower of date palm cv. Siwi.

Treatments Sugars concentration Total indole conc. Total phenol conc.

PBZ ABA Total Reducing Non-reducing

mg l�1 mg g�1 f.wt.

– – 7.941 7.189 0.712 1.030 0.232

0.5 – 2.908 1.676 1.174 1.446 0.874

1.0 – 3.556 1.557 1.899 0.514 0.071

2.0 – 5.148 2.255 2.750 0.869 0.137

– 0.5 1.494 0.215 1.217 1.484 0.136

– 1.0 3.764 0.765 2.861 1.046 0.069

– 2.0 10.870 5.419 5.178 1.348 0.139

LSD (5%) 0.159 0.112 0.210 0.477 0.056

Floral reversion of mature female inflorescence of date palm in vitro 131

(1998) obtained shoots directly from the petals by using veryyoung female inflorescences. Drira and Benbadis (1985)

obtained vegetative buds from carpels that reversed from thereproductive to vegetative phase. This deference in morpho-genetic behavior may be attributed to the age of the inflores-

cence and/or the experimental conditions. Heide (1968)reported that shoot bud formation on isolated Begoniacheimantha leaves was enhanced when the leaves were treated

with ABA, and inhibited when either auxins or gibberellinswas applied. In vitro applied ABA has induced terminalbud formation ex vitro in pine plants (Timmis andRitchie, 1988).

The present result indicated that adding ABA to the induc-tion media was more useful for direct organogenesis orembryogenesis. Manipulation of endogenous and/or exoge-

nous ABA levels increases the frequency of embryos reachingmaturity and can assist the handling of the large populationsof somatic embryos. This can be required for mass propaga-

tion (Ammirato, 1988). Kuol et al. (2002) found that, therewas a direct development of somatic embryos of Corydalis yan-husuo on the surface of converted primary somatic embryowhen cultured for two months on MS medium supplemented

with 2.0 mg l�1 ABA. Scanning electron micrograph of con-verted somatic embryo showed that, embryos at differentstages of development arise directly without an intervening cal-

lus phase.Mature inflorescence explants of spindle palm cultured on

medium contained lowest concentrations of ABA 0.01 mg l�1

gave the highest value of swelling and callus formation per-centage, whereas added the highest concentrations of ABA1.0 mg l�1 gave the lowest significant value (Zayed, 2011).

The present study reported a positive correlation betweenthe callus formation percent and increasing the level of PBZ.This observation could explain the decrease in the sugar con-tents in these cultures compared with control medium due to

that morphogenesis requiring high-energy. Immature femaleinflorescences of date palm contained much high levels of total,reducing and non-reducing sugars, while callus contains the

lowest level among the other developmental stages (Zayedand Abdelbar, 2015).

Catarina et al. (2003) found high level of total sugars in the

embryoides. This increase in total sugars could also haveoccurred as a consequence of the uptake and metabolism ofthe carbohydrates supplied exogenously. Cell division

demands a high amount of carbon in order to supply the

ATP necessary for cellular metabolism (Martin et al., 2000;Yoshida, 2003).

Non-reducing sugars reach the highest concentration whenABA concentration was increased to 2 mg l�1. These datainsure the previous data which showed that ABA may enhance

embryogenesis by promoting sucrose uptake from the medium(Saftner and wyes, 1984) or starch synthesis by controllinga-amylase activity (Higgins et al., 1982).

Moreover, Fortes and Pais (2000) explained the importantrole of starch accumulation/mobilization cycles in organogen-esis process of Humulus lupulus var. Nugget and they suggestedthat the starch is used both during organ initiation and later in

its development. The results obtained by authors revealed thatstarch accumulation in control explants was detected at alower extent than in explants cultured under organogenesis-

inducing conditions. However, when pre-nodular and nodularstructures were formed starch mobilization started in thesenodules before shoot regeneration.

Indole concentration was raised when tissues were culturedon induction media supplemented by ABA.

Regarding the total phenol concentration data clearlyreflected that media added with PBZ increased total phenols

within cultured explants after one sub-culture.The relationship between IAA and phenols has been dis-

cussed by many investigators (Lee and Skoog, 1965; Lee,

1980; Grambow and Langenbeck-Schwich, 1983), and it isinteresting to know how the plant cells have mechanisms forregulation of the endogenous IAA to its requirements. Phenols

were found to react with hydrogen peroxide produced duringIAA degradation, thereby protecting the cell from its toxiceffects. Relatively large amounts of natural inhibitors of IAA

oxidase have been reported to be present in meristematic andjuvenile tissues, but not in normal mature differentiated cellsuntil they are wounded (George et al., 2008).

Bernier et al. (1993) reported that, the increased sucrose

supply to the meristem precedes the activation of energy-consuming processes such as mitotic activation and thus doesnot result from a higher demand by the meristem. This sug-

gests a message-like role for sucrose.Meristems that would normally produce flowers or floral

parts can be induced to give vegetative shoots in vitro and is

reported in the literature. The exact origin of the shoots pro-duced is not always known but histological studies can assistin determination of origin (Guerra and Handro, 1998; Loutfi

and Chlyah, 1998).

132 E.M.M. Zayed et al.

In conclusion, an efficient and rapid protocol for plantregeneration from mature female flower of date palm trees ispossible by reversion mature flower to meristematic state.

The type and appropriate concentration of plant growth regu-lators is essential for success of the reversion process. In addi-tion, the most intensive induced callus and direct

organogenesis formation are noted with ABA.

References

Abahmane, L., 2011. Date palm micropropagation via organogenesis.

In: Jain, S.M. et al. (Eds.), Date Palm Biotechnology, pp. 69–90.

Altman, A., Goren, R., 1974. Growth and dormancy cycles in Citrus

bud cultures and their hormonal control. Physiol. Plant. 30, 240–

245.

Ammirato, P.V., 1988. Role of ABA in regulation of somatic

embryogenesis. HortScience 23, 520.

Battey, N.H., Lyndon, R.F., 1990. Reversion of flowering. Bot. Rev.

56, 162–189.

Bernier, G., Havelange, A., Houssa, C., Petitjean, A., Lejeune, P.,

1993. Physiological signals that induce flowering. Plant Cell 5,

1147–1155.

Bhaskaran, S., Smith, R., 1990. Regeneration in cereal tissue culture: a

review. Crop Sci. 30, 1328–1336.

Bhaskaran, S., Smith, R., 1992. Somatic embryogenesis from shoot tip

and immature inflorescence of Phoenix dactylifera L. cv. Barhee.

Plant Cell Rep. 12, 22–25.

Browning, G., Kuden, A., Blanke, P., 1992. Site of (2RS, 3RS)

Paclobutrazol promotion of axillary flower initiation in Pear cv.

Doyenne du Comice. J. Hort. Sci. 67 (1), 121–128.

Catarina, C.S., Randi, A.M., Viana, A.M., 2003. Growth and

accumulation of storage reserves by somatic embryos of Ocotea

catharinesisMez. (Lauraceae). Plant Cell Tiss. Org. Cult. 74, 67–71.

Chen, J.T., Chang, W.C., 2003. Effects of GA3, ancymidol, cycocel

and paclobutrazol on direct somatic embryogenesis of Oncidium

in vitro. Plant Cell Tiss. Org. Cult. 72, 105–108.

Dmitry, M., Filippov, M., Dolgov, S., 2009. Effects of daminozide on

somatic embryogenesis from immature and mature embryos of

wheat. Aust. J. Crop Sci. 3 (2), 83–94.

Drira, N., Benbadis, A., 1985. Vegetative multiplication of date palm

(Phoenix dactylifera L.) by reversion of in vitro cultured female

flower buds. J. Plant Physiol. 119 (3), 227–235.

Ezura, H., Harberd, N.P., 1995. Endogenous gibberellin levels

influente in vitro shoot regeneration in Arabidopsis thaliana (L.)

Heynh. Planta 197, 301–305.

Feng, X.R., Wolyn, D.J., 1993. High frequency production of

haploid embryos in asparagus anther culture Plant Cell Rep. 12,

281–285.

Fki, L., Bouaziza, N., Kriaa, W., Masmoudia, R.B., Gargouri-

Bouzidb, R., Rivalc, A., Driraa, N., 2011. Multiple bud cultures of

‘Barhee’ date palm (Phoenix dactylifera) and physiological status of

regenerated plants. J. Plant Physiol. 168, 1694–1700.

Fletcher, R.A., Gilley, A., Sankhla, N., Davis, T.D., 2000. Triazoles as

plant growth retardants and stress protectants. Horticult. Rev. 24,

55–138.

Fortes, A.M., Pais, M.S., 2000. Organogenesis from internode-derived

nodules of Humulus lupulus var. Nugget (Cannabinaceae): histo-

logical studies and changes in the starch content. Am. J. Bot. 87 (7),

971–979.

Gehlot, H.S., Upadhaya, A., Davis, T.D., Sankhla, N., 1989. Growth

and organogenesis is moth bean callus was affected by paclobu-

trazol. Plant Cell Physiol. 30, 933–936.

George, E.F., Hall, M.A., De Klerk, G.J.K., 2008. Plant Propagation

by Tissue Culture, third ed. Springer, Basingstoke, UK, pp. 227–

238.

Ghosh, A., Chikara, J., Chaudhary, D.R., Prakash, A.R., Boricha, G.,

Zala, A., 2010. Paclobutrazol arrests vegetative growth and unveils

unexpressed yield potential of Jatropha curca. J. Plant Growth

Regul. 29, 307–315.

Gomes da Cunha, A.C., Ferreira, A., 1996. Somatic embryogenesis,

organogenesis and callus growth kinetics of flax. Plant Cell, Tiss.

Org. Cult. 47, 1–8.

Grambow, H.J., Langenbeck-Schwich, B., 1983. The relationship

between oxidase activity, peroxidase activity, hydrogen peroxide

and phenolic compounds in the degradation of indole-3-acetic acid

in vitro. Planta 157, 131–137.

Guerra, M.P., Handro, W., 1998. Somatic embryogenesis and plant

regeneration in different organs of Euterpe edulis Mart. (Palmae) –

control and structural features. J. Plant. Res. 111 (1101), 65–71.

Hamama, L., Voisine, L., Naouar, A., Gala, R., Cesbron, D., Michel,

G., Leplat, F., Foucher, Fabrice, Hibrand-Saint Oyant, Laurence,

Dorion, N., 2012. Effect of GA3 and paclobutrazol on adventitious

shoot regeneration of two Pelargonium sp. Acta Hortic., 187–194

Heide, O.M., 1968. Stimulation of adventitious bud formation in

Begonia leaves by abscisic acid Nature 219, 960–961

Higgins, T.J.V., Jacobsen, J.V., Zwar, J.A., 1982. Gibberellic acid and

abscisic acid modulate protein synthesis and mRNA levels in barley

aleurone layers. Plant Mol. Biol. 1, 191–215.

Hutchinson, M.J., Krishna, R.S., Saxena, P.K., 1997. Inhibitory effect

of GA3 on the development of thidiazuron-induced somatic

embryogenesis in geranium (Pelargonium � hortorum Bailey) hypo-

cotyl cultures. Plant Cell Rep. 16, 435–438.

Isikawa, H., 1974. In vitro formation of adventitious buds and roots on

the hypocotyls of Cryptomeria japonica. Bot. Mag. 87, 73–77.

Jain, S.M., Al-Khayri, J.M., Johnson, D.V., 2011. Date Palm

Biotechnology. Springer.

Jimenez, V.M., Bangerth, F., 2001. Endogenous hormone levels in

explants and in embryogenic and non-embryogenic cultures of

carrot. Physiol. Plant. 111, 389–395.

Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill Book Co.,

New York, pp. 126–156.

Johnson, K.A., 1996. The application of in vitro technology to

Australian plants (with particular emphasis on the nutritional

requirements). In: Taji, A.M., Williams, R.R. (Eds.), Tissue

Culture of Australian Plants: Past, Present and Future. University

of New England Press, Armidale, Australia, pp. 16–55 (Chapter 2).

Kriaa, W., Sghaier, B., Masmoudi, F., Benjemaa, R., Driraa, N., 2012.

The date palm (Phoenix dactylifera L.) micropropagation using

completely mature female flowers. C.R. Biol. 335, 194–204.

Kuol, C.L., Sagare, A.P., Lo, S.F., Lee, C.Y., Chen, C.C., Tsay, H.S.,

2002. Abscisic acid promotes development of somatic embryos on

converted somatic embryos of Corydalis yanhusuo (Fumariaceae). J.

Plant Physiol. 159, 423–427.

Larsen, P., Harbo, A., Klungsour, S., Asheim, T.C., 1962. On the

biogenesis of some indole compounds in Acetobacter xylinum.

Physiol. Plant. 15, 552–655.

Lee, T.T., 1980. Effects of phenolic substances on metabolism of

exogenous indole-3-acetic acid in maize stems. Physiol. Plant. 50,

107–112.

Lee, T.T., Skoog, F., 1965. Effects of hydroxybenzoic acids on indole

acetic acid inactivation by tobacco callus extracts. Physiol. Plant.

18, 577–585.

Liu, M.C., 1993. Factors affecting induction, somatic embryogenesis

and plant regeneration of callus from cultured immature inflores-

cences of sugarcane. J. Plant Physiol. 141, 714–720.

Loutfi, K., Chlyah, H., 1998. Vegetative multiplication of date palms

from in vitro cultured inflorescences: effect of some growth

regulator combinations and organogenetic potential of various

cultivars. Agronomie 18 (8–9), 573–580.

Malik, E.P., Singh, M.B., 1980. Plant Enzymology and Histoenzy-

mology, first ed. Kalyani Publishers, New Delhi, p. 286.

Marshall, J.G., Rutledge, R.G., Blumwald, E., Dumbroff, E.B., 2000.

Reduction in turgid water volume in jack pine, white spruce and

black spruce in response to drought and paclobutrazol. Tree

Physiol. 20, 701–707.

Floral reversion of mature female inflorescence of date palm in vitro 133

Martin, L., Dorado, A., Villalobes, 2000. Differences in the contents of

total sugars, reducing sugars, starch and sucrose in embryogenic

and non-embryogenic calli from Medicago orborea L. Plant Sci.

154, 143–151.

Murashige, T., Skoog, F.A., 1962. A revised medium for rapid growth

and bioassays with tobacco tissue cultures Physiol. Plant. 15, 473–

479.

Pottino, B.G., 1981. Methods in Plant Tissue Culture. Dept. of Hort.,

Agric. College, Maryland Univ., Collage Park, Maryland, USA,

pp. 8–29.

Pouteau, S., Nicholls, D., Tooke, F., Coen, E., Battey, N., 1997. The

induction and maintenance of flowering in Impatiens. Development

124, 3343–3351.

Rademacher, A., 2000. Growth retardants effects on gibberellin

biosynthesis and other metabolic pathways. Annu. Rev. Plant

Physiol. Plant Mol. Biol. 51, 501–531.

Saftner, R.A., Wyes, R.E., 1984. Effect of plant hormones on sucrose

uptake by beet root tissue discs. Plant Physiol. 74, 951–955.

Sankhla, N., Davis, T.D., Gehlot, H.S., Upadhyaya, A., Sankhla, A.,

Sankhla, D., 1991. Growth and organogenesis in moth bean callus

cultures as influenced by triazole growth regulators and gibberellic

acid. J. Plant Growth Regul. 10 (1), 41–45.

Sass, J.E., 1951. Botanical Microtechnique, second ed. The Iowa State

College Press, pp. 5–77.

Senaratna, T., Bunn, E., Bishop, A., 2001. Triazole treatment of

explant source provides stress tolerance in progeny of Geranium

(Pelargonium hortorum Bailey) plants regenerated by somatic

embryogenesis. Plant Growth Regul. 36, 169–174.

Shales, O., Schales, S.S., 1945. A simple method for the determination

of glucose in the blood. Arch. Biochem. Biophys. 8, 285–292.

Shepard, J.F., 1980. Abscisic acid enhanced shoot initiation in

protoplast-derived calli of potato. Plant Sci. Lett. 18 (4), 327–333.

Smith, D.L., Aynsley, J.S., 1995. Field performance of tissue cultured

date palm (Phoenix dactylifera) clonally produced by somatic

embryogenesis. Principes 39, 47–52.

Snedecor, G.W., Cochran, W.C., 1989. Statistical Methods, eighth ed.

Iowa State University, Press, USA, p. 297.

Te-chato, S., Nujeen, P., Muangsorn, S., 2009. Paclobutrazol enhance

budbreak and flowering of Friederick’s dendrobium orchid in vitro.

J. Agric. Technol. 5 (1), 157–165.

Timmis, R., Ritchie, G.A., 1988. Epigenetic effects in clonal propa-

gation. In: Proc. Tenth North American For. Biol. Workshop,

Vancouver, BC, pp. 12–31.

Wang, L., Lin, C., 1992. The effect of paclobutrazol on physiological

and biochemical changes in the primary roots of pea. J. Exp. Bot.

43 (255), 1367–1372.

Wang, S.Y., Steffens, G.L., 1987. Effect of paclobutrazol on accumu-

lation of organic acids and total phenols in apple wood. J. Plant

Growth Regul. 6, 209–213.

Williams, R.R., Taji, A.M., Bolton, J.A., 1985. Specificity and

interaction among auxins, light and pH in rooting Australian

woody species in vitro. HortScience 20, 1052–1053.

Yates, P.J., Lenton, J.R., Goad, J.L., 1993. Effects of triazole inhibitors

of sterol biosynthesis on the fare and esterified sterol composition of

celery cell suspension cultures. Pestic. Sci. 39, 257–265.

Yoshida, S., 2003. Molecular regulation of leaf senescence. Curr. Opin.

Plant Biol. 6, 79–84.

Zayed, E.M.M., 2011. Propagation of Phoenix dactylifera L.,Chamaer-

ops humilis L. and Hyophorbe verschaffeltii L. palms by using tissue

culture technique. Ph.D. Thesis, Department of Horticulture,

Faculty of Agriculture, Cairo University, Egypt, pp. 130–136.

Zayed, E.M.M., Abdelbar, O.H., 2015. Morphogenesis of immature

female inflorescences of date palm in vitro. Ann. Agric. Sci. 60 (1),

113–120.